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Sample records for transcriptional activator proteins

  1. Adenovirus DNA binding protein inhibits SrCap-activated CBP and CREB-mediated transcription

    International Nuclear Information System (INIS)

    Xu Xiequn; Tarakanova, Vera; Chrivia, John; Yaciuk, Peter

    2003-01-01

    The SNF2-related CBP activator protein (SrCap) is a potent activator of transcription mediated by CBP and CREB. We have previously demonstrated that the Adenovirus 2 DNA Binding Protein (DBP) binds to SrCap and inhibits the transcription mediated by the carboxyl-terminal region of SrCap (amino acids 1275-2971). We report here that DBP inhibits the ability of full-length SrCap (1-2971) to activate transcription mediated by Gal-CREB and Gal-CBP. In addition, DBP also inhibits the ability of SrCap to enhance Protein Kinase A (PKA) activated transcription of the enkaphalin promoter. DBP was found to dramatically inhibit transcription of a mammalian two-hybrid system that was dependent on the interaction of SrCap and CBP binding domains. We also found that DBP has no effect on transcription mediated by a transcriptional activator that is not related to SrCap, indicating that our reported transcriptional inhibition is specific for SrCap and not due to nonspecific effects of DBP's DNA binding activity on the CAT reporter plasmid. Taken together, these results suggest a model in which DBP inhibits cellular transcription mediated by the interaction between SrCap and CBP

  2. Transcriptional activation of peroxisome proliferator-activated receptor-γ requires activation of both protein kinase A and Akt during adipocyte differentiation

    International Nuclear Information System (INIS)

    Kim, Sang-pil; Ha, Jung Min; Yun, Sung Ji; Kim, Eun Kyoung; Chung, Sung Woon; Hong, Ki Whan; Kim, Chi Dae; Bae, Sun Sik

    2010-01-01

    Research highlights: → Elevated cAMP activates both PKA and Epac. → PKA activates CREB transcriptional factor and Epac activates PI3K/Akt pathway via Rap1. → Akt modulates PPAR-γ transcriptional activity in concert with CREB. -- Abstract: Peroxisome proliferator-activated receptor-γ (PPAR-γ) is required for the conversion of pre-adipocytes. However, the mechanism underlying activation of PPAR-γ is unclear. Here we showed that cAMP-induced activation of protein kinase A (PKA) and Akt is essential for the transcriptional activation of PPAR-γ. Hormonal induction of adipogenesis was blocked by a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002), by a protein kinase A (PKA) inhibitor (H89), and by a Rap1 inhibitor (GGTI-298). Transcriptional activity of PPAR-γ was markedly enhanced by 3-isobutyl-1-methylxanthine (IBMX), but not insulin and dexamethasone. In addition, IBMX-induced PPAR-γ transcriptional activity was blocked by PI3K/Akt, PKA, or Rap1 inhibitors. 8-(4-Chlorophenylthio)-2'-O-methyl-cAMP (8-pCPT-2'-O-Me-cAMP) which is a specific agonist for exchanger protein directly activated by cAMP (Epac) significantly induced the activation of Akt. Furthermore, knock-down of Akt1 markedly attenuated PPAR-γ transcriptional activity. These results indicate that both PKA and Akt signaling pathways are required for transcriptional activation of PPAR-γ, suggesting post-translational activation of PPAR-γ might be critical step for adipogenic gene expression.

  3. The hepatitis B virus large surface protein (LHBs) is a transcriptional activator.

    Science.gov (United States)

    Hildt, E; Saher, G; Bruss, V; Hofschneider, P H

    1996-11-01

    It has been shown that a C-terminally truncated form of the middle-sized hepatitis B virus (HBV) surface protein (MHBst) functions as a transcriptional activator. This function is dependent on the cytosolic orientation of the N-terminal PreS2 domain of MHBst, but in the case of wild-type MHBs, the PreS2 domain is contranslationally translocated into the ER lumen. Recent reports demonstrated that the PreS2 domain of the large HBV surface protein (LHBs) initially remains on the cytosolic side of the ER membrane after translation. Therefore, the question arose as to whether the LHBs protein exhibits the same transcriptional activator function as MHBst. We show that LHBs, like MHBst, is indeed able to activate a variety of promoter elements. There is evidence for a PKC-dependent activation of AP-1 and NF-kappa B by LHBs. Downstream of the PKC the functionality of c-Raf-1 kinase is a prerequisite for LHBs-dependent activation of AP-1 and NF-kappa B since inhibition of c-Raf-1 kinase abolishes LHBs-dependent transcriptional activation of AP-1 and NF-kappa B.

  4. Transcription Factor Functional Protein-Protein Interactions in Plant Defense Responses

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    Murilo S. Alves

    2014-03-01

    Full Text Available Responses to biotic stress in plants lead to dramatic reprogramming of gene expression, favoring stress responses at the expense of normal cellular functions. Transcription factors are master regulators of gene expression at the transcriptional level, and controlling the activity of these factors alters the transcriptome of the plant, leading to metabolic and phenotypic changes in response to stress. The functional analysis of interactions between transcription factors and other proteins is very important for elucidating the role of these transcriptional regulators in different signaling cascades. In this review, we present an overview of protein-protein interactions for the six major families of transcription factors involved in plant defense: basic leucine zipper containing domain proteins (bZIP, amino-acid sequence WRKYGQK (WRKY, myelocytomatosis related proteins (MYC, myeloblastosis related proteins (MYB, APETALA2/ ETHYLENE-RESPONSIVE ELEMENT BINDING FACTORS (AP2/EREBP and no apical meristem (NAM, Arabidopsis transcription activation factor (ATAF, and cup-shaped cotyledon (CUC (NAC. We describe the interaction partners of these transcription factors as molecular responses during pathogen attack and the key components of signal transduction pathways that take place during plant defense responses. These interactions determine the activation or repression of response pathways and are crucial to understanding the regulatory networks that modulate plant defense responses.

  5. Hepatitis C virus core protein regulates p300/CBP co-activation function. Possible role in the regulation of NF-AT1 transcriptional activity

    International Nuclear Information System (INIS)

    Gomez-Gonzalo, Marta; Benedicto, Ignacio; Carretero, Marta; Lara-Pezzi, Enrique; Maldonado-Rodriguez, Alejandra; Moreno-Otero, Ricardo; Lai, Michael M.C.; Lopez-Cabrera, Manuel

    2004-01-01

    Hepatitis C virus (HCV) core is a viral structural protein; it also participates in some cellular processes, including transcriptional regulation. However, the mechanisms of core-mediated transcriptional regulation remain poorly understood. Oncogenic virus proteins often target p300/CBP, a known co-activator of a wide variety of transcription factors, to regulate the expression of cellular and viral genes. Here we demonstrate, for the first time, that HCV core protein interacts with p300/CBP and enhances both its acetyl-transferase and transcriptional activities. In addition, we demonstrate that nuclear core protein activates the NH 2 -terminal transcription activation domain (TAD) of NF-AT1 in a p300/CBP-dependent manner. We propose a model in which core protein regulates the co-activation function of p300/CBP and activates NF-AT1, and probably other p300/CBP-regulated transcription factors, by a novel mechanism involving the regulation of the acetylation state of histones and/or components of the transcriptional machinery

  6. Identification of the G13 (cAMP-response-element-binding protein-related protein) gene product related to activating transcription factor 6 as a transcriptional activator of the mammalian unfolded protein response.

    Science.gov (United States)

    Haze, K; Okada, T; Yoshida, H; Yanagi, H; Yura, T; Negishi, M; Mori, K

    2001-04-01

    Eukaryotic cells control the levels of molecular chaperones and folding enzymes in the endoplasmic reticulum (ER) by a transcriptional induction process termed the unfolded protein response (UPR). The mammalian UPR is mediated by the cis-acting ER stress response element consisting of 19 nt (CCAATN(9)CCACG), the CCACG part of which is considered to provide specificity. We recently identified the basic leucine zipper (bZIP) protein ATF6 as a mammalian UPR-specific transcription factor; ATF6 is activated by ER stress-induced proteolysis and binds directly to CCACG. Here we report that eukaryotic cells express another bZIP protein closely related to ATF6 in both structure and function. This protein encoded by the G13 (cAMP response element binding protein-related protein) gene is constitutively synthesized as a type II transmembrane glycoprotein anchored in the ER membrane and processed into a soluble form upon ER stress as occurs with ATF6. The proteolytic processing of ATF6 and the G13 gene product is accompanied by their relocation from the ER to the nucleus; their basic regions seem to function as a nuclear localization signal. Overexpression of the soluble form of the G13 product constitutively activates the UPR, whereas overexpression of a mutant lacking the activation domain exhibits a strong dominant-negative effect. Furthermore, the soluble forms of ATF6 and the G13 gene product are unable to bind to several point mutants of the cis-acting ER stress response element in vitro that hardly respond to ER stress in vivo. We thus concluded that the two related bZIP proteins are crucial transcriptional regulators of the mammalian UPR, and propose calling the ATF6 gene product ATF6alpha and the G13 gene product ATF6beta.

  7. AMP-Activated Protein Kinase Directly Phosphorylates and Destabilizes Hedgehog Pathway Transcription Factor GLI1 in Medulloblastoma

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    Yen-Hsing Li

    2015-07-01

    Full Text Available The Hedgehog (Hh pathway regulates cell differentiation and proliferation during development by controlling the Gli transcription factors. Cell fate decisions and progression toward organ and tissue maturity must be coordinated, and how an energy sensor regulates the Hh pathway is not clear. AMP-activated protein kinase (AMPK is an important sensor of energy stores and controls protein synthesis and other energy-intensive processes. AMPK is directly responsive to intracellular AMP levels, inhibiting a wide range of cell activities if ATP is low and AMP is high. Thus, AMPK can affect development by influencing protein synthesis and other processes needed for growth and differentiation. Activation of AMPK reduces GLI1 protein levels and stability, thus blocking Sonic-hedgehog-induced transcriptional activity. AMPK phosphorylates GLI1 at serines 102 and 408 and threonine 1074. Mutation of these three sites into alanine prevents phosphorylation by AMPK. This leads to increased GLI1 protein stability, transcriptional activity, and oncogenic potency.

  8. ZNF328, a novel human zinc-finger protein, suppresses transcriptional activities of SRE and AP-1

    International Nuclear Information System (INIS)

    Ou Ying; Wang Shenqiu; Cai Zhenyu; Wang Yuequn; Wang Canding; Li Yongqing; Li Fang; Yuan Wuzhou; Liu Bisheng; Wu Xiushan; Liu Mingyao

    2005-01-01

    The zinc finger proteins containing the Kruppel-associated box domain (KRAB-ZFPs) are the single largest class of transcription factors in human genome. Many of the KRAB-ZFPs are involved in cardiac development or cardiovascular diseases. Here, we have identified a novel human KRAB zinc finger gene, named ZNF328, from the human fetal heart cDNA library. The complete sequence of ZNF328 cDNA contains a 2376-bp open reading frame (ORF) and encodes a 792 amino acid protein with an N-terminal KRAB domain and classical zinc finger C 2 H 2 motifs in the C-terminus. Northern blot analysis indicates that the protein is expressed in most of the examined human adult and embryonic tissues. ZNF328 is a transcription suppressor when fused to Gal-4 DNA-binding domain and cotransfected with VP-16. Overexpression of ZNF328 in COS-7 cells inhibits the transcriptional activities of SRE and AP-1. Deletion analysis with a series of truncated fusion proteins indicates that the KRAB motif is a basal repression domain when cotransfected with VP-16. Similar results were obtained when the truncated fusion proteins were assayed for the transcriptional activities of SRE and AP-1. These results suggest that ZNF328 protein may act as a transcriptional repressor in mitogen-activated protein kinase (MAPK) signaling pathway to mediate cellular functions

  9. In vitro activation of transcription by the human T-cell leukemia virus type I Tax protein.

    Science.gov (United States)

    Matthews, M A; Markowitz, R B; Dynan, W S

    1992-05-01

    The human T-cell leukemia virus type I (HTLV-I) regulatory protein Tax activates transcription of the proviral long terminal repeats and a number of cellular promoters. We have developed an in vitro system to characterize the mechanism by which Tax interacts with the host cell transcription machinery. Tax was purified from cells infected with a baculovirus expression vector. Addition of these Tax preparations to nuclear extracts from uninfected human T lymphocytes activated transcription of the HTLV-I long terminal repeat approximately 10-fold. Transcription-stimulatory activity copurified with the immunoreactive 40-kDa Tax polypeptide on gel filtration chromatography, and, as expected, the effect of recombinant Tax was diminished in HTLV-I-infected T-lymphocyte extracts containing endogenous Tax. Tax-mediated transactivation in vivo has been previously shown to require 21-bp-repeat Tax-responsive elements (TxREs) in the promoter DNA. Stimulation of transcription in vitro was also strongly dependent on these sequences. To investigate the mechanism of Tax transactivation, cellular proteins that bind the 21-bp-repeat TxREs were prepared by DNA affinity chromatography. Recombinant Tax markedly increased the formation of a specific host protein-DNA complex detected in an electrophoretic mobility shift assay. These data suggest that Tax activates transcription through a direct interaction with cellular proteins that bind to the 21-bp-repeat TxREs.

  10. Hepatitis C virus nonstructural protein-5A activates sterol regulatory element-binding protein-1c through transcription factor Sp1

    Energy Technology Data Exchange (ETDEWEB)

    Xiang, Zhonghua; Qiao, Ling; Zhou, Yan [Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E3 (Canada); Babiuk, Lorne A. [University of Alberta, Edmonton, Alberta (Canada); Liu, Qiang, E-mail: qiang.liu@usask.ca [Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E3 (Canada)

    2010-11-19

    Research highlights: {yields} A chimeric subgenomic HCV replicon expresses HCV-3a NS5A in an HCV-1b backbone. {yields} HCV-3a NS5A increases mature SREBP-1c protein level. {yields} HCV-3a NS5A activates SREBP-1c transcription. {yields} Domain II of HCV-3a NS5A is more effective in SREBP-1c promoter activation. {yields} Transcription factor Sp1 is required for SREBP-1c activation by HCV-3a NS5A. -- Abstract: Steatosis is an important clinical manifestation of hepatitis C virus (HCV) infection. The molecular mechanisms of HCV-associated steatosis are not well understood. Sterol regulatory element-binding protein-1c (SREBP-1c) is a key transcription factor which activates the transcription of lipogenic genes. Here we showed that the nuclear, mature SREBP-1c level increases in the nucleus of replicon cells expressing HCV-3a nonstructural protein-5A (NS5A). We further showed that HCV-3a NS5A up-regulates SREBP-1c transcription. Additional analysis showed that transcriptional factor Sp1 is involved in SREBP-1c activation by HCV-3a NS5A because inhibition of Sp1 activity by mithramycin A or a dominant-negative Sp1 construct abrogated SREBP-1c promoter activation by HCV-3a NS5A. In addition, chromatin immunoprecipitation (ChIP) assay demonstrated enhanced binding of Sp1 on the SREBP-1c promoter in HCV-3a NS5A replicon cells. These results showed that HCV-3a NS5A activates SREBP-1c transcription through Sp1. Taken together, our results suggest that HCV-3a NS5A is a contributing factor for steatosis caused by HCV-3a infection.

  11. Hepatitis C virus nonstructural protein-5A activates sterol regulatory element-binding protein-1c through transcription factor Sp1

    International Nuclear Information System (INIS)

    Xiang, Zhonghua; Qiao, Ling; Zhou, Yan; Babiuk, Lorne A.; Liu, Qiang

    2010-01-01

    Research highlights: → A chimeric subgenomic HCV replicon expresses HCV-3a NS5A in an HCV-1b backbone. → HCV-3a NS5A increases mature SREBP-1c protein level. → HCV-3a NS5A activates SREBP-1c transcription. → Domain II of HCV-3a NS5A is more effective in SREBP-1c promoter activation. → Transcription factor Sp1 is required for SREBP-1c activation by HCV-3a NS5A. -- Abstract: Steatosis is an important clinical manifestation of hepatitis C virus (HCV) infection. The molecular mechanisms of HCV-associated steatosis are not well understood. Sterol regulatory element-binding protein-1c (SREBP-1c) is a key transcription factor which activates the transcription of lipogenic genes. Here we showed that the nuclear, mature SREBP-1c level increases in the nucleus of replicon cells expressing HCV-3a nonstructural protein-5A (NS5A). We further showed that HCV-3a NS5A up-regulates SREBP-1c transcription. Additional analysis showed that transcriptional factor Sp1 is involved in SREBP-1c activation by HCV-3a NS5A because inhibition of Sp1 activity by mithramycin A or a dominant-negative Sp1 construct abrogated SREBP-1c promoter activation by HCV-3a NS5A. In addition, chromatin immunoprecipitation (ChIP) assay demonstrated enhanced binding of Sp1 on the SREBP-1c promoter in HCV-3a NS5A replicon cells. These results showed that HCV-3a NS5A activates SREBP-1c transcription through Sp1. Taken together, our results suggest that HCV-3a NS5A is a contributing factor for steatosis caused by HCV-3a infection.

  12. The Transcriptional Repressive Activity of KRAB Zinc Finger Proteins Does Not Correlate with Their Ability to Recruit TRIM28.

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    Kristin E Murphy

    Full Text Available KRAB domain Zinc finger proteins are one of the most abundant families of transcriptional regulators in higher vertebrates. The prevailing view is that KRAB domain proteins function as potent transcriptional repressors by recruiting TRIM28 and promoting heterochromatin spreading. However, the extent to which all KRAB domain proteins are TRIM28-dependent transcriptional repressors is currently unclear. Our studies on mouse ZFP568 revealed that TRIM28 recruitment by KRAB domain proteins is not sufficient to warrant transcriptional repressive activity. By using luciferase reporter assays and yeast two-hybrid experiments, we tested the ability of ZFP568 and other mouse KRAB domain proteins to repress transcription and bind TRIM28. We found that some mouse KRAB domain proteins are poor transcriptional repressors despite their ability to recruit TRIM28, while others showed strong KRAB-dependent transcriptional repression, but no TRIM28 binding. Together, our results show that the transcriptional repressive activity of KRAB-ZNF proteins does not correlate with their ability to recruit TRIM28, and provide evidence that KRAB domains can regulate transcription in a TRIM28-independent fashion. Our findings challenge the current understanding of the molecular mechanisms used by KRAB domain proteins to control gene expression and highlight that a high percentage of KRAB domain proteins in the mouse genome differ from the consensus KRAB sequence at amino acid residues that are critical for TRIM28 binding and/or repressive activity.

  13. Ménage à trois: the complex relationships between mitogen-activated protein kinases, WRKY transcription factors, and VQ-motif-containing proteins.

    Science.gov (United States)

    Weyhe, Martin; Eschen-Lippold, Lennart; Pecher, Pascal; Scheel, Dierk; Lee, Justin

    2014-01-01

    Out of the 34 members of the VQ-motif-containing protein (VQP) family, 10 are phosphorylated by the mitogen-activated protein kinases (MAPKs), MPK3 and MPK6. Most of these MPK3/6-targeted VQPs (MVQs) interacted with specific sub-groups of WRKY transcription factors in a VQ-motif-dependent manner. In some cases, the MAPK appears to phosphorylate either the MVQ or the WRKY, while in other cases, both proteins have been reported to act as MAPK substrates. We propose a network of dynamic interactions between members from the MAPK, MVQ and WRKY families - either as binary or as tripartite interactions. The compositions of the WRKY-MVQ transcriptional protein complexes may change - for instance, through MPK3/6-mediated modulation of protein stability - and therefore control defense gene transcription.

  14. RNA-binding proteins involved in post-transcriptional regulation in bacteria

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    Elke eVan Assche

    2015-03-01

    Full Text Available Post-transcriptional regulation is a very important mechanism to control gene expression in changing environments. In the past decade, a lot of interest has been directed towards the role of small RNAs in bacterial post-transcriptional regulation. However, small RNAs are not the only molecules controlling gene expression at this level, RNA-binding proteins play an important role as well. CsrA and Hfq are the two best studied bacterial proteins of this type, but recently, additional proteins involved in post-transcriptional control have been identified. This review focuses on the general working mechanisms of post-transcriptionally active RNA-binding proteins, which include (i adaptation of the susceptibility of mRNAs and sRNAs to RNases, (ii modulating the accessibility of the ribosome binding site of mRNAs, (iii recruiting and assisting in the interaction of mRNAs with other molecules and (iv regulating transcription terminator / antiterminator formation, and gives an overview of both the well-studied and the newly identified proteins that are involved in post-transcriptional regulatory processes. Additionally, the post-transcriptional mechanisms by which the expression or the activity of these proteins is regulated, are described. For many of the newly identified proteins, however, mechanistic questions remain. Most likely, more post-transcriptionally active proteins will be identified in the future.

  15. Transcriptional switching by the MerR protein: Activation and repression mutants implicate distinct DNA and mercury(II) binding domains

    International Nuclear Information System (INIS)

    Shewchuk, L.M.; Helmann, J.D.; Ross, W.; Park, S.J.; Summers, A.O.; Walsh, C.T.

    1989-01-01

    Bacterial resistance to mercuric compounds is controlled by the MerR metalloregulatory protein. The MerR protein functions as both a transcriptional repressor and a mercuric ion dependent transcriptional activator. Chemical mutagenesis of the cloned merR structural gene has led to the identification of mutant proteins that are specifically deficient in transcriptional repression, activation, or both. Five mutant proteins have been overproduced, purified to homogeneity, and assayed for ability to dimerize, bind mer operator DNA, and bind mercuric ion. A mutation in the recognition helix of a proposed helix-turn-helix DNA binding motif (E22K) yields protein deficient in both activation and repression in vivo (a - r - ) and deficient in operator binding in vitro. In contrast, mutations in three of the four MerR cysteine residues are repression competent but activation deficient (a - r + ) in vivo. In vitro, the purified cysteine mutant proteins bind to the mer operator site with near wild-type affinity but are variable deficient in binding the in vivo inducer mercury(II) ion. A subset of the isolated proteins also appears compromised in their ability to form dimers at low protein concentrations. These data support a model in which DNA-bound MerR dimer binds one mercuric ion and transmits this occupancy information to a protein region involved in transcriptional activation

  16. Mutant Forms of the Azotobacter vinelandii Transcriptional Activator NifA Resistant to Inhibition by the NifL Regulatory Protein

    OpenAIRE

    Reyes-Ramirez, Francisca; Little, Richard; Dixon, Ray

    2002-01-01

    The Azotobacter vinelandii σ54-dependent transcriptional activator protein NifA is regulated by the NifL protein in response to redox, carbon, and nitrogen status. Under conditions inappropriate for nitrogen fixation, NifL inhibits transcription activation by NifA through the formation of the NifL-NifA protein complex. NifL inhibits the ATPase activity of the central AAA+ domain of NifA required to drive open complex formation by σ54-RNA polymerase and may also inhibit the activator-polymeras...

  17. ReTrOS: a MATLAB toolbox for reconstructing transcriptional activity from gene and protein expression data.

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    Minas, Giorgos; Momiji, Hiroshi; Jenkins, Dafyd J; Costa, Maria J; Rand, David A; Finkenstädt, Bärbel

    2017-06-26

    Given the development of high-throughput experimental techniques, an increasing number of whole genome transcription profiling time series data sets, with good temporal resolution, are becoming available to researchers. The ReTrOS toolbox (Reconstructing Transcription Open Software) provides MATLAB-based implementations of two related methods, namely ReTrOS-Smooth and ReTrOS-Switch, for reconstructing the temporal transcriptional activity profile of a gene from given mRNA expression time series or protein reporter time series. The methods are based on fitting a differential equation model incorporating the processes of transcription, translation and degradation. The toolbox provides a framework for model fitting along with statistical analyses of the model with a graphical interface and model visualisation. We highlight several applications of the toolbox, including the reconstruction of the temporal cascade of transcriptional activity inferred from mRNA expression data and protein reporter data in the core circadian clock in Arabidopsis thaliana, and how such reconstructed transcription profiles can be used to study the effects of different cell lines and conditions. The ReTrOS toolbox allows users to analyse gene and/or protein expression time series where, with appropriate formulation of prior information about a minimum of kinetic parameters, in particular rates of degradation, users are able to infer timings of changes in transcriptional activity. Data from any organism and obtained from a range of technologies can be used as input due to the flexible and generic nature of the model and implementation. The output from this software provides a useful analysis of time series data and can be incorporated into further modelling approaches or in hypothesis generation.

  18. The 42-kDa coat protein of Andean potato mottle virus acts as a transcriptional activator in yeast

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    Vidal M.S.

    2002-01-01

    Full Text Available Interactions of viral proteins play an important role in the virus life cycle, especially in capsid assembly. Andean potato mottle comovirus (APMoV is a plant RNA virus with a virion formed by two coat proteins (CP42 and CP22. Both APMoV coat protein open reading frames were cloned into pGBT9 and pGAD10, two-hybrid system vectors. HF7c yeast cells transformed with the p9CP42 construct grew on yeast dropout selection media lacking tryptophan and histidine. Clones also exhibited ß-galactosidase activity in both qualitative and quantitative assays. These results suggest that CP42 protein contains an amino acid motif able to activate transcription of His3 and lacZ reporter genes in Saccharomyces cerevisiae. Several deletions of the CP42 gene were cloned into the pGBT9 vector to locate the region involved in this activation. CP42 constructions lacking 12 residues from the C-terminal region and another one with 267 residues deleted from the N-terminus are still able to activate transcription of reporter genes. However, transcription activation was not observed with construction p9CP42deltaC57, which does not contain the last 57 amino acid residues. These results demonstrate that a transcription activation domain is present at the C-terminus of CP42 between residues 267 and 374.

  19. Protein kinase A activation enhances β-catenin transcriptional activity through nuclear localization to PML bodies.

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    Mei Zhang

    Full Text Available The Protein Kinase A (PKA and Wnt signaling cascades are fundamental pathways involved in cellular development and maintenance. In the osteoblast lineage, these pathways have been demonstrated functionally to be essential for the production of mineralized bone. Evidence for PKA-Wnt crosstalk has been reported both during tumorigenesis and during organogenesis, and the nature of the interaction is thought to rely on tissue and cell context. In this manuscript, we analyzed bone tumors arising from mice with activated PKA caused by mutation of the PKA regulatory subunit Prkar1a. In primary cells from these tumors, we observed relocalization of β-catenin to intranuclear punctuate structures, which were identified as PML bodies. Cellular redistribution of β-catenin could be recapitulated by pharmacologic activation of PKA. Using 3T3-E1 pre-osteoblasts as a model system, we found that PKA phosphorylation sites on β-catenin were required for nuclear re-localization. Further, β-catenin's transport to the nucleus was accompanied by an increase in canonical Wnt-dependent transcription, which also required the PKA sites. PKA-Wnt crosstalk in the cells was bi-directional, including enhanced interactions between β-catenin and the cAMP-responsive element binding protein (CREB and transcriptional crosstalk between the Wnt and PKA signaling pathways. Increases in canonical Wnt/β-catenin signaling were associated with a decrease in the activity of the non-canonical Wnt/Ror2 pathway, which has been shown to antagonize canonical Wnt signaling. Taken together, this study provides a new understanding of the complex regulation of the subcellular distribution of β-catenin and its differential protein-protein interaction that can be modulated by PKA signaling.

  20. Regulation of WRKY46 transcription factor function by mitogen-activated protein kinases in Arabidopsis thaliana.

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    Arsheed Hussain Sheikh

    2016-02-01

    Full Text Available AbstractMitogen-activated protein kinase (MAPK cascades are central signalling pathways activated in plants after sensing internal developmental and external stress cues. Knowledge about the downstream substrate proteins of MAPKs is still limited in plants. We screened Arabidopsis WRKY transcription factors as potential targets downstream of MAPKs, and concentrated on characterizing WRKY46 as a substrate of the MAPK, MPK3. Mass spectrometry revealed in vitro phosphorylation of WRKY46 at amino acid position S168 by MPK3. However, mutagenesis studies showed that a second phosphosite, S250, can also be phosphorylated. Elicitation with pathogen-associated molecular patterns (PAMPs, such as the bacterial flagellin-derived flg22 peptide led to in vivo destabilization of WRKY46 in Arabidopsis protoplasts. Mutation of either phosphorylation site reduced the PAMP-induced degradation of WRKY46. Furthermore, the protein for the double phosphosite mutant is expressed at higher levels compared to wild-type proteins or single phosphosite mutants. In line with its nuclear localization and predicted function as a transcriptional activator, overexpression of WRKY46 in protoplasts raised basal plant defence as reflected by the increase in promoter activity of the PAMP-responsive gene, NHL10, in a MAPK-dependent manner. Thus, MAPK-mediated regulation of WRKY46 is a mechanism to control plant defence.

  1. Cyclin D3 interacts with human activating transcription factor 5 and potentiates its transcription activity

    International Nuclear Information System (INIS)

    Liu Wenjin; Sun Maoyun; Jiang Jianhai; Shen Xiaoyun; Sun Qing; Liu Weicheng; Shen Hailian; Gu Jianxin

    2004-01-01

    The Cyclin D3 protein is a member of the D-type cyclins. Besides serving as cell cycle regulators, D-type cyclins have been reported to be able to interact with several transcription factors and modulate their transcriptional activations. Here we report that human activating transcription factor 5 (hATF5) is a new interacting partner of Cyclin D3. The interaction was confirmed by in vivo coimmunoprecipitation and in vitro binding analysis. Neither interaction between Cyclin D1 and hATF5 nor interaction between Cyclin D2 and hATF5 was observed. Confocal microscopy analysis showed that Cyclin D3 could colocalize with hATF5 in the nuclear region. Cyclin D3 could potentiate hATF5 transcriptional activity independently of its Cdk4 partner. But Cyclin D1 and Cyclin D2 had no effect on hATF5 transcriptional activity. These data provide a new clue to understand the new role of Cyclin D3 as a transcriptional regulator

  2. Selective activation of human heat shock gene transcription by nitrosourea antitumor drugs mediated by isocyanate-induced damage and activation of heat shock transcription factor.

    Science.gov (United States)

    Kroes, R A; Abravaya, K; Seidenfeld, J; Morimoto, R I

    1991-01-01

    Treatment of cultured human tumor cells with the chloroethylnitrosourea antitumor drug 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) selectively induces transcription and protein synthesis of a subset of the human heat shock or stress-induced genes (HSP90 and HSP70) with little effect on other stress genes or on expression of the c-fos, c-myc, or beta-actin genes. The active component of BCNU and related compounds appears to be the isocyanate moiety that causes carbamoylation of proteins and nucleic acids. Transcriptional activation of the human HSP70 gene by BCNU is dependent on the heat shock element and correlates with the level of heat shock transcription factor and its binding to the heat shock element in vivo. Unlike activation by heat or heavy metals, BCNU-mediated activation is strongly dependent upon new protein synthesis. This suggests that BCNU-induced, isocyanate-mediated damage to newly synthesized protein(s) may be responsible for activation of the heat shock transcription factor and increased transcription of the HSP90 and HSP70 genes. Images PMID:2052560

  3. Potential Role of Activating Transcription Factor 5 during Osteogenesis

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    Luisa Vicari

    2016-01-01

    Full Text Available Human adipose-derived stem cells are an abundant population of stem cells readily isolated from human adipose tissue that can differentiate into connective tissue lineages including bone, cartilage, fat, and muscle. Activating transcription factor 5 is a transcription factor of the ATF/cAMP response element-binding protein (CREB family. It is transcribed in two types of mRNAs (activating transcription factor 5 isoform 1 and activating transcription factor 5 isoform 2, encoding the same single 30-kDa protein. Although it is well demonstrated that it regulates the proliferation, differentiation, and apoptosis, little is known about its potential role in osteogenic differentiation. The aim of this study was to evaluate the expression levels of the two isoforms and protein during osteogenic differentiation of human adipose-derived stem cells. Our data indicate that activating transcription factor 5 is differentially expressed reaching a peak of expression at the stage of bone mineralization. These findings suggest that activating transcription factor 5 could play an interesting regulatory role during osteogenesis, which would provide a powerful tool to study bone physiology.

  4. Potential Role of Activating Transcription Factor 5 during Osteogenesis.

    Science.gov (United States)

    Vicari, Luisa; Calabrese, Giovanna; Forte, Stefano; Giuffrida, Raffaella; Colarossi, Cristina; Parrinello, Nunziatina Laura; Memeo, Lorenzo

    2016-01-01

    Human adipose-derived stem cells are an abundant population of stem cells readily isolated from human adipose tissue that can differentiate into connective tissue lineages including bone, cartilage, fat, and muscle. Activating transcription factor 5 is a transcription factor of the ATF/cAMP response element-binding protein (CREB) family. It is transcribed in two types of mRNAs (activating transcription factor 5 isoform 1 and activating transcription factor 5 isoform 2), encoding the same single 30-kDa protein. Although it is well demonstrated that it regulates the proliferation, differentiation, and apoptosis, little is known about its potential role in osteogenic differentiation. The aim of this study was to evaluate the expression levels of the two isoforms and protein during osteogenic differentiation of human adipose-derived stem cells. Our data indicate that activating transcription factor 5 is differentially expressed reaching a peak of expression at the stage of bone mineralization. These findings suggest that activating transcription factor 5 could play an interesting regulatory role during osteogenesis, which would provide a powerful tool to study bone physiology.

  5. Transcription and activity of antioxidant proteins after ionization irradiation of radiation-resistant and radiation-sensitive mice

    International Nuclear Information System (INIS)

    Hardmeier, R.

    1998-03-01

    The involvernent of antioxidant proteins catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH px), and thioredoxin (TRX) in radiobiological processes has been described at the enzyme activity level. We were interested in examining the transcription of these proteins in a mammalian system following ionizing irradiation. In order to answer the question whether radiation effects in sensitive mice (Balb/c) (RS) showed differences at the transcriptional level from radiation effects in resistant mice (C3H) (RR). We exposed the whole body of these strains to X/rays doses of 2, 4, and 6 Gy and sacrificed the animals 5, 15, and 30 minutes after irradiation. The mRNA was isolated from liver and hybrized with probes for antioxidant enzymes and thioredoxin, β-actin was used as a housekeeping gene control. Antioxidant enzyme activities were determined by standard assays. Parameters for aromatic hydroxylation (o-Tyr) and lipid peroxidation (MDA) were determined by HPLC methods. Antioxidant transcription was unchanged in contrast to antioxidant activities. SOD and CAT activities were elevated within 15 minutes in RR animals but not in RS at all radiation doses. Glutathione peroxidase activity was not different between RR and RS mice, and was only moderately elevated after irradiation. No significant differences were found between RR and RS animals at the oxidation level, although a radiation dose-dependent increase of oxidation products was detected in both groups. Quantification of thioredoxin mRNA revealed that RR mice transcribed this protein at a significantly higher level at an earlier time point (5 minutes) than did RS mice. This delay may well be responsible for the radioresistance although no quantitative differences were found. As unchanged transcription of antioxidant enzymes could not have been responsible for the increased antioxidant enzyme activities, preformed antioxidant enzymes may have been released by irradiation. This would be in agreement

  6. Modulation of Caenorhabditis elegans transcription factor activity by HIM-8 and the related Zinc-Finger ZIM proteins.

    Science.gov (United States)

    Sun, Hongliu; Nelms, Brian L; Sleiman, Sama F; Chamberlin, Helen M; Hanna-Rose, Wendy

    2007-10-01

    The previously reported negative regulatory activity of HIM-8 on the Sox protein EGL-13 is shared by the HIM-8-related ZIM proteins. Furthermore, mutation of HIM-8 can modulate the effects of substitution mutations in the DNA-binding domains of at least four other transcription factors, suggesting broad regulatory activity by HIM-8.

  7. Protein-protein interactions in the regulation of WRKY transcription factors.

    Science.gov (United States)

    Chi, Yingjun; Yang, Yan; Zhou, Yuan; Zhou, Jie; Fan, Baofang; Yu, Jing-Quan; Chen, Zhixiang

    2013-03-01

    It has been almost 20 years since the first report of a WRKY transcription factor, SPF1, from sweet potato. Great progress has been made since then in establishing the diverse biological roles of WRKY transcription factors in plant growth, development, and responses to biotic and abiotic stress. Despite the functional diversity, almost all analyzed WRKY proteins recognize the TTGACC/T W-box sequences and, therefore, mechanisms other than mere recognition of the core W-box promoter elements are necessary to achieve the regulatory specificity of WRKY transcription factors. Research over the past several years has revealed that WRKY transcription factors physically interact with a wide range of proteins with roles in signaling, transcription, and chromatin remodeling. Studies of WRKY-interacting proteins have provided important insights into the regulation and mode of action of members of the important family of transcription factors. It has also emerged that the slightly varied WRKY domains and other protein motifs conserved within each of the seven WRKY subfamilies participate in protein-protein interactions and mediate complex functional interactions between WRKY proteins and between WRKY and other regulatory proteins in the modulation of important biological processes. In this review, we summarize studies of protein-protein interactions for WRKY transcription factors and discuss how the interacting partners contribute, at different levels, to the establishment of the complex regulatory and functional network of WRKY transcription factors.

  8. Transcriptional activation of melanocortin 2 receptor accessory protein by PPARγ in adipocytes

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Nam Soo; Kim, Yoon-Jin [Department of Biology, Research Institute for Basic Science, Kyung Hee University, Seoul 130-701 (Korea, Republic of); Cho, Si Young [R and D Center, Amore Pacific Corporation, Yongin-si, Gyeonggi-do 446-729 (Korea, Republic of); Lee, Tae Ryong, E-mail: trlee@amorepacific.com [R and D Center, Amore Pacific Corporation, Yongin-si, Gyeonggi-do 446-729 (Korea, Republic of); Kim, Sang Hoon, E-mail: shkim@khu.ac.kr [Department of Biology, Research Institute for Basic Science, Kyung Hee University, Seoul 130-701 (Korea, Republic of)

    2013-09-27

    Highlights: •MRAP enhanced HSL expression. •ACTH-mediated MRAP reduced glycerol release. •PPARγ induced MRAP expression. •PPARγ bound to the MRAP promoter. -- Abstract: Adrenocorticotropic hormone (ACTH) in rodents decreases lipid accumulation and body weight. Melanocortin receptor 2 (MC2R) and MC2R accessory protein (MRAP) are specific receptors for ACTH in adipocytes. Peroxisome proliferator-activated receptor γ (PPARγ) plays a role in the transcriptional regulation of metabolic pathways such as adipogenesis and β-oxidation of fatty acids. In this study we investigated the transcriptional regulation of MRAP expression during differentiation of 3T3-L1 cells. Stimulation with ACTH affected lipolysis in murine mature adipocytes via MRAP. Putative peroxisome proliferator response element (PPRE) was identified in the MRAP promoter region. In chromatin immunoprecipitation and reporter assays, we observed binding of PPARγ to the MRAP promoter. The mutagenesis experiments showed that the −1209/−1198 region of the MRAP promoter could function as a PPRE site. These results suggest that PPARγ is required for transcriptional activation of the MRAP gene during adipogenesis, which contributes to understanding of the molecular mechanism of lipolysis in adipocytes.

  9. Transcriptional activation of melanocortin 2 receptor accessory protein by PPARγ in adipocytes

    International Nuclear Information System (INIS)

    Kim, Nam Soo; Kim, Yoon-Jin; Cho, Si Young; Lee, Tae Ryong; Kim, Sang Hoon

    2013-01-01

    Highlights: •MRAP enhanced HSL expression. •ACTH-mediated MRAP reduced glycerol release. •PPARγ induced MRAP expression. •PPARγ bound to the MRAP promoter. -- Abstract: Adrenocorticotropic hormone (ACTH) in rodents decreases lipid accumulation and body weight. Melanocortin receptor 2 (MC2R) and MC2R accessory protein (MRAP) are specific receptors for ACTH in adipocytes. Peroxisome proliferator-activated receptor γ (PPARγ) plays a role in the transcriptional regulation of metabolic pathways such as adipogenesis and β-oxidation of fatty acids. In this study we investigated the transcriptional regulation of MRAP expression during differentiation of 3T3-L1 cells. Stimulation with ACTH affected lipolysis in murine mature adipocytes via MRAP. Putative peroxisome proliferator response element (PPRE) was identified in the MRAP promoter region. In chromatin immunoprecipitation and reporter assays, we observed binding of PPARγ to the MRAP promoter. The mutagenesis experiments showed that the −1209/−1198 region of the MRAP promoter could function as a PPRE site. These results suggest that PPARγ is required for transcriptional activation of the MRAP gene during adipogenesis, which contributes to understanding of the molecular mechanism of lipolysis in adipocytes

  10. Targeted transcriptional repression using a chimeric TALE-SRDX repressor protein

    KAUST Repository

    Mahfouz, Magdy M.

    2011-12-14

    Transcriptional activator-like effectors (TALEs) are proteins secreted by Xanthomonas bacteria when they infect plants. TALEs contain a modular DNA binding domain that can be easily engineered to bind any sequence of interest, and have been used to provide user-selected DNA-binding modules to generate chimeric nucleases and transcriptional activators in mammalian cells and plants. Here we report the use of TALEs to generate chimeric sequence-specific transcriptional repressors. The dHax3 TALE was used as a scaffold to provide a DNA-binding module fused to the EAR-repression domain (SRDX) to generate a chimeric repressor that targets the RD29A promoter. The dHax3. SRDX protein efficiently repressed the transcription of the RD29A

  11. Targeted transcriptional repression using a chimeric TALE-SRDX repressor protein

    KAUST Repository

    Mahfouz, Magdy M.; Li, Lixin; Piatek, Marek J.; Fang, Xiaoyun; Mansour, Hicham; Bangarusamy, Dhinoth K.; Zhu, Jian-Kang

    2011-01-01

    Transcriptional activator-like effectors (TALEs) are proteins secreted by Xanthomonas bacteria when they infect plants. TALEs contain a modular DNA binding domain that can be easily engineered to bind any sequence of interest, and have been used to provide user-selected DNA-binding modules to generate chimeric nucleases and transcriptional activators in mammalian cells and plants. Here we report the use of TALEs to generate chimeric sequence-specific transcriptional repressors. The dHax3 TALE was used as a scaffold to provide a DNA-binding module fused to the EAR-repression domain (SRDX) to generate a chimeric repressor that targets the RD29A promoter. The dHax3. SRDX protein efficiently repressed the transcription of the RD29A

  12. Activation of transcriptional activities of AP-1 and SRE by a new zinc-finger protein ZNF641

    International Nuclear Information System (INIS)

    Qi Xingzhu; Li Yongqing; Xiao Jing; Yuan Wuzhou; Yan Yan; Wang Yuequn; Liang Shuyuan; Zhu Chuanbing; Chen Yingduan; Liu Mingyao; Wu Xiushan

    2006-01-01

    Mitogen-activated protein kinases (MAPKs) are evolutionarily conserved enzymes in cell signal transduction connecting cell-surface receptors to critical regulatory targets within cells and control cell survival, adaptation, and proliferation. Previous studies revealed that zinc-finger proteins are involved in the regulation of the MAPK signaling pathways. Here, we report the identification and characterization of a novel human zinc-finger protein, ZNF641. The cDNA of ZNF641 is 4.9 kb, encoding 438 amino acids in the nucleus. The protein is highly conserved in evolution across different vertebrate species from mouse to human. Northern blot analysis indicates that ZNF641 is expressed in most of the examined human tissues, with a high level in skeletal muscle. Overexpression of pCMV-Tag2B-ZNF641 in the COS-7 cells activates the transcriptional activities of AP-1 and SRE. Deletion analysis indicates that the linker between KRAB box and C 2 H 2 -type zinc-fingers represents the basal activation domain. These results suggest that ZNF641 may be a positive regulator in MAPK-mediated signaling pathways that lead to the activation of AP-1 and SRE

  13. cAMP response element binding protein (CREB activates transcription via two distinct genetic elements of the human glucose-6-phosphatase gene

    Directory of Open Access Journals (Sweden)

    Stefano Luisa

    2005-01-01

    Full Text Available Abstract Background The enzyme glucose-6-phosphatase catalyzes the dephosphorylation of glucose-6-phosphatase to glucose, the final step in the gluconeogenic and glycogenolytic pathways. Expression of the glucose-6-phosphatase gene is induced by glucocorticoids and elevated levels of intracellular cAMP. The effect of cAMP in regulating glucose-6-phosphatase gene transcription was corroborated by the identification of two genetic motifs CRE1 and CRE2 in the human and murine glucose-6-phosphatase gene promoter that resemble cAMP response elements (CRE. Results The cAMP response element is a point of convergence for many extracellular and intracellular signals, including cAMP, calcium, and neurotrophins. The major CRE binding protein CREB, a member of the basic region leucine zipper (bZIP family of transcription factors, requires phosphorylation to become a biologically active transcriptional activator. Since unphosphorylated CREB is transcriptionally silent simple overexpression studies cannot be performed to test the biological role of CRE-like sequences of the glucose-6-phosphatase gene. The use of a constitutively active CREB2/CREB fusion protein allowed us to uncouple the investigation of target genes of CREB from the variety of signaling pathways that lead to an activation of CREB. Here, we show that this constitutively active CREB2/CREB fusion protein strikingly enhanced reporter gene transcription mediated by either CRE1 or CRE2 derived from the glucose-6-phosphatase gene. Likewise, reporter gene transcription was enhanced following expression of the catalytic subunit of cAMP-dependent protein kinase (PKA in the nucleus of transfected cells. In contrast, activating transcription factor 2 (ATF2, known to compete with CREB for binding to the canonical CRE sequence 5'-TGACGTCA-3', did not transactivate reporter genes containing CRE1, CRE2, or both CREs derived from the glucose-6-phosphatase gene. Conclusions Using a constitutively active CREB2

  14. A comprehensive analysis of microProteins reveals their potentially widespread mechanism of transcriptional regulation

    NARCIS (Netherlands)

    Magnani, Enrico; de Klein, Niek; Nam, Hye-In; Kim, Jung-Gun; Pham, Kimberly; Fiume, Elisa; Mudgett, Mary Beth; Rhee, Seung Yon

    2014-01-01

    Truncated transcription factor-like proteins called microProteins (miPs) can modulate transcription factor activities, thereby increasing transcriptional regulatory complexity. To understand their prevalence, evolution, and function, we predicted over 400 genes that encode putative miPs from

  15. PPARγ activates ABCA1 gene transcription but reduces the level of ABCA1 protein in HepG2 cells

    International Nuclear Information System (INIS)

    Mogilenko, Denis A.; Shavva, Vladimir S.; Dizhe, Ella B.; Orlov, Sergey V.; Perevozchikov, Andrej P.

    2010-01-01

    Research highlights: → PPARγ activates ABCA1 gene expression but decreases ABCA1 protein content in human hepatoma cell line HepG2. → Treatment of HepG2 cells with PPARγ agonist GW1929 leads to dissociation of LXRβ from ABCA1-LXRβ complex. → Inhibition of protein kinases MEK1/2 abolishes PPARγ-mediated dissociation of LXRβ from ABCA1/LXRβ complex. → Activation of PPARγ leads to increasing of the level of LXRβ associated with LXRE within ABCA1 gene promoter. -- Abstract: Synthesis of ABCA1 protein in liver is necessary for high-density lipoproteins (HDL) formation in mammals. Nuclear receptor PPARγ is known as activator of ABCA1 expression, but details of PPARγ-mediated regulation of ABCA1 at both transcriptional and post-transcriptional levels in hepatocytes have not still been well elucidated. In this study we have shown, that PPARγ activates ABCA1 gene transcription in human hepatoma cells HepG2 through increasing of LXRβ binding with promoter region of ABCA1 gene. Treatment of HepG2 cells with PPARγ agonist GW1929 leads to dissociation of LXRβ from ABCA1/LXRβ complex and to nuclear translocation of this nuclear receptor resulting in reduction of ABCA1 protein level 24 h after treatment. Inhibition of protein kinases MEK1/2 abolishes PPARγ-mediated dissociation of LXRβ from ABCA1/LXRβ complex, but does not block PPARγ-dependent down-regulation of ABCA1 protein in HepG2 cells. These data suggest that PPARγ may be important for regulation of the level of hepatic ABCA1 protein and indicate the new interplays between PPARγ, LXRβ and MEK1/2 in regulation of ABCA1 mRNA and protein expression.

  16. Activating human genes with zinc finger proteins, transcription activator-like effectors and CRISPR/Cas9 for gene therapy and regenerative medicine.

    Science.gov (United States)

    Gersbach, Charles A; Perez-Pinera, Pablo

    2014-08-01

    New technologies have recently been developed to control the expression of human genes in their native genomic context by engineering synthetic transcription factors that can be targeted to any DNA sequence. The ability to precisely regulate any gene as it occurs naturally in the genome provides a means to address a variety of diseases and disorders. This approach also circumvents some of the traditional challenges of gene therapy. In this editorial, we review the technologies that have enabled targeted human gene activation, including the engineering of transcription factors based on zinc finger proteins, transcription activator-like effectors and the CRISPR/Cas9 system. Additionally, we highlight examples in which these methods have been developed for therapeutic applications and discuss challenges and opportunities.

  17. ZNF322, a novel human C2H2 Krueppel-like zinc-finger protein, regulates transcriptional activation in MAPK signaling pathways

    International Nuclear Information System (INIS)

    Li Yongqing; Wang Yuequn; Zhang Caibo; Yuan Wuzhou; Wang Jun; Zhu Chuanbing; Chen Lei; Huang Wen; Zeng Weiqi; Wu Xiushan; Liu Mingyao

    2004-01-01

    Cardiac differentiation involves a cascade of coordinated gene expression that regulates cell proliferation and matrix protein formation in a defined temporal-spatial manner. The C 2 H 2 zinc finger-containing transcription factors have been implicated as critical regulators of multiple cardiac-expressed genes and are important for human heart development and diseases. Here we have identified and characterized a novel zinc-finger gene named ZNF322 using degenerated primers from a human embryo heart cDNA library. The gene contains four exons and spans 23.2 kb in chromosome 6p22.1 region, and transcribes a 2.7 kb mRNA that encodes a protein with 402 amino acid residues. The predicted protein contains 9 tandem C 2 H 2 -type zinc-finger motifs. Northern blot analysis shows that ZNF322 is expressed in every human tissue examined at adult stage and during embryonic developmental stages from 80 days to 24 weeks. When overexpressed in COS-7 cells, ZNF322-EGFP fusion protein is detected in the nucleus and cytoplasm. Reporter gene assays show that ZNF322 is a transcriptional activator. Furthermore, overexpression of ZNF322 in COS-7 cells activates the transcriptional activity of SRE and AP-1. Together, these results suggest that ZNF322 is a member of the zinc-finger transcription factor family and may act as a positive regulator in gene transcription mediated by the MAPK signaling pathways

  18. Abscisic acid affects transcription of chloroplast genes via protein phosphatase 2C-dependent activation of nuclear genes: repression by guanosine-3'-5'-bisdiphosphate and activation by sigma factor 5.

    Science.gov (United States)

    Yamburenko, Maria V; Zubo, Yan O; Börner, Thomas

    2015-06-01

    Abscisic acid (ABA) represses the transcriptional activity of chloroplast genes (determined by run-on assays), with the exception of psbD and a few other genes in wild-type Arabidopsis seedlings and mature rosette leaves. Abscisic acid does not influence chloroplast transcription in the mutant lines abi1-1 and abi2-1 with constitutive protein phosphatase 2C (PP2C) activity, suggesting that ABA affects chloroplast gene activity by binding to the pyrabactin resistance (PYR)/PYR1-like or regulatory component of ABA receptor protein family (PYR/PYL/RCAR) and signaling via PP2Cs and sucrose non-fermenting protein-related kinases 2 (SnRK2s). Further we show by quantitative PCR that ABA enhances the transcript levels of RSH2, RSH3, PTF1 and SIG5. RelA/SpoT homolog 2 (RSH2) and RSH3 are known to synthesize guanosine-3'-5'-bisdiphosphate (ppGpp), an inhibitor of the plastid-gene-encoded chloroplast RNA polymerase. We propose, therefore, that ABA leads to an inhibition of chloroplast gene expression via stimulation of ppGpp synthesis. On the other hand, sigma factor 5 (SIG5) and plastid transcription factor 1 (PTF1) are known to be necessary for the transcription of psbD from a specific light- and stress-induced promoter (the blue light responsive promoter, BLRP). We demonstrate that ABA activates the psbD gene by stimulation of transcription initiation at BLRP. Taken together, our data suggest that ABA affects the transcription of chloroplast genes by a PP2C-dependent activation of nuclear genes encoding proteins involved in chloroplast transcription. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

  19. A compendium of transcription factor and Transcriptionally active protein coding gene families in cowpea (Vigna unguiculata L.).

    Science.gov (United States)

    Misra, Vikram A; Wang, Yu; Timko, Michael P

    2017-11-22

    Cowpea (Vigna unguiculata (L.) Walp.) is the most important food and forage legume in the semi-arid tropics of sub-Saharan Africa where approximately 80% of worldwide production takes place primarily on low-input, subsistence farm sites. Among the major goals of cowpea breeding and improvement programs are the rapid manipulation of agronomic traits for seed size and quality and improved resistance to abiotic and biotic stresses to enhance productivity. Knowing the suite of transcription factors (TFs) and transcriptionally active proteins (TAPs) that control various critical plant cellular processes would contribute tremendously to these improvement aims. We used a computational approach that employed three different predictive pipelines to data mine the cowpea genome and identified over 4400 genes representing 136 different TF and TAP families. We compare the information content of cowpea to two evolutionarily close species common bean (Phaseolus vulgaris), and soybean (Glycine max) to gauge the relative informational content. Our data indicate that correcting for genome size cowpea has fewer TF and TAP genes than common bean (4408 / 5291) and soybean (4408/ 11,065). Members of the GROWTH-REGULATING FACTOR (GRF) and Auxin/indole-3-acetic acid (Aux/IAA) gene families appear to be over-represented in the genome relative to common bean and soybean, whereas members of the MADS (Minichromosome maintenance deficient 1 (MCM1), AGAMOUS, DEFICIENS, and serum response factor (SRF)) and C2C2-YABBY appear to be under-represented. Analysis of the AP2-EREBP APETALA2-Ethylene Responsive Element Binding Protein (AP2-EREBP), NAC (NAM (no apical meristem), ATAF1, 2 (Arabidopsis transcription activation factor), CUC (cup-shaped cotyledon)), and WRKY families, known to be important in defense signaling, revealed changes and phylogenetic rearrangements relative to common bean and soybean that suggest these groups may have evolved different functions. The availability of detailed

  20. Triptolide inhibits transcription of hTERT through down-regulation of transcription factor specificity protein 1 in primary effusion lymphoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Long, Cong; Wang, Jingchao [Department of Pathogen Biology, School of Basic Medical Sciences, Wuhan University, Wuhan, 430071 (China); Guo, Wei [Department of Pathology and Physiology, School of Basic Medical Sciences, Wuhan University, Wuhan, 430071 (China); Wang, Huan; Wang, Chao; Liu, Yu [Department of Pathogen Biology, School of Basic Medical Sciences, Wuhan University, Wuhan, 430071 (China); Sun, Xiaoping, E-mail: xsun6@whu.edu.cn [Department of Pathogen Biology, School of Basic Medical Sciences, Wuhan University, Wuhan, 430071 (China); State Key Laboratory of Virology, Wuhan University, Wuhan, 430072 (China)

    2016-01-01

    Primary effusion lymphoma (PEL) is a rare and aggressive non-Hodgkin's lymphoma. Human telomerase reverse transcriptase (hTERT), a key component responsible for the regulation of telomerase activity, plays important roles in cellular immortalization and cancer development. Triptolide purified from Tripterygium extracts displays a broad-spectrum bioactivity profile, including immunosuppressive, anti-inflammatory, and anti-tumor. In this study, it is investigated whether triptolide reduces hTERT expression and suppresses its activity in PEL cells. The mRNA and protein levels of hTERT were examined by real time-PCR and Western blotting, respectively. The activity of hTERT promoter was determined by Dual luciferase reporter assay. Our results demonstrated that triptolide decreased expression of hTERT at both mRNA and protein levels. Further gene sequence analysis indicated that the activity of hTERT promoter was suppressed by triptolide. Triptolide also reduced the half-time of hTERT. Additionally, triptolide inhibited the expression of transcription factor specificity protein 1(Sp1) in PEL cells. Furthermore, knock-down of Sp1 by using specific shRNAs resulted in down-regulation of hTERT transcription and protein expression levels. Inhibition of Sp1 by specific shRNAs enhanced triptolide-induced cell growth inhibition and apoptosis. Collectively, our results demonstrate that the inhibitory effect of triptolide on hTERT transcription is possibly mediated by inhibition of transcription factor Sp1 in PEL cells. - Highlights: • Triptolide reduces expression of hTERT by decreasing its transcription level. • Triptolide reduces promoter activity and stability of hTERT. • Triptolide down-regulates expression of Sp1. • Special Sp1 shRNAs inhibit transcription and protein expression of hTERT. • Triptolide and Sp1 shRNA2 induce cell proliferation inhibition and apoptosis.

  1. TRIM45, a novel human RBCC/TRIM protein, inhibits transcriptional activities of ElK-1 and AP-1

    International Nuclear Information System (INIS)

    Wang Yuequn; Li Yongqing; Qi Xinzhu; Yuan Wuzhou; Ai Jianping; Zhu Chuanbing; Cao Lei; Yang Hong; Liu Fang; Wu Xiushan; Liu Mingyao

    2004-01-01

    The tripartite motif (TRIM) proteins play important roles in a variety of cellular functions including cell proliferation, differentiation, development, oncogenesis, and apoptosis. In this study, we report the identification and characterization of the human tripartite motif-containing protein 45 (TRIM45), a novel member of the TRIM family, from a human embryonic heart cDNA library. TRIM45 has a predicted 580 amino acid open reading frame, encoding a putative 64-kDa protein. The N-terminal region harbors a RING finger, two B-boxes, and a predicted α-helical coiled-coil domain, which together form the RBCC/TRIM motif found in a large family of proteins, whereas the C-terminal region contains a filamin-type immunoglobulin (IG-FLMN) domain. Northern blot analysis indicates that TRIM45 is expressed in a variety of human adult and embryonic tissues. In the cell, TRIM45 protein is expressed both in cytoplasm and in cell nucleus. Overexpression of TRIM45 in COS-7 cells inhibits the transcriptional activities of ElK-1 and AP-1. These results suggest that TRIM45 may act as a new transcriptional repressor in mitogen-activated protein kinase signaling pathway

  2. Inhibition of transcriptional activity of c-JUN by SIRT1

    International Nuclear Information System (INIS)

    Gao Zhanguo; Ye Jianping

    2008-01-01

    c-JUN is a major component of heterodimer transcription factor AP-1 (Activator Protein-1) that activates gene transcription in cell proliferation, inflammation and stress responses. SIRT1 (Sirtuin 1) is a histone deacetylase that controls gene transcription through modification of chromatin structure. However, it is not clear if SIRT1 regulates c-JUN activity in the control of gene transcription. Here, we show that SIRT1 associated with c-JUN in co-immunoprecipitation of whole cell lysate, and inhibited the transcriptional activity of c-JUN in the mammalian two hybridization system. SIRT1 was found in the AP-1 response element in the matrix metalloproteinase-9 (MMP9) promoter DNA leading to inhibition of histone 3 acetylation as shown in a ChIP assay. The SIRT1 signal was reduced by the AP-1 activator PMA, and induced by the SIRT1 activator Resveratrol in the promoter DNA. SIRT1-mediaetd inhibition of AP-1 was demonstrated in the MMP9 gene expression at the gene promoter, mRNA and protein levels. In mouse embryonic fibroblast (MEF) with SIRT1 deficiency (SIRT1 -/- ), mRNA and protein of MMP9 were increased in the basal condition, and the inhibitory activity of Resveratrol was significantly attenuated. Glucose-induced MMP9 expression was also inhibited by SIRT1 in response to Resveratrol. These data consistently suggest that SIRT1 directly inhibits the transcriptional activity of AP-1 by targeting c-JUN

  3. Polycomb group protein-mediated repression of transcription

    DEFF Research Database (Denmark)

    Morey, Lluís; Helin, Kristian

    2010-01-01

    The polycomb group (PcG) proteins are essential for the normal development of multicellular organisms. They form multi-protein complexes that work as transcriptional repressors of several thousand genes controlling differentiation pathways during development. How the PcG proteins work as transcri......The polycomb group (PcG) proteins are essential for the normal development of multicellular organisms. They form multi-protein complexes that work as transcriptional repressors of several thousand genes controlling differentiation pathways during development. How the PcG proteins work...... as transcriptional repressors is incompletely understood, but involves post-translational modifications of histones by two major PcG protein complexes: polycomb repressive complex 1 and polycomb repressive complex 2....

  4. Radiation activation of transcription factors in mammalian cells

    International Nuclear Information System (INIS)

    Kraemer, M.; Stein, B.; Mai, S.; Kunz, E.; Koenig, H.; Ponta, H.; Herrlich, P.; Rahmsdorf, H.J.; Loferer, H.; Grunicke, H.H.

    1990-01-01

    In mammalian cells radiation induces the enhanced transcription of several genes. The cis acting elements in the control region of inducible genes have been delimited by site directed mutagenesis. Several different elements have been found in different genes. They do not only activate gene transcription in response to radiation but also in response to growth factors and to tumor promoter phorbol esters. The transcription factors binding to these elements are present also in non-irradiated cells, but their DNA binding activity and their transactivating capability is increased upon irradiation. The signal chain linking the primary radiation induced signal (damaged DNA) to the activation of transcription factors involves the action of (a) protein kinase(s). (orig.)

  5. BASP1 is a transcriptional cosuppressor for the Wilms' tumor suppressor protein WT1

    DEFF Research Database (Denmark)

    Carpenter, Brian; Hill, Kathryn J; Charalambous, Marika

    2004-01-01

    The Wilms' tumor suppressor protein WT1 is a transcriptional regulator that plays a key role in the development of the kidneys. The transcriptional activation domain of WT1 is subject to regulation by a suppression region within the N terminus of WT1. Using a functional assay, we provide direct...... evidence that this requires a transcriptional cosuppressor, which we identify as brain acid soluble protein 1 (BASP1). WT1 and BASP1 associate within the nuclei of cells that naturally express both proteins. BASP1 can confer WT1 cosuppressor activity in transfection assays, and elimination of endogenous...

  6. The PDZ protein tax-interacting protein-1 inhibits beta-catenin transcriptional activity and growth of colorectal cancer cells.

    Science.gov (United States)

    Kanamori, Mutsumi; Sandy, Peter; Marzinotto, Stefania; Benetti, Roberta; Kai, Chikatoshi; Hayashizaki, Yoshihide; Schneider, Claudio; Suzuki, Harukazu

    2003-10-03

    Wnt signaling is essential during development while deregulation of this pathway frequently leads to the formation of various tumors including colorectal carcinomas. A key component of the pathway is beta-catenin that, in association with TCF-4, directly regulates the expression of Wnt-responsive genes. To identify novel binding partners of beta-catenin that may control its transcriptional activity, we performed a mammalian two-hybrid screen and isolated the Tax-interacting protein (TIP-1). The in vivo complex formation between beta-catenin and TIP-1 was verified by coimmunoprecipitation, and a direct physical association was revealed by glutathione S-transferase pull-down experiments in vitro. By using a panel of deletion mutants of both proteins, we demonstrate that the interaction is mediated by the PDZ (PSD-95/DLG/ZO-1 homology) domain of TIP-1 and requires primarily the last four amino acids of beta-catenin. TIP-1 overexpression resulted in a dose-dependent decrease in the transcriptional activity of beta-catenin when tested on the TOP/FOPFLASH reporter system. Conversely, siRNA-mediated knock-down of endogenous TIP-1 slightly increased endogenous beta-catenin transactivation function. Moreover, we show that overexpression of TIP-1 reduced the proliferation and anchorage-independent growth of colorectal cancer cells. These data suggest that TIP-1 may represent a novel regulatory element in the Wnt/beta-catenin signaling pathway.

  7. ZNF649, a novel Kruppel type zinc-finger protein, functions as a transcriptional suppressor

    International Nuclear Information System (INIS)

    Yang Hong; Yuan Wuzhou; Wang Ying; Zhu Chuanbing; Liu Bisheng; Wang Yuequn; Yang, Dan; Li Yongqing; Wang Canding; Wu Xiushan; Liu Mingyao

    2005-01-01

    Cardiac differentiation involves a cascade of coordinated gene expression that regulates cell proliferation and matrix protein formation in a defined temporo-spatial manner. Many of the KRAB-ZFPs are involved in cardiac development or cardiovascular diseases. Here we report the identification and characterization of a novel human zinc-finger gene named ZNF649. The cDNA of ZNF649 is 3176 bp, encoding a protein of 505 amino acids in the nuclei. Northern blot analysis indicates that ZNF649 is expressed in most of the examined human adult and embryonic tissues. ZNF649 is a transcription suppressor when fused to GAL-4 DNA-binding domain and cotransfected with VP-16. Overexpression of ZNF649 in COS-7 cells inhibits the transcriptional activities of SRE and AP-1. Deletion analysis with a series of truncated fusion proteins indicates that the KRAB motif is a basal repression domain when the truncated fusion proteins were assayed for the transcriptional activities of SRE and AP-1. These results suggest that ZNF649 protein may act as a transcriptional repressor in mitogen-activated protein kinase signaling pathway to mediate cellular functions

  8. Transcriptional activation of the mouse obese (ob) gene by CCAAT/enhancer binding protein alpha

    DEFF Research Database (Denmark)

    Hwang, C S; Mandrup, S; MacDougald, O A

    1996-01-01

    Like other adipocyte genes that are transcriptionally activated by CCAAT/enhancer binding protein alpha (C/EBP alpha) during preadipocyte differentiation, expression of the mouse obese (ob) gene is immediately preceded by the expression of C/EBP alpha. While the 5' flanking region of the mouse ob...... gene contains several consensus C/EBP binding sites, only one of these sites appears to be functional. DNase I cleavage inhibition patterns (footprinting) of the ob gene promoter revealed that recombinant C/EBP alpha, as well as a nuclear factor present in fully differentiated 3T3-L1 adipocytes...... to a consensus C/EBP binding site at nucleotides -55 to -47 generated a specific protein-oligonucleotide complex that was supershifted by antibody against C/EBP alpha. Probes corresponding to two upstream consensus C/EBP binding sites failed to generate protein-oligonucleotide complexes. Cotransfection of a C...

  9. Global RNA association with the transcriptionally active chromosome of chloroplasts.

    Science.gov (United States)

    Lehniger, Marie-Kristin; Finster, Sabrina; Melonek, Joanna; Oetke, Svenja; Krupinska, Karin; Schmitz-Linneweber, Christian

    2017-10-01

    Processed chloroplast RNAs are co-enriched with preparations of the chloroplast transcriptionally active chromosome. Chloroplast genomes are organized as a polyploid DNA-protein structure called the nucleoid. Transcriptionally active chloroplast DNA together with tightly bound protein factors can be purified by gel filtration as a functional entity called the transcriptionally active chromosome (TAC). Previous proteomics analyses of nucleoids and of TACs demonstrated a considerable overlap in protein composition including RNA binding proteins. Therefore the RNA content of TAC preparations from Nicotiana tabacum was determined using whole genome tiling arrays. A large number of chloroplast RNAs was found to be associated with the TAC. The pattern of RNAs attached to the TAC consists of RNAs produced by different chloroplast RNA polymerases and differs from the pattern of RNA found in input controls. An analysis of RNA splicing and RNA editing of selected RNA species demonstrated that TAC-associated RNAs are processed to a similar extent as the RNA in input controls. Thus, TAC fractions contain a specific subset of the processed chloroplast transcriptome.

  10. Transcriptional regulation of the Borrelia burgdorferi antigenically variable VlsE surface protein.

    Science.gov (United States)

    Bykowski, Tomasz; Babb, Kelly; von Lackum, Kate; Riley, Sean P; Norris, Steven J; Stevenson, Brian

    2006-07-01

    The Lyme disease agent Borrelia burgdorferi can persistently infect humans and other animals despite host active immune responses. This is facilitated, in part, by the vls locus, a complex system consisting of the vlsE expression site and an adjacent set of 11 to 15 silent vls cassettes. Segments of nonexpressed cassettes recombine with the vlsE region during infection of mammalian hosts, resulting in combinatorial antigenic variation of the VlsE outer surface protein. We now demonstrate that synthesis of VlsE is regulated during the natural mammal-tick infectious cycle, being activated in mammals but repressed during tick colonization. Examination of cultured B. burgdorferi cells indicated that the spirochete controls vlsE transcription levels in response to environmental cues. Analysis of PvlsE::gfp fusions in B. burgdorferi indicated that VlsE production is controlled at the level of transcriptional initiation, and regions of 5' DNA involved in the regulation were identified. Electrophoretic mobility shift assays detected qualitative and quantitative changes in patterns of protein-DNA complexes formed between the vlsE promoter and cytoplasmic proteins, suggesting the involvement of DNA-binding proteins in the regulation of vlsE, with at least one protein acting as a transcriptional activator.

  11. Characterization of DNA binding, transcriptional activation, and regulated nuclear association of recombinant human NFATp

    Directory of Open Access Journals (Sweden)

    Seto Anita G

    2000-11-01

    Full Text Available Abstract Background NFATp is one member of a family of transcriptional activators whose nuclear accumulation and hence transcriptional activity is regulated in mammalian cells. Human NFATp exists as a phosphoprotein in the cytoplasm of naive T cells. Upon antigen stimulation, NFATp is dephosphorylated, accumulates in nuclei, and functions to regulate transcription of genes including those encoding cytokines. While the properties of the DNA binding domain of NFATp have been investigated in detail, biochemical studies of the transcriptional activation and regulated association with nuclei have remained unexplored because of a lack of full length, purified recombinant NFATp. Results We developed methods for expressing and purifying full length recombinant human NFATp that has all of the properties known to be associated with native NFATp. The recombinant NFATp binds DNA on its own and cooperatively with AP-1 proteins, activates transcription in vitro, is phosphorylated, can be dephosphorylated by calcineurin, and exhibits regulated association with nuclei in vitro. Importantly, activation by recombinant NFATp in a reconstituted transcription system required regions of the protein outside of the central DNA binding domain. Conclusions We conclude that NFATp is a bona fide transcriptional activator. Moreover, the reagents and methods that we developed will facilitate future studies on the mechanisms of transcriptional activation and nuclear accumulation by NFATp, a member of an important family of transcriptional regulatory proteins.

  12. Heterochromatin protein 1 gamma and IκB kinase alpha interdependence during tumour necrosis factor gene transcription elongation in activated macrophages.

    Science.gov (United States)

    Thorne, James L; Ouboussad, Lylia; Lefevre, Pascal F

    2012-09-01

    IκB kinase α (IKKα) is part of the cytoplasmic IKK complex regulating nuclear factor-κB (NF-κB) release and translocation into the nucleus in response to pro-inflammatory signals. IKKα can also be recruited directly to the promoter of NF-κB-dependent genes by NF-κB where it phosphorylates histone H3 at serine 10, triggering recruitment of the bromodomain-containing protein 4 and the positive transcription elongation factor b. Herein, we report that IKKα travels with the elongating form of ribonucleic acid polymerase II together with heterochromatin protein 1 gamma (HP1γ) at NF-κB-dependent genes in activated macrophages. IKKα binds to and phosphorylates HP1γ, which in turn controls IKKα binding to chromatin and phosphorylation of the histone variant H3.3 at serine 31 within transcribing regions. Downstream of transcription end sites, IKKα accumulates with its inhibitor the CUE-domain containing protein 2, suggesting a link between IKKα inactivation and transcription termination.

  13. Cell Cycle-Dependent Recruitment of Polycomb Proteins to the ASNS Promoter Counteracts C/ebp-Mediated Transcriptional Activation in Bombyx mori

    Science.gov (United States)

    Li, Zhiqing; Cheng, Daojun; Mon, Hiroaki; Zhu, Li; Xu, Jian; Tatsuke, Tsuneyuki; Lee, Jae Man; Xia, Qingyou; Kusakabe, Takahiro

    2013-01-01

    Epigenetic modifiers and transcription factors contribute to developmentally programmed gene expression. Here, we establish a functional link between epigenetic regulation by Polycomb group (PcG) proteins and transcriptional regulation by C/ebp that orchestrates the correct expression of Bombyx mori asparagine synthetase (BmASNS), a gene involved in the biosynthesis of asparagine. We show that the cis-regulatory elements of YY1-binding motifs and the CpG island present on the BmASNS promoter are required for the recruitment of PcG proteins and the subsequent deposition of the epigenetic repression mark H3K27me3. RNAi-mediated knockdown of PcG genes leads to derepression of the BmASNS gene via the recruitment of activators, including BmC/ebp, to the promoter. Intriguingly, we find that PcG proteins and BmC/ebp can dynamically modulate the transcriptional output of the BmASNS target in a cell cycle-dependent manner. It will be essential to suppress BmASNS expression by PcG proteins at the G2/M phase of the cell cycle in the presence of BmC/ebp activator. Thus, our results provide a novel insight into the molecular mechanism underlying the recruitment and regulation of the PcG system at a discrete gene locus in Bombyx mori. PMID:23382816

  14. High-mobility group (HMG) protein HMG-1 and TATA-binding protein-associated factor TAF(II)30 affect estrogen receptor-mediated transcriptional activation.

    Science.gov (United States)

    Verrier, C S; Roodi, N; Yee, C J; Bailey, L R; Jensen, R A; Bustin, M; Parl, F F

    1997-07-01

    The estrogen receptor (ER) belongs to a family of ligand-inducible nuclear receptors that exert their effects by binding to cis-acting DNA elements in the regulatory region of target genes. The detailed mechanisms by which ER interacts with the estrogen response element (ERE) and affects transcription still remain to be elucidated. To study the ER-ERE interaction and transcription initiation, we employed purified recombinant ER expressed in both the baculovirus-Sf9 and his-tagged bacterial systems. The effect of high-mobility group (HMG) protein HMG-1 and purified recombinant TATA-binding protein-associated factor TAF(II)30 on ER-ERE binding and transcription initiation were assessed by electrophoretic mobility shift assay and in vitro transcription from an ERE-containing template (pERE2LovTATA), respectively. We find that purified, recombinant ER fails to bind to ERE in spite of high ligand-binding activity and electrophoretic and immunological properties identical to ER in MCF-7 breast cancer cells. HMG-1 interacts with ER and promotes ER-ERE binding in a concentration- and time-dependent manner. The effectiveness of HMG-1 to stimulate ER-ERE binding in the electrophoretic mobility shift assay depends on the sequence flanking the ERE consensus as well as the position of the latter in the oligonucleotide. We find that TAF(II)30 has no effect on ER-ERE binding either alone or in combination with ER and HMG-1. Although HMG-1 promotes ER-ERE binding, it fails to stimulate transcription initiation either in the presence or absence of hormone. In contrast, TAF(II)30, while not affecting ER-ERE binding, stimulates transcription initiation 20-fold in the presence of HMG-1. These results indicate that HMG-1 and TAF(II)30 act in sequence, the former acting to promote ER-ERE binding followed by the latter to stimulate transcription initiation.

  15. The adenovirus oncoprotein E1a stimulates binding of transcription factor ETF to transcriptionally activate the p53 gene.

    Science.gov (United States)

    Hale, T K; Braithwaite, A W

    1999-08-20

    Expression of the tumor suppressor protein p53 plays an important role in regulating the cellular response to DNA damage. During adenovirus infection, levels of p53 protein also increase. It has been shown that this increase is due not only to increased stability of the p53 protein but to the transcriptional activation of the p53 gene during infection. We demonstrate here that the E1a proteins of adenovirus are responsible for activating the mouse p53 gene and that both major E1a proteins, 243R and 289R, are required for complete activation. E1a brings about the binding of two cellular transcription factors to the mouse p53 promoter. One of these, ETF, binds to three upstream sites in the p53 promoter and one downstream site, whereas E2F binds to one upstream site in the presence of E1a. Our studies indicate that E2F binding is not essential for activation of the p53 promoter but that ETF is. Our data indicate the ETF site located downstream of the start site of transcription is the key site in conferring E1a responsiveness on the p53 promoter.

  16. A Herpesviral Immediate Early Protein Promotes Transcription Elongation of Viral Transcripts.

    Science.gov (United States)

    Fox, Hannah L; Dembowski, Jill A; DeLuca, Neal A

    2017-06-13

    Herpes simplex virus 1 (HSV-1) genes are transcribed by cellular RNA polymerase II (RNA Pol II). While four viral immediate early proteins (ICP4, ICP0, ICP27, and ICP22) function in some capacity in viral transcription, the mechanism by which ICP22 functions remains unclear. We observed that the FACT complex (comprised of SSRP1 and Spt16) was relocalized in infected cells as a function of ICP22. ICP22 was also required for the association of FACT and the transcription elongation factors SPT5 and SPT6 with viral genomes. We further demonstrated that the FACT complex interacts with ICP22 throughout infection. We therefore hypothesized that ICP22 recruits cellular transcription elongation factors to viral genomes for efficient transcription elongation of viral genes. We reevaluated the phenotype of an ICP22 mutant virus by determining the abundance of all viral mRNAs throughout infection by transcriptome sequencing (RNA-seq). The accumulation of almost all viral mRNAs late in infection was reduced compared to the wild type, regardless of kinetic class. Using chromatin immunoprecipitation sequencing (ChIP-seq), we mapped the location of RNA Pol II on viral genes and found that RNA Pol II levels on the bodies of viral genes were reduced in the ICP22 mutant compared to wild-type virus. In contrast, the association of RNA Pol II with transcription start sites in the mutant was not reduced. Taken together, our results indicate that ICP22 plays a role in recruiting elongation factors like the FACT complex to the HSV-1 genome to allow for efficient viral transcription elongation late in viral infection and ultimately infectious virion production. IMPORTANCE HSV-1 interacts with many cellular proteins throughout productive infection. Here, we demonstrate the interaction of a viral protein, ICP22, with a subset of cellular proteins known to be involved in transcription elongation. We determined that ICP22 is required to recruit the FACT complex and other transcription

  17. Enterovirus type 71 2A protease functions as a transcriptional activator in yeast

    Directory of Open Access Journals (Sweden)

    Lai Meng-Jiun

    2010-08-01

    Full Text Available Abstract Enterovirus type 71 (EV71 2A protease exhibited strong transcriptional activity in yeast cells. The transcriptional activity of 2A protease was independent of its protease activity. EV71 2A protease retained its transcriptional activity after truncation of 40 amino acids at the N-terminus but lost this activity after truncation of 60 amino acids at the N-terminus or deletion of 20 amino acids at the C-terminus. Thus, the acidic domain at the C-terminus of this protein is essential for its transcriptional activity. Indeed, deletion of amino acids from 146 to 149 (EAME in this acidic domain lost the transcriptional activity of EV71 2A protein though still retained its protease activity. EV71 2A protease was detected both in the cytoplasm and nucleus using confocal microscopy analysis. Coxsackie virus B3 2A protease also exhibited transcriptional activity in yeast cells. As expected, an acidic domain in the C-terminus of Coxsackie virus B3 2A protease was also identified. Truncation of this acidic domain resulted in the loss of transcriptional activity. Interestingly, this acidic region of poliovirus 2A protease is critical for viral RNA replication. The transcriptional activity of the EV71 or Coxsackie virus B3 2A protease should play a role in viral replication and/or pathogenesis.

  18. SR proteins in vertical integration of gene expression from transcription to RNA processing to translation.

    Science.gov (United States)

    Zhong, Xiang-Yang; Wang, Pingping; Han, Joonhee; Rosenfeld, Michael G; Fu, Xiang-Dong

    2009-07-10

    SR proteins have been studied extensively as a family of RNA-binding proteins that participate in both constitutive and regulated pre-mRNA splicing in mammalian cells. However, SR proteins were first discovered as factors that interact with transcriptionally active chromatin. Recent studies have now uncovered properties that connect these once apparently disparate functions, showing that a subset of SR proteins seem to bind directly to the histone 3 tail, play an active role in transcriptional elongation, and colocalize with genes that are engaged in specific intra- and interchromosome interactions for coordinated regulation of gene expression in the nucleus. These transcription-related activities are also coupled with a further expansion of putative functions of specific SR protein family members in RNA metabolism downstream of mRNA splicing, from RNA export to stability control to translation. These findings, therefore, highlight the broader roles of SR proteins in vertical integration of gene expression and provide mechanistic insights into their contributions to genome stability and proper cell-cycle progression in higher eukaryotic cells.

  19. Genetic variation shapes protein networks mainly through non-transcriptional mechanisms.

    Directory of Open Access Journals (Sweden)

    Eric J Foss

    2011-09-01

    Full Text Available Networks of co-regulated transcripts in genetically diverse populations have been studied extensively, but little is known about the degree to which these networks cause similar co-variation at the protein level. We quantified 354 proteins in a genetically diverse population of yeast segregants, which allowed for the first time construction of a coherent protein co-variation matrix. We identified tightly co-regulated groups of 36 and 93 proteins that were made up predominantly of genes involved in ribosome biogenesis and amino acid metabolism, respectively. Even though the ribosomal genes were tightly co-regulated at both the protein and transcript levels, genetic regulation of proteins was entirely distinct from that of transcripts, and almost no genes in this network showed a significant correlation between protein and transcript levels. This result calls into question the widely held belief that in yeast, as opposed to higher eukaryotes, ribosomal protein levels are regulated primarily by regulating transcript levels. Furthermore, although genetic regulation of the amino acid network was more similar for proteins and transcripts, regression analysis demonstrated that even here, proteins vary predominantly as a result of non-transcriptional variation. We also found that cis regulation, which is common in the transcriptome, is rare at the level of the proteome. We conclude that most inter-individual variation in levels of these particular high abundance proteins in this genetically diverse population is not caused by variation of their underlying transcripts.

  20. Transcriptional activation of NAD+-dependent protein deacetylase SIRT1 by nuclear receptor TLX.

    Science.gov (United States)

    Iwahara, Naotoshi; Hisahara, Shin; Hayashi, Takashi; Horio, Yoshiyuki

    2009-09-04

    An orphan nuclear receptor TLX is a transcriptional repressor that promotes the proliferation and self-renewal of neural precursor cells (NPCs). SIRT1, an NAD(+)-dependent protein deacetylase, is highly expressed in the NPCs and participates in neurogenesis. Here, we found that TLX colocalized with SIRT1 and knockdown of TLX by small interfering RNAs decreased SIRT1 levels in NPCs. TLX increased the SIRT1 expression by binding to the newly identified TLX-activating element in the SIRT1 gene promoter in HEK293 cells. Thus, TLX is an inducer of SIRT1 and may contribute to neurogenesis both as a transactivator and as a repressor.

  1. Transcriptional activation of NAD+-dependent protein deacetylase SIRT1 by nuclear receptor TLX

    International Nuclear Information System (INIS)

    Iwahara, Naotoshi; Hisahara, Shin; Hayashi, Takashi; Horio, Yoshiyuki

    2009-01-01

    An orphan nuclear receptor TLX is a transcriptional repressor that promotes the proliferation and self-renewal of neural precursor cells (NPCs). SIRT1, an NAD + -dependent protein deacetylase, is highly expressed in the NPCs and participates in neurogenesis. Here, we found that TLX colocalized with SIRT1 and knockdown of TLX by small interfering RNAs decreased SIRT1 levels in NPCs. TLX increased the SIRT1 expression by binding to the newly identified TLX-activating element in the SIRT1 gene promoter in HEK293 cells. Thus, TLX is an inducer of SIRT1 and may contribute to neurogenesis both as a transactivator and as a repressor.

  2. Emerging functions of ribosomal proteins in gene-specific transcription and translation

    International Nuclear Information System (INIS)

    Lindstroem, Mikael S.

    2009-01-01

    Ribosomal proteins have remained highly conserved during evolution presumably reflecting often critical functions in ribosome biogenesis or mature ribosome function. In addition, several ribosomal proteins possess distinct extra-ribosomal functions in apoptosis, DNA repair and transcription. An increasing number of ribosomal proteins have been shown to modulate the trans-activation function of important regulatory proteins such as NF-κB, p53, c-Myc and nuclear receptors. Furthermore, a subset of ribosomal proteins can bind directly to untranslated regions of mRNA resulting in transcript-specific translational control outside of the ribosome itself. Collectively, these findings suggest that ribosomal proteins may have a wider functional repertoire within the cell than previously thought. The future challenge is to identify and validate these novel functions in the background of an often essential primary function in ribosome biogenesis and cell growth.

  3. The predictive nature of transcript expression levels on protein expression in adult human brain.

    Science.gov (United States)

    Bauernfeind, Amy L; Babbitt, Courtney C

    2017-04-24

    Next generation sequencing methods are the gold standard for evaluating expression of the transcriptome. When determining the biological implications of such studies, the assumption is often made that transcript expression levels correspond to protein levels in a meaningful way. However, the strength of the overall correlation between transcript and protein expression is inconsistent, particularly in brain samples. Following high-throughput transcriptomic (RNA-Seq) and proteomic (liquid chromatography coupled with tandem mass spectrometry) analyses of adult human brain samples, we compared the correlation in the expression of transcripts and proteins that support various biological processes, molecular functions, and that are located in different areas of the cell. Although most categories of transcripts have extremely weak predictive value for the expression of their associated proteins (R 2 values of < 10%), transcripts coding for protein kinases and membrane-associated proteins, including those that are part of receptors or ion transporters, are among those that are most predictive of downstream protein expression levels. The predictive value of transcript expression for corresponding proteins is variable in human brain samples, reflecting the complex regulation of protein expression. However, we found that transcriptomic analyses are appropriate for assessing the expression levels of certain classes of proteins, including those that modify proteins, such as kinases and phosphatases, regulate metabolic and synaptic activity, or are associated with a cellular membrane. These findings can be used to guide the interpretation of gene expression results from primate brain samples.

  4. Repressive effects of resveratrol on androgen receptor transcriptional activity.

    Directory of Open Access Journals (Sweden)

    Wen-feng Shi

    2009-10-01

    Full Text Available The chemopreventive effects of resveratrol (RSV on prostate cancer have been well established; the androgen receptor (AR plays pivotal roles in prostatic tumorigenesis. However, the exact underlying molecular mechanisms about the effects of RSV on AR have not been fully elucidated. A model system is needed to determine whether and how RSV represses AR transcriptional activity.The AR cDNA was first cloned into the retroviral vector pOZ-N and then integrated into the genome of AR-negative HeLa cells to generate the AR(+ cells. The constitutively expressed AR was characterized by monitoring hormone-stimulated nuclear translocation, DNA binding, and transcriptional activation, with the AR(- cells serving as controls. AR(+ cells were treated with RSV, and both AR protein levels and AR transcriptional activity were measured simultaneously. Chromatin immunoprecipitation (ChIP assays were used to detect the effects of RSV on the recruitment of AR to its cognate element (ARE.AR in the AR (+ stable cell line functions in a manner similar to that of endogenously expressed AR. Using this model system we clearly demonstrated that RSV represses AR transcriptional activity independently of any effects on AR protein levels. However, neither the hormone-mediated nucleus translocation nor the AR/ARE interaction was affected by RSV treatment.We demonstrated unambiguously that RSV regulates AR target gene expression, at least in part, by repressing AR transcriptional activity. Repressive effects of RSV on AR activity result from mechanisms other than the affects of AR nuclear translocation or DNA binding.

  5. Large-scale transcriptome data reveals transcriptional activity of fission yeast LTR retrotransposons

    DEFF Research Database (Denmark)

    Mourier, Tobias; Willerslev, Eske

    2010-01-01

    of transcriptional activity are observed from both strands of solitary LTR sequences. Transcriptome data collected during meiosis suggests that transcription of solitary LTRs is correlated with the transcription of nearby protein-coding genes. CONCLUSIONS: Presumably, the host organism negatively regulates...

  6. An upstream activation element exerting differential transcriptional activation on an archaeal promoter

    DEFF Research Database (Denmark)

    Peng, Nan; Xia, Qiu; Chen, Zhengjun

    2009-01-01

    S gene encoding an arabinose binding protein was characterized using an Sulfolobus islandicus reporter gene system. The minimal active araS promoter (P(araS)) was found to be 59 nucleotides long and harboured four promoter elements: an ara-box, an upstream transcription factor B-responsive element (BRE......), a TATA-box and a proximal promoter element, each of which contained important nucleotides that either greatly decreased or completely abolished promoter activity upon mutagenesis. The basal araS promoter was virtually inactive due to intrinsically weak BRE element, and the upstream activating sequence...... (UAS) ara-box activated the basal promoter by recruiting transcription factor B to its BRE. While this UAS ensured a general expression from an inactive or weak basal promoter in the presence of other tested carbon resources, it exhibited a strong arabinose-responsive transcriptional activation. To our...

  7. Bicarbonate-mediated transcriptional activation of divergent operons by the virulence regulatory protein, RegA, from Citrobacter rodentium.

    Science.gov (United States)

    Yang, Ji; Hart, Emily; Tauschek, Marija; Price, G Dean; Hartland, Elizabeth L; Strugnell, Richard A; Robins-Browne, Roy M

    2008-04-01

    Regulation of virulence gene expression plays a central role in the pathogenesis of enteric bacteria as they encounter diverse environmental conditions in the gastrointestinal tract of their hosts. In this study, we investigated environmental regulation of two putative virulence determinants adcA and kfc by RegA, an AraC/XylS-like regulator, from Citrobacter rodentium, and identified bicarbonate as the environmental signal which induced transcription of adcA and kfc through RegA. Primer extension experiments showed that adcA and kfc were divergently transcribed from sigma(70) promoters. In vivo and in vitro experiments demonstrated that bicarbonate facilitated and stabilized the binding of RegA to an operator located between the two promoters. The interaction of RegA with its DNA target resulted in the formation of a nucleosome-like structure, which evidently displaced the histone-like proteins, H-NS and StpA, from the adcA and kfc promoter regions, leading to transcriptional derepression. In addition, our results indicated that RegA also behaved as a Class I activator by directly stimulating transcription initiation by RNA polymerase. This is the first report to describe the molecular mechanism by which an environmental chemical stimulates transcription of virulence-associated genes of an enteric pathogen through an AraC/XlyS-like activator.

  8. Proteins mediating DNA loops effectively block transcription.

    Science.gov (United States)

    Vörös, Zsuzsanna; Yan, Yan; Kovari, Daniel T; Finzi, Laura; Dunlap, David

    2017-07-01

    Loops are ubiquitous topological elements formed when proteins simultaneously bind to two noncontiguous DNA sites. While a loop-mediating protein may regulate initiation at a promoter, the presence of the protein at the other site may be an obstacle for RNA polymerases (RNAP) transcribing a different gene. To test whether a DNA loop alters the extent to which a protein blocks transcription, the lac repressor (LacI) was used. The outcome of in vitro transcription along templates containing two LacI operators separated by 400 bp in the presence of LacI concentrations that produced both looped and unlooped molecules was visualized with scanning force microscopy (SFM). An analysis of transcription elongation complexes, moving for 60 s at an average of 10 nt/s on unlooped DNA templates, revealed that they more often surpassed LacI bound to the lower affinity O2 operator than to the highest affinity Os operator. However, this difference was abrogated in looped DNA molecules where LacI became a strong roadblock independently of the affinity of the operator. Recordings of transcription elongation complexes, using magnetic tweezers, confirmed that they halted for several minutes upon encountering a LacI bound to a single operator. The average pause lifetime is compatible with RNAP waiting for LacI dissociation, however, the LacI open conformation visualized in the SFM images also suggests that LacI could straddle RNAP to let it pass. Independently of the mechanism by which RNAP bypasses the LacI roadblock, the data indicate that an obstacle with looped topology more effectively interferes with transcription. © 2017 The Authors Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.

  9. Transcriptional inhibition by the retinoblastoma protein

    DEFF Research Database (Denmark)

    Fattaey, A; Helin, K; Harlow, E

    1993-01-01

    The retinoblastoma protein, pRB, appears to play a key role in coordinating the regulation of cell cycle position and transcriptional events. pRB undergoes specific cell-cycle-dependent phosphorylation, being underphosphorylated in G1 and heavily phosphorylated in S, G2, and M. The underphosphory......The retinoblastoma protein, pRB, appears to play a key role in coordinating the regulation of cell cycle position and transcriptional events. pRB undergoes specific cell-cycle-dependent phosphorylation, being underphosphorylated in G1 and heavily phosphorylated in S, G2, and M......-mediated transcription would be lost by mutation in the retinoblastoma gene in human tumours, by pRB's interaction with DNA tumour virus oncoproteins, or by phosphorylation during the cell cycle....

  10. Post-translational regulation of Oct4 transcriptional activity.

    Directory of Open Access Journals (Sweden)

    Jonathan P Saxe

    Full Text Available Oct4 is a key component of the molecular circuitry which regulates embryonic stem cell proliferation and differentiation. It is essential for maintenance of undifferentiated, pluripotent cell populations, and accomplishes these tasks by binding DNA in multiple heterodimer and homodimer configurations. Very little is known about how formation of these complexes is regulated, or the mechanisms through which Oct4 proteins respond to complex extracellular stimuli which regulate pluripotency. Here, we provide evidence for a phosphorylation-based mechanism which regulates specific Oct4 homodimer conformations. Point mutations of a putative phosphorylation site can specifically abrogate transcriptional activity of a specific homodimer assembly, with little effect on other configurations. Moreover, we performed bioinformatic predictions to identify a subset of Oct4 target genes which may be regulated by this specific assembly, and show that altering Oct4 protein levels affects transcription of Oct4 target genes which are regulated by this assembly but not others. Finally, we identified several signaling pathways which may mediate this phosphorylation and act in combination to regulate Oct4 transcriptional activity and protein stability. These results provide a mechanism for rapid and reversible alteration of Oct4 transactivation potential in response to extracellular signals.

  11. Detecting Differential Transcription Factor Activity from ATAC-Seq Data

    Directory of Open Access Journals (Sweden)

    Ignacio J. Tripodi

    2018-05-01

    Full Text Available Transcription factors are managers of the cellular factory, and key components to many diseases. Many non-coding single nucleotide polymorphisms affect transcription factors, either by directly altering the protein or its functional activity at individual binding sites. Here we first briefly summarize high-throughput approaches to studying transcription factor activity. We then demonstrate, using published chromatin accessibility data (specifically ATAC-seq, that the genome-wide profile of TF recognition motifs relative to regions of open chromatin can determine the key transcription factor altered by a perturbation. Our method of determining which TFs are altered by a perturbation is simple, is quick to implement, and can be used when biological samples are limited. In the future, we envision that this method could be applied to determine which TFs show altered activity in response to a wide variety of drugs and diseases.

  12. Post-Transcriptional Regulation Prevents Accumulation of Glutathione Reductase Protein and Activity in the Bundle Sheath Cells of Maize1

    Science.gov (United States)

    Pastori, Gabriela M.; Mullineaux, Philip M.; Foyer, Christine H.

    2000-01-01

    Glutathione reductase (GR; EC 1.6.4.2) activity was assayed in bundle sheath and mesophyll cells of maize (Zea mays L. var H99) from plants grown at 20°C, 18°C, and 15°C. The purity of each fraction was determined by measuring the associated activity of the compartment-specific marker enzymes, Rubisco and phosphoenolpyruvate carboxylase, respectively. GR activity and the abundance of GR protein and mRNA increased in plants grown at 15°C and 18°C compared with those grown at 20°C. In all cases GR activity was found only in mesophyll fractions of the leaves, with no GR activity being detectable in bundle sheath extracts. Immunogold labeling with GR-specific antibodies showed that the GR protein was exclusively localized in the mesophyll cells of leaves at all growth temperatures, whereas GR transcripts (as determined by in situ hybridization techniques) were observed in both cell types. These results indicate that post-transcriptional regulation prevents GR accumulation in the bundle sheath cells of maize leaves. The resulting limitation on the capacity for regeneration of reduced glutathione in this compartment may contribute to the extreme chilling sensitivity of maize leaves. PMID:10712529

  13. Regulatory Interactions of Csr Components: the RNA Binding Protein CsrA Activates csrB Transcription in Escherichia coli

    OpenAIRE

    Gudapaty, Seshagirirao; Suzuki, Kazushi; Wang, Xin; Babitzke, Paul; Romeo, Tony

    2002-01-01

    The global regulator CsrA (carbon storage regulator) of Escherichia coli is a small RNA binding protein that represses various metabolic pathways and processes that are induced in the stationary phase of growth, while it activates certain exponential phase functions. Both repression and activation by CsrA involve posttranscriptional mechanisms, in which CsrA binding to mRNA leads to decreased or increased transcript stability, respectively. CsrA also binds to a small untranslated RNA, CsrB, f...

  14. Dual functions of Rift Valley fever virus NSs protein: inhibition of host mRNA transcription and post-transcriptional downregulation of protein kinase PKR.

    Science.gov (United States)

    Ikegami, Tetsuro; Narayanan, Krishna; Won, Sungyong; Kamitani, Wataru; Peters, C J; Makino, Shinji

    2009-09-01

    Rift Valley fever virus (RVFV), which belongs to the genus Phlebovirus, family Bunyaviridae, is a negative-stranded RNA virus carrying a single-stranded, tripartite RNA genome. RVFV is an important zoonotic pathogen transmitted by mosquitoes and causes large outbreaks among ruminants and humans in Africa and the Arabian Peninsula. Human patients develop an acute febrile illness, followed by a fatal hemorrhagic fever, encephalitis, or ocular diseases. A viral nonstructural protein, NSs, is a major viral virulence factor. Past studies showed that NSs suppresses the transcription of host mRNAs, including interferon-beta mRNAs. Here we demonstrated that the NSs protein induced post-transcriptional downregulation of dsRNA-dependent protein kinase (PKR), to prevent phosphorylation of eIF2alpha and promoted viral translation in infected cells. These two biological activities of the NSs most probably have a synergistic effect in suppressing host innate immune functions and facilitate efficient viral replication in infected mammalian hosts.

  15. TcoF-DB: dragon database for human transcription co-factors and transcription factor interacting proteins

    KAUST Repository

    Schaefer, Ulf; Schmeier, Sebastian; Bajic, Vladimir B.

    2010-01-01

    The initiation and regulation of transcription in eukaryotes is complex and involves a large number of transcription factors (TFs), which are known to bind to the regulatory regions of eukaryotic DNA. Apart from TF-DNA binding, protein-protein interaction involving TFs is an essential component of the machinery facilitating transcriptional regulation. Proteins that interact with TFs in the context of transcription regulation but do not bind to the DNA themselves, we consider transcription co-factors (TcoFs). The influence of TcoFs on transcriptional regulation and initiation, although indirect, has been shown to be significant with the functionality of TFs strongly influenced by the presence of TcoFs. While the role of TFs and their interaction with regulatory DNA regions has been well-studied, the association between TFs and TcoFs has so far been given less attention. Here, we present a resource that is comprised of a collection of human TFs and the TcoFs with which they interact. Other proteins that have a proven interaction with a TF, but are not considered TcoFs are also included. Our database contains 157 high-confidence TcoFs and additionally 379 hypothetical TcoFs. These have been identified and classified according to the type of available evidence for their involvement in transcriptional regulation and their presence in the cell nucleus. We have divided TcoFs into four groups, one of which contains high-confidence TcoFs and three others contain TcoFs which are hypothetical to different extents. We have developed the Dragon Database for Human Transcription Co-Factors and Transcription Factor Interacting Proteins (TcoF-DB). A web-based interface for this resource can be freely accessed at http://cbrc.kaust.edu.sa/tcof/ and http://apps.sanbi.ac.za/tcof/. © The Author(s) 2010.

  16. TcoF-DB: dragon database for human transcription co-factors and transcription factor interacting proteins

    KAUST Repository

    Schaefer, Ulf

    2010-10-21

    The initiation and regulation of transcription in eukaryotes is complex and involves a large number of transcription factors (TFs), which are known to bind to the regulatory regions of eukaryotic DNA. Apart from TF-DNA binding, protein-protein interaction involving TFs is an essential component of the machinery facilitating transcriptional regulation. Proteins that interact with TFs in the context of transcription regulation but do not bind to the DNA themselves, we consider transcription co-factors (TcoFs). The influence of TcoFs on transcriptional regulation and initiation, although indirect, has been shown to be significant with the functionality of TFs strongly influenced by the presence of TcoFs. While the role of TFs and their interaction with regulatory DNA regions has been well-studied, the association between TFs and TcoFs has so far been given less attention. Here, we present a resource that is comprised of a collection of human TFs and the TcoFs with which they interact. Other proteins that have a proven interaction with a TF, but are not considered TcoFs are also included. Our database contains 157 high-confidence TcoFs and additionally 379 hypothetical TcoFs. These have been identified and classified according to the type of available evidence for their involvement in transcriptional regulation and their presence in the cell nucleus. We have divided TcoFs into four groups, one of which contains high-confidence TcoFs and three others contain TcoFs which are hypothetical to different extents. We have developed the Dragon Database for Human Transcription Co-Factors and Transcription Factor Interacting Proteins (TcoF-DB). A web-based interface for this resource can be freely accessed at http://cbrc.kaust.edu.sa/tcof/ and http://apps.sanbi.ac.za/tcof/. © The Author(s) 2010.

  17. The retinoblastoma protein binds to a family of E2F transcription factors

    DEFF Research Database (Denmark)

    Lees, J A; Saito, M; Vidal, M

    1993-01-01

    E2F is a transcription factor that helps regulate the expression of a number of genes that are important in cell proliferation. Recently, several laboratories have isolated a cDNA clone that encodes an E2F-like protein, known as E2F-1. Subsequent characterization of this protein showed that it had...... the properties of E2F, but it was difficult to account for all of the suggested E2F activities through the function of this one protein. Using low-stringency hybridization, we have isolated cDNA clones that encode two additional E2F-like proteins, called E2F-2 and E2F-3. The chromosomal locations of the genes...... protein in vivo. Finally, E2F-2 and E2F-3 were able to activate transcription of E2F-responsive genes in a manner that was dependent upon the presence of at least one functional E2F binding site. These observations suggest that the E2F activities described previously result from the combined action...

  18. A NodD-like protein activates transcription of genes involved with naringenin degradation in a flavonoid-dependent manner in Herbaspirillum seropedicae.

    Science.gov (United States)

    Wassem, R; Marin, A M; Daddaoua, A; Monteiro, R A; Chubatsu, L S; Ramos, J L; Deakin, W J; Broughton, W J; Pedrosa, F O; Souza, E M

    2017-03-01

    Herbaspirillum seropedicae is an associative, endophytic non-nodulating diazotrophic bacterium that colonises several grasses. An ORF encoding a LysR-type transcriptional regulator, very similar to NodD proteins of rhizobia, was identified in its genome. This nodD-like gene, named fdeR, is divergently transcribed from an operon encoding enzymes involved in flavonoid degradation (fde operon). Apigenin, chrysin, luteolin and naringenin strongly induce transcription of the fde operon, but not that of the fdeR, in an FdeR-dependent manner. The intergenic region between fdeR and fdeA contains several generic LysR consensus sequences (T-N 11 -A) and we propose a binding site for FdeR, which is conserved in other bacteria. DNase I foot-printing revealed that the interaction with the FdeR binding site is modified by the four flavonoids that stimulate transcription of the fde operon. Moreover, FdeR binds naringenin and chrysin as shown by isothermal titration calorimetry. Interestingly, FdeR also binds in vitro to the nod-box from the nodABC operon of Rhizobium sp. NGR234 and is able to activate its transcription in vivo. These results show that FdeR exhibits two features of rhizobial NodD proteins: nod-box recognition and flavonoid-dependent transcription activation, but its role in H. seropedicae and related organisms seems to have evolved to control flavonoid metabolism. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

  19. Modifiers of notch transcriptional activity identified by genome-wide RNAi

    Directory of Open Access Journals (Sweden)

    Firnhaber Christopher B

    2010-10-01

    Full Text Available Abstract Background The Notch signaling pathway regulates a diverse array of developmental processes, and aberrant Notch signaling can lead to diseases, including cancer. To obtain a more comprehensive understanding of the genetic network that integrates into Notch signaling, we performed a genome-wide RNAi screen in Drosophila cell culture to identify genes that modify Notch-dependent transcription. Results Employing complementary data analyses, we found 399 putative modifiers: 189 promoting and 210 antagonizing Notch activated transcription. These modifiers included several known Notch interactors, validating the robustness of the assay. Many novel modifiers were also identified, covering a range of cellular localizations from the extracellular matrix to the nucleus, as well as a large number of proteins with unknown function. Chromatin-modifying proteins represent a major class of genes identified, including histone deacetylase and demethylase complex components and other chromatin modifying, remodeling and replacement factors. A protein-protein interaction map of the Notch-dependent transcription modifiers revealed that a large number of the identified proteins interact physically with these core chromatin components. Conclusions The genome-wide RNAi screen identified many genes that can modulate Notch transcriptional output. A protein interaction map of the identified genes highlighted a network of chromatin-modifying enzymes and remodelers that regulate Notch transcription. Our results open new avenues to explore the mechanisms of Notch signal regulation and the integration of this pathway into diverse cellular processes.

  20. The putative Agrobacterium transcriptional activator-like virulence protein VirD5 may target T-complex to prevent the degradation of coat proteins in the plant cell nucleus.

    Science.gov (United States)

    Wang, Yafei; Peng, Wei; Zhou, Xu; Huang, Fei; Shao, Lingyun; Luo, Meizhong

    2014-09-01

    Agrobacterium exports at least five virulence proteins (VirE2, VirE3, VirF, VirD2, VirD5) into host cells and hijacks some host plant factors to facilitate its transformation process. Random DNA binding selection assays (RDSAs), electrophoretic mobility shift assays (EMSAs) and yeast one-hybrid systems were used to identify protein-bound DNA elements. Bimolecular fluorescence complementation, glutathione S-transferase pull-down and yeast two-hybrid assays were used to detect protein interactions. Protoplast transformation, coprecipitation, competitive binding and cell-free degradation assays were used to analyze the relationships among proteins. We found that Agrobacterium VirD5 exhibits transcriptional activation activity in yeast, is located in the plant cell nucleus, and forms homodimers. A specific VirD5-bound DNA element designated D5RE (VirD5 response element) was identified. VirD5 interacted directly with Arabidopsis VirE2 Interacting Protein 1 (AtVIP1). However, the ternary complex of VirD5-AtVIP1-VirE2 could be detected, whereas that of VirD5-AtVIP1-VBF (AtVIP1 Binding F-box protein) could not. We demonstrated that VirD5 competes with VBF for binding to AtVIP1 and stabilizes AtVIP1 and VirE2 in the cell-free degradation system. Our results indicated that VirD5 may act as both a transcriptional activator-like effector to regulate host gene expression and a protector preventing the coat proteins of the T-complex from being quickly degraded by the host's ubiquitin proteasome system (UPS). © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

  1. Activation of the polyomavirus enhancer by a murine activator protein 1 (AP1) homolog and two contiguous proteins.

    OpenAIRE

    Martin, M E; Piette, J; Yaniv, M; Tang, W J; Folk, W R

    1988-01-01

    The polyomavirus enhancer is composed of multiple DNA sequence elements serving as binding sites for proteins present in mouse nuclear extracts that activate transcription and DNA replication. We have identified three such proteins and their binding sites and correlate them with enhancer function. Mutation of nucleotide (nt) 5140 in the enhancer alters the binding site (TGACTAA, nt 5139-5145) for polyomavirus enhancer A binding protein 1 (PEA1), a murine homolog of the human transcription fac...

  2. Two familial ALS proteins function in prevention/repair of transcription-associated DNA damage.

    Science.gov (United States)

    Hill, Sarah J; Mordes, Daniel A; Cameron, Lisa A; Neuberg, Donna S; Landini, Serena; Eggan, Kevin; Livingston, David M

    2016-11-29

    Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron dysfunction disease that leads to paralysis and death. There is currently no established molecular pathogenesis pathway. Multiple proteins involved in RNA processing are linked to ALS, including FUS and TDP43, and we propose a disease mechanism in which loss of function of at least one of these proteins leads to an accumulation of transcription-associated DNA damage contributing to motor neuron cell death and progressive neurological symptoms. In support of this hypothesis, we find that FUS or TDP43 depletion leads to increased sensitivity to a transcription-arresting agent due to increased DNA damage. Thus, these proteins normally contribute to the prevention or repair of transcription-associated DNA damage. In addition, both FUS and TDP43 colocalize with active RNA polymerase II at sites of DNA damage along with the DNA damage repair protein, BRCA1, and FUS and TDP43 participate in the prevention or repair of R loop-associated DNA damage, a manifestation of aberrant transcription and/or RNA processing. Gaining a better understanding of the role(s) that FUS and TDP43 play in transcription-associated DNA damage could shed light on the mechanisms underlying ALS pathogenesis.

  3. Fungal mediator tail subunits contain classical transcriptional activation domains.

    Science.gov (United States)

    Liu, Zhongle; Myers, Lawrence C

    2015-04-01

    Classical activation domains within DNA-bound eukaryotic transcription factors make weak interactions with coactivator complexes, such as Mediator, to stimulate transcription. How these interactions stimulate transcription, however, is unknown. The activation of reporter genes by artificial fusion of Mediator subunits to DNA binding domains that bind to their promoters has been cited as evidence that the primary role of activators is simply to recruit Mediator. We have identified potent classical transcriptional activation domains in the C termini of several tail module subunits of Saccharomyces cerevisiae, Candida albicans, and Candida dubliniensis Mediator, while their N-terminal domains are necessary and sufficient for their incorporation into Mediator but do not possess the ability to activate transcription when fused to a DNA binding domain. This suggests that Mediator fusion proteins actually are functioning in a manner similar to that of a classical DNA-bound activator rather than just recruiting Mediator. Our finding that deletion of the activation domains of S. cerevisiae Med2 and Med3, as well as C. dubliniensis Tlo1 (a Med2 ortholog), impairs the induction of certain genes shows these domains function at native promoters. Activation domains within coactivators are likely an important feature of these complexes and one that may have been uniquely leveraged by a common fungal pathogen. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  4. O-GlcNAc transferase regulates transcriptional activity of human Oct4.

    Science.gov (United States)

    Constable, Sandii; Lim, Jae-Min; Vaidyanathan, Krithika; Wells, Lance

    2017-10-01

    O-linked β-N-acetylglucosamine (O-GlcNAc) is a single sugar modification found on many different classes of nuclear and cytoplasmic proteins. Addition of this modification, by the enzyme O-linked N-acetylglucosamine transferase (OGT), is dynamic and inducible. One major class of proteins modified by O-GlcNAc is transcription factors. O-GlcNAc regulates transcription factor properties through a variety of different mechanisms including localization, stability and transcriptional activation. Maintenance of embryonic stem (ES) cell pluripotency requires tight regulation of several key transcription factors, many of which are modified by O-GlcNAc. Octamer-binding protein 4 (Oct4) is one of the key transcription factors required for pluripotency of ES cells and more recently, the generation of induced pluripotent stem (iPS) cells. The action of Oct4 is modulated by the addition of several post-translational modifications, including O-GlcNAc. Previous studies in mice found a single site of O-GlcNAc addition responsible for transcriptional regulation. This study was designed to determine if this mechanism is conserved in humans. We mapped 10 novel sites of O-GlcNAc attachment on human Oct4, and confirmed a role for OGT in transcriptional activation of Oct4 at a site distinct from that found in mouse that allows distinction between different Oct4 target promoters. Additionally, we uncovered a potential new role for OGT that does not include its catalytic function. These results confirm that human Oct4 activity is being regulated by OGT by a mechanism that is distinct from mouse Oct4. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  5. HuR represses Wnt/β-catenin-mediated transcriptional activity by promoting cytoplasmic localization of β-catenin

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Inae; Hur, Jung; Jeong, Sunjoo, E-mail: sjsj@dankook.ac.kr

    2015-01-30

    Highlights: • Wnt signaling as well as β-catenin overexpression enhance HuR cytoplasmic export. • HuR overexpression promotes cytoplasmic localization of β-catenin from the perinuclear fraction. • Wnt/β-catenin-mediated transcriptional activity is repressesed by HuR. - Abstract: β-Catenin is the key transcriptional activator of canonical Wnt signaling in the nucleus; thus, nuclear accumulation of β-catenin is a critical step for expressing target genes. β-Catenin accumulates in the nucleus of cancer cells where it activates oncogenic target genes. Hu antigen R (HuR) is a RNA binding protein that regulates multiple post-transcriptional processes including RNA stability. Thus, cytoplasmic HuR protein may be involved in tumorigenesis by stabilizing oncogenic transcripts, but the molecular mechanism remains unclear. Here, we observed that Wnt/β-catenin signaling induced export of the HuR protein, whereas HuR overexpression promoted accumulation of the β-catenin protein in the cytoplasm. Thus, Wnt/β-catenin-mediated transcriptional activity in the nucleus was reduced by overexpressing HuR. These results suggest novel and uncharacterized cytoplasmic β-catenin functions related to HuR-mediated RNA metabolism in cancer cells.

  6. HuR represses Wnt/β-catenin-mediated transcriptional activity by promoting cytoplasmic localization of β-catenin

    International Nuclear Information System (INIS)

    Kim, Inae; Hur, Jung; Jeong, Sunjoo

    2015-01-01

    Highlights: • Wnt signaling as well as β-catenin overexpression enhance HuR cytoplasmic export. • HuR overexpression promotes cytoplasmic localization of β-catenin from the perinuclear fraction. • Wnt/β-catenin-mediated transcriptional activity is repressesed by HuR. - Abstract: β-Catenin is the key transcriptional activator of canonical Wnt signaling in the nucleus; thus, nuclear accumulation of β-catenin is a critical step for expressing target genes. β-Catenin accumulates in the nucleus of cancer cells where it activates oncogenic target genes. Hu antigen R (HuR) is a RNA binding protein that regulates multiple post-transcriptional processes including RNA stability. Thus, cytoplasmic HuR protein may be involved in tumorigenesis by stabilizing oncogenic transcripts, but the molecular mechanism remains unclear. Here, we observed that Wnt/β-catenin signaling induced export of the HuR protein, whereas HuR overexpression promoted accumulation of the β-catenin protein in the cytoplasm. Thus, Wnt/β-catenin-mediated transcriptional activity in the nucleus was reduced by overexpressing HuR. These results suggest novel and uncharacterized cytoplasmic β-catenin functions related to HuR-mediated RNA metabolism in cancer cells

  7. DREAM Controls the On/Off Switch of Specific Activity-Dependent Transcription Pathways

    Science.gov (United States)

    Mellström, Britt; Sahún, Ignasi; Ruiz-Nuño, Ana; Murtra, Patricia; Gomez-Villafuertes, Rosa; Savignac, Magali; Oliveros, Juan C.; Gonzalez, Paz; Kastanauskaite, Asta; Knafo, Shira; Zhuo, Min; Higuera-Matas, Alejandro; Errington, Michael L.; Maldonado, Rafael; DeFelipe, Javier; Jefferys, John G. R.; Bliss, Tim V. P.; Dierssen, Mara

    2014-01-01

    Changes in nuclear Ca2+ homeostasis activate specific gene expression programs and are central to the acquisition and storage of information in the brain. DREAM (downstream regulatory element antagonist modulator), also known as calsenilin/KChIP-3 (K+ channel interacting protein 3), is a Ca2+-binding protein that binds DNA and represses transcription in a Ca2+-dependent manner. To study the function of DREAM in the brain, we used transgenic mice expressing a Ca2+-insensitive/CREB-independent dominant active mutant DREAM (daDREAM). Using genome-wide analysis, we show that DREAM regulates the expression of specific activity-dependent transcription factors in the hippocampus, including Npas4, Nr4a1, Mef2c, JunB, and c-Fos. Furthermore, DREAM regulates its own expression, establishing an autoinhibitory feedback loop to terminate activity-dependent transcription. Ablation of DREAM does not modify activity-dependent transcription because of gene compensation by the other KChIP family members. The expression of daDREAM in the forebrain resulted in a complex phenotype characterized by loss of recurrent inhibition and enhanced long-term potentiation (LTP) in the dentate gyrus and impaired learning and memory. Our results indicate that DREAM is a major master switch transcription factor that regulates the on/off status of specific activity-dependent gene expression programs that control synaptic plasticity, learning, and memory. PMID:24366545

  8. Cooperative activation of transcription by autoimmune regulator AIRE and CBP

    International Nuclear Information System (INIS)

    Pitkaenen, J.; Rebane, A.; Rowell, J.; Murumaegi, A.; Stroebel, P.; Moell, K.; Saare, M.; Heikkilae, J.; Doucas, V.; Marx, A.; Peterson, P.

    2005-01-01

    Autoimmune regulator (AIRE) is a transcriptional regulator that is believed to control the expression of tissue-specific genes in the thymus. Mutated AIRE is responsible for onset of the hereditary autoimmune disease APECED. AIRE is able to form nuclear bodies (NBs) and interacts with the ubiquitous transcriptional coactivator CBP. In this paper, we show that CBP and AIRE synergistically activate transcription on different promoter reporters whereas AIRE gene mutation R257X, found in APECED patients, interferes with this coactivation effect. Furthermore, the overexpression of AIRE and CBP collaboratively enhance endogenous IFNβ mRNA expression. The immunohistochemical studies suggest that CBP, depending on the balance of nuclear proteins, is a component of AIRE NBs. We also show that AIRE NBs are devoid of active chromatin and, therefore, not sites of transcription. In addition, we demonstrate by 3D analyses that AIRE and CBP, when colocalizing, are located spatially differently within AIRE NBs. In conclusion, our data suggest that AIRE activates transcription of the target genes, i.e., autoantigens in collaboration with CBP and that this activation occurs outside of AIRE NBs

  9. The Drosophila melanogaster DmCK2beta transcription unit encodes for functionally non-redundant protein isoforms.

    Science.gov (United States)

    Jauch, Eike; Wecklein, Heike; Stark, Felix; Jauch, Mandy; Raabe, Thomas

    2006-06-07

    Genes encoding for the two evolutionary highly conserved subunits of a heterotetrameric protein kinase CK2 holoenzyme are present in all examined eukaryotic genomes. Depending on the organism, multiple transcription units encoding for a catalytically active CK2alpha subunit and/or a regulatory CK2beta subunit may exist. The phosphotransferase activity of members of the protein kinase CK2alpha family is thought to be independent of second messengers but is modulated by interaction with CK2beta-like proteins. In the genome of Drosophila melanogaster, one gene encoding for a CK2alpha subunit and three genes encoding for CK2beta-like proteins are present. The X-linked DmCK2beta transcription unit encodes for several CK2beta protein isoforms due to alternative splicing of its primary transcript. We addressed the question whether CK2beta-like proteins are redundant in function. Our in vivo experiments show that variations of the very C-terminal tail of CK2beta isoforms encoded by the X-linked DmCK2beta transcription unit influence their functional properties. In addition, we find that CK2beta-like proteins encoded by the autosomal D. melanogaster genes CK2betates and CK2beta' cannot fully substitute for a loss of CK2beta isoforms encoded by DmCK2beta.

  10. The Gcn4 transcription factor reduces protein synthesis capacity and extends yeast lifespan.

    Science.gov (United States)

    Mittal, Nitish; Guimaraes, Joao C; Gross, Thomas; Schmidt, Alexander; Vina-Vilaseca, Arnau; Nedialkova, Danny D; Aeschimann, Florian; Leidel, Sebastian A; Spang, Anne; Zavolan, Mihaela

    2017-09-06

    In Saccharomyces cerevisiae, deletion of large ribosomal subunit protein-encoding genes increases the replicative lifespan in a Gcn4-dependent manner. However, how Gcn4, a key transcriptional activator of amino acid biosynthesis genes, increases lifespan, is unknown. Here we show that Gcn4 acts as a repressor of protein synthesis. By analyzing the messenger RNA and protein abundance, ribosome occupancy and protein synthesis rate in various yeast strains, we demonstrate that Gcn4 is sufficient to reduce protein synthesis and increase yeast lifespan. Chromatin immunoprecipitation reveals Gcn4 binding not only at genes that are activated, but also at genes, some encoding ribosomal proteins, that are repressed upon Gcn4 overexpression. The promoters of repressed genes contain Rap1 binding motifs. Our data suggest that Gcn4 is a central regulator of protein synthesis under multiple perturbations, including ribosomal protein gene deletions, calorie restriction, and rapamycin treatment, and provide an explanation for its role in longevity and stress response.The transcription factor Gcn4 is known to regulate yeast amino acid synthesis. Here, the authors show that Gcn4 also acts as a repressor of protein biosynthesis in a range of conditions that enhance yeast lifespan, such as ribosomal protein knockout, calorie restriction or mTOR inhibition.

  11. Structural studies of bacterial transcriptional regulatory proteins by multidimensional heteronuclear NMR

    Energy Technology Data Exchange (ETDEWEB)

    Volkman, Brian Finley [Univ. of California, Berkeley, CA (United States)

    1995-02-01

    Nuclear magnetic resonance spectroscopy was used to elucidate detailed structural information for peptide and protein molecules. A small peptide was designed and synthesized, and its three-dimensional structure was calculated using distance information derived from two-dimensional NMR measurements. The peptide was used to induce antibodies in mice, and the cross-reactivity of the antibodies with a related protein was analyzed with enzyme-linked immunosorbent assays. Two proteins which are involved in regulation of transcription in bacteria were also studied. The ferric uptake regulation (Fur) protein is a metal-dependent repressor which controls iron uptake in bacteria. Two- and three-dimensional NMR techniques, coupled with uniform and selective isotope labeling allowed the nearly complete assignment of the resonances of the metal-binding domain of the Fur protein. NTRC is a transcriptional enhancer binding protein whose N-terminal domain is a "receiver domain" in the family of "two-component" regulatory systems. Phosphorylation of the N-terminal domain of NTRC activates the initiation of transcription of aeries encoding proteins involved in nitrogen regulation. Three- and four-dimensional NMR spectroscopy methods have been used to complete the resonance assignments and determine the solution structure of the N-terminal receiver domain of the NTRC protein. Comparison of the solution structure of the NTRC receiver domain with the crystal structures of the homologous protein CheY reveals a very similar fold, with the only significant difference being the position of helix 4 relative to the rest of the protein. The determination of the structure of the NTRC receiver domain is the first step toward understanding a mechanism of signal transduction which is common to many bacterial regulatory systems.

  12. G-actin sequestering protein thymosin-β4 regulates the activity of myocardin-related transcription factor.

    Science.gov (United States)

    Morita, Tsuyoshi; Hayashi, Ken'ichiro

    2013-08-02

    Myocardin-related transcription factors (MRTFs) are robust coactivators of serum response factor (SRF). MRTFs contain three copies of the RPEL motif at their N-terminus, and they bind to monomeric globular actin (G-actin). Previous studies illustrate that G-actin binding inhibits MRTF activity by preventing the MRTFs nuclear accumulation. In the living cells, the majority of G-actin is sequestered by G-actin binding proteins that prevent spontaneous actin polymerization. Here, we demonstrate that the most abundant G-actin sequestering protein thymosin-β4 (Tβ4) was involved in the regulation of subcellular localization and activity of MRTF-A. Tβ4 competed with MRTF-A for G-actin binding; thus, interfering with G-actin-MRTF-A complex formation. Tβ4 overexpression induced the MRTF-A nuclear accumulation and activation of MRTF-SRF signaling. The activation rate of MRTF-A by the Tβ4 mutant L17A, whose affinity for G-actin is very low, was lower than that by wild-type Tβ4. In contrast, the β-actin mutant 3DA, which has a lower affinity for Tβ4, more effectively suppressed MRTF-A activity than wild-type β-actin. Furthermore, ectopic Tβ4 increased the endogenous expression of SRF-dependent actin cytoskeletal genes. Thus, Tβ4 is an important MRTF regulator that controls the G-actin-MRTFs interaction. Copyright © 2013 Elsevier Inc. All rights reserved.

  13. Maribavir Inhibits Epstein-Barr Virus Transcription through the EBV Protein Kinase

    Science.gov (United States)

    Whitehurst, Christopher B.; Sanders, Marcia K.; Law, Mankit; Wang, Fu-Zhang; Xiong, Jie; Dittmer, Dirk P.

    2013-01-01

    Maribavir (MBV) inhibits Epstein-Barr virus (EBV) replication and the enzymatic activity of the viral protein kinase BGLF4. MBV also inhibits expression of multiple EBV transcripts during EBV lytic infection. Here we demonstrate, with the use of a BGLF4 knockout virus, that effects of MBV on transcription take place primarily through inhibition of BGLF4. MBV inhibits viral genome copy numbers and infectivity to levels similar to and exceeding levels produced by BGLF4 knockout virus. PMID:23449792

  14. Malondialdehyde inhibits an AMPK-mediated nuclear translocation and repression activity of ALDH2 in transcription

    International Nuclear Information System (INIS)

    Choi, Ji-Woong; Kim, Jae-Hwan; Cho, Sung-Chun; Ha, Moon-Kyung; Song, Kye-Yong; Youn, Hong-Duk; Park, Sang Chul

    2011-01-01

    Research highlights: → ALDH2 is an MDA-modified protein in old rat kidney tissues. → AMPK associates with ALDH2 and triggers the nuclear localization of ALDH2. → ALDH2 serves as a general transcriptional repressor by associating with HDACs. → MDA inhibits the AMPK-mediated translocation of ALDH2 and its repression activity. -- Abstract: Aging process results from deleterious damages by reactive oxygen species, in particular, various metabolic aldehydes. Aldehyde dehydrogenase 2 (ALDH2) is one of metabolic enzymes detoxifying various aldehydes under oxidative conditions. AMP-activated protein kinase (AMPK) plays a key role in controlling metabolic process. However, little was known about the relationship of ALDH2 with AMPK under oxidative conditions. Here, we, by using MDA-specific monoclonal antibody, screened the tissues of young and old rats for MDA-modified proteins and identified an ALDH2 as a prominent MDA-modified protein band in the old rat kidney tissue. ALDH2 associates with AMPK and is phosphorylated by AMPK. In addition, AICAR, an activator of AMP-activated protein kinase, induces the nuclear translocation of ALDH2. ALDH2 in nucleus is involved in general transcription repression by association with histone deacetylases. Furthermore, MDA modification inhibited the translocation of ALDH2 and the association with AMPK, and ultimately led to de-repression of transcription in the reporter system analysis. In this study, we have demonstrated that ALDH2 acts as a transcriptional repressor in response to AMPK activation, and MDA modifies ALDH2 and inhibits repressive activity of ALDH2 in general transcription. We thus suggest that increasing amount of MDA during aging process may interrupt the nuclear function of ALDH2, modulated by AMPK.

  15. Malondialdehyde inhibits an AMPK-mediated nuclear translocation and repression activity of ALDH2 in transcription

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Ji-Woong [Department of Biomedical Sciences and Biochemistry and Molecular Biology, Seoul National University College of Medicine, 28 Yongon-dong, Chongro-gu, Seoul 110-799 (Korea, Republic of); Aging and Apoptosis Research Center (AARC), Seoul National University College of Medicine, 28 Yongon-dong, Chongro-gu, Seoul 110-799, (Korea, Republic of); Kim, Jae-Hwan [Department of Biomedical Sciences and Biochemistry and Molecular Biology, Seoul National University College of Medicine, 28 Yongon-dong, Chongro-gu, Seoul 110-799 (Korea, Republic of); Cho, Sung-Chun; Ha, Moon-Kyung [Department of Biomedical Sciences and Biochemistry and Molecular Biology, Seoul National University College of Medicine, 28 Yongon-dong, Chongro-gu, Seoul 110-799 (Korea, Republic of); Aging and Apoptosis Research Center (AARC), Seoul National University College of Medicine, 28 Yongon-dong, Chongro-gu, Seoul 110-799, (Korea, Republic of); Song, Kye-Yong [Department of Pathology, Chung-Ang University College of Medicine, Seoul 156-756 (Korea, Republic of); Youn, Hong-Duk, E-mail: hdyoun@snu.ac.kr [Department of Biomedical Sciences and Biochemistry and Molecular Biology, Seoul National University College of Medicine, 28 Yongon-dong, Chongro-gu, Seoul 110-799 (Korea, Republic of); Park, Sang Chul, E-mail: scpark@snu.ac.kr [Department of Biomedical Sciences and Biochemistry and Molecular Biology, Seoul National University College of Medicine, 28 Yongon-dong, Chongro-gu, Seoul 110-799 (Korea, Republic of); Aging and Apoptosis Research Center (AARC), Seoul National University College of Medicine, 28 Yongon-dong, Chongro-gu, Seoul 110-799, (Korea, Republic of)

    2011-01-07

    Research highlights: {yields} ALDH2 is an MDA-modified protein in old rat kidney tissues. {yields} AMPK associates with ALDH2 and triggers the nuclear localization of ALDH2. {yields} ALDH2 serves as a general transcriptional repressor by associating with HDACs. {yields} MDA inhibits the AMPK-mediated translocation of ALDH2 and its repression activity. -- Abstract: Aging process results from deleterious damages by reactive oxygen species, in particular, various metabolic aldehydes. Aldehyde dehydrogenase 2 (ALDH2) is one of metabolic enzymes detoxifying various aldehydes under oxidative conditions. AMP-activated protein kinase (AMPK) plays a key role in controlling metabolic process. However, little was known about the relationship of ALDH2 with AMPK under oxidative conditions. Here, we, by using MDA-specific monoclonal antibody, screened the tissues of young and old rats for MDA-modified proteins and identified an ALDH2 as a prominent MDA-modified protein band in the old rat kidney tissue. ALDH2 associates with AMPK and is phosphorylated by AMPK. In addition, AICAR, an activator of AMP-activated protein kinase, induces the nuclear translocation of ALDH2. ALDH2 in nucleus is involved in general transcription repression by association with histone deacetylases. Furthermore, MDA modification inhibited the translocation of ALDH2 and the association with AMPK, and ultimately led to de-repression of transcription in the reporter system analysis. In this study, we have demonstrated that ALDH2 acts as a transcriptional repressor in response to AMPK activation, and MDA modifies ALDH2 and inhibits repressive activity of ALDH2 in general transcription. We thus suggest that increasing amount of MDA during aging process may interrupt the nuclear function of ALDH2, modulated by AMPK.

  16. The Small Protein HemP Is a Transcriptional Activator for the Hemin Uptake Operon in Burkholderia multivorans ATCC 17616.

    Science.gov (United States)

    Sato, Takuya; Nonoyama, Shouta; Kimura, Akane; Nagata, Yuji; Ohtsubo, Yoshiyuki; Tsuda, Masataka

    2017-08-15

    Iron and heme play very important roles in various metabolic functions in bacteria, and their intracellular homeostasis is maintained because high concentrations of free forms of these molecules greatly facilitate the Fenton reaction-mediated production of large amounts of reactive oxygen species that severely damage various biomolecules. The ferric uptake regulator (Fur) from Burkholderia multivorans ATCC 17616 is an iron-responsive global transcriptional regulator, and its fur deletant exhibits pleiotropic phenotypes. In this study, we found that the phenotypes of the fur deletant were suppressed by an additional mutation in hemP The transcription of hemP was negatively regulated by Fur under iron-replete conditions and was constitutive in the fur deletant. Growth of a hemP deletant was severely impaired in a medium containing hemin as the sole iron source, demonstrating the important role of HemP in hemin utilization. HemP was required as a transcriptional activator that specifically binds the promoter-containing region upstream of a Fur-repressive hmuRSTUV operon, which encodes the proteins for hemin uptake. A hmuR deletant was still able to grow using hemin as the sole iron source, albeit at a rate clearly lower than that of the wild-type strain. These results strongly suggested (i) the involvement of HmuR in hemin uptake and (ii) the presence in ATCC 17616 of at least part of other unknown hemin uptake systems whose expression depends on the HemP function. Our in vitro analysis also indicated high-affinity binding of HemP to hemin, and such a property might modulate transcriptional activation of the hmu operon. IMPORTANCE Although the hmuRSTUV genes for the utilization of hemin as a sole iron source have been identified in a few Burkholderia strains, the regulatory expression of these genes has remained unknown. Our analysis in this study using B. multivorans ATCC 17616 showed that its HemP protein is required for expression of the hmuRSTUV operon, and the

  17. The HIV-1 transcriptional activator Tat has potent nucleic acid chaperoning activities in vitro.

    Science.gov (United States)

    Kuciak, Monika; Gabus, Caroline; Ivanyi-Nagy, Roland; Semrad, Katharina; Storchak, Roman; Chaloin, Olivier; Muller, Sylviane; Mély, Yves; Darlix, Jean-Luc

    2008-06-01

    The human immunodeficiency virus type 1 (HIV-1) is a primate lentivirus that causes the acquired immunodeficiency syndrome (AIDS). In addition to the virion structural proteins and enzyme precursors, that are Gag, Env and Pol, HIV-1 encodes several regulatory proteins, notably a small nuclear transcriptional activator named Tat. The Tat protein is absolutely required for virus replication since it controls proviral DNA transcription to generate the full-length viral mRNA. Tat can also regulate mRNA capping and splicing and was recently found to interfere with the cellular mi- and siRNA machinery. Because of its extensive interplay with nucleic acids, and its basic and disordered nature we speculated that Tat had nucleic acid-chaperoning properties. This prompted us to examine in vitro the nucleic acid-chaperoning activities of Tat and Tat peptides made by chemical synthesis. Here we report that Tat has potent nucleic acid-chaperoning activities according to the standard DNA annealing, DNA and RNA strand exchange, RNA ribozyme cleavage and trans-splicing assays. The active Tat(44-61) peptide identified here corresponds to the smallest known sequence with DNA/RNA chaperoning properties.

  18. Sequence motifs in MADS transcription factors responsible for specificity and diversification of protein-protein interaction.

    Directory of Open Access Journals (Sweden)

    Aalt D J van Dijk

    Full Text Available Protein sequences encompass tertiary structures and contain information about specific molecular interactions, which in turn determine biological functions of proteins. Knowledge about how protein sequences define interaction specificity is largely missing, in particular for paralogous protein families with high sequence similarity, such as the plant MADS domain transcription factor family. In comparison to the situation in mammalian species, this important family of transcription regulators has expanded enormously in plant species and contains over 100 members in the model plant species Arabidopsis thaliana. Here, we provide insight into the mechanisms that determine protein-protein interaction specificity for the Arabidopsis MADS domain transcription factor family, using an integrated computational and experimental approach. Plant MADS proteins have highly similar amino acid sequences, but their dimerization patterns vary substantially. Our computational analysis uncovered small sequence regions that explain observed differences in dimerization patterns with reasonable accuracy. Furthermore, we show the usefulness of the method for prediction of MADS domain transcription factor interaction networks in other plant species. Introduction of mutations in the predicted interaction motifs demonstrated that single amino acid mutations can have a large effect and lead to loss or gain of specific interactions. In addition, various performed bioinformatics analyses shed light on the way evolution has shaped MADS domain transcription factor interaction specificity. Identified protein-protein interaction motifs appeared to be strongly conserved among orthologs, indicating their evolutionary importance. We also provide evidence that mutations in these motifs can be a source for sub- or neo-functionalization. The analyses presented here take us a step forward in understanding protein-protein interactions and the interplay between protein sequences and

  19. Structural and functional analysis of VQ motif-containing proteins in Arabidopsis as interacting proteins of WRKY transcription factors.

    Science.gov (United States)

    Cheng, Yuan; Zhou, Yuan; Yang, Yan; Chi, Ying-Jun; Zhou, Jie; Chen, Jian-Ye; Wang, Fei; Fan, Baofang; Shi, Kai; Zhou, Yan-Hong; Yu, Jing-Quan; Chen, Zhixiang

    2012-06-01

    WRKY transcription factors are encoded by a large gene superfamily with a broad range of roles in plants. Recently, several groups have reported that proteins containing a short VQ (FxxxVQxLTG) motif interact with WRKY proteins. We have recently discovered that two VQ proteins from Arabidopsis (Arabidopsis thaliana), SIGMA FACTOR-INTERACTING PROTEIN1 and SIGMA FACTOR-INTERACTING PROTEIN2, act as coactivators of WRKY33 in plant defense by specifically recognizing the C-terminal WRKY domain and stimulating the DNA-binding activity of WRKY33. In this study, we have analyzed the entire family of 34 structurally divergent VQ proteins from Arabidopsis. Yeast (Saccharomyces cerevisiae) two-hybrid assays showed that Arabidopsis VQ proteins interacted specifically with the C-terminal WRKY domains of group I and the sole WRKY domains of group IIc WRKY proteins. Using site-directed mutagenesis, we identified structural features of these two closely related groups of WRKY domains that are critical for interaction with VQ proteins. Quantitative reverse transcription polymerase chain reaction revealed that expression of a majority of Arabidopsis VQ genes was responsive to pathogen infection and salicylic acid treatment. Functional analysis using both knockout mutants and overexpression lines revealed strong phenotypes in growth, development, and susceptibility to pathogen infection. Altered phenotypes were substantially enhanced through cooverexpression of genes encoding interacting VQ and WRKY proteins. These findings indicate that VQ proteins play an important role in plant growth, development, and response to environmental conditions, most likely by acting as cofactors of group I and IIc WRKY transcription factors.

  20. Pokemon decreases the transcriptional activity of RARα in the absence of ligand.

    Science.gov (United States)

    Yang, Yutao; Li, Yueting; Di, Fei; Cui, Jiajun; Wang, Yue; David Xu, Zhi-Qing

    2016-12-20

    Pokemon is a transcriptional repressor that belongs to the POZ and Krüppel (POK) protein family. In this study, we investigated the potential interaction between Pokemon and retinoic acid receptor alpha (RARα) and determined the role of Pokemon in regulation of RARα transcriptional activity in the absence of ligand. We found that Pokemon could directly interact with RARα. Moreover, we demonstrated that Pokemon could decrease the transcriptional activity of RARα in the absence of ligand. Furthermore, we showed that Pokemon could repress the transcriptional activity of RARα by increasing the recruitment of nuclear receptor co-repressor (NCoR) and silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) to the retinoic acid response element (RARE) element. Taken together, these data suggest that Pokemon is a novel partner of RARα that acts as a co-repressor to regulate RARα transcriptional activity in the absence of ligand.

  1. EBV tegument protein BNRF1 disrupts DAXX-ATRX to activate viral early gene transcription.

    Directory of Open Access Journals (Sweden)

    Kevin Tsai

    2011-11-01

    Full Text Available Productive infection by herpesviruses involve the disabling of host-cell intrinsic defenses by viral encoded tegument proteins. Epstein-Barr Virus (EBV typically establishes a non-productive, latent infection and it remains unclear how it confronts the host-cell intrinsic defenses that restrict viral gene expression. Here, we show that the EBV major tegument protein BNRF1 targets host-cell intrinsic defense proteins and promotes viral early gene activation. Specifically, we demonstrate that BNRF1 interacts with the host nuclear protein Daxx at PML nuclear bodies (PML-NBs and disrupts the formation of the Daxx-ATRX chromatin remodeling complex. We mapped the Daxx interaction domain on BNRF1, and show that this domain is important for supporting EBV primary infection. Through reverse transcription PCR and infection assays, we show that BNRF1 supports viral gene expression upon early infection, and that this function is dependent on the Daxx-interaction domain. Lastly, we show that knockdown of Daxx and ATRX induces reactivation of EBV from latently infected lymphoblastoid cell lines (LCLs, suggesting that Daxx and ATRX play a role in the regulation of viral chromatin. Taken together, our data demonstrate an important role of BNRF1 in supporting EBV early infection by interacting with Daxx and ATRX; and suggest that tegument disruption of PML-NB-associated antiviral resistances is a universal requirement for herpesvirus infection in the nucleus.

  2. EBV Tegument Protein BNRF1 Disrupts DAXX-ATRX to Activate Viral Early Gene Transcription

    Science.gov (United States)

    Tsai, Kevin; Thikmyanova, Nadezhda; Wojcechowskyj, Jason A.; Delecluse, Henri-Jacques; Lieberman, Paul M.

    2011-01-01

    Productive infection by herpesviruses involve the disabling of host-cell intrinsic defenses by viral encoded tegument proteins. Epstein-Barr Virus (EBV) typically establishes a non-productive, latent infection and it remains unclear how it confronts the host-cell intrinsic defenses that restrict viral gene expression. Here, we show that the EBV major tegument protein BNRF1 targets host-cell intrinsic defense proteins and promotes viral early gene activation. Specifically, we demonstrate that BNRF1 interacts with the host nuclear protein Daxx at PML nuclear bodies (PML-NBs) and disrupts the formation of the Daxx-ATRX chromatin remodeling complex. We mapped the Daxx interaction domain on BNRF1, and show that this domain is important for supporting EBV primary infection. Through reverse transcription PCR and infection assays, we show that BNRF1 supports viral gene expression upon early infection, and that this function is dependent on the Daxx-interaction domain. Lastly, we show that knockdown of Daxx and ATRX induces reactivation of EBV from latently infected lymphoblastoid cell lines (LCLs), suggesting that Daxx and ATRX play a role in the regulation of viral chromatin. Taken together, our data demonstrate an important role of BNRF1 in supporting EBV early infection by interacting with Daxx and ATRX; and suggest that tegument disruption of PML-NB-associated antiviral resistances is a universal requirement for herpesvirus infection in the nucleus. PMID:22102817

  3. Transcription elongation factor GreA has functional chaperone activity.

    Science.gov (United States)

    Li, Kun; Jiang, Tianyi; Yu, Bo; Wang, Limin; Gao, Chao; Ma, Cuiqing; Xu, Ping; Ma, Yanhe

    2012-01-01

    Bacterial GreA is an indispensable factor in the RNA polymerase elongation complex. It plays multiple roles in transcriptional elongation, and may be implicated in resistance to various stresses. In this study, we show that Escherichia coli GreA inhibits aggregation of several substrate proteins under heat shock condition. GreA can also effectively promote the refolding of denatured proteins. These facts reveal that GreA has chaperone activity. Distinct from many molecular chaperones, GreA does not form stable complexes with unfolded substrates. GreA overexpression confers the host cells with enhanced resistance to heat shock and oxidative stress. Moreover, GreA expression in the greA/greB double mutant could suppress the temperature-sensitive phenotype, and dramatically alleviate the in vivo protein aggregation. The results suggest that bacterial GreA may act as chaperone in vivo. These results suggest that GreA, in addition to its function as a transcription factor, is involved in protection of cellular proteins against aggregation.

  4. Resveratrol stimulates c-Fos gene transcription via activation of ERK1/2 involving multiple genetic elements.

    Science.gov (United States)

    Thiel, Gerald; Rössler, Oliver G

    2018-06-05

    The polyphenol resveratrol is found in many plant and fruits and is a constituent of our diet. Resveratrol has been proposed to have chemopreventive and anti-inflammatory activities. On the cellular level, resveratrol activates stimulus-regulated transcription factors. To identify resveratrol-responsive elements within a natural gene promoter, the molecular pathway leading to c-Fos gene expression by resveratrol was dissected. The c-Fos gene encodes a basic region leucine zipper transcription factor and is a prototype of an immediate-early gene that is regulated by a wide range of signaling molecules. We analyzed chromatin-integrated c-Fos promoter-luciferase reporter genes where transcription factor binding sites were destroyed by point mutations or deletion mutagenesis. The results show that mutation of the binding sites for serum response factor (SRF), activator protein-1 (AP-1) and cAMP response element binding protein (CREB) significantly reduced reporter gene transcription following stimulation of the cells with resveratrol. Inactivation of the binding sites for signal transducer and activator of transcription (STAT) or ternary complex factors did not influence resveratrol-regulated c-Fos promoter activity. Thus, the c-Fos promoter contains three resveratrol-responsive elements, the cAMP response element (CRE), and the binding sites for SRF and AP-1. Moreover, we show that the transcriptional activation potential of the c-Fos protein is increased in resveratrol-stimulated cells, indicating that the biological activity of c-Fos is elevated by resveratrol stimulation. Pharmacological and genetic experiments revealed that the protein kinase ERK1/2 is the signal transducer that connects resveratrol treatment with the c-Fos gene. Copyright © 2018 Elsevier B.V. All rights reserved.

  5. SUMO-, MAPK- and resistance protein-signaling converge at transcription complexes that regulate plant innate immunity

    NARCIS (Netherlands)

    Burg, van den H.A.; Takken, F.L.W.

    2010-01-01

    Upon pathogen perception plant innate immune receptors activate various signaling pathways that trigger host defenses. PAMP-triggered defense signaling requires mitogen-activated protein kinase (MAPK) pathways, which modulate the activity of transcription factors through phosphorylation. Here, we

  6. SUMO-, MAPK-, and resistance protein-signaling converge at transcription complexes that regulate plant innate immunity

    NARCIS (Netherlands)

    van den Burg, H.A.; Takken, F.L.W.

    2010-01-01

    Upon pathogen perception plant innate immune receptors activate various signaling pathways that trigger host defenses. PAMP-triggered defense signaling requires mitogen-activated protein kinase (MAPK) pathways, which modulate the activity of transcription factors through phosphorylation. Here, we

  7. In silico and biological survey of transcription-associated proteins implicated in the transcriptional machinery during the erythrocytic development of Plasmodium falciparum

    Directory of Open Access Journals (Sweden)

    Bischoff Emmanuel

    2010-01-01

    Full Text Available Abstract Background Malaria is the most important parasitic disease in the world with approximately two million people dying every year, mostly due to Plasmodium falciparum infection. During its complex life cycle in the Anopheles vector and human host, the parasite requires the coordinated and modulated expression of diverse sets of genes involved in epigenetic, transcriptional and post-transcriptional regulation. However, despite the availability of the complete sequence of the Plasmodium falciparum genome, we are still quite ignorant about Plasmodium mechanisms of transcriptional gene regulation. This is due to the poor prediction of nuclear proteins, cognate DNA motifs and structures involved in transcription. Results A comprehensive directory of proteins reported to be potentially involved in Plasmodium transcriptional machinery was built from all in silico reports and databanks. The transcription-associated proteins were clustered in three main sets of factors: general transcription factors, chromatin-related proteins (structuring, remodelling and histone modifying enzymes, and specific transcription factors. Only a few of these factors have been molecularly analysed. Furthermore, from transcriptome and proteome data we modelled expression patterns of transcripts and corresponding proteins during the intra-erythrocytic cycle. Finally, an interactome of these proteins based either on in silico or on 2-yeast-hybrid experimental approaches is discussed. Conclusion This is the first attempt to build a comprehensive directory of potential transcription-associated proteins in Plasmodium. In addition, all complete transcriptome, proteome and interactome raw data were re-analysed, compared and discussed for a better comprehension of the complex biological processes of Plasmodium falciparum transcriptional regulation during the erythrocytic development.

  8. THRAP3 interacts with and inhibits the transcriptional activity of SOX9 during chondrogenesis.

    Science.gov (United States)

    Sono, Takashi; Akiyama, Haruhiko; Miura, Shigenori; Deng, Jian Min; Shukunami, Chisa; Hiraki, Yuji; Tsushima, Yu; Azuma, Yoshiaki; Behringer, Richard R; Matsuda, Shuichi

    2018-07-01

    Sex-determining region Y (Sry)-box (Sox)9 is required for chondrogenesis as a transcriptional activator of genes related to chondrocyte proliferation, differentiation, and cartilage-specific extracellular matrix. Although there have been studies investigating the Sox9-dependent transcriptional complexes, not all their components have been identified. In the present study, we demonstrated that thyroid hormone receptor-associated protein (THRAP)3 is a component of a SOX9 transcriptional complex by liquid chromatography mass spectrometric analysis of FLAG-tagged Sox9-binding proteins purified from FLAG-HA-tagged Sox9 knock-in mice. Thrap3 knockdown in ATDC5 chondrogenic cells increased the expression of Collagen type II alpha 1 chain (Col2a1) without affecting Sox9 expression. THRAP3 and SOX9 overexpression reduced Col2a1 levels to a greater degree than overexpression of SOX9 alone. The negative regulation of SOX9 transcriptional activity by THRAP3 was mediated by interaction between the proline-, glutamine-, and serine-rich domain of SOX9 and the innominate domain of THRAP3. These results indicate that THRAP3 negatively regulates SOX9 transcriptional activity as a cofactor of a SOX9 transcriptional complex during chondrogenesis.

  9. A pp32-retinoblastoma protein complex modulates androgen receptor-mediated transcription and associates with components of the splicing machinery

    International Nuclear Information System (INIS)

    Adegbola, Onikepe; Pasternack, Gary R.

    2005-01-01

    We have previously shown pp32 and the retinoblastoma protein interact. pp32 and the retinoblastoma protein are nuclear receptor transcriptional coregulators: the retinoblastoma protein is a coactivator for androgen receptor, the major regulator of prostate cancer growth, while pp32, which is highly expressed in prostate cancer, is a corepressor of the estrogen receptor. We now show pp32 increases androgen receptor-mediated transcription and the retinoblastoma protein modulates this activity. Using affinity purification and mass spectrometry, we identify members of the pp32-retinoblastoma protein complex as PSF and nonO/p54nrb, proteins implicated in coordinate regulation of nuclear receptor-mediated transcription and splicing. We show that the pp32-retinoblastoma protein complex is modulated during TPA-induced K562 differentiation. Present evidence suggests that nuclear receptors assemble multiprotein complexes to coordinately regulate transcription and mRNA processing. Our results suggest that pp32 and the retinoblastoma protein may be part of a multiprotein complex that coordinately regulates nuclear receptor-mediated transcription and mRNA processing

  10. COBRA1 inhibits AP-1 transcriptional activity in transfected cells

    International Nuclear Information System (INIS)

    Zhong Hongjun; Zhu Jianhua; Zhang Hao; Ding Lihua; Sun Yan; Huang Cuifen; Ye Qinong

    2004-01-01

    Mutations in the breast cancer susceptibility gene (BRCA1) account for a significant proportion of hereditary breast and ovarian cancers. Cofactor of BRCA1 (COBRA1) was isolated as a BRCA1-interacting protein and exhibited a similar chromatin reorganizing activity to that of BRCA1. However, the biological role of COBRA1 remains largely unexplored. Here, we report that ectopic expression of COBRA1 inhibited activator protein 1 (AP-1) transcriptional activity in transfected cells in a dose-dependent manner, whereas reduction of endogenous COBRA1 with a small interfering RNA significantly enhanced AP-1-mediated transcriptional activation. COBRA1 physically interacted with the AP-1 family members, c-Jun and c-Fos, and the middle region of COBRA1 bound to c-Fos. Lack of c-Fos binding site in the COBRA1 completely abolished the COBRA1 inhibition of AP-1 trans-activation. These findings suggest that COBRA1 may directly modulate AP-1 pathway and, therefore, may play important roles in cell proliferation, differentiation, apoptosis, and oncogenesis

  11. G-actin sequestering protein thymosin-β4 regulates the activity of myocardin-related transcription factor

    International Nuclear Information System (INIS)

    Morita, Tsuyoshi; Hayashi, Ken’ichiro

    2013-01-01

    Highlights: •Tβ4 competed with MRTF-A for G-actin binding. •Tβ4 activated the MRTF–SRF signaling pathway. •Tβ4 increased the endogenous expression of SRF-dependent genes. -- Abstract: Myocardin-related transcription factors (MRTFs) are robust coactivators of serum response factor (SRF). MRTFs contain three copies of the RPEL motif at their N-terminus, and they bind to monomeric globular actin (G-actin). Previous studies illustrate that G-actin binding inhibits MRTF activity by preventing the MRTFs nuclear accumulation. In the living cells, the majority of G-actin is sequestered by G-actin binding proteins that prevent spontaneous actin polymerization. Here, we demonstrate that the most abundant G-actin sequestering protein thymosin-β4 (Tβ4) was involved in the regulation of subcellular localization and activity of MRTF-A. Tβ4 competed with MRTF-A for G-actin binding; thus, interfering with G-actin–MRTF-A complex formation. Tβ4 overexpression induced the MRTF-A nuclear accumulation and activation of MRTF–SRF signaling. The activation rate of MRTF-A by the Tβ4 mutant L17A, whose affinity for G-actin is very low, was lower than that by wild-type Tβ4. In contrast, the β-actin mutant 3DA, which has a lower affinity for Tβ4, more effectively suppressed MRTF-A activity than wild-type β-actin. Furthermore, ectopic Tβ4 increased the endogenous expression of SRF-dependent actin cytoskeletal genes. Thus, Tβ4 is an important MRTF regulator that controls the G-actin–MRTFs interaction

  12. G-actin sequestering protein thymosin-β4 regulates the activity of myocardin-related transcription factor

    Energy Technology Data Exchange (ETDEWEB)

    Morita, Tsuyoshi, E-mail: tsuyo@nbiochem.med.osaka-u.ac.jp; Hayashi, Ken’ichiro

    2013-08-02

    Highlights: •Tβ4 competed with MRTF-A for G-actin binding. •Tβ4 activated the MRTF–SRF signaling pathway. •Tβ4 increased the endogenous expression of SRF-dependent genes. -- Abstract: Myocardin-related transcription factors (MRTFs) are robust coactivators of serum response factor (SRF). MRTFs contain three copies of the RPEL motif at their N-terminus, and they bind to monomeric globular actin (G-actin). Previous studies illustrate that G-actin binding inhibits MRTF activity by preventing the MRTFs nuclear accumulation. In the living cells, the majority of G-actin is sequestered by G-actin binding proteins that prevent spontaneous actin polymerization. Here, we demonstrate that the most abundant G-actin sequestering protein thymosin-β4 (Tβ4) was involved in the regulation of subcellular localization and activity of MRTF-A. Tβ4 competed with MRTF-A for G-actin binding; thus, interfering with G-actin–MRTF-A complex formation. Tβ4 overexpression induced the MRTF-A nuclear accumulation and activation of MRTF–SRF signaling. The activation rate of MRTF-A by the Tβ4 mutant L17A, whose affinity for G-actin is very low, was lower than that by wild-type Tβ4. In contrast, the β-actin mutant 3DA, which has a lower affinity for Tβ4, more effectively suppressed MRTF-A activity than wild-type β-actin. Furthermore, ectopic Tβ4 increased the endogenous expression of SRF-dependent actin cytoskeletal genes. Thus, Tβ4 is an important MRTF regulator that controls the G-actin–MRTFs interaction.

  13. SUMOylation of the KRAB zinc-finger transcription factor PARIS/ZNF746 regulates its transcriptional activity

    International Nuclear Information System (INIS)

    Nishida, Tamotsu; Yamada, Yoshiji

    2016-01-01

    Parkin-interacting substrate (PARIS), a member of the family of Krüppel-associated box (KRAB)-containing zinc-finger transcription factors, is a substrate of the ubiquitin E3 ligase parkin. PARIS represses the expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), although the underlying mechanisms remain largely unknown. In the present study, we demonstrate that PARIS can be SUMOylated, and its SUMOylation plays a role in the repression of PGC-1a promoter activity. Protein inhibitor of activated STAT y (PIASy) was identified as an interacting protein of PARIS and shown to enhance its SUMOylation. PIASy repressed PGC-1a promoter activity, and this effect was attenuated by PARIS in a manner dependent on its SUMOylation status. Co-expression of SUMO-1 with PIASy completely repressed PGC-1a promoter activity independently of PARIS expression. PARIS-mediated PGC-1a promoter repression depended on the activity of histone deacetylases (HDAC), whereas PIASy repressed the PGC-1a promoter in an HDAC-independent manner. Taken together, these results suggest that PARIS and PIASy modulate PGC-1a gene transcription through distinct molecular mechanisms. -- Highlights: •PARIS can be SUMOylated in vivo and in vitro. •SUMOylation of PARIS functions in the repression of PGC-1a promoter activity. •PIASy interacts with PARIS and enhances its SUMOylation. •PIASy influences PARIS-mediated repression of PGC-1a promoter activity.

  14. SUMOylation of the KRAB zinc-finger transcription factor PARIS/ZNF746 regulates its transcriptional activity

    Energy Technology Data Exchange (ETDEWEB)

    Nishida, Tamotsu, E-mail: nishida@gene.mie-u.ac.jp; Yamada, Yoshiji

    2016-05-13

    Parkin-interacting substrate (PARIS), a member of the family of Krüppel-associated box (KRAB)-containing zinc-finger transcription factors, is a substrate of the ubiquitin E3 ligase parkin. PARIS represses the expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), although the underlying mechanisms remain largely unknown. In the present study, we demonstrate that PARIS can be SUMOylated, and its SUMOylation plays a role in the repression of PGC-1a promoter activity. Protein inhibitor of activated STAT y (PIASy) was identified as an interacting protein of PARIS and shown to enhance its SUMOylation. PIASy repressed PGC-1a promoter activity, and this effect was attenuated by PARIS in a manner dependent on its SUMOylation status. Co-expression of SUMO-1 with PIASy completely repressed PGC-1a promoter activity independently of PARIS expression. PARIS-mediated PGC-1a promoter repression depended on the activity of histone deacetylases (HDAC), whereas PIASy repressed the PGC-1a promoter in an HDAC-independent manner. Taken together, these results suggest that PARIS and PIASy modulate PGC-1a gene transcription through distinct molecular mechanisms. -- Highlights: •PARIS can be SUMOylated in vivo and in vitro. •SUMOylation of PARIS functions in the repression of PGC-1a promoter activity. •PIASy interacts with PARIS and enhances its SUMOylation. •PIASy influences PARIS-mediated repression of PGC-1a promoter activity.

  15. TALE factors poise promoters for activation by Hox proteins.

    Science.gov (United States)

    Choe, Seong-Kyu; Ladam, Franck; Sagerström, Charles G

    2014-01-27

    Hox proteins form complexes with TALE cofactors from the Pbx and Prep/Meis families to control transcription, but it remains unclear how Hox:TALE complexes function. Examining a Hoxb1b:TALE complex that regulates zebrafish hoxb1a transcription, we find maternally deposited TALE proteins at the hoxb1a promoter already during blastula stages. These TALE factors recruit histone-modifying enzymes to promote an active chromatin profile at the hoxb1a promoter and also recruit RNA polymerase II (RNAPII) and P-TEFb. However, in the presence of TALE factors, RNAPII remains phosphorylated on serine 5 and hoxb1a transcription is inefficient. By gastrula stages, Hoxb1b binds together with TALE factors to the hoxb1a promoter. This triggers P-TEFb-mediated transitioning of RNAPII to the serine 2-phosphorylated form and efficient hoxb1a transcription. We conclude that TALE factors access promoters during early embryogenesis to poise them for activation but that Hox proteins are required to trigger efficient transcription. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. Down syndrome critical region 2 protein inhibits the transcriptional activity of peroxisome proliferator-activated receptor β in HEK293 cells

    International Nuclear Information System (INIS)

    Song, Hae Jin; Park, Joongkyu; Seo, Su Ryeon; Kim, Jongsun; Paik, Seung R.; Chung, Kwang Chul

    2008-01-01

    Down syndrome is mainly caused by a trisomy of chromosome 21. The Down syndrome critical region 2 (DSCR2) gene is located within a part of chromosome 21, the Down syndrome critical region (DSCR). To investigate the function of DSCR2, we sought to identify DSCR2-interacting proteins using yeast two-hybrid assays. A human fetal brain cDNA library was screened, and DSCR2 was found to interact with a member of the nuclear receptor superfamily, peroxisome proliferator-activated receptor β, (PPARβ). A co-immunoprecipitation assay demonstrated that DSCR2 physically interacts with PPARβ in mammalian HEK293 cells. DSCR2 also inhibited the ligand-induced transcriptional activity of PPARβ. Furthermore, PPARβ also decreased the solubility of DSCR2, which increased levels of insoluble DSCR2

  17. A super-family of transcriptional activators regulates bacteriophage packaging and lysis in Gram-positive bacteria

    Science.gov (United States)

    Quiles-Puchalt, Nuria; Tormo-Más, María Ángeles; Campoy, Susana; Toledo-Arana, Alejandro; Monedero, Vicente; Lasa, Íñigo; Novick, Richard P.; Christie, Gail E.; Penadés, José R.

    2013-01-01

    The propagation of bacteriophages and other mobile genetic elements requires exploitation of the phage mechanisms involved in virion assembly and DNA packaging. Here, we identified and characterized four different families of phage-encoded proteins that function as activators required for transcription of the late operons (morphogenetic and lysis genes) in a large group of phages infecting Gram-positive bacteria. These regulators constitute a super-family of proteins, here named late transcriptional regulators (Ltr), which share common structural, biochemical and functional characteristics and are unique to this group of phages. They are all small basic proteins, encoded by genes present at the end of the early gene cluster in their respective phage genomes and expressed under cI repressor control. To control expression of the late operon, the Ltr proteins bind to a DNA repeat region situated upstream of the terS gene, activating its transcription. This involves the C-terminal part of the Ltr proteins, which control specificity for the DNA repeat region. Finally, we show that the Ltr proteins are the only phage-encoded proteins required for the activation of the packaging and lysis modules. In summary, we provide evidence that phage packaging and lysis is a conserved mechanism in Siphoviridae infecting a wide variety of Gram-positive bacteria. PMID:23771138

  18. The PDZ-binding motif of Yes-associated protein is required for its co-activation of TEAD-mediated CTGF transcription and oncogenic cell transforming activity

    International Nuclear Information System (INIS)

    Shimomura, Tadanori; Miyamura, Norio; Hata, Shoji; Miura, Ryota; Hirayama, Jun; Nishina, Hiroshi

    2014-01-01

    Highlights: •Loss of the PDZ-binding motif inhibits constitutively active YAP (5SA)-induced oncogenic cell transformation. •The PDZ-binding motif of YAP promotes its nuclear localization in cultured cells and mouse liver. •Loss of the PDZ-binding motif inhibits YAP (5SA)-induced CTGF transcription in cultured cells and mouse liver. -- Abstract: YAP is a transcriptional co-activator that acts downstream of the Hippo signaling pathway and regulates multiple cellular processes, including proliferation. Hippo pathway-dependent phosphorylation of YAP negatively regulates its function. Conversely, attenuation of Hippo-mediated phosphorylation of YAP increases its ability to stimulate proliferation and eventually induces oncogenic transformation. The C-terminus of YAP contains a highly conserved PDZ-binding motif that regulates YAP’s functions in multiple ways. However, to date, the importance of the PDZ-binding motif to the oncogenic cell transforming activity of YAP has not been determined. In this study, we disrupted the PDZ-binding motif in the YAP (5SA) protein, in which the sites normally targeted by Hippo pathway-dependent phosphorylation are mutated. We found that loss of the PDZ-binding motif significantly inhibited the oncogenic transformation of cultured cells induced by YAP (5SA). In addition, the increased nuclear localization of YAP (5SA) and its enhanced activation of TEAD-dependent transcription of the cell proliferation gene CTGF were strongly reduced when the PDZ-binding motif was deleted. Similarly, in mouse liver, deletion of the PDZ-binding motif suppressed nuclear localization of YAP (5SA) and YAP (5SA)-induced CTGF expression. Taken together, our results indicate that the PDZ-binding motif of YAP is critical for YAP-mediated oncogenesis, and that this effect is mediated by YAP’s co-activation of TEAD-mediated CTGF transcription

  19. The PDZ-binding motif of Yes-associated protein is required for its co-activation of TEAD-mediated CTGF transcription and oncogenic cell transforming activity

    Energy Technology Data Exchange (ETDEWEB)

    Shimomura, Tadanori; Miyamura, Norio; Hata, Shoji; Miura, Ryota; Hirayama, Jun, E-mail: hirayama.dbio@mri.tmd.ac.jp; Nishina, Hiroshi, E-mail: nishina.dbio@mri.tmd.ac.jp

    2014-01-17

    Highlights: •Loss of the PDZ-binding motif inhibits constitutively active YAP (5SA)-induced oncogenic cell transformation. •The PDZ-binding motif of YAP promotes its nuclear localization in cultured cells and mouse liver. •Loss of the PDZ-binding motif inhibits YAP (5SA)-induced CTGF transcription in cultured cells and mouse liver. -- Abstract: YAP is a transcriptional co-activator that acts downstream of the Hippo signaling pathway and regulates multiple cellular processes, including proliferation. Hippo pathway-dependent phosphorylation of YAP negatively regulates its function. Conversely, attenuation of Hippo-mediated phosphorylation of YAP increases its ability to stimulate proliferation and eventually induces oncogenic transformation. The C-terminus of YAP contains a highly conserved PDZ-binding motif that regulates YAP’s functions in multiple ways. However, to date, the importance of the PDZ-binding motif to the oncogenic cell transforming activity of YAP has not been determined. In this study, we disrupted the PDZ-binding motif in the YAP (5SA) protein, in which the sites normally targeted by Hippo pathway-dependent phosphorylation are mutated. We found that loss of the PDZ-binding motif significantly inhibited the oncogenic transformation of cultured cells induced by YAP (5SA). In addition, the increased nuclear localization of YAP (5SA) and its enhanced activation of TEAD-dependent transcription of the cell proliferation gene CTGF were strongly reduced when the PDZ-binding motif was deleted. Similarly, in mouse liver, deletion of the PDZ-binding motif suppressed nuclear localization of YAP (5SA) and YAP (5SA)-induced CTGF expression. Taken together, our results indicate that the PDZ-binding motif of YAP is critical for YAP-mediated oncogenesis, and that this effect is mediated by YAP’s co-activation of TEAD-mediated CTGF transcription.

  20. Enhanced oxygen consumption in Herbaspirillum seropedicae fnr mutants leads to increased NifA mediated transcriptional activation.

    Science.gov (United States)

    Batista, Marcelo Bueno; Wassem, Roseli; Pedrosa, Fábio de Oliveira; de Souza, Emanuel Maltempi; Dixon, Ray; Monteiro, Rose Adele

    2015-05-07

    Orthologous proteins of the Crp/Fnr family have been previously implicated in controlling expression and/or activity of the NifA transcriptional activator in some diazotrophs. This study aimed to address the role of three Fnr-like proteins from H. seropedicae SmR1 in controlling NifA activity and consequent NifA-mediated transcription activation. The activity of NifA-dependent transcriptional fusions (nifA::lacZ and nifB::lacZ) was analysed in a series of H. seropedicae fnr deletion mutant backgrounds. We found that combined deletions in both the fnr1 and fnr3 genes lead to higher expression of both the nifA and nifB genes and also an increased level of nifH transcripts. Expression profiles of nifB under different oxygen concentrations, together with oxygen consumption measurements suggest that the triple fnr mutant has higher respiratory activity when compared to the wild type, which we believe to be responsible for greater stability of the oxygen sensitive NifA protein. This conclusion was further substantiated by measuring the levels of NifA protein and its activity in fnr deletion strains in comparison with the wild-type. Fnr proteins are indirectly involved in controlling the activity of NifA in H. seropedicae, probably as a consequence of their influence on respiratory activity in relation to oxygen availability. Additionally we can suggest that there is some redundancy in the physiological function of the three Fnr paralogs in this organism, since altered respiration and effects on NifA activity are only observed in deletion strains lacking both fnr1 and fnr3.

  1. RNA-guided Transcriptional Regulation in Plants via dCas9 Chimeric Proteins

    KAUST Repository

    Baazim, Hatoon

    2014-05-01

    Developing targeted genome regulation approaches holds much promise for accelerating trait discovery and development in agricultural biotechnology. Clustered Regularly Interspaced Palindromic Repeats (CRISPRs)/CRISPR associated (Cas) system provides bacteria and archaea with an adaptive molecular immunity mechanism against invading nucleic acids through phages and conjugative plasmids. The type II CRISPR/Cas system has been adapted for genome editing purposes across a variety of cell types and organisms. Recently, the catalytically inactive Cas9 (dCas9) protein combined with guide RNAs (gRNAs) were used as a DNA-targeting platform to modulate the expression patterns in bacterial, yeast and human cells. Here, we employed this DNA-targeting system for targeted transcriptional regulation in planta by developing chimeric dCas9-based activators and repressors. For example, we fused to the C-terminus of dCas9 with the activation domains of EDLL and TAL effectors, respectively, to generate transcriptional activators, and the SRDX repression domain to generate transcriptional repressor. Our data demonstrate that the dCas9:EDLL and dCas9:TAD activators, guided by gRNAs complementary to promoter elements, induce strong transcriptional activation on episomal targets in plant cells. Moreover, our data suggest that the dCas9:SRDX repressor and the dCas9:EDLL and dCas9:TAD activators are capable of markedly repressing or activating, respectively, the transcription of an endogenous genomic target. Our data indicate that the CRISPR/dCas9:TFs DNA targeting system can be used in plants as a functional genomic tool and for biotechnological applications.

  2. Drosophila C-terminal binding protein, dCtBP is required for sensory organ prepattern and sharpens proneural transcriptional activity of the GATA factor Pnr.

    Science.gov (United States)

    Biryukova, Inna; Heitzler, Pascal

    2008-11-01

    The peripheral nervous system is required for animals to detect and to relay environmental stimuli to central nervous system for the information processing. In Drosophila, the precise spatial and temporal expression of two proneural genes achaete (ac) and scute (sc), is necessary for development of the sensory organs. Here we present an evidence that the transcription co-repressor, dCtBP acts as a negative regulator of sensory organ prepattern. Loss of dCtBP function mutant exhibits ectopic sensory organs, while overexpression of dCtBP results in a dramatic loss of sensory organs. These phenotypes are correlated with mis-emerging of sensory organ precursors and perturbated expression of proneural transcription activator Ac. Mammalian CtBP-1 was identified via interaction with the consensus motif PXDLSX(K/R) of adenovirus E1A oncoprotein. We demonstrated that dCtBP binds directly to PLDLS motif of Drosophila Friend of GATA-1 protein, U-shaped and sharpens the adult sensory organ development. Moreover, we found that dCtBP mediates multivalent interaction with the GATA transcriptional activator Pannier and acts as a direct co-repressor of the Pannier-mediated activation of proneural genes. We demonstrated that Pannier genetically interacts with dCtBP-interacting protein HDAC1, suggesting that the dCtBP-dependent regulation of Pannier activity could utilize a repressive mechanism involving alteration of local chromatine structure.

  3. CCAAT/enhancer-binding protein delta activates insulin-like growth factor-I gene transcription in osteoblasts. Identification of a novel cyclic AMP signaling pathway in bone

    Science.gov (United States)

    Umayahara, Y.; Ji, C.; Centrella, M.; Rotwein, P.; McCarthy, T. L.

    1997-01-01

    Insulin-like growth factor-I (IGF-I) plays a key role in skeletal growth by stimulating bone cell replication and differentiation. We previously showed that prostaglandin E2 (PGE2) and other cAMP-activating agents enhanced IGF-I gene transcription in cultured primary rat osteoblasts through promoter 1, the major IGF-I promoter, and identified a short segment of the promoter, termed HS3D, that was essential for hormonal regulation of IGF-I gene expression. We now demonstrate that CCAAT/enhancer-binding protein (C/EBP) delta is a major component of a PGE2-stimulated DNA-protein complex involving HS3D and find that C/EBPdelta transactivates IGF-I promoter 1 through this site. Competition gel shift studies first indicated that a core C/EBP half-site (GCAAT) was required for binding of a labeled HS3D oligomer to osteoblast nuclear proteins. Southwestern blotting and UV-cross-linking studies showed that the HS3D probe recognized a approximately 35-kDa nuclear protein, and antibody supershift assays indicated that C/EBPdelta comprised most of the PGE2-activated gel-shifted complex. C/EBPdelta was detected by Western immunoblotting in osteoblast nuclear extracts after treatment of cells with PGE2. An HS3D oligonucleotide competed effectively with a high affinity C/EBP site from the rat albumin gene for binding to osteoblast nuclear proteins. Co-transfection of osteoblast cell cultures with a C/EBPdelta expression plasmid enhanced basal and PGE2-activated IGF-I promoter 1-luciferase activity but did not stimulate a reporter gene lacking an HS3D site. By contrast, an expression plasmid for the related protein, C/EBPbeta, did not alter basal IGF-I gene activity but did increase the response to PGE2. In osteoblasts and in COS-7 cells, C/EBPdelta, but not C/EBPbeta, transactivated a reporter gene containing four tandem copies of HS3D fused to a minimal promoter; neither transcription factor stimulated a gene with four copies of an HS3D mutant that was unable to bind osteoblast

  4. DMPD: Post-transcriptional regulation of proinflammatory proteins. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 15075353 Post-transcriptional regulation of proinflammatory proteins. Anderson P, P...l) (.csml) Show Post-transcriptional regulation of proinflammatory proteins. PubmedID 15075353 Title Post-tr...anscriptional regulation of proinflammatory proteins. Authors Anderson P, Phillip

  5. Cisplatin Induces Cytotoxicity through the Mitogen-Activated Protein Kinase Pathways ana Activating Transcription Factor 3

    Directory of Open Access Journals (Sweden)

    Carly St. Germain

    2010-07-01

    Full Text Available The mechanisms underlying the proapoptotic effect of the chemotherapeutic agent, cisplatin, are largely undefined. Understanding the mechanisms regulating cisplatin cytotoxicity may uncover strategies to enhance the efficacy of this important therapeutic agent. This study evaluates the role of activating transcription factor 3 (ATF3 as a mediator of cisplatin-induced cytotoxicity. Cytotoxic doses of cisplatin and carboplatin treatments consistently induced ATF3 expression in five tumor-derived cell lines. Characterization of this induction revealed a p53, BRCA1, and integrated stress response-independent mechanism, all previously implicated in stress-mediated ATF3 induction. Analysis of mitogenactivated protein kinase (MAPK pathway involvement in ATF3 induction by cisplatin revealed a MAPK-dependent mechanism. Cisplatin treatment combined with specific inhibitors to each MAPK pathway (c-Jun N-terminal kinase, extracellularsignal-regulated kinase, and p38 resulted in decreasedATF3 induction at the protein level. MAPK pathway inhibition led to decreased ATF3 messenger RNA expression and reduced cytotoxic effects of cisplatin as measured by the 3-(4,5-dimethylthiazol-2-ylF2,5-diphenyltetrazolium bromide cell viability assay. In A549 lung carcinoma cells, targeting ATF3 with specific small hairpin RNA also attenuated the cytotoxic effects of cisplatin. Similarly, ATF3-/murine embryonic fibroblasts (MEFs were shown to be less sensitive to cisplatin-induced cytotoxicity compared with ATF3+/+ MEFs. This study identifies cisplatin as a MAPK pathway-dependent inducer of ATF3, whose expression influences cisplatin’s cytotoxic effects.

  6. Exploring cellular memory molecules marking competent and active transcriptions

    Directory of Open Access Journals (Sweden)

    Liu De-Pei

    2007-05-01

    Full Text Available Abstract Background Development in higher eukaryotes involves programmed gene expression. Cell type-specific gene expression is established during this process and is inherited in succeeding cell cycles. Higher eukaryotes have evolved elegant mechanisms by which committed gene-expression states are transmitted through numerous cell divisions. Previous studies have shown that both DNase I-sensitive sites and the basal transcription factor TFIID remain on silenced mitotic chromosomes, suggesting that certain trans-factors might act as bookmarks, maintaining the information and transmitting it to the next generation. Results We used the mouse globin gene clusters as a model system to examine the retention of active information on M-phase chromosomes and its contribution to the persistence of transcriptional competence of these gene clusters in murine erythroleukemia cells. In cells arrested in mitosis, the erythroid-specific activator NF-E2p45 remained associated with its binding sites on the globin gene loci, while the other major erythroid factor, GATA-1, was removed from chromosome. Moreover, despite mitotic chromatin condensation, the distant regulatory regions and promoters of transcriptionally competent globin gene loci are marked by a preserved histone code consisting in active histone modifications such as H3 acetylation, H3-K4 dimethylation and K79 dimethylation. Further analysis showed that other active genes are also locally marked by the preserved active histone code throughout mitotic inactivation of transcription. Conclusion Our results imply that certain kinds of specific protein factors and active histone modifications function as cellular memory markers for both competent and active genes during mitosis, and serve as a reactivated core for the resumption of transcription when the cells exit mitosis.

  7. Transcriptional regulation by Polycomb group proteins

    DEFF Research Database (Denmark)

    Di Croce, Luciano; Helin, Kristian

    2013-01-01

    Polycomb group (PcG) proteins are epigenetic regulators of transcription that have key roles in stem-cell identity, differentiation and disease. Mechanistically, they function within multiprotein complexes, called Polycomb repressive complexes (PRCs), which modify histones (and other proteins......) and silence target genes. The dynamics of PRC1 and PRC2 components has been the focus of recent research. Here we discuss our current knowledge of the PRC complexes, how they are targeted to chromatin and how the high diversity of the PcG proteins allows these complexes to influence cell identity....

  8. Transcription activator-like effector-mediated regulation of gene expression based on the inducible packaging and delivery via designed extracellular vesicles

    International Nuclear Information System (INIS)

    Lainšček, Duško; Lebar, Tina; Jerala, Roman

    2017-01-01

    Transcription activator-like effector (TALE) proteins present a powerful tool for genome editing and engineering, enabling introduction of site-specific mutations, gene knockouts or regulation of the transcription levels of selected genes. TALE nucleases or TALE-based transcription regulators are introduced into mammalian cells mainly via delivery of the coding genes. Here we report an extracellular vesicle-mediated delivery of TALE transcription regulators and their ability to upregulate the reporter gene in target cells. Designed transcriptional activator TALE-VP16 fused to the appropriate dimerization domain was enriched as a cargo protein within extracellular vesicles produced by mammalian HEK293 cells stimulated by Ca-ionophore and using blue light- or rapamycin-inducible dimerization systems. Blue light illumination or rapamycin increased the amount of the TALE-VP16 activator in extracellular vesicles and their addition to the target cells resulted in an increased expression of the reporter gene upon addition of extracellular vesicles to the target cells. This technology therefore represents an efficient delivery for the TALE-based transcriptional regulators. - Highlights: • Inducible dimerization enriched cargo proteins within extracellular vesicles (EV). • Farnesylation surpassed LAMP-1 fusion proteins for the EV packing. • Extracellular vesicles were able to deliver TALE regulators to mammalian cells. • TALE mediated transcriptional activation was achieved by designed EV.

  9. Antisense RNA Controls LRP1 Sense Transcript Expression through Interaction with a Chromatin-Associated Protein, HMGB2

    Directory of Open Access Journals (Sweden)

    Yasunari Yamanaka

    2015-05-01

    Full Text Available Long non-coding RNAs (lncRNAs, including natural antisense transcripts (NATs, are expressed more extensively than previously anticipated and have widespread roles in regulating gene expression. Nevertheless, the molecular mechanisms of action of the majority of NATs remain largely unknown. Here, we identify a NAT of low-density lipoprotein receptor-related protein 1 (Lrp1, referred to as Lrp1-AS, that negatively regulates Lrp1 expression. We show that Lrp1-AS directly binds to high-mobility group box 2 (Hmgb2 and inhibits the activity of Hmgb2 to enhance Srebp1a-dependent transcription of Lrp1. Short oligonucleotides targeting Lrp1-AS inhibit the interaction of antisense transcript and Hmgb2 protein and increase Lrp1 expression by enhancing Hmgb2 activity. Quantitative RT-PCR analysis of brain tissue samples from Alzheimer’s disease patients and aged-matched controls revealed upregulation of LRP1-AS and downregulation of LRP1. Our data suggest a regulatory mechanism whereby a NAT interacts with a ubiquitous chromatin-associated protein to modulate its activity in a locus-specific fashion.

  10. A Member of the p38 Mitogen-Activated Protein Kinase Family Is Responsible for Transcriptional Induction of Dopa decarboxylase in the Epidermis of Drosophila melanogaster during the Innate Immune Response▿ †

    Science.gov (United States)

    Davis, Monica M.; Primrose, David A.; Hodgetts, Ross B.

    2008-01-01

    Drosophila innate immunity is controlled primarily by the activation of IMD (immune deficiency) or Toll signaling leading to the production of antimicrobial peptides (AMPs). IMD signaling also activates the JUN N-terminal kinase (JNK) cascade, which is responsible for immune induction of non-antimicrobial peptide immune gene transcription though the transcription factor AP-1. Transcription of the Dopa decarboxylase (Ddc) gene is induced in response to gram-negative and gram-positive septic injury, but not aseptic wounding. Transcription is induced throughout the epidermis and not specifically at the site of infection. Ddc transcripts are detectible within 2 h and remain high for several hours following infection with either gram-negative or gram-positive bacteria. Using Ddc-green fluorescent protein (GFP) reporter gene constructs, we show that a conserved consensus AP-1 binding site upstream of the Ddc transcription start site is required for induction. However, neither the Toll, IMD, nor JNK pathway is involved. Rather, Ddc transcription depends on a previously uncharacterized member of the p38 mitogen-activated protein kinase family, p38c. We propose that the involvement of DDC in a new pathway involved in Drosophila immunity increases the levels of dopamine, which is metabolized to produce reactive quinones that exert an antimicrobial effect on invading bacteria. PMID:18519585

  11. Simplified Method for Predicting a Functional Class of Proteins in Transcription Factor Complexes

    KAUST Repository

    Piatek, Marek J.

    2013-07-12

    Background:Initiation of transcription is essential for most of the cellular responses to environmental conditions and for cell and tissue specificity. This process is regulated through numerous proteins, their ligands and mutual interactions, as well as interactions with DNA. The key such regulatory proteins are transcription factors (TFs) and transcription co-factors (TcoFs). TcoFs are important since they modulate the transcription initiation process through interaction with TFs. In eukaryotes, transcription requires that TFs form different protein complexes with various nuclear proteins. To better understand transcription regulation, it is important to know the functional class of proteins interacting with TFs during transcription initiation. Such information is not fully available, since not all proteins that act as TFs or TcoFs are yet annotated as such, due to generally partial functional annotation of proteins. In this study we have developed a method to predict, using only sequence composition of the interacting proteins, the functional class of human TF binding partners to be (i) TF, (ii) TcoF, or (iii) other nuclear protein. This allows for complementing the annotation of the currently known pool of nuclear proteins. Since only the knowledge of protein sequences is required in addition to protein interaction, the method should be easily applicable to many species.Results:Based on experimentally validated interactions between human TFs with different TFs, TcoFs and other nuclear proteins, our two classification systems (implemented as a web-based application) achieve high accuracies in distinguishing TFs and TcoFs from other nuclear proteins, and TFs from TcoFs respectively.Conclusion:As demonstrated, given the fact that two proteins are capable of forming direct physical interactions and using only information about their sequence composition, we have developed a completely new method for predicting a functional class of TF interacting protein partners

  12. Regulation of human protein S gene (PROS1) transcription

    NARCIS (Netherlands)

    Wolf, Cornelia de

    2006-01-01

    This thesis describes the investigation of the transcriptional regulation of the gene for anticoagulant plasma Protein S, PROS1. Protein S is a cofactor for Protein C in the Protein C anticoagulant pathway. The coagulation cascade is negatively regulated by this pathway through inactivation of

  13. Menin and RNF20 recruitment is associated with dynamic histone modifications that regulate signal transducer and activator of transcription 1 (STAT1-activated transcription of the interferon regulatory factor 1 gene (IRF1

    Directory of Open Access Journals (Sweden)

    Buro Lauren J

    2010-09-01

    Full Text Available Abstract Background Signal transducer and activator of transcription (STAT activation of gene expression is both rapid and transient, and when properly executed it affects growth, differentiation, homeostasis and the immune response, but when dysregulated it contributes to human disease. Transcriptional activation is regulated by alterations to the chromatin template. However, the role of histone modification at gene loci that are activated for transcription in response to STAT signaling is poorly defined. Results Using chromatin immunoprecipitation, we profiled several histone modifications during STAT1 activation of the interferon regulatory factor 1 gene (IRF1. Methylated lysine histone proteins H3K4me2, H3K4me3, H3K79me3, H3K36me3 and monoubiquitinated histone ubH2B are dynamic and correlate with interferon (IFNγ induction of STAT1 activity. Chemical inhibition of H3K4 methylation downregulates IRF1 transcription and decreases RNA polymerase II (Pol II occupancy at the IRF1 promoter. MEN1, a component of a complex proteins associated with Set1 (COMPASS-like complex and the hBRE1 component, RNF20, are localized to IRF1 in the uninduced state and are further recruited when IRF1 is activated. RNAi-mediated depletion of RNF20 lowers both ubH2B and H3K4me3, but surprisingly, upregulates IFNγ induced IRF1 transcription. The dynamics of phosphorylation in the C-terminal domain (CTD of Pol II are disrupted during gene activation as well. Conclusions H2B monoubiquitination promotes H3K4 methylation, but the E3 ubiquitin ligase, RNF20, is repressive of inducible transcription at the IRF1 gene locus, suggesting that ubH2B can, directly or indirectly, affect Pol II CTD phosphorylation cycling to exert control on ongoing transcription.

  14. Use of green fluorescent fusion protein to track activation of the transcription factor osterix during early osteoblast differentiation

    International Nuclear Information System (INIS)

    Tai Guangping; Christodoulou, Ioannis; Bishop, Anne E.; Polak, Julia M.

    2005-01-01

    Osterix (Osx) is a transcription factor required for the differentiation of preosteoblasts into fully functioning osteoblasts. However, the pattern of Osx activation during preosteoblast differentiation and maturation has not been clearly defined. Our aim was to study Osx activation during these processes in osteoblasts differentiating from murine and human embryonic stem cells (ESC). To do this, we constructed an Osx-GFP fusion protein reporter system to track Osx translocation within the cells. The distribution of Osx-GFP at representative stages of differentiation was also investigated by screening primary osteoblasts, mesenchymal stem cells, synoviocytes, and pre-adipocytes. Our experiments revealed that Osx-GFP protein was detectable in the cytoplasm of cultured, differentiated ESC 4 days after plating of enzymatically dispersed embryoid bodies. Osterix-GFP protein became translocated into the nucleus on day 7 following transfer of differentiated ESC to osteogenic medium. After 14 days of differentiation, cells showing nuclear translocation of Osx-GFP formed rudimentary bone nodules that continued to increase in number over the following weeks (through day 21). We also found that Osx translocated into the nuclei of mesenchymal stem cells (C3H10T1/2) and pre-osteoblasts (MC3T3-E1) and showed partial activation in pre-adipocytes (MC3T3-L1). These data suggest that Osx activation occurs at a very early point in the differentiation of the mesenchymal-osteoblastic lineage

  15. Mitotic Transcriptional Activation: Clearance of Actively Engaged Pol II via Transcriptional Elongation Control in Mitosis.

    Science.gov (United States)

    Liang, Kaiwei; Woodfin, Ashley R; Slaughter, Brian D; Unruh, Jay R; Box, Andrew C; Rickels, Ryan A; Gao, Xin; Haug, Jeffrey S; Jaspersen, Sue L; Shilatifard, Ali

    2015-11-05

    Although it is established that some general transcription factors are inactivated at mitosis, many details of mitotic transcription inhibition (MTI) and its underlying mechanisms are largely unknown. We have identified mitotic transcriptional activation (MTA) as a key regulatory step to control transcription in mitosis for genes with transcriptionally engaged RNA polymerase II (Pol II) to activate and transcribe until the end of the gene to clear Pol II from mitotic chromatin, followed by global impairment of transcription reinitiation through MTI. Global nascent RNA sequencing and RNA fluorescence in situ hybridization demonstrate the existence of transcriptionally engaged Pol II in early mitosis. Both genetic and chemical inhibition of P-TEFb in mitosis lead to delays in the progression of cell division. Together, our study reveals a mechanism for MTA and MTI whereby transcriptionally engaged Pol II can progress into productive elongation and finish transcription to allow proper cellular division. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Design, Assembly, and Characterization of TALE-Based Transcriptional Activators and Repressors.

    Science.gov (United States)

    Thakore, Pratiksha I; Gersbach, Charles A

    2016-01-01

    Transcription activator-like effectors (TALEs) are modular DNA-binding proteins that can be fused to a variety of effector domains to regulate the epigenome. Nucleotide recognition by TALE monomers follows a simple cipher, making this a powerful and versatile method to activate or repress gene expression. Described here are methods to design, assemble, and test TALE transcription factors (TALE-TFs) for control of endogenous gene expression. In this protocol, TALE arrays are constructed by Golden Gate cloning and tested for activity by transfection and quantitative RT-PCR. These methods for engineering TALE-TFs are useful for studies in reverse genetics and genomics, synthetic biology, and gene therapy.

  17. Controlling cellular P-TEFb activity by the HIV-1 transcriptional transactivator Tat.

    Directory of Open Access Journals (Sweden)

    Lisa Muniz

    Full Text Available The human immunodeficiency virus 1 (HIV-1 transcriptional transactivator (Tat is essential for synthesis of full-length transcripts from the integrated viral genome by RNA polymerase II (Pol II. Tat recruits the host positive transcription elongation factor b (P-TEFb to the HIV-1 promoter through binding to the transactivator RNA (TAR at the 5'-end of the nascent HIV transcript. P-TEFb is a general Pol II transcription factor; its cellular activity is controlled by the 7SK small nuclear RNA (snRNA and the HEXIM1 protein, which sequester P-TEFb into transcriptionally inactive 7SK/HEXIM/P-TEFb snRNP. Besides targeting P-TEFb to HIV transcription, Tat also increases the nuclear level of active P-TEFb through promoting its dissociation from the 7SK/HEXIM/P-TEFb RNP by an unclear mechanism. In this study, by using in vitro and in vivo RNA-protein binding assays, we demonstrate that HIV-1 Tat binds with high specificity and efficiency to an evolutionarily highly conserved stem-bulge-stem motif of the 5'-hairpin of human 7SK snRNA. The newly discovered Tat-binding motif of 7SK is structurally and functionally indistinguishable from the extensively characterized Tat-binding site of HIV TAR and importantly, it is imbedded in the HEXIM-binding elements of 7SK snRNA. We show that Tat efficiently replaces HEXIM1 on the 7SK snRNA in vivo and therefore, it promotes the disassembly of the 7SK/HEXIM/P-TEFb negative transcriptional regulatory snRNP to augment the nuclear level of active P-TEFb. This is the first demonstration that HIV-1 specifically targets an important cellular regulatory RNA, most probably to promote viral transcription and replication. Demonstration that the human 7SK snRNA carries a TAR RNA-like Tat-binding element that is essential for the normal transcriptional regulatory function of 7SK questions the viability of HIV therapeutic approaches based on small drugs blocking the Tat-binding site of HIV TAR.

  18. Dissection of TALE-dependent gene activation reveals that they induce transcription cooperatively and in both orientations.

    Science.gov (United States)

    Streubel, Jana; Baum, Heidi; Grau, Jan; Stuttman, Johannes; Boch, Jens

    2017-01-01

    Plant-pathogenic Xanthomonas bacteria inject transcription activator-like effector proteins (TALEs) into host cells to specifically induce transcription of plant genes and enhance susceptibility. Although the DNA-binding mode is well-understood it is still ambiguous how TALEs initiate transcription and whether additional promoter elements are needed to support this. To systematically dissect prerequisites for transcriptional initiation the activity of one TALE was compared on different synthetic Bs4 promoter fragments. In addition, a large collection of artificial TALEs spanning the OsSWEET14 promoter was compared. We show that the presence of a TALE alone is not sufficient to initiate transcription suggesting the requirement of additional supporting promoter elements. At the OsSWEET14 promoter TALEs can initiate transcription from various positions, in a synergistic manner of multiple TALEs binding in parallel to the promoter, and even by binding in reverse orientation. TALEs are known to shift the transcriptional start site, but our data show that this shift depends on the individual position of a TALE within a promoter context. Our results implicate that TALEs function like classical enhancer-binding proteins and initiate transcription in both orientations which has consequences for in planta target gene prediction and design of artificial activators.

  19. Altered association of transcriptionally active DNA with the nuclear-matrix after heat shock

    NARCIS (Netherlands)

    Sakkers, RJ; Brunsting, JF; Filon, AR; Kampinga, HH; Konings, AWT; Mullenders, LHF

    Purpose: Exposure of human cells to heat leads to denaturation and aggregation of proteins. Within the nucleus, it has been suggested that protein aggregation is linked to the: selective inhibition by hyperthermia of nucleotide excision repair in transcriptionally active genes. Tn this study it was

  20. E2F1 transcription is induced by genotoxic stress through ATM/ATR activation.

    Science.gov (United States)

    Carcagno, Abel L; Ogara, María F; Sonzogni, Silvina V; Marazita, Mariela C; Sirkin, Pablo F; Ceruti, Julieta M; Cánepa, Eduardo T

    2009-05-01

    E2F1, a member of the E2F family of transcription factors, plays a critical role in controlling both cell cycle progression and apoptotic cell death in response to DNA damage and oncogene activation. Following genotoxic stresses, E2F1 protein is stabilized by phosphorylation and acetylation driven to its accumulation. The aim of the present work was to examine whether the increase in E2F1 protein levels observed after DNA damage is only a reflection of an increase in E2F1 protein stability or is also the consequence of enhanced transcription of the E2F1 gene. The data presented here demonstrates that UV light and other genotoxics induce the transcription of E2F1 gene in an ATM/ATR dependent manner, which results in increasing E2F1 mRNA and protein levels. After genotoxic stress, transcription of cyclin E, an E2F1 target gene, was significantly induced. This induction was the result of two well-differentiated effects, one of them dependent on de novo protein synthesis and the other on the protein stabilization. Our results strongly support a transcriptional effect of DNA damaging agents on E2F1 expression. The results presented herein uncover a new mechanism involving E2F1 in response to genotoxic stress.

  1. Expression, processing and transcriptional regulation of granulysin in short-term activated human lymphocytes

    Directory of Open Access Journals (Sweden)

    Groscurth Peter

    2007-06-01

    Full Text Available Abstract Background Granulysin, a cytotoxic protein expressed in human natural killer cells and activated T lymphocytes, exhibits cytolytic activity against a variety of intracellular microbes. Expression and transcription have been partially characterised in vitro and four transcripts (NKG5, 519, 520, and 522 were identified. However, only a single protein product of 15 kDa was found, which is subsequently processed to an active 9 kDa protein. Results In this study we investigated generation of granulysin in lymphokine activated killer (LAK cells and antigen (Listeria specific T-cells. Semiquantitative RT-PCR revealed NKG5 to be the most prominent transcript. It was found to be up-regulated in a time-dependent manner in LAK cells and antigen specific T-cells and their subsets. Two isoforms of 519 mRNA were up-regulated under IL-2 and antigen stimulation. Moreover, two novel transcripts, without any known function, comprising solely parts of the 5 prime region of the primary transcript, were detected. A significant increase of granulysin expressing LAK cells as well as antigen specific T-cells was shown by fluorescence microscopy. On the subset level, increase in CD4+ granulysin expressing cells was found only under antigen stimulation. Immunoblotting showed the 15 kDa form of granulysin to be present in the first week of stimulation either with IL-2 or with bacterial antigen. Substantial processing to the 9 kDa form was detected during the first week in LAK cells and in the second week in antigen specific T-cells. Conclusion This first comprehensive study of granulysin gene regulation in primary cultured human lymphocytes shows that the regulation of granulysin synthesis in response to IL-2 or bacterial antigen stimulation occurs at several levels: RNA expression, extensive alternative splicing and posttranslational processing.

  2. An essential nuclear protein in trypanosomes is a component of mRNA transcription/export pathway.

    Directory of Open Access Journals (Sweden)

    Mariana Serpeloni

    Full Text Available In eukaryotic cells, different RNA species are exported from the nucleus via specialized pathways. The mRNA export machinery is highly integrated with mRNA processing, and includes a different set of nuclear transport adaptors as well as other mRNA binding proteins, RNA helicases, and NPC-associated proteins. The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease, a widespread and neglected human disease which is endemic to Latin America. Gene expression in Trypanosoma has unique characteristics, such as constitutive polycistronic transcription of protein-encoding genes and mRNA processing by trans-splicing. In general, post-transcriptional events are the major points for regulation of gene expression in these parasites. However, the export pathway of mRNA from the nucleus is poorly understood. The present study investigated the function of TcSub2, which is a highly conserved protein ortholog to Sub2/ UAP56, a component of the Transcription/Export (TREX multiprotein complex connecting transcription with mRNA export in yeast/human. Similar to its orthologs, TcSub2 is a nuclear protein, localized in dispersed foci all over the nuclei -except the fibrillar center of nucleolus- and at the interface between dense and non-dense chromatin areas, proposing the association of TcSub2 with transcription/processing sites. These findings were analyzed further by BrUTP incorporation assays and confirmed that TcSub2 is physically associated with active RNA polymerase II (RNA pol II, but not RNA polymerase I (RNA pol I or Spliced Leader (SL transcription, demonstrating participation particularly in nuclear mRNA metabolism in T. cruzi. The double knockout of the TcSub2 gene is lethal in T. cruzi, suggesting it has an essential function. Alternatively, RNA interference assays were performed in Trypanosoma brucei. It allowed demonstrating that besides being an essential protein, its knockdown causes mRNA accumulation in the nucleus and

  3. microProtein Prediction Program (miP3) : A Software for Predicting microProteins and Their Target Transcription Factors

    NARCIS (Netherlands)

    de Klein, Niek; Magnani, Enrico; Banf, Michael; Rhee, Seung Yon

    2015-01-01

    An emerging concept in transcriptional regulation is that a class of truncated transcription factors (TFs), called microProteins (miPs), engages in protein-protein interactions with TF complexes and provides feedback controls. A handful of miP examples have been described in the literature but the

  4. Cellular reprogramming through mitogen-activated protein kinases

    Directory of Open Access Journals (Sweden)

    Justin eLee

    2015-10-01

    Full Text Available Mitogen-activated protein kinase (MAPK cascades are conserved eukaryote signaling modules where MAPKs, as the final kinases in the cascade, phosphorylate protein substrates to regulate cellular processes. While some progress in the identification of MAPK substrates has been made in plants, the knowledge on the spectrum of substrates and their mechanistic action is still fragmentary. In this focused review, we discuss the biological implications of the data in our original paper (Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana; Frontiers in Plant Science 5: 554 in the context of related research. In our work, we mimicked in vivo activation of two stress-activated MAPKs, MPK3 and MPK6, through transgenic manipulation of Arabidopsis thaliana and used phosphoproteomics analysis to identify potential novel MAPK substrates. Here, we plotted the identified putative MAPK substrates (and downstream phosphoproteins as a global protein clustering network. Based on a highly stringent selection confidence level, the core networks highlighted a MAPK-induced cellular reprogramming at multiple levels of gene and protein expression – including transcriptional, post-transcriptional, translational, post-translational (such as protein modification, folding and degradation steps, and also protein re-compartmentalization. Additionally, the increase in putative substrates/phosphoproteins of energy metabolism and various secondary metabolite biosynthesis pathways coincides with the observed accumulation of defense antimicrobial substances as detected by metabolome analysis. Furthermore, detection of protein networks in phospholipid or redox elements suggests activation of downstream signaling events. Taken in context with other studies, MAPKs are key regulators that reprogram cellular events to orchestrate defense signaling in eukaryotes.

  5. Retrotransposon-centered analysis of piRNA targeting shows a shift from active to passive retrotransposon transcription in developing mouse testes

    Directory of Open Access Journals (Sweden)

    Mourier Tobias

    2011-09-01

    Full Text Available Abstract Background Piwi-associated RNAs (piRNAs bind transcripts from retrotransposable elements (RTE in mouse germline cells and seemingly act as guides for genomic methylation, thereby repressing the activity of RTEs. It is currently unknown if and how Piwi proteins distinguish RTE transcripts from other cellular RNAs. During germline development, the main target of piRNAs switch between different types of RTEs. Using the piRNA targeting of RTEs as an indicator of RTE activity, and considering the entire population of genomic RTE loci along with their age and location, this study aims at further elucidating the dynamics of RTE activity during mouse germline development. Results Due to the inherent sequence redundancy between RTE loci, assigning piRNA targeting to specific loci is problematic. This limits the analysis, although certain features of piRNA targeting of RTE loci are apparent. As expected, young RTEs display a much higher level of piRNA targeting than old RTEs. Further, irrespective of age, RTE loci near protein-coding coding genes are targeted to a greater extent than RTE loci far from genes. During development, a shift in piRNA targeting is observed, with a clear increase in the relative piRNA targeting of RTEs residing within boundaries of protein-coding gene transcripts. Conclusions Reanalyzing published piRNA sequences and taking into account the features of individual RTE loci provide novel insight into the activity of RTEs during development. The obtained results are consistent with some degree of proportionality between what transcripts become substrates for Piwi protein complexes and the level by which the transcripts are present in the cell. A transition from active transcription of RTEs to passive co-transcription of RTE sequences residing within protein-coding transcripts appears to take place in postnatal development. Hence, the previously reported increase in piRNA targeting of SINEs in postnatal testis development

  6. Structural and Functional Analysis of VQ Motif-Containing Proteins in Arabidopsis as Interacting Proteins of WRKY Transcription Factors1[W][OA

    Science.gov (United States)

    Cheng, Yuan; Zhou, Yuan; Yang, Yan; Chi, Ying-Jun; Zhou, Jie; Chen, Jian-Ye; Wang, Fei; Fan, Baofang; Shi, Kai; Zhou, Yan-Hong; Yu, Jing-Quan; Chen, Zhixiang

    2012-01-01

    WRKY transcription factors are encoded by a large gene superfamily with a broad range of roles in plants. Recently, several groups have reported that proteins containing a short VQ (FxxxVQxLTG) motif interact with WRKY proteins. We have recently discovered that two VQ proteins from Arabidopsis (Arabidopsis thaliana), SIGMA FACTOR-INTERACTING PROTEIN1 and SIGMA FACTOR-INTERACTING PROTEIN2, act as coactivators of WRKY33 in plant defense by specifically recognizing the C-terminal WRKY domain and stimulating the DNA-binding activity of WRKY33. In this study, we have analyzed the entire family of 34 structurally divergent VQ proteins from Arabidopsis. Yeast (Saccharomyces cerevisiae) two-hybrid assays showed that Arabidopsis VQ proteins interacted specifically with the C-terminal WRKY domains of group I and the sole WRKY domains of group IIc WRKY proteins. Using site-directed mutagenesis, we identified structural features of these two closely related groups of WRKY domains that are critical for interaction with VQ proteins. Quantitative reverse transcription polymerase chain reaction revealed that expression of a majority of Arabidopsis VQ genes was responsive to pathogen infection and salicylic acid treatment. Functional analysis using both knockout mutants and overexpression lines revealed strong phenotypes in growth, development, and susceptibility to pathogen infection. Altered phenotypes were substantially enhanced through cooverexpression of genes encoding interacting VQ and WRKY proteins. These findings indicate that VQ proteins play an important role in plant growth, development, and response to environmental conditions, most likely by acting as cofactors of group I and IIc WRKY transcription factors. PMID:22535423

  7. An activator of transcription regulates phage TP901-1 late gene expression

    DEFF Research Database (Denmark)

    Brøndsted, Lone; Pedersen, Margit; Hammer, Karin

    2001-01-01

    bp contains both the promoter and the region necessary for activation by ORF29. The transcriptional start site of the promoter was identified by primer extension to position 13073 on the TP901-1 genome, thus located 87 bp downstream of orf29 in a 580-bp intergenic region between orf29 and orf30....... Furthermore, the region located -85 to -61 bp upstream of the start site was shown to be necessary for promoter activity. During infection, the transcript arising from the late promoter is fully induced at 40 min postinfection, and our results suggest that a certain level of ORF29 must he reached in order...... to activate transcription of the promoter. Several lactococcal bacteriophages encode ORF29 homologous proteins, indicating that late transcription may be controlled by a similar mechanism in these phages. With the identification of this novel regulator, our results suggest that within the P335 group...

  8. The intracellular immune receptor Rx1 regulates the DNA-binding activity of a Golden2-like transcription factor

    NARCIS (Netherlands)

    Townsend, Philip D.; Dixon, Christopher H.; Slootweg, Erik J.; Sukarta, Octavina C.A.; Yang, Ally W.H.; Hughes, Timothy R.; Sharples, Gary J.; Palsson, Lars-Olof; Takken, Frank L.W.; Goverse, Aska; Cann, Martin J.

    2018-01-01

    Plant NLR proteins enable the immune system to recognise and respond to pathogen attack. An early consequence of immune activation is transcriptional reprogramming and some NLRs have been shown to act in the nucleus and interact with transcription factors. The Rx1 NLR protein of potato is further

  9. Human herpesvirus 6B U19 protein is a PML-regulated transcriptional activator that localizes to nuclear foci in a PML-independent manner

    DEFF Research Database (Denmark)

    Kofod-Olsen, Emil; Ross-Hansen, Katrine; Mikkelsen, Jacob Giehm

    2008-01-01

    Human herpesvirus 6B (HHV-6B) contains an IE-B domain spanning open reading frames U16/17-U19, based on homology with human cytomegalovirus. Here, the protein product, U19, of the HHV-6B U19 gene is identified as a 47 kDa transcriptional activator. HHV-6B infection or overexpression of U19...

  10. Novel isoforms of the TFIID subunit TAF4 modulate nuclear receptor-mediated transcriptional activity

    International Nuclear Information System (INIS)

    Brunkhorst, Adrian; Neuman, Toomas; Hall, Anita; Arenas, Ernest; Bartfai, Tamas; Hermanson, Ola; Metsis, Madis

    2004-01-01

    The transcription factor TFIID consists of TATA-binding protein (TBP) and TBP-associated factors (TAFs). TAFs are essential for modulation of transcriptional activity but the regulation of TAFs is complex and many important aspects remain unclear. In this study, we have identified and characterized five novel truncated forms of the TFIID subunit TAF4 (TAF II 135). Analysis of the mouse gene structure revealed that all truncations were the results of alternative splicing and resulted in the loss of domains or parts of domains implicated in TAF4 functional interactions. Results from transcriptional assays showed that several of the TAF4 isoforms exerted dominant negative effects on TAF4 activity in nuclear receptor-mediated transcriptional activation. In addition, alternative TAF4 isoforms could be detected in specific cell types. Our results indicate an additional level of complexity in TAF4-mediated regulation of transcription and suggest context-specific roles for these new TAF4 isoforms in transcriptional regulation in vivo

  11. Distinct structural features of TFAM drive mitochondrial DNA packaging versus transcriptional activation.

    Science.gov (United States)

    Ngo, Huu B; Lovely, Geoffrey A; Phillips, Rob; Chan, David C

    2014-01-01

    TFAM (transcription factor A, mitochondrial) is a DNA-binding protein that activates transcription at the two major promoters of mitochondrial DNA (mtDNA)--the light strand promoter (LSP) and the heavy strand promoter 1 (HSP1). Equally important, it coats and packages the mitochondrial genome. TFAM has been shown to impose a U-turn on LSP DNA; however, whether this distortion is relevant at other sites is unknown. Here we present crystal structures of TFAM bound to HSP1 and to nonspecific DNA. In both, TFAM similarly distorts the DNA into a U-turn. Yet, TFAM binds to HSP1 in the opposite orientation from LSP explaining why transcription from LSP requires DNA bending, whereas transcription at HSP1 does not. Moreover, the crystal structures reveal dimerization of DNA-bound TFAM. This dimerization is dispensable for DNA bending and transcriptional activation but is important in DNA compaction. We propose that TFAM dimerization enhances mitochondrial DNA compaction by promoting looping of the DNA.

  12. Monitoring of transcriptional regulation in Pichia pastoris under protein production conditions

    Directory of Open Access Journals (Sweden)

    Bhattacharyya Anamitra

    2007-06-01

    Full Text Available Abstract Background It has become evident that host cells react to recombinant protein production with a variety of metabolic and intrinsic stresses such as the unfolded protein response (UPR pathway. Additionally, environmental conditions such as growth temperature may have a strong impact on cell physiology and specific productivity. However, there is little information about the molecular reactions of the host cells on a genomic level, especially in context to recombinant protein secretion. For the first time, we monitored transcriptional regulation of a subset of marker genes in the common production host Pichia pastoris to gain insights into the general physiological status of the cells under protein production conditions, with the main focus on secretion stress related genes. Results Overexpression of the UPR activating transcription factor Hac1p was employed to identify UPR target genes in P. pastoris and the responses were compared to those known for Saccharomyces cerevisiae. Most of the folding/secretion related genes showed similar regulation patterns in both yeasts, whereas genes associated with the general stress response were differentially regulated. Secretion of an antibody Fab fragment led to induction of UPR target genes in P. pastoris, however not to the same magnitude as Hac1p overproduction. Overexpression of S. cerevisiae protein disulfide isomerase (PDI1 enhances Fab secretion rates 1.9 fold, but did not relief UPR stress. Reduction of cultivation temperature from 25°C to 20°C led to a 1.4-fold increase of specific product secretion rate in chemostat cultivations, although the transcriptional levels of the product genes (Fab light and heavy chain were significantly reduced at the lower temperature. A subset of folding related genes appeared to be down-regulated at the reduced temperature, whereas transcription of components of the ER associated degradation and the secretory transport was enhanced. Conclusion Monitoring of

  13. Direct modulation of T-box riboswitch-controlled transcription by protein synthesis inhibitors.

    Science.gov (United States)

    Stamatopoulou, Vassiliki; Apostolidi, Maria; Li, Shuang; Lamprinou, Katerina; Papakyriakou, Athanasios; Zhang, Jinwei; Stathopoulos, Constantinos

    2017-09-29

    Recently, it was discovered that exposure to mainstream antibiotics activate numerous bacterial riboregulators that control antibiotic resistance genes including metabolite-binding riboswitches and other transcription attenuators. However, the effects of commonly used antibiotics, many of which exhibit RNA-binding properties, on the widespread T-box riboswitches, remain unknown. In Staphylococcus aureus, a species-specific glyS T-box controls the supply of glycine for both ribosomal translation and cell wall synthesis, making it a promising target for next-generation antimicrobials. Here, we report that specific protein synthesis inhibitors could either significantly increase T-box-mediated transcription antitermination, while other compounds could suppress it, both in vitro and in vivo. In-line probing of the full-length T-box combined with molecular modelling and docking analyses suggest that the antibiotics that promote transcription antitermination stabilize the T-box:tRNA complex through binding specific positions on stem I and the Staphylococcal-specific stem Sa. By contrast, the antibiotics that attenuate T-box transcription bind to other positions on stem I and do not interact with stem Sa. Taken together, our results reveal that the transcription of essential genes controlled by T-box riboswitches can be directly modulated by commonly used protein synthesis inhibitors. These findings accentuate the regulatory complexities of bacterial response to antimicrobials that involve multiple riboregulators. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  14. Transcript and protein expression profile of PF11_0394, a Plasmodium falciparum protein expressed in salivary gland sporozoites

    Directory of Open Access Journals (Sweden)

    Schlarman Maggie S

    2012-03-01

    Full Text Available Abstract Background Plasmodium falciparum malaria is a significant problem around the world today, thus there is still a need for new control methods to be developed. Because the sporozoite displays dual infectivity for both the mosquito salivary glands and vertebrate host tissue, it is a good target for vaccine development. Methods The P. falciparum gene, PF11_0394, was chosen as a candidate for study due to its potential role in the invasion of host tissues. This gene, which was selected using a data mining approach from PlasmoDB, is expressed both at the transcriptional and protein levels in sporozoites and likely encodes a putative surface protein. Using reverse transcription-polymerase chain reaction (RT-PCR and green fluorescent protein (GFP-trafficking studies, a transcript and protein expression profile of PF11_0394 was determined. Results The PF11_0394 protein has orthologs in other Plasmodium species and Apicomplexans, but none outside of the group Apicomplexa. PF11_0394 transcript was found to be present during both the sporozoite and erythrocytic stages of the parasite life cycle, but no transcript was detected during axenic exoerythrocytic stages. Despite the presence of transcript throughout several life cycle stages, the PF11_0394 protein was only detected in salivary gland sporozoites. Conclusions PF11_0394 appears to be a protein uniquely detected in salivary gland sporozoites. Even though a specific function of PF11_0394 has not been determined in P. falciparum biology, it could be another candidate for a new vaccine.

  15. Development of novel metabolite-responsive transcription factors via transposon-mediated protein fusion.

    Science.gov (United States)

    Younger, Andrew K D; Su, Peter Y; Shepard, Andrea J; Udani, Shreya V; Cybulski, Thaddeus R; Tyo, Keith E J; Leonard, Joshua N

    2018-02-01

    Naturally evolved metabolite-responsive biosensors enable applications in metabolic engineering, ranging from screening large genetic libraries to dynamically regulating biosynthetic pathways. However, there are many metabolites for which a natural biosensor does not exist. To address this need, we developed a general method for converting metabolite-binding proteins into metabolite-responsive transcription factors-Biosensor Engineering by Random Domain Insertion (BERDI). This approach takes advantage of an in vitro transposon insertion reaction to generate all possible insertions of a DNA-binding domain into a metabolite-binding protein, followed by fluorescence activated cell sorting to isolate functional biosensors. To develop and evaluate the BERDI method, we generated a library of candidate biosensors in which a zinc finger DNA-binding domain was inserted into maltose binding protein, which served as a model well-studied metabolite-binding protein. Library diversity was characterized by several methods, a selection scheme was deployed, and ultimately several distinct and functional maltose-responsive transcriptional biosensors were identified. We hypothesize that the BERDI method comprises a generalizable strategy that may ultimately be applied to convert a wide range of metabolite-binding proteins into novel biosensors for applications in metabolic engineering and synthetic biology. © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  16. First functional polymorphism in CFTR promoter that results in decreased transcriptional activity and Sp1/USF binding

    International Nuclear Information System (INIS)

    Taulan, M.; Lopez, E.; Guittard, C.; Rene, C.; Baux, D.; Altieri, J.P.; DesGeorges, M.; Claustres, M.; Romey, M.C.

    2007-01-01

    Growing evidences show that functionally relevant polymorphisms in various promoters alter both transcriptional activity and affinities of existing protein-DNA interactions, and thus influence disease progression in humans. We previously reported the -94G>T CFTR promoter variant in a female CF patient in whom any known disease-causing mutation has been detected. To investigate whether the -94G>T could be a regulatory variant, we have proceeded to in silico analyses and functional studies including EMSA and reporter gene assays. Our data indicate that the promoter variant decreases basal CFTR transcriptional activity in different epithelial cells and alters binding affinities of both Sp1 and USF nuclear proteins to the CFTR promoter. The present report provides evidence for the first functional polymorphism that negatively affects the CFTR transcriptional activity and demonstrates a cooperative role of Sp1 and USF transcription factors in transactivation of the CFTR gene promoter

  17. Resveratrol upregulates Egr-1 expression and activity involving extracellular signal-regulated protein kinase and ternary complex factors

    Energy Technology Data Exchange (ETDEWEB)

    Rössler, Oliver G.; Glatzel, Daniel; Thiel, Gerald, E-mail: gerald.thiel@uks.eu

    2015-03-01

    Many intracellular functions have been attributed to resveratrol, a polyphenolic phytoalexin found in grapes and in other plants. Here, we show that resveratrol induces the expression of the transcription factor Egr-1 in human embryonic kidney cells. Using a chromosomally embedded Egr-1-responsive reporter gene, we show that the Egr-1 activity was significantly elevated in resveratrol-treated cells, indicating that the newly synthesized Egr-1 protein was biologically active. Stimulus-transcription coupling leading to the resveratrol-induced upregulation of Egr-1 expression and activity requires the protein kinases Raf and extracellular signal-regulated protein kinase ERK, while MAP kinase phosphatase-1 functions as a nuclear shut-off device that interrupts the signaling cascade connecting resveratrol stimulation with enhanced Egr-1 expression. On the transcriptional level, Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, connects the intracellular signaling cascade elicited by resveratrol with transcription of the Egr-1 gene. These data were corroborated by the observation that stimulation of the cells with resveratrol increased the transcriptional activation potential of Elk-1. The SRE as well as the GC-rich DNA binding site of Egr-1 function as resveratrol-responsive elements. Thus, resveratrol regulates gene transcription via activation of the stimulus-regulated protein kinases Raf and ERK and the stimulus-responsive transcription factors TCF and Egr-1. - Highlights: • The plant polyphenol resveratrol upregulates Egr-1 expression and activity. • The stimulation of Egr-1 requires the protein kinases ERK and Raf. • Resveratrol treatment upregulates the transcriptional activation potential of Elk-1. • Resveratrol-induced stimulation of Egr-1 requires ternary complex factors. • Two distinct resveratrol-responsive elements were identified.

  18. Resveratrol upregulates Egr-1 expression and activity involving extracellular signal-regulated protein kinase and ternary complex factors

    International Nuclear Information System (INIS)

    Rössler, Oliver G.; Glatzel, Daniel; Thiel, Gerald

    2015-01-01

    Many intracellular functions have been attributed to resveratrol, a polyphenolic phytoalexin found in grapes and in other plants. Here, we show that resveratrol induces the expression of the transcription factor Egr-1 in human embryonic kidney cells. Using a chromosomally embedded Egr-1-responsive reporter gene, we show that the Egr-1 activity was significantly elevated in resveratrol-treated cells, indicating that the newly synthesized Egr-1 protein was biologically active. Stimulus-transcription coupling leading to the resveratrol-induced upregulation of Egr-1 expression and activity requires the protein kinases Raf and extracellular signal-regulated protein kinase ERK, while MAP kinase phosphatase-1 functions as a nuclear shut-off device that interrupts the signaling cascade connecting resveratrol stimulation with enhanced Egr-1 expression. On the transcriptional level, Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, connects the intracellular signaling cascade elicited by resveratrol with transcription of the Egr-1 gene. These data were corroborated by the observation that stimulation of the cells with resveratrol increased the transcriptional activation potential of Elk-1. The SRE as well as the GC-rich DNA binding site of Egr-1 function as resveratrol-responsive elements. Thus, resveratrol regulates gene transcription via activation of the stimulus-regulated protein kinases Raf and ERK and the stimulus-responsive transcription factors TCF and Egr-1. - Highlights: • The plant polyphenol resveratrol upregulates Egr-1 expression and activity. • The stimulation of Egr-1 requires the protein kinases ERK and Raf. • Resveratrol treatment upregulates the transcriptional activation potential of Elk-1. • Resveratrol-induced stimulation of Egr-1 requires ternary complex factors. • Two distinct resveratrol-responsive elements were identified

  19. Possible linkage of SP6 transcriptional activity with amelogenesis by protein stabilization.

    Science.gov (United States)

    Utami, Trianna W; Miyoshi, Keiko; Hagita, Hiroko; Yanuaryska, Ryna Dwi; Horiguchi, Taigo; Noma, Takafumi

    2011-01-01

    Ameloblasts produce enamel matrix proteins such as amelogenin, ameloblastin, and amelotin during tooth development. The molecular mechanisms of ameloblast differentiation (amelogenesis) are currently not well understood. SP6 is a transcription factor of the Sp/KLF family that was recently found to regulate cell proliferation in a cell-type-specific manner. Sp6-deficient mice demonstrate characteristic tooth anomalies such as delayed eruption of the incisors and supernumerary teeth with disorganized amelogenesis. However, it remains unclear how Sp6 controls amelogenesis. In this study, we used SP6 high producer cells to identify SP6 target genes. Based on the observations that long-term culture of SP6 high producer cells reduced SP6 protein expression but not Sp6 mRNA expression, we found that SP6 is short lived and specifically degraded through a proteasome pathway. We established an in vitro inducible SP6 expression system coupled with siRNA knockdown and found a possible linkage between SP6 and amelogenesis through the regulation of amelotin and Rock1 gene expression by microarray analysis. Our findings suggest that the regulation of SP6 protein stability is one of the crucial steps in amelogenesis.

  20. Possible Linkage of SP6 Transcriptional Activity with Amelogenesis by Protein Stabilization

    Directory of Open Access Journals (Sweden)

    Trianna W. Utami

    2011-01-01

    Full Text Available Ameloblasts produce enamel matrix proteins such as amelogenin, ameloblastin, and amelotin during tooth development. The molecular mechanisms of ameloblast differentiation (amelogenesis are currently not well understood. SP6 is a transcription factor of the Sp/KLF family that was recently found to regulate cell proliferation in a cell-type-specific manner. Sp6-deficient mice demonstrate characteristic tooth anomalies such as delayed eruption of the incisors and supernumerary teeth with disorganized amelogenesis. However, it remains unclear how Sp6 controls amelogenesis. In this study, we used SP6 high producer cells to identify SP6 target genes. Based on the observations that long-term culture of SP6 high producer cells reduced SP6 protein expression but not Sp6 mRNA expression, we found that SP6 is short lived and specifically degraded through a proteasome pathway. We established an in vitro inducible SP6 expression system coupled with siRNA knockdown and found a possible linkage between SP6 and amelogenesis through the regulation of amelotin and Rock1 gene expression by microarray analysis. Our findings suggest that the regulation of SP6 protein stability is one of the crucial steps in amelogenesis.

  1. TAF(II)250: a transcription toolbox.

    Science.gov (United States)

    Wassarman, D A; Sauer, F

    2001-08-01

    Activation of RNA-polymerase-II-dependent transcription involves conversion of signals provided by gene-specific activator proteins into the synthesis of messenger RNA. This conversion requires dynamic structural changes in chromatin and assembly of general transcription factors (GTFs) and RNA polymerase II at core promoter sequence elements surrounding the transcription start site of genes. One hallmark of transcriptional activation is the interaction of DNA-bound activators with coactivators such as the TATA-box binding protein (TBP)-associated factors (TAF(II)s) within the GTF TFIID. TAF(II)250 possesses a variety of activities that are likely to contribute to the initial steps of RNA polymerase II transcription. TAF(II)250 is a scaffold for assembly of other TAF(II)s and TBP into TFIID, TAF(II)250 binds activators to recruit TFIID to particular promoters, TAF(II)250 regulates binding of TBP to DNA, TAF(II)250 binds core promoter initiator elements, TAF(II)250 binds acetylated lysine residues in core histones, and TAF(II)250 possesses protein kinase, ubiquitin-activating/conjugating and acetylase activities that modify histones and GTFs. We speculate that these activities achieve two goals--(1) they aid in positioning and stabilizing TFIID at particular promoters, and (2) they alter chromatin structure at the promoter to allow assembly of GTFs--and we propose a model for how TAF(II)250 converts activation signals into active transcription.

  2. Locked and proteolysis-based transcription activator-like effector (TALE) regulation.

    Science.gov (United States)

    Lonzarić, Jan; Lebar, Tina; Majerle, Andreja; Manček-Keber, Mateja; Jerala, Roman

    2016-02-18

    Development of orthogonal, designable and adjustable transcriptional regulators is an important goal of synthetic biology. Their activity has been typically modulated through stimulus-induced oligomerization or interaction between the DNA-binding and activation/repression domain. We exploited a feature of the designable Transcription activator-like effector (TALE) DNA-binding domain that it winds around the DNA which allows to topologically prevent it from binding by intramolecular cyclization. This new approach was investigated through noncovalent ligand-induced cyclization or through a covalent split intein cyclization strategy, where the topological inhibition of DNA binding by cyclization and its restoration by a proteolytic release of the topologic constraint was expected. We show that locked TALEs indeed have diminished DNA binding and regain full transcriptional activity by stimulation with the rapamycin ligand or site-specific proteolysis of the peptide linker, with much higher level of activation than rapamycin-induced heterodimerization. Additionally, we demonstrated reversibility, activation of genomic targets and implemented logic gates based on combinations of protein cyclization, proteolytic cleavage and ligand-induced dimerization, where the strongest fold induction was achieved by the proteolytic cleavage of a repression domain from a linear TALE. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  3. A Transcription Activator-Like Effector (TALE) Toolbox for Genome Engineering

    Science.gov (United States)

    Sanjana, Neville E.; Cong, Le; Zhou, Yang; Cunniff, Margaret M.; Feng, Guoping; Zhang, Feng

    2013-01-01

    Transcription activator-like effectors (TALEs) are a class of naturally occurring DNA binding proteins found in the plant pathogen Xanthomonas sp. The DNA binding domain of each TALE consists of tandem 34-amino acid repeat modules that can be rearranged according to a simple cipher to target new DNA sequences. Customized TALEs can be used for a wide variety of genome engineering applications, including transcriptional modulation and genome editing. Here we describe a toolbox for rapid construction of custom TALE transcription factors (TALE-TFs) and nucleases (TALENs) using a hierarchical ligation procedure. This toolbox facilitates affordable and rapid construction of custom TALE-TFs and TALENs within one week and can be easily scaled up to construct TALEs for multiple targets in parallel. We also provide details for testing the activity in mammalian cells of custom TALE-TFs and TALENs using, respectively, qRT-PCR and Surveyor nuclease. The TALE toolbox described here will enable a broad range of biological applications. PMID:22222791

  4. Role of nuclear factor of activated T-cells and activator protein-1 in the inhibition of interleukin-2 gene transcription by cannabinol in EL4 T-cells.

    Science.gov (United States)

    Yea, S S; Yang, K H; Kaminski, N E

    2000-02-01

    We previously reported that immunosuppressive cannabinoids inhibited interleukin (IL)-2 steady-state mRNA expression and secretion by phorbol-12-myristate-13-acetate plus ionomycin-activated mouse splenocytes and EL4 murine T-cells. Here we show that inhibition of IL-2 production by cannabinol, a modest central nervous system-active cannabinoid, is mediated through the inhibition of IL-2 gene transcription. Moreover, electrophoretic mobility shift assays demonstrated that cannabinol markedly inhibited the DNA binding activity of nuclear factor of activated T-cells (NF-AT) and activator protein-1 (AP-1) in a time- and concentration-dependent manner in activated EL4 cells. The inhibitory effects produced by cannabinol on AP-1 DNA binding were quite transient, showing partial recovery by 240 min after cell activation and no effect on the activity of a reporter gene under the control of AP-1. Conversely, cannabinol-mediated inhibition of NF-AT was robust and sustained as demonstrated by an NF-AT-regulated reporter gene. Collectively, these results suggest that decreased IL-2 production by cannabinol in EL4 cells is due to the inhibition of transcriptional activation of the IL-2 gene and is mediated, at least in part, through a transient inhibition of AP-1 and a sustained inhibition of NF-AT.

  5. Differential regulation of the transcriptional activity of the glucocorticoid receptor through site-specific phosphorylation

    Directory of Open Access Journals (Sweden)

    Raj Kumar

    2008-08-01

    Full Text Available Raj Kumar1, William J Calhoun21Division of Gastroenterology; 2Division of Allergy, Pulmonary, Immunology, Critical Care, and Sleep (APICS, Department of Internal Medicine, University of Texas Medical Branch, Galveston, TX, USAAbstract: Post-translational modifications such as phosphorylation are known to play an important role in the gene regulation by the transcription factors including the nuclear hormone receptor superfamily of which the glucocorticoid receptor (GR is a member. Protein phosphorylation often switches cellular activity from one state to another. Like many other transcription factors, the GR is a phosphoprotein, and phosphorylation plays an important role in the regulation of GR activity. Cell signaling pathways that regulate phosphorylation of the GR and its associated proteins are important determinants of GR function under various physiological conditions. While the role of many phosphorylation sites in the GR is still not fully understood, the role of others is clearer. Several aspects of transcription factor function, including DNA binding affinity, interaction of transactivation domains with the transcription initiation complex, and shuttling between the cytoplasmic compartments, have all been linked to site-specific phosphorylation. All major phosphorylation sites in the human GR are located in the N-terminal domain including the major transactivation domain, AF1. Available literature clearly indicates that many of these potential phosphorylation sites are substrates for multiple kinases, suggesting the potential for a very complex regulatory network. Phosphorylated GR interacts favorably with critical coregulatory proteins and subsequently enhances transcriptional activity. In addition, the activities and specificities of coregulators may be subject to similar regulation by phosphorylation. Regulation of the GR activity due to phosphorylation appears to be site-specific and dependent upon specific cell signaling cascade

  6. Transcriptional regulation of human FE65, a ligand of Alzheimer's disease amyloid precursor protein, by Sp1.

    LENUS (Irish Health Repository)

    Yu, Hoi-Tin

    2010-03-01

    FE65 is a neuronal-enriched adaptor protein that binds to the Alzheimer\\'s disease amyloid precursor protein (APP). FE65 forms a transcriptionally active complex with the APP intracellular domain (AICD). The precise gene targets for this complex are unclear but several Alzheimer\\'s disease-linked genes have been proposed. Additionally, evidence suggests that FE65 influences APP metabolism. The mechanism by which FE65 expression is regulated is as yet unknown. To gain insight into the regulatory mechanism, we cloned a 1.6 kb fragment upstream of the human FE65 gene and found that it possesses particularly strong promoter activity in neurones. To delineate essential regions in the human FE65 promoter, a series of deletion mutants were generated. The minimal FE65 promoter was located between -100 and +5, which contains a functional Sp1 site. Overexpression of the transcription factor Sp1 potentiates the FE65 promoter activity. Conversely, suppression of the FE65 promoter was observed in cells either treated with an Sp1 inhibitor or in which Sp1 was knocked down. Furthermore, reduced levels of Sp1 resulted in downregulation of endogenous FE65 mRNA and protein. These findings reveal that Sp1 plays a crucial role in transcriptional control of the human FE65 gene.

  7. A transcriptional coregulator, SPIN·DOC, attenuates the coactivator activity of Spindlin1.

    Science.gov (United States)

    Bae, Narkhyun; Gao, Min; Li, Xu; Premkumar, Tolkappiyan; Sbardella, Gianluca; Chen, Junjie; Bedford, Mark T

    2017-12-22

    Spindlin1 (SPIN1) is a transcriptional coactivator with critical functions in embryonic development and emerging roles in cancer. SPIN1 harbors three Tudor domains, two of which engage the tail of histone H3 by reading the H3-Lys-4 trimethylation and H3-Arg-8 asymmetric dimethylation marks. To gain mechanistic insight into how SPIN1 functions as a transcriptional coactivator, here we purified its interacting proteins. We identified an uncharacterized protein (C11orf84), which we renamed SPIN1 docking protein (SPIN·DOC), that directly binds SPIN1 and strongly disrupts its histone methylation reading ability, causing it to disassociate from chromatin. The Spindlin family of coactivators has five related members (SPIN1, 2A, 2B, 3, and 4), and we found that all of them bind SPIN·DOC. It has been reported previously that SPIN1 regulates gene expression in the Wnt signaling pathway by directly interacting with transcription factor 4 (TCF4). We observed here that SPIN·DOC associates with TCF4 in a SPIN1-dependent manner and dampens SPIN1 coactivator activity in TOPflash reporter assays. Furthermore, knockdown and overexpression experiments indicated that SPIN·DOC represses the expression of a number of SPIN1-regulated genes, including those encoding ribosomal RNA and the cytokine IL1B. In conclusion, we have identified SPIN·DOC as a transcriptional repressor that binds SPIN1 and masks its ability to engage the H3-Lys-4 trimethylation activation mark. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Identification of target genes of transcription factor activator protein 2 gamma in breast cancer cells

    International Nuclear Information System (INIS)

    Ailan, He; Shuanglin, Xiang; Xiangwen, Xiao; Daolong, Ren; Lu, Gan; Xiaofeng, Ding; Xi, Qiao; Xingwang, Hu; Rushi, Liu; Jian, Zhang

    2009-01-01

    Activator protein 2 gamma (AP-2γ) is a member of the transcription factor activator protein-2 (AP-2) family, which is developmentally regulated and plays a role in human neoplasia. AP-2γ has been found to be overexpressed in most breast cancers, and have a dual role to inhibit tumor initiation and promote tumor progression afterwards during mammary tumorigensis. To identify the gene targets that mediate its effects, we performed chromatin immunoprecipitation (ChIP) to isolate AP-2γ binding sites on genomic DNA from human breast cancer cell line MDA-MB-453. 20 novel DNA fragments proximal to potential AP-2γ targets were obtained. They are categorized into functional groups of carcinogenesis, metabolism and others. A combination of sequence analysis, reporter gene assays, quantitative real-time PCR, electrophoretic gel mobility shift assays and immunoblot analysis further confirmed the four AP-2γ target genes in carcinogenesis group: ErbB2, CDH2, HPSE and IGSF11. Our results were consistent with the previous reports that ErbB2 was the target gene of AP-2γ. Decreased expression and overexpression of AP-2γ in human breast cancer cells significantly altered the expression of these four genes, indicating that AP-2γ directly regulates them. This suggested that AP-2γ can coordinate the expression of a network of genes, involving in carcinogenesis, especially in breast cancer. They could serve as therapeutic targets against breast cancers in the future

  9. A damage-responsive DNA binding protein regulates transcription of the yeast DNA repair gene PHR1

    International Nuclear Information System (INIS)

    Sebastian, J.; Sancar, G.B.

    1991-01-01

    The PHR1 gene of Saccharomyces cerevisiae encodes the DNA repair enzyme photolyase. Transcription of PHR1 increases in response to treatment of cells with 254-nm radiation and chemical agents that damage DNA. The authors here the identification of a damage-responsive DNA binding protein, termed photolyase regulatory protein (PRP), and its cognate binding site, termed the PHR1 transcription after DNA damage. PRP activity, monitored by electrophoretic-mobility-shift assay, was detected in cells during normal growth but disappeared within 30 min after irradiation. Copper-phenanthroline footprinting of PRP-DNA complexes revealed that PRP protects a 39-base-pair region of PHR1 5' flanking sequence beginning 40 base pairs upstream from the coding sequence. Thus these observations establish that PRP is a damage-responsive repressor of PHR1 transcription

  10. Mutational analysis of the activator of late transcription, Alt , in the lactococcal bacteriophage TP901-1

    DEFF Research Database (Denmark)

    Pedersen, Margit; Hammer, Karin

    2007-01-01

    An activator protein, Alt, synthesized during the early state of lytic infection is required for transcription of the late operon in the lactococcal phage TP901-1. In order to identify amino acid residues in the Alt protein required for activation of the TP901-1 late promoter, Plate, hydroxylamine...

  11. ERalpha and ERbeta expression and transcriptional activity are differentially regulated by HDAC inhibitors

    Science.gov (United States)

    Duong, Vanessa; Licznar, Anne; Margueron, Raphaël; Boulle, Nathalie; Busson, Muriel; Lacroix, Matthieu; Katzenellenbogen, Benita S.; Cavaillès, Vincent; Lazennec, Gwendal

    2006-01-01

    The proliferative action of ERα largely accounts for the carcinogenic activity of estrogens. By contrast, recent data show that ERβ displays tumor-suppressor properties, thus supporting the interest to identify compounds which could increase its activity. Here, we show that histone deacetylase inhibitors (HDI) up-regulated ERβ protein levels, whereas it decreased ERα expression. Part of this regulation took place at the mRNA level through a mechanism independent of de novo protein synthesis. In addition, we found that, in various cancer cells, the treatment with different HDI enhanced the ligand-dependent activity of ERβ more strongly than that of ERα. On the other hand, in MDA-MB231 and HeLa cells, the expression of ERs modified the transcriptional response to HDI. The use of deletion mutants of both receptors demonstrated that AF1 domain of the receptors was required. Finally, we show that ERβ expression led to a dramatic increased in the antiproliferative activity of HDI, which correlated with a modification of the transcription of genes involved in cell cycle control by HDI. Altogether, these data demonstrate that the interference of ERβ and HDAC on the control of transcription and cell proliferation constitute a promising approach for cancer therapy. PMID:16158045

  12. Transcriptional Repression and Protein Degradation of the Ca2+-Activated K+ Channel KCa1.1 by Androgen Receptor Inhibition in Human Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Anowara Khatun

    2018-04-01

    Full Text Available The large-conductance Ca2+-activated K+ channel KCa1.1 plays an important role in the promotion of breast cancer cell proliferation and metastasis. The androgen receptor (AR is proposed as a therapeutic target for AR-positive advanced triple-negative breast cancer. We herein investigated the effects of a treatment with antiandrogens on the functional activity, activation kinetics, transcriptional expression, and protein degradation of KCa1.1 in human breast cancer MDA-MB-453 cells using real-time PCR, Western blotting, voltage-sensitive dye imaging, and whole-cell patch clamp recording. A treatment with the antiandrogen bicalutamide or enzalutamide for 48 h significantly suppressed (1 depolarization responses induced by paxilline (PAX, a specific KCa1.1 blocker and (2 PAX-sensitive outward currents induced by the depolarizing voltage step. The expression levels of KCa1.1 transcripts and proteins were significantly decreased in MDA-MB-453 cells, and the protein degradation of KCa1.1 mainly contributed to reductions in KCa1.1 activity. Among the eight regulatory β and γ subunits, LRRC26 alone was expressed at high levels in MDA-MB-453 cells and primary and metastatic breast cancer tissues, whereas no significant changes were observed in the expression levels of LRRC26 and activation kinetics of PAX-sensitive outward currents in MDA-MB-453 cells by the treatment with antiandrogens. The treatment with antiandrogens up-regulated the expression of the ubiquitin E3 ligases, FBW7, MDM2, and MDM4 in MDA-MB-453 cells, and the protein degradation of KCa1.1 was significantly inhibited by the respective siRNA-mediated blockade of FBW7 and MDM2. Based on these results, we concluded that KCa1.1 is an androgen-responsive gene in AR-positive breast cancer cells, and its down-regulation through enhancements in its protein degradation by FBW7 and/or MDM2 may contribute, at least in part, to the antiproliferative and antimetastatic effects of antiandrogens in

  13. Basic leucine zipper protein Cnc-C is a substrate and transcriptional regulator of the Drosophila 26S proteasome.

    Science.gov (United States)

    Grimberg, Kristian Björk; Beskow, Anne; Lundin, Daniel; Davis, Monica M; Young, Patrick

    2011-02-01

    While the 26S proteasome is a key proteolytic complex, little is known about how proteasome levels are maintained in higher eukaryotic cells. Here we describe an RNA interference (RNAi) screen of Drosophila melanogaster that was used to identify transcription factors that may play a role in maintaining levels of the 26S proteasome. We used an RNAi library against 993 Drosophila transcription factor genes to identify genes whose suppression in Schneider 2 cells stabilized a ubiquitin-green fluorescent protein reporter protein. This screen identified Cnc (cap 'n' collar [CNC]; basic region leucine zipper) as a candidate transcriptional regulator of proteasome component expression. In fact, 20S proteasome activity was reduced in cells depleted of cnc. Immunoblot assays against proteasome components revealed a general decline in both 19S regulatory complex and 20S proteasome subunits after RNAi depletion of this transcription factor. Transcript-specific silencing revealed that the longest of the seven transcripts for the cnc gene, cnc-C, was needed for proteasome and p97 ATPase production. Quantitative reverse transcription-PCR confirmed the role of Cnc-C in activation of transcription of genes encoding proteasome components. Expression of a V5-His-tagged form of Cnc-C revealed that the transcription factor is itself a proteasome substrate that is stabilized when the proteasome is inhibited. We propose that this single cnc gene in Drosophila resembles the ancestral gene family of mammalian nuclear factor erythroid-derived 2-related transcription factors, which are essential in regulating oxidative stress and proteolysis.

  14. Human herpesvirus 6B U19 protein is a PML-regulated transcriptional activator that localizes to nuclear foci in a PML-independent manner

    DEFF Research Database (Denmark)

    Kofod-Olsen, Emil; Ross-Hansen, Katrine; Mikkelsen, Jacob Giehm

    2008-01-01

    Human herpesvirus 6B (HHV-6B) contains an IE-B domain spanning open reading frames U16/17-U19, based on homology with human cytomegalovirus. Here, the protein product, U19, of the HHV-6B U19 gene is identified as a 47 kDa transcriptional activator. HHV-6B infection or overexpression of U19...... transactivated the RANTES promoter. Mutational analysis of the promoter indicated that transactivation was not critically dependent on the promoter sites CRE, NF-kappaB, ISRE or NF-IL6. ND10 are nuclear substructures that are involved in several cellular regulatory pathways, including those controlling gene...... structure, U19 also localized to the centre of ND10. Knockdown of PML by small interfering RNA did not prevent U19 localization to ND10-like foci, but instead led to a fourfold increase in U19-induced transcription from the RANTES promoter. Generation of four truncated U19 proteins indicated that the N...

  15. The DNA-mimic antirestriction proteins ArdA ColIB-P9, Arn T4, and Ocr T7 as activators of H-NS-dependent gene transcription.

    Science.gov (United States)

    Melkina, Olga E; Goryanin, Ignatiy I; Zavilgelsky, Gennadii B

    2016-11-01

    The antirestriction proteins ArdA ColIb-P9, Arn T4 and Ocr T7 specifically inhibit type I and type IV restriction enzymes and belong to the family of DNA-mimic proteins because their three-dimensional structure is similar to the double-helical B-form DNA. It is proposed that the DNA-mimic proteins are able to bind nucleoid protein H-NS and alleviate H-NS-silencing of the transcription of bacterial genes. Escherichia coli lux biosensors were constructed by inserting H-NS-dependent promoters into a vector, thereby placing each fragment upstream of the promoterless Photorhabdus luminescens luxCDABE operon. It was demonstrated that the DNA-mimic proteins ArdA, Arn and Ocr activate the transcription of H-NS-dependent promoters of the lux operon of marine luminescent bacteria (mesophilic Aliivibrio fischeri and psychrophilic Aliivibrio logei), and the dps gene from E. coli. It was also demonstrated that the ArdA antirestriction protein, the genes of which are located on transmissive plasmids ColIb-P9, R64, PK101, decreases levels of H-NS silencing of the PluxC promoter during conjugation in the recipient bacteria. Copyright © 2016 Elsevier GmbH. All rights reserved.

  16. Fe65 does not stabilize AICD during activation of transcription in a luciferase assay

    International Nuclear Information System (INIS)

    Huysseune, Sandra; Kienlen-Campard, Pascal; Octave, Jean-Noel

    2007-01-01

    The APP intracellular domain (AICD) could be involved in signaling via interaction with the adaptor protein Fe65, and with the histone acetyl transferase Tip60. However, the real function of AICD and Fe65 in regulation of transcription remains controversial. In this study, the human APPGal4 fusion protein was expressed in CHO cells and the transcriptional activity of AICDGal4 was measured in a luciferase-based reporter assay. AICDGal4 was stabilized by expression of Fe65 and levels of AICDGal4 controlled luciferase activity. On the contrary, when human APP was expressed in CHO cells, coexpression of Fe65 increased luciferase activity without affecting the amount of AICD fragment. AICD produced from APP was protected from degradation by orthophenanthroline, but not by lactacystine, indicating that AICD is not a substrate of the chymotryptic activity of the proteasome. It is concluded that Fe65 can control luciferase activity without stabilizing the labile AICD fragment

  17. Hypoxia-Inducible Factor 1 Is an Inductor of Transcription Factor Activating Protein 2 Epsilon Expression during Chondrogenic Differentiation

    Directory of Open Access Journals (Sweden)

    Stephan Niebler

    2015-01-01

    Full Text Available The transcription factor AP-2ε (activating enhancer-binding protein epsilon is expressed in cartilage of humans and mice. However, knowledge about regulatory mechanisms influencing AP-2ε expression is limited. Using quantitative real time PCR, we detected a significant increase in AP-2ε mRNA expression comparing initial and late stages of chondrogenic differentiation processes in vitro and in vivo. Interestingly, in these samples the expression pattern of the prominent hypoxia marker gene angiopoietin-like 4 (Angptl4 strongly correlated with that of AP-2ε suggesting that hypoxia might represent an external regulator of AP-2ε expression in mammals. In order to show this, experiments directly targeting the activity of hypoxia-inducible factor-1 (HIF1, the complex mediating responses to oxygen deprivation, were performed. While the HIF1-activating compounds 2,2′-dipyridyl and desferrioxamine resulted in significantly enhanced mRNA concentration of AP-2ε, siRNA against HIF1α led to a significantly reduced expression rate of AP-2ε. Additionally, we detected a significant upregulation of the AP-2ε mRNA level after oxygen deprivation. In sum, these different experimental approaches revealed a novel role for the HIF1 complex in the regulation of the AP-2ε gene in cartilaginous cells and underlined the important role of hypoxia as an important external regulatory stimulus during chondrogenic differentiation modulating the expression of downstream transcription factors.

  18. CMYB1 Encoding a MYB Transcriptional Activator Is Involved in Abiotic Stress and Circadian Rhythm in Rice

    Directory of Open Access Journals (Sweden)

    Min Duan

    2014-01-01

    Full Text Available Through analysis of cold-induced transcriptome, a novel gene encoding a putative MYB transcription factor was isolated and designated Cold induced MYB 1 (CMYB1. Tissue-specific gene expression analysis revealed that CMYB1 was highly expressed in rice stems and nodes. qRT-PCR assay indicated that CMYB1 was dramatically induced by cold stress (>100-folds and induced by exogenous ABA and osmotic stress. Interestingly, CMYB1 showed rhythmic expression profile in rice leaves at different developmental stages. Subcellular localization assay suggested that CMYB1-GFP (green fluorescent protein fusion protein was localized in the nuclei. Moreover, CMYB1 exhibited the transcriptional activation activity when transiently expressed in rice protoplast cells. Taken together, CMYB1 probably functions as a transcriptional activator in mediating stress and rhythm responsive gene expression in rice.

  19. Regulation of protein quality control by UBE4B and LSD1 through p53-mediated transcription.

    Directory of Open Access Journals (Sweden)

    Goran Periz

    2015-04-01

    Full Text Available Protein quality control is essential for clearing misfolded and aggregated proteins from the cell, and its failure is associated with many neurodegenerative disorders. Here, we identify two genes, ufd-2 and spr-5, that when inactivated, synergistically and robustly suppress neurotoxicity associated with misfolded proteins in Caenorhabditis elegans. Loss of human orthologs ubiquitination factor E4 B (UBE4B and lysine-specific demethylase 1 (LSD1, respectively encoding a ubiquitin ligase and a lysine-specific demethylase, promotes the clearance of misfolded proteins in mammalian cells by activating both proteasomal and autophagic degradation machineries. An unbiased search in this pathway reveals a downstream effector as the transcription factor p53, a shared substrate of UBE4B and LSD1 that functions as a key regulator of protein quality control to protect against proteotoxicity. These studies identify a new protein quality control pathway via regulation of transcription factors and point to the augmentation of protein quality control as a wide-spectrum antiproteotoxicity strategy.

  20. Contraction-induced interleukin-6 gene transcription in skeletal muscle is regulated by c-Jun terminal kinase/activator protein-1.

    Science.gov (United States)

    Whitham, Martin; Chan, M H Stanley; Pal, Martin; Matthews, Vance B; Prelovsek, Oja; Lunke, Sebastian; El-Osta, Assam; Broenneke, Hella; Alber, Jens; Brüning, Jens C; Wunderlich, F Thomas; Lancaster, Graeme I; Febbraio, Mark A

    2012-03-30

    Exercise increases the expression of the prototypical myokine IL-6, but the precise mechanism by which this occurs has yet to be identified. To mimic exercise conditions, C2C12 myotubes were mechanically stimulated via electrical pulse stimulation (EPS). We compared the responses of EPS with the pharmacological Ca(2+) carrier calcimycin (A23187) because contraction induces marked increases in cytosolic Ca(2+) levels or the classical IκB kinase/NFκB inflammatory response elicited by H(2)O(2). We demonstrate that, unlike H(2)O(2)-stimulated increases in IL-6 mRNA, neither calcimycin- nor EPS-induced IL-6 mRNA expression is under the transcriptional control of NFκB. Rather, we show that EPS increased the phosphorylation of JNK and the reporter activity of the downstream transcription factor AP-1. Furthermore, JNK inhibition abolished the EPS-induced increase in IL-6 mRNA and protein expression. Finally, we observed an exercise-induced increase in both JNK phosphorylation and IL-6 mRNA expression in the skeletal muscles of mice after 30 min of treadmill running. Importantly, exercise did not increase IL-6 mRNA expression in skeletal muscle-specific JNK-deficient mice. These data identify a novel contraction-mediated transcriptional regulatory pathway for IL-6 in skeletal muscle.

  1. Signal transducer and activator of transcription 5 activation is sufficient to drive transcriptional induction of cyclin D2 gene and proliferation of rat pancreatic beta-cells

    DEFF Research Database (Denmark)

    Friedrichsen, Birgitte N; Richter, Henrijette E; Hansen, Johnny A

    2003-01-01

    in a time-dependent manner by hGH in INS-1 cells. Inhibition of protein synthesis by coincubation with cycloheximide did not affect the hGH-induced increase of cyclin D2 mRNA levels at 4 h. Expression of a dominant negative STAT5 mutant, STAT5aDelta749, partially inhibited cyclin D2 protein levels. INS-1...... cells transiently transfected with a cyclin D2 promoter-reporter construct revealed a 3- to 5-fold increase of transcriptional activity in response to hGH stimulation. Furthermore, coexpression of a constitutive active STAT5 mutant (either CA-STAT5a or CA-STAT5b) was sufficient to drive transactivation...

  2. Transcription of human resistin gene involves an interaction of Sp1 with peroxisome proliferator-activating receptor gamma (PPARgamma.

    Directory of Open Access Journals (Sweden)

    Anil K Singh

    2010-03-01

    Full Text Available Resistin is a cysteine rich protein, mainly expressed and secreted by circulating human mononuclear cells. While several factors responsible for transcription of mouse resistin gene have been identified, not much is known about the factors responsible for the differential expression of human resistin.We show that the minimal promoter of human resistin lies within approximately 80 bp sequence upstream of the transcriptional start site (-240 whereas binding sites for cRel, CCAAT enhancer binding protein alpha (C/EBP-alpha, activating transcription factor 2 (ATF-2 and activator protein 1 (AP-1 transcription factors, important for induced expression, are present within sequences up to -619. Specificity Protein 1(Sp1 binding site (-276 to -295 is also present and an interaction of Sp1 with peroxisome proliferator activating receptor gamma (PPARgamma is necessary for constitutive expression in U937 cells. Indeed co-immunoprecipitation assay demonstrated a direct physical interaction of Sp1 with PPARgamma in whole cell extracts of U937 cells. Phorbol myristate acetate (PMA upregulated the expression of resistin mRNA in U937 cells by increasing the recruitment of Sp1, ATF-2 and PPARgamma on the resistin gene promoter. Furthermore, PMA stimulation of U937 cells resulted in the disruption of Sp1 and PPARgamma interaction. Chromatin immunoprecipitation (ChIP assay confirmed the recruitment of transcription factors phospho ATF-2, Sp1, Sp3, PPARgamma, chromatin modifier histone deacetylase 1 (HDAC1 and the acetylated form of histone H3 but not cRel, C/EBP-alpha and phospho c-Jun during resistin gene transcription.Our findings suggest a complex interplay of Sp1 and PPARgamma along with other transcription factors that drives the expression of resistin in human monocytic U937 cells.

  3. Phorbol-ester-induced activation of the NF-κB transcription factor involves dissociation of an apparently cytoplasmic NF-κB/inhibitor complex

    International Nuclear Information System (INIS)

    Baeuerle, P.A.; Lenardo, M.; Pierce, J.W.; Baltimore, D.

    1988-01-01

    There is increasing evidence that inducible transcription of genes is mediated through the induction of the activity of trans-acting protein factors. The NF-κB transcription factor provides a model system to study the posttranslational activation of a phorbol-ester-inducible transcription factor. The finding that NF-κB activity is undectable in subcellular fractions from unstimulated cells suggests that NF-κB exists as an inactive precursor. The authors showed that NF-κB is detectable in two different forms. After selective removal of endogenous NF-κB, they demonstrate the existence of a protein inhibitor in cytosolic fractions of unstimulated cells that is able in vitro to convert NF-κB into an inactive desoxycholate-dependent form. The data are consistent with a molecular mechanism of inducible gene expression by which an apparently cytoplasmic transcription factor-inhibitor complex is dissociated by the action of TPA-activated protein kinase C

  4. Repression of class I transcription by cadmium is mediated by the protein phosphatase 2A

    Science.gov (United States)

    Zhou, Lei; Le Roux, Gwenaëlle; Ducrot, Cécile; Chédin, Stéphane; Labarre, Jean; Riva, Michel; Carles, Christophe

    2013-01-01

    Toxic metals are part of our environment, and undue exposure to them leads to a variety of pathologies. In response, most organisms adapt their metabolism and have evolved systems to limit this toxicity and to acquire tolerance. Ribosome biosynthesis being central for protein synthesis, we analyzed in yeast the effects of a moderate concentration of cadmium (Cd2+) on Pol I transcription that represents >60% of the transcriptional activity of the cells. We show that Cd2+ rapidly and drastically shuts down the expression of the 35S rRNA. Repression does not result from a poisoning of any of the components of the class I transcriptional machinery by Cd2+, but rather involves a protein phosphatase 2A (PP2A)-dependent cellular signaling pathway that targets the formation/dissociation of the Pol I–Rrn3 complex. We also show that Pol I transcription is repressed by other toxic metals, such as Ag+ and Hg2+, which likewise perturb the Pol I–Rrn3 complex, but through PP2A-independent mechanisms. Taken together, our results point to a central role for the Pol I–Rrn3 complex as molecular switch for regulating Pol I transcription in response to toxic metals. PMID:23640330

  5. Insulators target active genes to transcription factories and polycomb-repressed genes to polycomb bodies.

    Directory of Open Access Journals (Sweden)

    Hua-Bing Li

    2013-04-01

    Full Text Available Polycomb bodies are foci of Polycomb proteins in which different Polycomb target genes are thought to co-localize in the nucleus, looping out from their chromosomal context. We have shown previously that insulators, not Polycomb response elements (PREs, mediate associations among Polycomb Group (PcG targets to form Polycomb bodies. Here we use live imaging and 3C interactions to show that transgenes containing PREs and endogenous PcG-regulated genes are targeted by insulator proteins to different nuclear structures depending on their state of activity. When two genes are repressed, they co-localize in Polycomb bodies. When both are active, they are targeted to transcription factories in a fashion dependent on Trithorax and enhancer specificity as well as the insulator protein CTCF. In the absence of CTCF, assembly of Polycomb bodies is essentially reduced to those representing genomic clusters of Polycomb target genes. The critical role of Trithorax suggests that stable association with a specialized transcription factory underlies the cellular memory of the active state.

  6. A Herpesviral Immediate Early Protein Promotes Transcription Elongation of Viral Transcripts

    Directory of Open Access Journals (Sweden)

    Hannah L. Fox

    2017-06-01

    Full Text Available Herpes simplex virus 1 (HSV-1 genes are transcribed by cellular RNA polymerase II (RNA Pol II. While four viral immediate early proteins (ICP4, ICP0, ICP27, and ICP22 function in some capacity in viral transcription, the mechanism by which ICP22 functions remains unclear. We observed that the FACT complex (comprised of SSRP1 and Spt16 was relocalized in infected cells as a function of ICP22. ICP22 was also required for the association of FACT and the transcription elongation factors SPT5 and SPT6 with viral genomes. We further demonstrated that the FACT complex interacts with ICP22 throughout infection. We therefore hypothesized that ICP22 recruits cellular transcription elongation factors to viral genomes for efficient transcription elongation of viral genes. We reevaluated the phenotype of an ICP22 mutant virus by determining the abundance of all viral mRNAs throughout infection by transcriptome sequencing (RNA-seq. The accumulation of almost all viral mRNAs late in infection was reduced compared to the wild type, regardless of kinetic class. Using chromatin immunoprecipitation sequencing (ChIP-seq, we mapped the location of RNA Pol II on viral genes and found that RNA Pol II levels on the bodies of viral genes were reduced in the ICP22 mutant compared to wild-type virus. In contrast, the association of RNA Pol II with transcription start sites in the mutant was not reduced. Taken together, our results indicate that ICP22 plays a role in recruiting elongation factors like the FACT complex to the HSV-1 genome to allow for efficient viral transcription elongation late in viral infection and ultimately infectious virion production.

  7. The metal-responsive transcription factor-1 contributes to HIF-1 activation during hypoxic stress

    International Nuclear Information System (INIS)

    Murphy, Brian J.; Sato, Barbara G.; Dalton, Timothy P.; Laderoute, Keith R.

    2005-01-01

    Hypoxia-inducible factor-1 (HIF-1), the major transcriptional regulator of the mammalian cellular response to low oxygen (hypoxia), is embedded within a complex network of signaling pathways. We have been investigating the importance of another stress-responsive transcription factor, MTF-1, for the adaptation of cells to hypoxia. This article reports that MTF-1 plays a central role in hypoxic cells by contributing to HIF-1 activity. Loss of MTF-1 in transformed Mtf1 null mouse embryonic fibroblasts (MEFs) results in an attenuation of nuclear HIF-1α protein accumulation, HIF-1 transcriptional activity, and expression of an established HIF-1 target gene, glucose transporter-1 (Glut1). Mtf1 null (Mtf1 KO) MEFs also have constitutively higher levels of both glutathione (GSH) and the rate-limiting enzyme involved in GSH synthesis-glutamate cysteine ligase catalytic subunit-than wild type cells. The altered cellular redox state arising from increased GSH may perturb oxygen-sensing mechanisms in hypoxic Mtf1 KO cells and decrease the accumulation of HIF-1α protein. Together, these novel findings define a role for MTF-1 in the regulation of HIF-1 activity

  8. The oncoprotein HBXIP upregulates PDGFB via activating transcription factor Sp1 to promote the proliferation of breast cancer cells

    International Nuclear Information System (INIS)

    Zhang, Yingyi; Zhao, Yu; Li, Leilei; Shen, Yu; Cai, Xiaoli; Zhang, Xiaodong; Ye, Lihong

    2013-01-01

    Highlights: •HBXIP is able to upregulate the expression of PDGFB in breast cancer cells. •HBXIP serves as a coactivator of activating transcription factor Sp1. •HBXIP stimulates the PDGFB promoter via activating transcription factor Sp1. •HBXIP promotes the proliferation of breast cancer cell via upregulating PDGFB. -- Abstract: We have reported that the oncoprotein hepatitis B virus X-interacting protein (HBXIP) acts as a novel transcriptional coactivator to promote proliferation and migration of breast cancer cells. Previously, we showed that HBXIP was able to activate nuclear factor-κB (NF-κB) in breast cancer cells. As an oncogene, the platelet-derived growth factor beta polypeptide (PDGFB) plays crucial roles in carcinogenesis. In the present study, we found that both HBXIP and PDGFB were highly expressed in breast cancer cell lines. Interestingly, HBXIP was able to increase transcriptional activity of NF-κB through PDGFB, suggesting that HBXIP is associated with PDGFB in the cells. Moreover, HBXIP was able to upregulate PDGFB at the levels of mRNA, protein and promoter in the cells. Then, we identified that HBXIP stimulated the promoter of PDGFB through activating transcription factor Sp1. In function, HBXIP enhanced the proliferation of breast cancer cells through PDGFB in vitro. Thus, we conclude that HBXIP upregulates PDGFB via activating transcription factor Sp1 to promote proliferation of breast cancer cells

  9. The oncoprotein HBXIP upregulates PDGFB via activating transcription factor Sp1 to promote the proliferation of breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yingyi; Zhao, Yu; Li, Leilei; Shen, Yu; Cai, Xiaoli [Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071 (China); Zhang, Xiaodong, E-mail: zhangxd@nankai.edu.cn [Department of Cancer Research, Institute for Molecular Biology, College of Life Sciences, Nankai University, Tianjin 300071 (China); Ye, Lihong, E-mail: yelihong@nankai.edu.cn [Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071 (China)

    2013-05-03

    Highlights: •HBXIP is able to upregulate the expression of PDGFB in breast cancer cells. •HBXIP serves as a coactivator of activating transcription factor Sp1. •HBXIP stimulates the PDGFB promoter via activating transcription factor Sp1. •HBXIP promotes the proliferation of breast cancer cell via upregulating PDGFB. -- Abstract: We have reported that the oncoprotein hepatitis B virus X-interacting protein (HBXIP) acts as a novel transcriptional coactivator to promote proliferation and migration of breast cancer cells. Previously, we showed that HBXIP was able to activate nuclear factor-κB (NF-κB) in breast cancer cells. As an oncogene, the platelet-derived growth factor beta polypeptide (PDGFB) plays crucial roles in carcinogenesis. In the present study, we found that both HBXIP and PDGFB were highly expressed in breast cancer cell lines. Interestingly, HBXIP was able to increase transcriptional activity of NF-κB through PDGFB, suggesting that HBXIP is associated with PDGFB in the cells. Moreover, HBXIP was able to upregulate PDGFB at the levels of mRNA, protein and promoter in the cells. Then, we identified that HBXIP stimulated the promoter of PDGFB through activating transcription factor Sp1. In function, HBXIP enhanced the proliferation of breast cancer cells through PDGFB in vitro. Thus, we conclude that HBXIP upregulates PDGFB via activating transcription factor Sp1 to promote proliferation of breast cancer cells.

  10. TAF4 nucleates a core subcomplex of TFIID and mediates activated transcription from a TATA-less promoter

    OpenAIRE

    Wright, Kevin J.; Marr, Michael T.; Tjian, Robert

    2006-01-01

    Activator-dependent recruitment of TFIID initiates formation of the transcriptional preinitiation complex. TFIID binds core promoter DNA elements and directs the assembly of other general transcription factors, leading to binding of RNA polymerase II and activation of RNA synthesis. How TATA box-binding protein (TBP) and the TBP-associated factors (TAFs) are assembled into a functional TFIID complex with promoter recognition and coactivator activities in vivo remains unknown. Here, we use RNA...

  11. Activation of the unfolded protein response during anoxia exposure in the turtle Trachemys scripta elegans.

    Science.gov (United States)

    Krivoruchko, Anastasia; Storey, Kenneth B

    2013-02-01

    Red-eared slider turtles, Trachemys scripta elegans, can survive for several weeks without oxygen when submerged in cold water. We hypothesized that anaerobiosis is aided by adaptive up-regulation of the unfolded protein response (UPR), a stress-responsive pathway that is activated by accumulation of unfolded proteins in the endoplasmic reticulum (ER) and functions to restore ER homeostasis. RT-PCR, western immunoblotting and DNA-binding assays were used to quantify the responses and/or activation status of UPR-responsive genes and proteins in turtle tissues after animal exposure to 5 or 20 h of anoxic submergence at 4 °C. The phosphorylation state of protein kinase-like ER kinase (PERK) (a UPR-regulated kinase) and eukaryotic initiation factor 2 (eIF2α) increased by 1.43-2.50 fold in response to anoxia in turtle heart, kidney, and liver. Activation of the PERK-regulated transcription factor, activating transcription factor 4 (ATF4), during anoxia was documented by elevated atf4 transcripts and total ATF4 protein (1.60-2.43 fold), increased nuclear ATF4 content, and increased DNA-binding activity (1.44-2.32 fold). ATF3 and GADD34 (downstream targets of ATF4) also increased by 1.38-3.32 fold in heart and liver under anoxia, and atf3 transcripts were also elevated in heart. Two characteristic chaperones of the UPR, GRP78, and GRP94, also responded positively to anoxia with strong increases in both the transcript and protein levels. The data demonstrate that the UPR is activated in turtle heart, kidney, and liver in response to anoxia, suggesting that this pathway mediates an integrated stress response to protect tissues during oxygen deprivation.

  12. Cross activity of orthologous WRKY transcription factors in wheat and Arabidopsis

    NARCIS (Netherlands)

    Poietti, S.; Bertini, L.; Ent, S. van der; Leon Reyes, H.A.; Pieterse, C.M.J.; Tucci, M.; Caporale, C.; Caruso, C.

    2011-01-01

    WRKY proteins are transcription factors involved in many plant processes including plant responses to pathogens. Here, the cross activity of TaWRKY78 from the monocot wheat and AtWRKY20 from the dicot Arabidopsis on the cognate promoters of the orthologous PR4-type genes wPR4e and AtHEL of wheat and

  13. Direct activation of human and mouse Oct4 genes using engineered TALE and Cas9 transcription factors.

    Science.gov (United States)

    Hu, Jiabiao; Lei, Yong; Wong, Wing-Ki; Liu, Senquan; Lee, Kai-Chuen; He, Xiangjun; You, Wenxing; Zhou, Rui; Guo, Jun-Tao; Chen, Xiongfong; Peng, Xianlu; Sun, Hao; Huang, He; Zhao, Hui; Feng, Bo

    2014-04-01

    The newly developed transcription activator-like effector protein (TALE) and clustered regularly interspaced short palindromic repeats/Cas9 transcription factors (TF) offered a powerful and precise approach for modulating gene expression. In this article, we systematically investigated the potential of these new tools in activating the stringently silenced pluripotency gene Oct4 (Pou5f1) in mouse and human somatic cells. First, with a number of TALEs and sgRNAs targeting various regions in the mouse and human Oct4 promoters, we found that the most efficient TALE-VP64s bound around -120 to -80 bp, while highly effective sgRNAs targeted from -147 to -89-bp upstream of the transcription start sites to induce high activity of luciferase reporters. In addition, we observed significant transcriptional synergy when multiple TFs were applied simultaneously. Although individual TFs exhibited marginal activity to up-regulate endogenous gene expression, optimized combinations of TALE-VP64s could enhance endogenous Oct4 transcription up to 30-fold in mouse NIH3T3 cells and 20-fold in human HEK293T cells. More importantly, the enhancement of OCT4 transcription ultimately generated OCT4 proteins. Furthermore, examination of different epigenetic modifiers showed that histone acetyltransferase p300 could enhance both TALE-VP64 and sgRNA/dCas9-VP64 induced transcription of endogenous OCT4. Taken together, our study suggested that engineered TALE-TF and dCas9-TF are useful tools for modulating gene expression in mammalian cells.

  14. Leptin upregulates telomerase activity and transcription of human telomerase reverse transcriptase in MCF-7 breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Ren, He, E-mail: herenrh@yahoo.com.cn [Key Laboratory of Breast Cancer Prevention and Therapy, Tianjin Medical University, Ministry of Education, Tianjin Medical University Cancer Hospital, Tianjin (China); Zhao, Tiansuo; Wang, Xiuchao; Gao, Chuntao; Wang, Jian; Yu, Ming [Key Laboratory of Breast Cancer Prevention and Therapy, Tianjin Medical University, Ministry of Education, Tianjin Medical University Cancer Hospital, Tianjin (China); Hao, Jihui, E-mail: jihuihao@yahoo.com [Key Laboratory of Breast Cancer Prevention and Therapy, Tianjin Medical University, Ministry of Education, Tianjin Medical University Cancer Hospital, Tianjin (China)

    2010-03-26

    The aim was to analyze the mechanism of leptin-induced activity of telomerase in MCF-7 breast cancer cells. We found that leptin activated telomerase in a dose-dependent manner; leptin upregulated the expression of Human Telomerase Reverse Transcriptase (hTERT) at mRNA and protein levels; blockade of signal transducer and activator of transcription 3 (STAT3) phosphorylation significantly counteracted leptin-induced hTERT transcription and protein expression; chromatin immunoprecipitation analysis showed that leptin enhanced the binding of STAT3 to the hTERT promoter. This study uncovers a new mechanism of the proliferative effect of leptin on breast cancer cells and provides a new explanation of obesity-related breast cancer.

  15. The metazoan Mediator co-activator complex as an integrative hub for transcriptional regulation.

    Science.gov (United States)

    Malik, Sohail; Roeder, Robert G

    2010-11-01

    The Mediator is an evolutionarily conserved, multiprotein complex that is a key regulator of protein-coding genes. In metazoan cells, multiple pathways that are responsible for homeostasis, cell growth and differentiation converge on the Mediator through transcriptional activators and repressors that target one or more of the almost 30 subunits of this complex. Besides interacting directly with RNA polymerase II, Mediator has multiple functions and can interact with and coordinate the action of numerous other co-activators and co-repressors, including those acting at the level of chromatin. These interactions ultimately allow the Mediator to deliver outputs that range from maximal activation of genes to modulation of basal transcription to long-term epigenetic silencing.

  16. Transcriptional activity of Pax3 is co-activated by TAZ

    International Nuclear Information System (INIS)

    Murakami, Masao; Tominaga, Junji; Makita, Ryosuke; Uchijima, Yasunobu; Kurihara, Yukiko; Nakagawa, Osamu; Asano, Tomoichiro; Kurihara, Hiroki

    2006-01-01

    Pax3 is a transcription factor which functions in embryonic development and human diseases. In a yeast two-hybrid screen with full-length Pax3 as bait, we isolated a clone encoding transcriptional co-activator with PDZ-binding motif (TAZ) from an E10.5 mouse embryo cDNA library. Co-immunoprecipitation and nuclear co-localization of TAZ with Pax3 suggest that their association is functionally relevant. In situ hybridization revealed TAZ and Pax3 expression to partially overlap in the paraxial mesoderm, limb buds, and the neural tube. In C2C12 myoblast cells and NIH3T3 cells, TAZ enhanced the transcriptional activity of Pax3 on artificial and microphthalmia-associated transcription factor promoter-luciferase constructs, suggesting that TAZ can function as a co-activator of Pax3. Functional interaction between Pax3 and TAZ may provide a clue to clarifying the mechanism by which Pax3 serves as a transcriptional activator during embryogenesis

  17. Examination of transcript amounts and activity of protein kinase CK2 in muscle lysates of different types of human muscle pathologies.

    Science.gov (United States)

    Heuss, Dieter; Klascinski, Janine; Schubert, Steffen W; Moriabadi, Tehmur; Lochmüller, Hanns; Hashemolhosseini, Said

    2008-09-01

    Motoneurons release the heparansulfate proteoglycan agrin and thereby activate the muscle-specific receptor tyrosine kinase (MuSK), which is the main organizer of subsynaptic specializations at the neuromuscular junction. Recently, we showed that (1) the protein kinase CK2 interacts with the intracellular region of MuSK; (2) the CK2 protein is enriched and co-localized with MuSK at postsynaptic specializations; (3) CK2-mediated phosphorylation of serine residues within a specific MuSK epitope, named the kinase insert, regulates acetylcholine receptor (AChR) clustering; (4) muscle-specific CK2beta knockout mice develop a myasthenic phenotype due to impaired muscle endplate structure and function (see Genes Dev 20(13):1800-1816, 2006). Here, we investigated for the first time if CK2 is modulated in biopsies from human patients. To this end, we measured transcript amounts of the subunits CK2alpha and CK2beta and determined holoenzyme CK2 activity in 34 muscle biopsies of human patients with different muscle pathologies.

  18. The Brakeless co-regulator can directly activate and repress transcription in early Drosophila embryos.

    Science.gov (United States)

    Crona, Filip; Holmqvist, Per-Henrik; Tang, Min; Singla, Bhumica; Vakifahmetoglu-Norberg, Helin; Fantur, Katrin; Mannervik, Mattias

    2015-11-01

    The Brakeless protein performs many important functions during Drosophila development, but how it controls gene expression is poorly understood. We previously showed that Brakeless can function as a transcriptional co-repressor. In this work, we perform transcriptional profiling of brakeless mutant embryos. Unexpectedly, the majority of affected genes are down-regulated in brakeless mutants. We demonstrate that genomic regions in close proximity to some of these genes are occupied by Brakeless, that over-expression of Brakeless causes a reciprocal effect on expression of these genes, and that Brakeless remains an activator of the genes upon fusion to an activation domain. Together, our results show that Brakeless can both repress and activate gene expression. A yeast two-hybrid screen identified the Mediator complex subunit Med19 as interacting with an evolutionarily conserved part of Brakeless. Both down- and up-regulated Brakeless target genes are also affected in Med19-depleted embryos, but only down-regulated targets are influenced in embryos depleted of both Brakeless and Med19. Our data provide support for a Brakeless activator function that regulates transcription by interacting with Med19. We conclude that the transcriptional co-regulator Brakeless can either activate or repress transcription depending on context. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Zipper plot: visualizing transcriptional activity of genomic regions.

    Science.gov (United States)

    Avila Cobos, Francisco; Anckaert, Jasper; Volders, Pieter-Jan; Everaert, Celine; Rombaut, Dries; Vandesompele, Jo; De Preter, Katleen; Mestdagh, Pieter

    2017-05-02

    Reconstructing transcript models from RNA-sequencing (RNA-seq) data and establishing these as independent transcriptional units can be a challenging task. Current state-of-the-art tools for long non-coding RNA (lncRNA) annotation are mainly based on evolutionary constraints, which may result in false negatives due to the overall limited conservation of lncRNAs. To tackle this problem we have developed the Zipper plot, a novel visualization and analysis method that enables users to simultaneously interrogate thousands of human putative transcription start sites (TSSs) in relation to various features that are indicative for transcriptional activity. These include publicly available CAGE-sequencing, ChIP-sequencing and DNase-sequencing datasets. Our method only requires three tab-separated fields (chromosome, genomic coordinate of the TSS and strand) as input and generates a report that includes a detailed summary table, a Zipper plot and several statistics derived from this plot. Using the Zipper plot, we found evidence of transcription for a set of well-characterized lncRNAs and observed that fewer mono-exonic lncRNAs have CAGE peaks overlapping with their TSSs compared to multi-exonic lncRNAs. Using publicly available RNA-seq data, we found more than one hundred cases where junction reads connected protein-coding gene exons with a downstream mono-exonic lncRNA, revealing the need for a careful evaluation of lncRNA 5'-boundaries. Our method is implemented using the statistical programming language R and is freely available as a webtool.

  20. Differential Regulation of Telomerase Reverse Transcriptase Promoter Activation and Protein Degradation by Histone Deacetylase Inhibition.

    Science.gov (United States)

    Qing, Hua; Aono, Jun; Findeisen, Hannes M; Jones, Karrie L; Heywood, Elizabeth B; Bruemmer, Dennis

    2016-06-01

    Telomerase reverse transcriptase (TERT) maintains telomeres and is rate limiting for replicative life span. While most somatic tissues silence TERT transcription resulting in telomere shortening, cells derived from cancer or cardiovascular diseases express TERT and activate telomerase. In the present study, we demonstrate that histone deacetylase (HDAC) inhibition induces TERT transcription and promoter activation. At the protein level in contrast, HDAC inhibition decreases TERT protein abundance through enhanced degradation, which decreases telomerase activity and induces senescence. Finally, we demonstrate that HDAC inhibition decreases TERT expression during vascular remodeling in vivo. These data illustrate a differential regulation of TERT transcription and protein stability by HDAC inhibition and suggest that TERT may constitute an important target for the anti-proliferative efficacy of HDAC inhibitors. © 2015 Wiley Periodicals, Inc.

  1. The leucine zipper domains of the transcription factors GCN4 and c-Jun have ribonuclease activity.

    Directory of Open Access Journals (Sweden)

    Yaroslav Nikolaev

    Full Text Available Basic-region leucine zipper (bZIP proteins are one of the largest transcription factor families that regulate a wide range of cellular functions. Owing to the stability of their coiled coil structure leucine zipper (LZ domains of bZIP factors are widely employed as dimerization motifs in protein engineering studies. In the course of one such study, the X-ray structure of the retro-version of the LZ moiety of yeast transcriptional activator GCN4 suggested that this retro-LZ may have ribonuclease activity. Here we show that not only the retro-LZ but also the authentic LZ of GCN4 has weak but distinct ribonuclease activity. The observed cleavage of RNA is unspecific, it is not suppressed by the ribonuclease A inhibitor RNasin and involves the breakage of 3',5'-phosphodiester bonds with formation of 2',3'-cyclic phosphates as the final products as demonstrated by HPLC/electrospray ionization mass spectrometry. Several mutants of the GCN4 leucine zipper are catalytically inactive, providing important negative controls and unequivocally associating the enzymatic activity with the peptide under study. The leucine zipper moiety of the human factor c-Jun as well as the entire c-Jun protein are also shown to catalyze degradation of RNA. The presented data, which was obtained in the test-tube experiments, adds GCN4 and c-Jun to the pool of proteins with multiple functions (also known as moonlighting proteins. If expressed in vivo, the endoribonuclease activity of these bZIP-containing factors may represent a direct coupling between transcription activation and controlled RNA turnover. As an additional result of this work, the retro-leucine zipper of GCN4 can be added to the list of functional retro-peptides.

  2. Activation of the transcription factor carbohydrate-responsive element-binding protein by glucose leads to increased pancreatic beta cell differentiation in rats.

    Science.gov (United States)

    Soggia, A; Flosseau, K; Ravassard, P; Szinnai, G; Scharfmann, R; Guillemain, G

    2012-10-01

    Pancreatic cell development is a tightly controlled process. Although information is available regarding the mesodermal signals that control pancreatic development, little is known about the role of environmental factors such as nutrients, including glucose, on pancreatic development. We previously showed that glucose and its metabolism through the hexosamine biosynthesis pathway (HBP) promote pancreatic endocrine cell differentiation. Here, we analysed the role of the transcription factor carbohydrate-responsive element-binding protein (ChREBP) in this process. This transcription factor is activated by glucose, and has been recently described as a target of the HBP. We used an in vitro bioassay in which pancreatic endocrine and exocrine cells develop from rat embryonic pancreas in a way that mimics in vivo pancreatic development. Using this model, gain-of-function and loss-of-function experiments were undertaken. ChREBP was produced in the endocrine lineage during pancreatic development, its abundance increasing with differentiation. When rat embryonic pancreases were cultured in the presence of glucose or xylitol, the production of ChREBP targets was induced. Concomitantly, beta cell differentiation was enhanced. On the other hand, when embryonic pancreases were cultured with inhibitors decreasing ChREBP activity or an adenovirus producing a dominant-negative ChREBP, beta cell differentiation was reduced, indicating that ChREBP activity was necessary for proper beta cell differentiation. Interestingly, adenovirus producing a dominant-negative ChREBP also reduced the positive effect of N-acetylglucosamine, a substrate of the HBP acting on beta cell differentiation. Our work supports the idea that glucose, through the transcription factor ChREBP, controls beta cell differentiation from pancreatic progenitors.

  3. Comparative transcriptional and translational analysis of leptospiral outer membrane protein expression in response to temperature.

    Science.gov (United States)

    Lo, Miranda; Cordwell, Stuart J; Bulach, Dieter M; Adler, Ben

    2009-12-08

    Leptospirosis is a global zoonosis affecting millions of people annually. Transcriptional changes in response to temperature were previously investigated using microarrays to identify genes potentially expressed upon host entry. Past studies found that various leptospiral outer membrane proteins are differentially expressed at different temperatures. However, our microarray studies highlighted a divergence between protein abundance and transcript levels for some proteins. Given the abundance of post-transcriptional expression control mechanisms, this finding highlighted the importance of global protein analysis systems. To complement our previous transcription study, we evaluated differences in the proteins of the leptospiral outer membrane fraction in response to temperature upshift. Outer membrane protein-enriched fractions from Leptospira interrogans grown at 30 degrees C or overnight upshift to 37 degrees C were isolated and the relative abundance of each protein was determined by iTRAQ analysis coupled with two-dimensional liquid chromatography and tandem mass spectrometry (2-DLC/MS-MS). We identified 1026 proteins with 99% confidence; 27 and 66 were present at elevated and reduced abundance respectively. Protein abundance changes were compared with transcriptional differences determined from the microarray studies. While there was some correlation between the microarray and iTRAQ data, a subset of genes that showed no differential expression by microarray was found to encode temperature-regulated proteins. This set of genes is of particular interest as it is likely that regulation of their expression occurs post-transcriptionally, providing an opportunity to develop hypotheses about the molecular dynamics of the outer membrane of Leptospira in response to changing environments. This is the first study to compare transcriptional and translational responses to temperature shift in L. interrogans. The results thus provide an insight into the mechanisms used by L

  4. Comparative transcriptional and translational analysis of leptospiral outer membrane protein expression in response to temperature.

    Directory of Open Access Journals (Sweden)

    Miranda Lo

    Full Text Available BACKGROUND: Leptospirosis is a global zoonosis affecting millions of people annually. Transcriptional changes in response to temperature were previously investigated using microarrays to identify genes potentially expressed upon host entry. Past studies found that various leptospiral outer membrane proteins are differentially expressed at different temperatures. However, our microarray studies highlighted a divergence between protein abundance and transcript levels for some proteins. Given the abundance of post-transcriptional expression control mechanisms, this finding highlighted the importance of global protein analysis systems. METHODOLOGY/PRINCIPAL FINDINGS: To complement our previous transcription study, we evaluated differences in the proteins of the leptospiral outer membrane fraction in response to temperature upshift. Outer membrane protein-enriched fractions from Leptospira interrogans grown at 30 degrees C or overnight upshift to 37 degrees C were isolated and the relative abundance of each protein was determined by iTRAQ analysis coupled with two-dimensional liquid chromatography and tandem mass spectrometry (2-DLC/MS-MS. We identified 1026 proteins with 99% confidence; 27 and 66 were present at elevated and reduced abundance respectively. Protein abundance changes were compared with transcriptional differences determined from the microarray studies. While there was some correlation between the microarray and iTRAQ data, a subset of genes that showed no differential expression by microarray was found to encode temperature-regulated proteins. This set of genes is of particular interest as it is likely that regulation of their expression occurs post-transcriptionally, providing an opportunity to develop hypotheses about the molecular dynamics of the outer membrane of Leptospira in response to changing environments. CONCLUSIONS/SIGNIFICANCE: This is the first study to compare transcriptional and translational responses to temperature

  5. Polyphenol Compound as a Transcription Factor Inhibitor.

    Science.gov (United States)

    Park, Seyeon

    2015-10-30

    A target-based approach has been used to develop novel drugs in many therapeutic fields. In the final stage of intracellular signaling, transcription factor-DNA interactions are central to most biological processes and therefore represent a large and important class of targets for human therapeutics. Thus, we focused on the idea that the disruption of protein dimers and cognate DNA complexes could impair the transcriptional activation and cell transformation regulated by these proteins. Historically, natural products have been regarded as providing the primary leading compounds capable of modulating protein-protein or protein-DNA interactions. Although their mechanism of action is not fully defined, polyphenols including flavonoids were found to act mostly as site-directed small molecule inhibitors on signaling. There are many reports in the literature of screening initiatives suggesting improved drugs that can modulate the transcription factor interactions responsible for disease. In this review, we focus on polyphenol compound inhibitors against dimeric forms of transcription factor components of intracellular signaling pathways (for instance, c-jun/c-fos (Activator Protein-1; AP-1), c-myc/max, Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and β-catenin/T cell factor (Tcf)).

  6. Nuclear cereblon modulates transcriptional activity of Ikaros and regulates its downstream target, enkephalin, in human neuroblastoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Wada, Takeyoshi [Faculty of Science and Engineering, Waseda University, TWIns, 2-2 Wakamatsu, Shinjuku, Tokyo, 162-8480 (Japan); Asahi, Toru [Faculty of Science and Engineering, Waseda University, TWIns, 2-2 Wakamatsu, Shinjuku, Tokyo, 162-8480 (Japan); Research Organization for Nano & Life Innovation, Waseda University #03C309, TWIns, 2-2 Wakamatsu, Shinjuku, Tokyo, 162-8480 (Japan); Sawamura, Naoya, E-mail: naoya.sawamura@gmail.com [Faculty of Science and Engineering, Waseda University, TWIns, 2-2 Wakamatsu, Shinjuku, Tokyo, 162-8480 (Japan); Research Organization for Nano & Life Innovation, Waseda University #03C309, TWIns, 2-2 Wakamatsu, Shinjuku, Tokyo, 162-8480 (Japan)

    2016-08-26

    The gene coding cereblon (CRBN) was originally identified in genetic linkage analysis of mild autosomal recessive nonsyndromic intellectual disability. CRBN has broad localization in both the cytoplasm and nucleus. However, the significance of nuclear CRBN remains unknown. In the present study, we aimed to elucidate the role of CRBN in the nucleus. First, we generated a series of CRBN deletion mutants and determined the regions responsible for the nuclear localization. Only CRBN protein lacking the N-terminal region was localized outside of the nucleus, suggesting that the N-terminal region is important for its nuclear localization. CRBN was also identified as a thalidomide-binding protein and component of the cullin-4-containing E3 ubiquitin ligase complex. Thalidomide has been reported to be involved in the regulation of the transcription factor Ikaros by CRBN-mediated degradation. To investigate the nuclear functions of CRBN, we performed co-immunoprecipitation experiments and evaluated the binding of CRBN to Ikaros. As a result, we found that CRBN was associated with Ikaros protein, and the N-terminal region of CRBN was required for Ikaros binding. In luciferase reporter gene experiments, CRBN modulated transcriptional activity of Ikaros. Furthermore, we found that CRBN modulated Ikaros-mediated transcriptional repression of the proenkephalin gene by binding to its promoter region. These results suggest that CRBN binds to Ikaros via its N-terminal region and regulates transcriptional activities of Ikaros and its downstream target, enkephalin. - Highlights: • We found that CRBN is a nucleocytoplasmic shutting protein and identified the key domain for nucleocytoplasmic shuttling. • CRBN associates with the transcription factor Ikaros via the N-terminal domain. • CRBN modulates Ikaros-mediated transcriptional regulation and its downstream target, enkephalin.

  7. Nuclear cereblon modulates transcriptional activity of Ikaros and regulates its downstream target, enkephalin, in human neuroblastoma cells

    International Nuclear Information System (INIS)

    Wada, Takeyoshi; Asahi, Toru; Sawamura, Naoya

    2016-01-01

    The gene coding cereblon (CRBN) was originally identified in genetic linkage analysis of mild autosomal recessive nonsyndromic intellectual disability. CRBN has broad localization in both the cytoplasm and nucleus. However, the significance of nuclear CRBN remains unknown. In the present study, we aimed to elucidate the role of CRBN in the nucleus. First, we generated a series of CRBN deletion mutants and determined the regions responsible for the nuclear localization. Only CRBN protein lacking the N-terminal region was localized outside of the nucleus, suggesting that the N-terminal region is important for its nuclear localization. CRBN was also identified as a thalidomide-binding protein and component of the cullin-4-containing E3 ubiquitin ligase complex. Thalidomide has been reported to be involved in the regulation of the transcription factor Ikaros by CRBN-mediated degradation. To investigate the nuclear functions of CRBN, we performed co-immunoprecipitation experiments and evaluated the binding of CRBN to Ikaros. As a result, we found that CRBN was associated with Ikaros protein, and the N-terminal region of CRBN was required for Ikaros binding. In luciferase reporter gene experiments, CRBN modulated transcriptional activity of Ikaros. Furthermore, we found that CRBN modulated Ikaros-mediated transcriptional repression of the proenkephalin gene by binding to its promoter region. These results suggest that CRBN binds to Ikaros via its N-terminal region and regulates transcriptional activities of Ikaros and its downstream target, enkephalin. - Highlights: • We found that CRBN is a nucleocytoplasmic shutting protein and identified the key domain for nucleocytoplasmic shuttling. • CRBN associates with the transcription factor Ikaros via the N-terminal domain. • CRBN modulates Ikaros-mediated transcriptional regulation and its downstream target, enkephalin.

  8. A novel glutamine-rich putative transcriptional adaptor protein (TIG-1), preferentially expressed in placental and bone-marrow tissues.

    Science.gov (United States)

    Abraham, S; Solomon, W B

    2000-09-19

    We used a subtractive hybridization protocol to identify novel expressed sequence tags (ESTs) corresponding to mRNAs whose expression was induced upon exposure of the human leukemia cell line K562 to the phorbol ester 12-O-tetradecanolyphorbol-13-acetate (TPA). The complete open reading frame of one of the novel ESTs, named TIG-1, was obtained by screening K562 cell and placental cDNA libraries. The deduced open reading frame of the TIG-1 cDNA encodes for a glutamine repeat-rich protein with a predicted molecular weight of 63kDa. The predicted open reading frame also contains a consensus bipartite nuclear localization signal, though no specific DNA-binding domain is found. The corresponding TIG-1 mRNA is ubiquitously expressed. Placental tissue expresses the TIG-1 mRNA 200 times more than the lowest expressing tissues such as kidney and lung. There is also preferential TIG-1 mRNA expression in cells of bone-marrow lineage.In-vitro transcription/translation of the TIG-1 cDNA yielded a polypeptide with an apparent molecular weight of 97kDa. Using polyclonal antibodies obtained from a rabbit immunized with the carboxy-terminal portion of bacterially expressed TIG-1 protein, a polypeptide with molecular weight of 97kDa was identified by Western blot analyses of protein lysates obtained from K562 cells. Cotransfection assays of K562 cells, using a GAL4-TIG-1 fusion gene and GAL4 operator-CAT, indicate that the TIG-1 protein may have transcriptional regulatory activity when tethered to DNA. We hypothesize that this novel glutamine-rich protein participates in a protein complex that regulates gene transcription. It has been demonstrated by Naar et al. (Naar, A.M., Beaurang, P.A., Zhou, S., Abraham, S., Solomon, W.B., Tjian, R., 1999, Composite co-activator ARC mediates chromatin-directed transcriptional activation. Nature 398, 828-830) that the amino acid sequences of peptide fragments obtained from a polypeptide found in a complex of proteins that alters chromatin

  9. A calcineurin inhibitory protein overexpressed in Down's syndrome interacts with the product of a ubiquitously expressed transcript

    Directory of Open Access Journals (Sweden)

    H.C.S. Silveira

    2004-06-01

    Full Text Available The Down's syndrome candidate region 1 (DSCR1 protein, encoded by a gene located in the human chromosome 21, interacts with calcineurin and is overexpressed in Down's syndrome patients. As an approach to clarifying a putative function for this protein, in the present study we used the yeast two-hybrid system to identify DSCR1 partners. The two-hybrid system is a method that allows the identification of protein-protein interactions through reconstitution of the activity of the yeast GAL 4 transcriptional activator. The gene DSCR1 fused to the GAL 4 binding domain (BD was used to screen a human fetal brain cDNA library cloned in fusion with the GAL 4 activation domain (AD. Three positive clones were found and sequence analysis revealed that all the plasmids coded for the ubiquitously expressed transcript (UXT. UXT, which is encoded in human Xp11, is a 157-amino acid protein present in both cytosol and nucleus of the cells. This positive interaction of DSCR1 and UXT was confirmed in vivo by mating the yeast strain AH109 (MATaexpressing AD-UXT with the strain Y187 (MATalpha expressing BD-DSCR1, and in vitro by co-immunoprecipitation experiments. These results may help elucidate a new function for DSCR1 and its participation in Down's syndrome pathogenesis.

  10. Inter-domain cross-talk controls the NifA protein activity of Herbaspirillum seropedicae.

    Science.gov (United States)

    Monteiro, R A; de Souza, E M; Wassem, R; Yates, M G; Pedrosa, F O; Chubatsu, L S

    2001-11-09

    Herbaspirillum seropedicae is an endophytic diazotroph, which colonizes sugar cane, wheat, rice and maize. The activity of NifA, a transcriptional activator of nif genes in H. seropedicae, is controlled by ammonium ions through a mechanism involving its N-terminal domain. Here we show that this domain interacts specifically in vitro with the N-truncated NifA protein, as revealed by protection against proteolysis, and this interaction caused an inhibitory effect on both the ATPase and DNA-binding activities of the N-truncated NifA protein. We suggest that the N-terminal domain inhibits NifA-dependent transcriptional activation by an inter-domain cross-talk between the catalytic domain of the NifA protein and its regulatory N-terminal domain in response to fixed nitrogen.

  11. HSF1 transcriptional activity mediates alcohol induction of Vamp2 expression and GABA release

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    Florence P. Varodayan

    2013-12-01

    Full Text Available Many central synapses are highly sensitive to alcohol, and it is now accepted that short-term alterations in synaptic function may lead to longer term changes in circuit function. The regulation of postsynaptic receptors by alcohol has been well studied, but the mechanisms underlying the effects of alcohol on the presynaptic terminal are relatively unexplored. To identify a pathway by which alcohol regulates neurotransmitter release, we recently investigated the mechanism by which ethanol induces the Vamp2 gene, but not Vamp1, in mouse primary cortical cultures. These two genes encode isoforms of synaptobrevin, a vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE protein required for synaptic vesicle fusion. We found that alcohol activates the transcription factor heat shock factor 1 (HSF1 to induce Vamp2 gene expression, while Vamp1 mRNA levels remain unaffected. As the Vamp2 gene encodes a SNARE protein, we then investigated whether ethanol exposure and HSF1 transcriptional activity alter neurotransmitter release using electrophysiology. We found that alcohol increased the frequency of γ-aminobutyric acid (GABA-mediated miniature IPSCs via HSF1, but had no effect on mEPSCs. Overall, these data indicate that alcohol induces HSF1 transcriptional activity to trigger a specific coordinated adaptation in GABAergic presynaptic terminals. This mechanism could explain some of the changes in synaptic function that occur soon after alcohol exposure, and may underlie some of the more enduring effects of chronic alcohol intake on local circuit function.

  12. The Tax oncogene enhances ELL incorporation into p300 and P-TEFb containing protein complexes to activate transcription.

    Science.gov (United States)

    Fufa, Temesgen D; Byun, Jung S; Wakano, Clay; Fernandez, Alfonso G; Pise-Masison, Cynthia A; Gardner, Kevin

    2015-09-11

    The eleven-nineteen lysine-rich leukemia protein (ELL) is a key regulator of RNA polymerase II mediated transcription. ELL facilitates RNA polymerase II transcription pause site entry and release by dynamically interacting with p300 and the positive transcription elongation factor b (P-TEFb). In this study, we investigated the role of ELL during the HTLV-1 Tax oncogene induced transactivation. We show that ectopic expression of Tax enhances ELL incorporation into p300 and P-TEFb containing transcriptional complexes and the subsequent recruitment of these complexes to target genes in vivo. Depletion of ELL abrogates Tax induced transactivation of the immediate early genes Fos, Egr2 and NF-kB, suggesting that ELL is an essential cellular cofactor of the Tax oncogene. Thus, our study identifies a novel mechanism of ELL-dependent transactivation of immediate early genes by Tax and provides the rational for further defining the genome-wide targets of Tax and ELL. Published by Elsevier Inc.

  13. Synthetic Promoters and Transcription Factors for Heterologous Protein Expression in Saccharomyces cerevisiae

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    Fabian Machens

    2017-10-01

    Full Text Available Orthogonal systems for heterologous protein expression as well as for the engineering of synthetic gene regulatory circuits in hosts like Saccharomyces cerevisiae depend on synthetic transcription factors (synTFs and corresponding cis-regulatory binding sites. We have constructed and characterized a set of synTFs based on either transcription activator-like effectors or CRISPR/Cas9, and corresponding small synthetic promoters (synPs with minimal sequence identity to the host’s endogenous promoters. The resulting collection of functional synTF/synP pairs confers very low background expression under uninduced conditions, while expression output upon induction of the various synTFs covers a wide range and reaches induction factors of up to 400. The broad spectrum of expression strengths that is achieved will be useful for various experimental setups, e.g., the transcriptional balancing of expression levels within heterologous pathways or the construction of artificial regulatory networks. Furthermore, our analyses reveal simple rules that enable the tuning of synTF expression output, thereby allowing easy modification of a given synTF/synP pair. This will make it easier for researchers to construct tailored transcriptional control systems.

  14. Overexpression of the transcription factor Yap1 modifies intracellular redox conditions and enhances recombinant protein secretion

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    Marizela Delic

    2014-10-01

    Full Text Available Oxidative folding of secretory proteins in the endoplasmic reticulum (ER is a redox active process, which also impacts the redox conditions in the cytosol. As the transcription factor Yap1 is involved in the transcriptional response to oxidative stress, we investigate its role upon the production of secretory proteins, using the yeast Pichia pastoris as model, and report a novel important role of Yap1 during oxidative protein folding. Yap1 is needed for the detoxification of reactive oxygen species (ROS caused by increased oxidative protein folding. Constitutive co-overexpression of PpYAP1 leads to increased levels of secreted recombinant protein, while a lowered Yap1 function leads to accumulation of ROS and strong flocculation. Transcriptional analysis revealed that more than 150 genes were affected by overexpression of YAP1, in particular genes coding for antioxidant enzymes or involved in oxidation-reduction processes. By monitoring intracellular redox conditions within the cytosol and the ER using redox-sensitive roGFP1 variants, we could show that overexpression of YAP1 restores cellular redox conditions of protein-secreting P. pastoris by reoxidizing the cytosolic redox state to the levels of the wild type. These alterations are also reflected by increased levels of oxidized intracellular glutathione (GSSG in the YAP1 co-overexpressing strain. Taken together, these data indicate a strong impact of intracellular redox balance on the secretion of (recombinant proteins without affecting protein folding per se. Re-establishing suitable redox conditions by tuning the antioxidant capacity of the cell reduces metabolic load and cell stress caused by high oxidative protein folding load, thereby increasing the secretion capacity.

  15. Glucocorticoids facilitate the transcription from the human cytomegalovirus major immediate early promoter in glucocorticoid receptor- and nuclear factor-I-like protein-dependent manner

    International Nuclear Information System (INIS)

    Inoue-Toyoda, Maki; Kato, Kohsuke; Nagata, Kyosuke; Yoshikawa, Hiroyuki

    2015-01-01

    Human cytomegalovirus (HCMV) is a common and usually asymptomatic virus agent in healthy individuals. Initiation of HCMV productive infection depends on expression of the major immediate early (MIE) genes. The transcription of HCMV MIE genes is regulated by a diverse set of transcription factors. It was previously reported that productive HCMV infection is triggered probably by elevation of the plasma hydroxycorticoid level. However, it is poorly understood whether the transcription of MIE genes is directly regulated by glucocorticoid. Here, we found that the dexamethasone (DEX), a synthetic glucocorticoid, facilitates the transcription of HCMV MIE genes through the MIE promoter and enhancer in a glucocorticoid receptor (GR)-dependent manner. By competitive EMSA and reporter assays, we revealed that an NF-I like protein is involved in DEX-mediated transcriptional activation of the MIE promoter. Thus, this study supports a notion that the increased level of hydroxycorticoid in the third trimester of pregnancy reactivates HCMV virus production from the latent state. - Highlights: • DEX facilitates the transcription from the HCMV MIE promoter. • GR is involved in DEX-dependent transcription from the HCMV MIE promoter. • A 17 bp repeat is responsible for the HCMV MIE promoter activation by DEX. • An NF-I-like protein is involved in the HCMV MIE promoter activation by DEX

  16. HCV core protein induces hepatic lipid accumulation by activating SREBP1 and PPARγ

    International Nuclear Information System (INIS)

    Kim, Kook Hwan; Hong, Sung Pyo; Kim, KyeongJin; Park, Min Jung; Kim, Kwang Jin; Cheong, JaeHun

    2007-01-01

    Hepatic steatosis is a common feature in patients with chronic hepatitis C virus (HCV) infection. HCV core protein plays an important role in the development of hepatic steatosis in HCV infection. Because SREBP1 (sterol regulatory element binding protein 1) and PPARγ (peroxisome proliferators-activated receptor γ) are involved in the regulation of lipid metabolism of hepatocyte, we sought to determine whether HCV core protein may impair the expression and activity of SREBP1 and PPARγ. In this study, it was demonstrated that HCV core protein increases the gene expression of SREBP1 not only in Chang liver, Huh7, and HepG2 cells transiently transfected with HCV core protein expression plasmid, but also in Chang liver-core stable cells. Furthermore, HCV core protein enhanced the transcriptional activity of SREBP1. In addition, HCV core protein elevated PPARγ transcriptional activity. However, HCV core protein had no effect on PPARγ gene expression. Finally, we showed that HCV core protein stimulates the genes expression of lipogenic enzyme and fatty acid uptake associated protein. Therefore, our finding provides a new insight into the mechanism of hepatic steatosis by HCV infection

  17. Direct transcriptional activation of BT genes by NLP transcription factors is a key component of the nitrate response in Arabidopsis.

    Science.gov (United States)

    Sato, Takeo; Maekawa, Shugo; Konishi, Mineko; Yoshioka, Nozomi; Sasaki, Yuki; Maeda, Haruna; Ishida, Tetsuya; Kato, Yuki; Yamaguchi, Junji; Yanagisawa, Shuichi

    2017-01-29

    Nitrate modulates growth and development, functioning as a nutrient signal in plants. Although many changes in physiological processes in response to nitrate have been well characterized as nitrate responses, the molecular mechanisms underlying the nitrate response are not yet fully understood. Here, we show that NLP transcription factors, which are key regulators of the nitrate response, directly activate the nitrate-inducible expression of BT1 and BT2 encoding putative scaffold proteins with a plant-specific domain structure in Arabidopsis. Interestingly, the 35S promoter-driven expression of BT2 partially rescued growth inhibition caused by reductions in NLP activity in Arabidopsis. Furthermore, simultaneous disruption of BT1 and BT2 affected nitrate-dependent lateral root development. These results suggest that direct activation of BT1 and BT2 by NLP transcriptional activators is a key component of the molecular mechanism underlying the nitrate response in Arabidopsis. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. The flavonoid fisetin promotes osteoblasts differentiation through Runx2 transcriptional activity.

    Science.gov (United States)

    Léotoing, Laurent; Davicco, Marie-Jeanne; Lebecque, Patrice; Wittrant, Yohann; Coxam, Véronique

    2014-06-01

    Flavonoids represent a group of polyphenolic compounds commonly found in daily nutrition with proven health benefits. Among this group, the flavonol fisetin has been previously shown to protect bone by repressing osteoclast differentiation. In the present study, we investigated the role of fisetin in regulating osteoblasts physiology. In vivo mice treated with LPSs exhibited osteoporosis features associated with a dramatic repression of osteoblast marker expression. In this model, inhibition of osteocalcin and type I collagen alpha 1 transcription was partially countered by a daily consumption of fisetin. Interestingly, in vitro, fisetin promoted both osteoblast alkaline phosphatase activity and mineralization process. To decipher how fisetin may exert its positive effect on osteoblastogenesis, we analyzed its ability to control the runt-related transcription factor 2 (Runx2), a key organizer in developing and maturing osteoblasts. While fisetin did not impact Runx2 mRNA and protein levels, it upregulated its transcriptional activity. Actually, fisetin stimulated the luciferase activity of a reporter plasmid driven by the osteocalcin gene promoter that contains Runx2 binding sites and promoted the mRNA expression of osteocalcin and type I collagen alpha 1 targets. Bone sparing properties of fisetin also rely on its positive influence on osteoblast differentiation and activity. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Prospects of engineering thermotolerance in crops through modulation of heat stress transcription factor and heat shock protein networks.

    Science.gov (United States)

    Fragkostefanakis, Sotirios; Röth, Sascha; Schleiff, Enrico; Scharf, Klaus-Dieter

    2015-09-01

    Cell survival under high temperature conditions involves the activation of heat stress response (HSR), which in principle is highly conserved among different organisms, but shows remarkable complexity and unique features in plant systems. The transcriptional reprogramming at higher temperatures is controlled by the activity of the heat stress transcription factors (Hsfs). Hsfs allow the transcriptional activation of HSR genes, among which heat shock proteins (Hsps) are best characterized. Hsps belong to multigene families encoding for molecular chaperones involved in various processes including maintenance of protein homeostasis as a requisite for optimal development and survival under stress conditions. Hsfs form complex networks to activate downstream responses, but are concomitantly subjected to cell-type-dependent feedback regulation through factor-specific physical and functional interactions with chaperones belonging to Hsp90, Hsp70 and small Hsp families. There is increasing evidence that the originally assumed specialized function of Hsf/chaperone networks in the HSR turns out to be a complex central stress response system that is involved in the regulation of a broad variety of other stress responses and may also have substantial impact on various developmental processes. Understanding in detail the function of such regulatory networks is prerequisite for sustained improvement of thermotolerance in important agricultural crops. © 2014 John Wiley & Sons Ltd.

  20. Atf4 regulates chondrocyte proliferation and differentiation during endochondral ossification by activating Ihh transcription.

    Science.gov (United States)

    Wang, Weiguang; Lian, Na; Li, Lingzhen; Moss, Heather E; Wang, Weixi; Perrien, Daniel S; Elefteriou, Florent; Yang, Xiangli

    2009-12-01

    Activating transcription factor 4 (Atf4) is a leucine-zipper-containing protein of the cAMP response element-binding protein (CREB) family. Ablation of Atf4 (Atf4(-/-)) in mice leads to severe skeletal defects, including delayed ossification and low bone mass, short stature and short limbs. Atf4 is expressed in proliferative and prehypertrophic growth plate chondrocytes, suggesting an autonomous function of Atf4 in chondrocytes during endochondral ossification. In Atf4(-/-) growth plate, the typical columnar structure of proliferative chondrocytes is disturbed. The proliferative zone is shortened, whereas the hypertrophic zone is transiently expanded. The expression of Indian hedgehog (Ihh) is markedly decreased, whereas the expression of other chondrocyte marker genes, such as type II collagen (Col2a1), PTH/PTHrP receptor (Pth1r) and type X collagen (Col10a1), is normal. Furthermore, forced expression of Atf4 in chondrocytes induces endogenous Ihh mRNA, and Atf4 directly binds to the Ihh promoter and activates its transcription. Supporting these findings, reactivation of Hh signaling pharmacologically in mouse limb explants corrects the Atf4(-/-) chondrocyte proliferation and short limb phenotypes. This study thus identifies Atf4 as a novel transcriptional activator of Ihh in chondrocytes that paces longitudinal bone growth by controlling growth plate chondrocyte proliferation and differentiation.

  1. Trans-activation of the 5' to 3' viral DNA strand transfer by nucleocapsid protein during reverse transcription of HIV1 RNA.

    Science.gov (United States)

    Darlix, J L; Vincent, A; Gabus, C; de Rocquigny, H; Roques, B

    1993-08-01

    Two DNA strand transfer reactions take place during reverse transcription of the retroviral genome. The first transfer, that of the minus-strand strong stop DNA from the 5' end of the viral RNA to the 3' end, has been studied in vitro with two RNAs mimicking the 5' and 3' regions of the HIV1 genome and with nucleocapsid protein, NCp7, and reverse transcriptase. The results show that NCp7 strongly activates the 5' to 3' DNA strand transfer during reverse transcription while a basic peptide resembling NCp7 is inactive. Activation of the first transfer by several NCp7 derived peptides and the influence of the terminal redundancies (R) present at the 5' and 3' ends of HIV1 RNA were also examined. The first transfer is optimal in the presence of intact NCp7 and necessitates R on both the 5' and 3' RNAs. Sequencing of full length viral DNA products reveals approximately 40% misincorporations at the first nucleotide beyond the transfer point. If such base misincorporations occur during proviral DNA synthesis with possible homologous recombinations it may well contribute to the high level of genetic variability of HIV.

  2. In vitro synthesis of biologically active transcripts of tomato black ring virus satellite RNA.

    Science.gov (United States)

    Greif, C; Hemmer, O; Demangeat, G; Fritsch, C

    1990-04-01

    Synthetic transcripts of tomato black ring virus satellite RNA (TBRV satRNA), isolate L, were prepared from cDNA cloned in the Bluescribe transcription vector. Transcripts with 49 (T49L) or two (T2GL) extra nucleotides at their 5' ends and 42 extra nucleotides at their 3' ends were able to induce, but to different extents, the synthesis in vitro of the satRNA-encoded 48K protein. However, when inoculated into Chenopodium quinoa together with TBRV L genomic RNAs, only T2GL was biologically active, in the presence or absence of a 5' cap analogue in the transcription reactions. Analysis of the 5' and 3' termini of the satRNA isolated from plants showed that nonviral extensions were not maintained in the transcript progeny.

  3. A light- and calcium-gated transcription factor for imaging and manipulating activated neurons.

    Science.gov (United States)

    Wang, Wenjing; Wildes, Craig P; Pattarabanjird, Tanyaporn; Sanchez, Mateo I; Glober, Gordon F; Matthews, Gillian A; Tye, Kay M; Ting, Alice Y

    2017-09-01

    Activity remodels neurons, altering their molecular, structural, and electrical characteristics. To enable the selective characterization and manipulation of these neurons, we present FLARE, an engineered transcription factor that drives expression of fluorescent proteins, opsins, and other genetically encoded tools only in the subset of neurons that experienced activity during a user-defined time window. FLARE senses the coincidence of elevated cytosolic calcium and externally applied blue light, which together produce translocation of a membrane-anchored transcription factor to the nucleus to drive expression of any transgene. In cultured rat neurons, FLARE gives a light-to-dark signal ratio of 120 and a high- to low-calcium signal ratio of 10 after 10 min of stimulation. Opsin expression permitted functional manipulation of FLARE-marked neurons. In adult mice, FLARE also gave light- and motor-activity-dependent transcription in the cortex. Due to its modular design, minute-scale temporal resolution, and minimal dark-state leak, FLARE should be useful for the study of activity-dependent processes in neurons and other cells that signal with calcium.

  4. Polyphenol Compound as a Transcription Factor Inhibitor

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    Seyeon Park

    2015-10-01

    Full Text Available A target-based approach has been used to develop novel drugs in many therapeutic fields. In the final stage of intracellular signaling, transcription factor–DNA interactions are central to most biological processes and therefore represent a large and important class of targets for human therapeutics. Thus, we focused on the idea that the disruption of protein dimers and cognate DNA complexes could impair the transcriptional activation and cell transformation regulated by these proteins. Historically, natural products have been regarded as providing the primary leading compounds capable of modulating protein–protein or protein-DNA interactions. Although their mechanism of action is not fully defined, polyphenols including flavonoids were found to act mostly as site-directed small molecule inhibitors on signaling. There are many reports in the literature of screening initiatives suggesting improved drugs that can modulate the transcription factor interactions responsible for disease. In this review, we focus on polyphenol compound inhibitors against dimeric forms of transcription factor components of intracellular signaling pathways (for instance, c-jun/c-fos (Activator Protein-1; AP-1, c-myc/max, Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB and β-catenin/T cell factor (Tcf.

  5. Mycobacterium tuberculosis cAMP Receptor Protein (Rv3676) Differs from the Escherichia coli Paradigm in Its cAMP Binding and DNA Binding Properties and Transcription Activation Properties*

    Science.gov (United States)

    Stapleton, Melanie; Haq, Ihtshamul; Hunt, Debbie M.; Arnvig, Kristine B.; Artymiuk, Peter J.; Buxton, Roger S.; Green, Jeffrey

    2010-01-01

    The pathogen Mycobacterium tuberculosis produces a burst of cAMP upon infection of macrophages. Bacterial cyclic AMP receptor proteins (CRP) are transcription factors that respond to cAMP by binding at target promoters when cAMP concentrations increase. Rv3676 (CRPMt) is a CRP family protein that regulates expression of genes (rpfA and whiB1) that are potentially involved in M. tuberculosis persistence and/or emergence from the dormant state. Here, the CRPMt homodimer is shown to bind two molecules of cAMP (one per protomer) at noninteracting sites. Furthermore, cAMP binding by CRPMt was relatively weak, entropy driven, and resulted in a relatively small enhancement in DNA binding. Tandem CRPMt-binding sites (CRP1 at −58.5 and CRP2 at −37.5) were identified at the whiB1 promoter (PwhiB1). In vitro transcription reactions showed that CRP1 is an activating site and that CRP2, which was only occupied in the presence of cAMP or at high CRPMt concentrations in the absence of cAMP, is a repressing site. Binding of CRPMt to CRP1 was not essential for open complex formation but was required for transcription activation. Thus, these data suggest that binding of CRPMt to the PwhiB1 CRP1 site activates transcription at a step after open complex formation. In contrast, high cAMP concentrations allowed occupation of both CRP1 and CRP2 sites, resulting in inhibition of open complex formation. Thus, M. tuberculosis CRP has evolved several distinct characteristics, compared with the Escherichia coli CRP paradigm, to allow it to regulate gene expression against a background of high concentrations of cAMP. PMID:20028978

  6. Hyperosmotic stress strongly potentiates serum response factor (SRF)-dependent transcriptional activity in ehrlich lettré ascites cells through a mechanism involving p38 mitogen-activated protein kinase

    DEFF Research Database (Denmark)

    Gorbatenko, Andrej; Wiwel, Maria; Klingberg, Henrik

    2011-01-01

    Long-term osmotic stress results in altered gene transcription, however, with the exception of the TonE/TonEBP system, the underlying mechanisms are poorly understood. We previously showed that upon osmotic shrinkage of Ehrlich Lettré Ascites (ELA) fibroblasts, the MEK1-ERK1/2 pathway is transien......Long-term osmotic stress results in altered gene transcription, however, with the exception of the TonE/TonEBP system, the underlying mechanisms are poorly understood. We previously showed that upon osmotic shrinkage of Ehrlich Lettré Ascites (ELA) fibroblasts, the MEK1-ERK1/2 pathway......) and cAMP response element-binding protein (CREB) are differentially regulated in ELA cells. SRF Ser103 phosphorylation and SRF-dependent transcriptional activity were strongly augmented 5–30¿min and 24¿h, respectively, after hyperosmotic stress (50% increase in extracellular ionic strength), in a p38...... dephosphorylated within 5¿min of shrinkage. MSK1 phosphorylation recovered within 30¿min in a p38-MAPK-dependent manner. CREB was transiently dephosphorylated after shrinkage in a manner exacerbated by p38 MAPK inhibition or MSK1 knockdown, but unaffected by inhibition of RSK. In conclusion, in ELA cells...

  7. The effects of Ankaferd® Blood Stopper on transcription factors in HUVEC and the erythrocyte protein profile

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    Erkan Yılmaz

    2011-12-01

    Full Text Available Objective: Ankaferd® Blood Stopper (ABS is an herbal extract that has historically been used as a hemostatic agent in traditional Turkish medicine. ABS is comprised of a standardized herbal mixture of T. vulgaris, G. glabra, V. vinifera, A. officinarum, and U. dioica. ABS’s basic mechanism of action is the formation of an encapsulated protein web, which represents the focal point for vital erythrocyte masses. The hemostatic effects of ABS have been observed in vitro and in vivo. ABS was registered as a hemostatic agent for external hemorrhages and dental bleeding following phase I randomized, double-blind crossover placebo-controlled clinical research, and safety and efficacy reports. In terms of the potential use of ABS, transcription factors may be novel factors that play a role in the hemostatic and other pleiotropic effects of ABS. Materials and Methods: Hence, the present study aimed to investigate the effects of ABS on endothelium, and possible transcription factor changes in HUVEC (human umbilical vein endothelial cells and the erythrocyte membrane profile. ABS (5 μL and 50 μL was administered to HUVEC (in 75 cm2; ~75% fullness for 5 min and 15 min. Results: ABS caused significant increases in the level of activation of the following transcription factors; AP2, AR, CRE/ATF1, CREB, E2F1-5, E2F6, EGR, GATA, HNF-1, ISRE, Myc-Max, NF-1, NFkB, p53, PPAR, SMAD 2/3, SP1, TRE/AP1, and YY1. Following erythrocyte membrane isolation, protein complexes were undissolved, but denatured. The protein complex formed was resistant to heat and detergent. Trypsin and sonication were used in order to break this complex; the complex dissolved and erythrocyte membrane proteins were released in SDS-PAGE.Conclusion: ABS established a very fast and solid protein web, and increased the level of transcription factor activation. Therefore the cellular effects of ABS could be related to different intracellular biological pathways.

  8. The Enzyme-Like Domain of Arabidopsis Nuclear β-Amylases Is Critical for DNA Sequence Recognition and Transcriptional Activation.

    Science.gov (United States)

    Soyk, Sebastian; Simková, Klára; Zürcher, Evelyne; Luginbühl, Leonie; Brand, Luise H; Vaughan, Cara K; Wanke, Dierk; Zeeman, Samuel C

    2014-04-01

    Plant BZR1-BAM transcription factors contain a β-amylase (BAM)-like domain, characteristic of proteins involved in starch breakdown. The enzyme-derived domains appear to be noncatalytic, but they determine the function of the two Arabidopsis thaliana BZR1-BAM isoforms (BAM7 and BAM8) during transcriptional initiation. Removal or swapping of the BAM domains demonstrates that the BAM7 BAM domain restricts DNA binding and transcriptional activation, while the BAM8 BAM domain allows both activities. Furthermore, we demonstrate that BAM7 and BAM8 interact on the protein level and cooperate during transcriptional regulation. Site-directed mutagenesis of residues in the BAM domain of BAM8 shows that its function as a transcriptional activator is independent of catalysis but requires an intact substrate binding site, suggesting it may bind a ligand. Microarray experiments with plants overexpressing truncated versions lacking the BAM domain indicate that the pseudo-enzymatic domain increases selectivity for the preferred cis-regulatory element BBRE (BZR1-BAM Responsive Element). Side specificity toward the G-box may allow crosstalk to other signaling networks. This work highlights the importance of the enzyme-derived domain of BZR1-BAMs, supporting their potential role as metabolic sensors. © 2014 American Society of Plant Biologists. All rights reserved.

  9. A single amino acid change within the R2 domain of the VvMYB5b transcription factor modulates affinity for protein partners and target promoters selectivity

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    Granier Thierry

    2011-08-01

    Full Text Available Abstract Background Flavonoid pathway is spatially and temporally controlled during plant development and the transcriptional regulation of the structural genes is mostly orchestrated by a ternary protein complex that involves three classes of transcription factors (R2-R3-MYB, bHLH and WDR. In grapevine (Vitis vinifera L., several MYB transcription factors have been identified but the interactions with their putative bHLH partners to regulate specific branches of the flavonoid pathway are still poorly understood. Results In this work, we describe the effects of a single amino acid substitution (R69L located in the R2 domain of VvMYB5b and predicted to affect the formation of a salt bridge within the protein. The activity of the mutated protein (name VvMYB5bL, the native protein being referred as VvMYB5bR was assessed in different in vivo systems: yeast, grape cell suspensions, and tobacco. In the first two systems, VvMYB5bL exhibited a modified trans-activation capability. Moreover, using yeast two-hybrid assay, we demonstrated that modification of VvMYB5b transcriptional properties impaired its ability to correctly interact with VvMYC1, a grape bHLH protein. These results were further substantiated by overexpression of VvMYB5bR and VvMYB5bL genes in tobacco. Flowers from 35S::VvMYB5bL transgenic plants showed a distinct phenotype in comparison with 35S::VvMYB5bR and the control plants. Finally, significant differences in transcript abundance of flavonoid metabolism genes were observed along with variations in pigments accumulation. Conclusions Taken together, our findings indicate that VvMYB5bL is still able to bind DNA but the structural consequences linked to the mutation affect the capacity of the protein to activate the transcription of some flavonoid genes by modifying the interaction with its co-partner(s. In addition, this study underlines the importance of an internal salt bridge for protein conformation and thus for the establishment

  10. Diffusion-weighted magnetic resonance imaging reflects activation of signal transducer and activator of transcription 3 during focal cerebral ischemia/reperfusion

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    Wen-juan Wu

    2017-01-01

    Full Text Available Signal transducer and activator of transcription (STAT is a unique protein family that binds to DNA, coupled with tyrosine phosphorylation signaling pathways, acting as a transcriptional regulator to mediate a variety of biological effects. Cerebral ischemia and reperfusion can activate STATs signaling pathway, but no studies have confirmed whether STAT activation can be verified by diffusion-weighted magnetic resonance imaging (DWI in rats after cerebral ischemia/reperfusion. Here, we established a rat model of focal cerebral ischemia injury using the modified Longa method. DWI revealed hyperintensity in parts of the left hemisphere before reperfusion and a low apparent diffusion coefficient. STAT3 protein expression showed no significant change after reperfusion, but phosphorylated STAT3 expression began to increase after 30 minutes of reperfusion and peaked at 24 hours. Pearson correlation analysis showed that STAT3 activation was correlated positively with the relative apparent diffusion coefficient and negatively with the DWI abnormal signal area. These results indicate that DWI is a reliable representation of the infarct area and reflects STAT phosphorylation in rat brain following focal cerebral ischemia/reperfusion.

  11. Msn2p/Msn4p act as a key transcriptional activator of yeast cytoplasmic thiol peroxidase II.

    Science.gov (United States)

    Hong, Seung-Keun; Cha, Mee-Kyung; Choi, Yong-Soo; Kim, Won-Cheol; Kim, Il-Han

    2002-04-05

    We observed that the transcription of Saccharomyces cerevisiae cytoplasmic thiol peroxidase type II (cTPx II) (YDR453C) is regulated in response to various stresses (e.g. oxidative stress, carbon starvation, and heat-shock). It has been suggested that both transcription-activating proteins, Yap1p and Skn7p, regulate the transcription of cTPx II upon exposure to oxidative stress. However, a dramatic loss of transcriptional response to various stresses in yeast mutant strains lacking both Msn2p and Msn4p suggests that the transcription factors act as a principal transcriptional activator. In addition to two Yap1p response elements (YREs), TTACTAA and TTAGTAA, the presence of two stress response elements (STREs) (CCCCT) in the upstream sequence of cTPx II also suggests that Msn2p/Msn4p could control stress-induced expression of cTPx II. Analysis of the transcriptional activity of site-directed mutagenesis of the putative STREs (STRE1 and STRE2) and YREs (TRE1 and YRE2) in terms of the activity of a lacZ reporter gene under control of the cTPx II promoter indicates that STRE2 acts as a principal binding element essential for transactivation of the cTPx II promoter. The transcriptional activity of the cTPx II promoter was exponentially increased after postdiauxic growth. The transcriptional activity of the cTPx II promoter is greatly increased by rapamycin. Deletion of Tor1, Tor2, Ras1, and Ras2 resulted in a considerable induction when compared with their parent strains, suggesting that the transcription of cTPx II is under negative control of the Ras/cAMP and target of rapamycin signaling pathways. Taken together, these results suggest that cTPx II is a target of Msn2p/Msn4p transcription factors under negative control of the Ras-protein kinase A and target of rapamycin signaling pathways. Furthermore, the accumulation of cTPx II upon exposure to oxidative stress and during the postdiauxic shift suggests an important antioxidant role in stationary phase yeast cells.

  12. Two transcription products of the vesicular stomatitis virus genome may control L-cell protein synthesis

    International Nuclear Information System (INIS)

    Dunigan, D.D.; Lucas-Lenard, J.M.

    1983-01-01

    When mouse L-cells are infected with vesicular stomatitis virus, there is a decrease in the rate of protein synthesis ranging from 20 to 85% of that in mock-infected cells. Vesicular stomatitis virus, irradiated with increasing doses of UV light, eventually loses this capacity to inhibit protein synthesis. The UV inactivation curve was biphasic, suggesting that transcription of two regions of the viral genome is necessary for the virus to become inactivated in this capacity. The first transcription produced corresponded to about 373 nucleotides, and the second corresponded to about 42 nucleotides. Inhibition of transcription of the larger product by irradiating the virus with low doses of UV light left a residual inhibition of protein synthesis consisting of approximately 60 to 65% of the total inhibition. This residual inhibition could be obviated by irradiating the virus with a UV dose of greater than 20,000 ergs/mm 2 and was thus considered to represent the effect of the smaller transcription product. In the R1 mutant of another author, the inhibition of transcription of the larger product sufficed to restore protein synthesis to the mock-infected level, suggesting that the smaller transcription product is nonfunctional with respect to protein synthesis inhibition. Extracts from cells infected with virus irradiated with low doses of UV light showed a protein synthesis capacity quite similar to that of their in vivo counterparts, indicating that these extracts closely reflect the in vivo effects of virus infection

  13. The cellular transcription factor CREB corresponds to activating transcription factor 47 (ATF-47) and forms complexes with a group of polypeptides related to ATF-43.

    OpenAIRE

    Hurst, H C; Masson, N; Jones, N C; Lee, K A

    1990-01-01

    Promoter elements containing the sequence motif CGTCA are important for a variety of inducible responses at the transcriptional level. Multiple cellular factors specifically bind to these elements and are encoded by a multigene family. Among these factors, polypeptides termed activating transcription factor 43 (ATF-43) and ATF-47 have been purified from HeLa cells and a factor referred to as cyclic AMP response element-binding protein (CREB) has been isolated from PC12 cells and rat brain. We...

  14. Exosome proteomics reveals transcriptional regulator proteins with potential to mediate downstream pathways.

    Science.gov (United States)

    Ung, Timothy H; Madsen, Helen J; Hellwinkel, Justin E; Lencioni, Alex M; Graner, Michael W

    2014-11-01

    Exosomes are virus-sized, membrane-enclosed vesicles with origins in the cellular endosomal system, but are released extracellularly. As a population, these tiny vesicles carry relatively enormous amounts of information in their protein, lipid and nucleic acid content, and the vesicles can have profound impacts on recipient cells. This review employs publically-available data combined with gene ontology applications to propose a novel concept, that exosomes transport transcriptional and translational machinery that may have direct impacts on gene expression in recipient cells. Here, we examine the previously published proteomic contents of medulloblastoma-derived exosomes, focusing on transcriptional regulators; we found that there are numerous proteins that may have potential roles in transcriptional and translational regulation with putative influence on downstream, cancer-related pathways. We expanded this search to all of the proteins in the Vesiclepedia database; using gene ontology approaches, we see that these regulatory factors are implicated in many of the processes involved in cancer initiation and progression. This information suggests that some of the effects of exosomes on recipient cells may be due to the delivery of protein factors that can directly and fundamentally change the transcriptional landscape of the cells. Within a tumor environment, this has potential to tilt the advantage towards the cancer. © 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

  15. Activating RNAs associate with Mediator to enhance chromatin architecture and transcription

    OpenAIRE

    Lai, Fan; Orom, Ulf A; Cesaroni, Matteo; Beringer, Malte; Taatjes, Dylan J; Blobel, Gerd A.; Shiekhattar, Ramin

    2013-01-01

    Recent advances in genomic research have revealed the existence of a large number of transcripts devoid of protein-coding potential in multiple organisms 1-8 . While the functional role for long non-coding RNAs (lncRNAs) has been best defined in epigenetic phenomena such as X inactivation and imprinting, different classes of lncRNAs may have varied biological functions 8-13 . We and others have identified a class of lncRNAs, termed ncRNA-activating (ncRNA-a), that function to activate their n...

  16. HuR/ELAVL1 RNA binding protein modulates interleukin-8 induction by muco-active ribotoxin deoxynivalenol

    International Nuclear Information System (INIS)

    Choi, Hye Jin; Yang, Hyun; Park, Seong Hwan; Moon, Yuseok

    2009-01-01

    HuR/Elav-like RNA binding protein 1 (ELAVL1) positively regulates mRNA stability of AU-rich elements (ARE)-containing transcript such as pro-inflammatory cytokines. Ribotoxic stresses can trigger the production of pro-inflammatory mediators by enhancing mRNA stability and the transcriptional activity. We investigated the effects of ribotoxin deoxynivalenol (DON) on HuR translocation and its involvement in the regulation of the pro-inflammatory interleukin-8 (IL-8) mRNA stability. Exposure to the muco-active DON induced nuclear export of both endogenous and exogenous HuR RNA binding protein in human intestinal epithelial cells. Moreover, the interference with HuR protein production suppressed ribotoxic DON-induced IL-8 secretion and its mRNA stability. Cytoplasmic HuR protein interacted with IL-8 mRNA and the complex stabilization was due to the presence of 3'-untranslated region of the transcript. Partly in terms of IL-8-modulating transcription factors, HuR protein was demonstrated to be positively and negatively associated with DON-induced early growth response gene 1 (EGR-1) and activating transcription factor 3 (ATF3), respectively. HuR was a critical mechanistic link between ribotoxic stress and the pro-inflammatory cytokine production, and may have a broader functional significance with regard to mucosal insults since ribotoxic stress responses are also produced upon interactions with the diverse environment of gut.

  17. MiT/TFE transcription factors are activated during mitophagy downstream of Parkin and Atg5.

    Science.gov (United States)

    Nezich, Catherine L; Wang, Chunxin; Fogel, Adam I; Youle, Richard J

    2015-08-03

    The kinase PINK1 and ubiquitin ligase Parkin can regulate the selective elimination of damaged mitochondria through autophagy (mitophagy). Because of the demand on lysosomal function by mitophagy, we investigated a role for the transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, in this process. We show that during mitophagy TFEB translocates to the nucleus and displays transcriptional activity in a PINK1- and Parkin-dependent manner. MITF and TFE3, homologues of TFEB belonging to the same microphthalmia/transcription factor E (MiT/TFE) family, are similarly regulated during mitophagy. Unlike TFEB translocation after starvation-induced mammalian target of rapamycin complex 1 inhibition, Parkin-mediated TFEB relocalization required Atg9A and Atg5 activity. However, constitutively active Rag guanosine triphosphatases prevented TFEB translocation during mitophagy, suggesting cross talk between these two MiT/TFE activation pathways. Analysis of clustered regularly interspaced short palindromic repeats-generated TFEB/MITF/TFE3/TFEC single, double, and triple knockout cell lines revealed that these proteins partly facilitate Parkin-mediated mitochondrial clearance. These results illuminate a pathway leading to MiT/TFE transcription factor activation, distinct from starvation-induced autophagy, which occurs during mitophagy.

  18. IQCJ-SCHIP1, a novel fusion transcript encoding a calmodulin-binding IQ motif protein

    International Nuclear Information System (INIS)

    Kwasnicka-Crawford, Dorota A.; Carson, Andrew R.; Scherer, Stephen W.

    2006-01-01

    The existence of transcripts that span two adjacent, independent genes is considered rare in the human genome. This study characterizes a novel human fusion gene named IQCJ-SCHIP1. IQCJ-SCHIP1 is the longest isoform of a complex transcriptional unit that bridges two separate genes that encode distinct proteins, IQCJ, a novel IQ motif containing protein and SCHIP1, a schwannomin interacting protein that has been previously shown to interact with the Neurofibromatosis type 2 (NF2) protein. IQCJ-SCHIP1 is located on the chromosome 3q25 and comprises a 1692-bp transcript encompassing 11 exons spanning 828 kb of the genomic DNA. We show that IQCJ-SCHIP1 mRNA is highly expressed in the brain. Protein encoded by the IQCJ-SCHIP1 gene was localized to cytoplasm and actin-rich regions and in differentiated PC12 cells was also seen in neurite extensions

  19. Rift Valley fever virus NSs inhibits host transcription independently of the degradation of dsRNA-dependent protein kinase PKR.

    Science.gov (United States)

    Kalveram, Birte; Lihoradova, Olga; Indran, Sabarish V; Lokugamage, Nandadeva; Head, Jennifer A; Ikegami, Tetsuro

    2013-01-20

    Rift Valley fever virus (RVFV) encodes one major virulence factor, the NSs protein. NSs suppresses host general transcription, including interferon (IFN)-β mRNA synthesis, and promotes degradation of the dsRNA-dependent protein kinase (PKR). We generated a novel RVFV mutant (rMP12-NSsR173A) specifically lacking the function to promote PKR degradation. rMP12-NSsR173A infection induces early phosphorylation of eIF2α through PKR activation, while retaining the function to inhibit host general transcription including IFN-β gene inhibition. MP-12 NSs but not R173A NSs binds to wt PKR. R173A NSs formed filamentous structure in nucleus in a mosaic pattern, which was distinct from MP-12 NSs filament pattern. Due to early phosphorylation of eIF2α, rMP12-NSsR173A could not efficiently accumulate viral proteins. Our results suggest that NSs-mediated host general transcription suppression occurs independently of PKR degradation, while the PKR degradation is important to inhibit the phosphorylation of eIF2α in infected cells undergoing host general transcription suppression. Copyright © 2012 Elsevier Inc. All rights reserved.

  20. Epstein-Barr virus (EBV) LMP2A alters normal transcriptional regulation following B-cell receptor activation

    International Nuclear Information System (INIS)

    Portis, Toni; Longnecker, Richard

    2004-01-01

    The latent membrane protein 2A (LMP2A) of Epstein-Barr virus (EBV) is an important mediator of viral latency in infected B-lymphocytes. LMP2A inhibits B-cell receptor (BCR) signaling in vitro and allows for the survival of BCR-negative B cells in vivo. In this study, we compared gene transcription in BCR-activated B cells from non-transgenic and LMP2A Tg6 transgenic mice. We found that the transcriptional induction and down-regulation of many genes that normally occurs in B cells following BCR activation did not occur in B cells from LMP2A Tg6 transgenic mice. Furthermore, LMP2A induced the expression of various transcription factors and genes associated with DNA/RNA metabolism, which may allow for the altered transcriptional regulation observed in BCR-activated B cells from LMP2A Tg6 mice. These results suggest that LMP2A may inhibit the downstream effects of BCR signaling by directly or indirectly altering gene transcription to ensure EBV persistence in infected B cells

  1. Transcriptional co-activator PGC-1 alpha drives the formation of slow-twitch muscle fibres.

    Science.gov (United States)

    Lin, Jiandie; Wu, Hai; Tarr, Paul T; Zhang, Chen-Yu; Wu, Zhidan; Boss, Olivier; Michael, Laura F; Puigserver, Pere; Isotani, Eiji; Olson, Eric N; Lowell, Bradford B; Bassel-Duby, Rhonda; Spiegelman, Bruce M

    2002-08-15

    The biochemical basis for the regulation of fibre-type determination in skeletal muscle is not well understood. In addition to the expression of particular myofibrillar proteins, type I (slow-twitch) fibres are much higher in mitochondrial content and are more dependent on oxidative metabolism than type II (fast-twitch) fibres. We have previously identified a transcriptional co-activator, peroxisome-proliferator-activated receptor-gamma co-activator-1 (PGC-1 alpha), which is expressed in several tissues including brown fat and skeletal muscle, and that activates mitochondrial biogenesis and oxidative metabolism. We show here that PGC-1 alpha is expressed preferentially in muscle enriched in type I fibres. When PGC-1 alpha is expressed at physiological levels in transgenic mice driven by a muscle creatine kinase (MCK) promoter, a fibre type conversion is observed: muscles normally rich in type II fibres are redder and activate genes of mitochondrial oxidative metabolism. Notably, putative type II muscles from PGC-1 alpha transgenic mice also express proteins characteristic of type I fibres, such as troponin I (slow) and myoglobin, and show a much greater resistance to electrically stimulated fatigue. Using fibre-type-specific promoters, we show in cultured muscle cells that PGC-1 alpha activates transcription in cooperation with Mef2 proteins and serves as a target for calcineurin signalling, which has been implicated in slow fibre gene expression. These data indicate that PGC-1 alpha is a principal factor regulating muscle fibre type determination.

  2. Thyroid hormone and retinoic acid nuclear receptors: specific ligand-activated transcription factors

    International Nuclear Information System (INIS)

    Brtko, J.

    1998-01-01

    Transcriptional regulation by both the thyroid hormone and the vitamin A-derived 'retinoid hormones' is a critical component in controlling many aspects of higher vertebrate development and metabolism. Their functions are mediated by nuclear receptors, which comprise a large super-family of ligand-inducible transcription factors. Both the thyroid hormone and the retinoids are involved in a complex arrangement of physiological and development responses in many tissues of higher vertebrates. The functions of 3,5,3'-triiodothyronine (T 3 ), the thyromimetically active metabolite of thyroxine as well as all-trans retinoic acid, the biologically active vitamin A metabolite are mediated by nuclear receptor proteins that are members of the steroid/thyroid/retinoid hormone receptor family. The functions of all members of the receptor super family are discussed. (authors)

  3. Modulation of Epstein–Barr Virus Nuclear Antigen 2-dependent transcription by protein arginine methyltransferase 5

    International Nuclear Information System (INIS)

    Liu, Cheng-Der; Cheng, Chi-Ping; Fang, Jia-Shih; Chen, Ling-Chih; Zhao, Bo; Kieff, Elliott; Peng, Chih-Wen

    2013-01-01

    Highlights: ► Catalytic active PRMT5 substantially binds to the EBNA2 RG domain. ► PRMT5 augments the EBNA2-dependent transcription. ► PRMT5 triggers the symmetric dimethylation of the EBNA2 RG domain. ► PRMT5 enhances the promoter occupancy of EBNA2 on its target promoters. -- Abstract: Epstein–Barr Virus Nuclear Antigen (EBNA) 2 features an Arginine–Glycine repeat (RG) domain at amino acid positions 335–360, which is a known target for protein arginine methyltransferaser 5 (PRMT5). In this study, we performed protein affinity pull-down assays to demonstrate that endogenous PRMT5 derived from lymphoblastoid cells specifically associated with the protein bait GST-E2 RG. Transfection of a plasmid expressing PRMT5 induced a 2.5- to 3-fold increase in EBNA2-dependent transcription of both the LMP1 promoter in AKATA cells, which contain the EBV genome endogenously, and a Cp-Luc reporter plasmid in BJAB cells, which are EBV negative. Furthermore, we showed that there was a 2-fold enrichment of EBNA2 occupancy in target promoters in the presence of exogenous PRMT5. Taken together, we show that PRMT5 triggers the symmetric dimethylation of EBNA2 RG domain to coordinate with EBNA2-mediated transcription. This modulation suggests that PRMT5 may play a role in latent EBV infection

  4. Modulation of Epstein–Barr Virus Nuclear Antigen 2-dependent transcription by protein arginine methyltransferase 5

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Cheng-Der; Cheng, Chi-Ping; Fang, Jia-Shih; Chen, Ling-Chih [Department of Life Sciences, Tzu-Chi University, 701 Chung-Yang Rd. Sec 3, Hualien 97004, Taiwan (China); Zhao, Bo; Kieff, Elliott [Department of Medicine and Microbiology and Molecular Genetics, Channing Laboratory, Brigham and Women’s Hospital and Harvard Medical School, 181 Longwood Ave., Boston 02115, MA (United States); Peng, Chih-Wen, E-mail: pengcw@mail.tcu.edu.tw [Department of Life Sciences, Tzu-Chi University, 701 Chung-Yang Rd. Sec 3, Hualien 97004, Taiwan (China)

    2013-01-18

    Highlights: ► Catalytic active PRMT5 substantially binds to the EBNA2 RG domain. ► PRMT5 augments the EBNA2-dependent transcription. ► PRMT5 triggers the symmetric dimethylation of the EBNA2 RG domain. ► PRMT5 enhances the promoter occupancy of EBNA2 on its target promoters. -- Abstract: Epstein–Barr Virus Nuclear Antigen (EBNA) 2 features an Arginine–Glycine repeat (RG) domain at amino acid positions 335–360, which is a known target for protein arginine methyltransferaser 5 (PRMT5). In this study, we performed protein affinity pull-down assays to demonstrate that endogenous PRMT5 derived from lymphoblastoid cells specifically associated with the protein bait GST-E2 RG. Transfection of a plasmid expressing PRMT5 induced a 2.5- to 3-fold increase in EBNA2-dependent transcription of both the LMP1 promoter in AKATA cells, which contain the EBV genome endogenously, and a Cp-Luc reporter plasmid in BJAB cells, which are EBV negative. Furthermore, we showed that there was a 2-fold enrichment of EBNA2 occupancy in target promoters in the presence of exogenous PRMT5. Taken together, we show that PRMT5 triggers the symmetric dimethylation of EBNA2 RG domain to coordinate with EBNA2-mediated transcription. This modulation suggests that PRMT5 may play a role in latent EBV infection.

  5. The effect of aluminium-stress and exogenous spermidine on chlorophyll degradation, glutathione reductase activity and the photosystem II D1 protein gene (psbA) transcript level in lichen Xanthoria parietina.

    Science.gov (United States)

    Sen, Gulseren; Eryilmaz, Isil Ezgi; Ozakca, Dilek

    2014-02-01

    In this study, the effects of short-term aluminium toxicity and the application of spermidine on the lichen Xanthoria parietina were investigated at the physiological and transcriptional levels. Our results suggest that aluminium stress leads to physiological processes in a dose-dependent manner through differences in lipid peroxidation rate, chlorophyll content and glutathione reductase (EC 1.6.4.2) activity in aluminium and spermidine treated samples. The expression of the photosystem II D1 protein (psbA) gene was quantified using semi-quantitative RT-PCR. Increased glutathione reductase activity and psbA mRNA transcript levels were observed in the X. parietina thalli that were treated with spermidine before aluminium-stress. The results showed that the application of spermidine could mitigate aluminium-induced lipid peroxidation and chlorophyll degradation on lichen X. parietina thalli through an increase in psbA transcript levels and activity of glutathione reductase (GR) enzymes. Copyright © 2013 Elsevier Ltd. All rights reserved.

  6. Novel activation domain derived from Che-1 cofactor coupled with the artificial protein Jazz drives utrophin upregulation.

    Science.gov (United States)

    Desantis, Agata; Onori, Annalisa; Di Certo, Maria Grazia; Mattei, Elisabetta; Fanciulli, Maurizio; Passananti, Claudio; Corbi, Nicoletta

    2009-02-01

    Our aim is to upregulate the expression level of the dystrophin related gene utrophin in Duchenne muscular dystrophy, thus complementing the lack of dystrophin functions. To this end, we have engineered synthetic zinc finger based transcription factors. We have previously shown that the artificial three-zinc finger protein named Jazz fused with the Vp16 activation domain, is able to bind utrophin promoter A and to increase the endogenous level of utrophin in transgenic mice. Here, we report on an innovative artificial protein, named CJ7, that consists of Jazz DNA binding domain fused to a novel activation domain derived from the regulatory multivalent adaptor protein Che-1/AATF. This transcriptional activation domain is 100 amino acids in size and it is very powerful as compared to the Vp16 activation domain. We show that CJ7 protein efficiently promotes transcription and accumulation of the acetylated form of histone H3 on the genomic utrophin promoter locus.

  7. BACH transcription factors in innate and adaptive immunity.

    Science.gov (United States)

    Igarashi, Kazuhiko; Kurosaki, Tomohiro; Roychoudhuri, Rahul

    2017-07-01

    BTB and CNC homology (BACH) proteins are transcriptional repressors of the basic region leucine zipper (bZIP) transcription factor family. Recent studies indicate widespread roles of BACH proteins in controlling the development and function of the innate and adaptive immune systems, including the differentiation of effector and memory cells of the B and T cell lineages, CD4 + regulatory T cells and macrophages. Here, we emphasize similarities at a molecular level in the cell-type-specific activities of BACH factors, proposing that competitive interactions of BACH proteins with transcriptional activators of the bZIP family form a common mechanistic theme underlying their diverse actions. The findings contribute to a general understanding of how transcriptional repressors shape lineage commitment and cell-type-specific functions through repression of alternative lineage programmes.

  8. A Protein Complex Required for Polymerase V Transcripts and RNA- Directed DNA Methylation in Arabidopsis

    KAUST Repository

    Law, Julie A.; Ausí n, Israel; Johnson, Lianna M.; Vashisht, Ajay  A Amar; Zhu, Jian-Kang; Wohlschlegel, James  A A.; Jacobsen, Steven E.

    2010-01-01

    DNA methylation is an epigenetic modification associated with gene silencing. In Arabidopsis, DNA methylation is established by DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2), which is targeted by small interfering RNAs through a pathway termed RNA-directed DNA methylation (RdDM) [1, 2]. Recently, RdDM was shown to require intergenic noncoding (IGN) transcripts that are dependent on the Pol V polymerase. These transcripts are proposed to function as scaffolds for the recruitment of downstream RdDM proteins, including DRM2, to loci that produce both siRNAs and IGN transcripts [3]. However, the mechanism(s) through which Pol V is targeted to specific genomic loci remains largely unknown. Through affinity purification of two known RdDM components, DEFECTIVE IN RNA-DIRECTED DNA METHYLATION 1 (DRD1) [4] and DEFECTIVE IN MERISTEM SILENCING 3 (DMS3) [5, 6], we found that they copurify with each other and with a novel protein, RNA-DIRECTED DNA METHYLATION 1 (RDM1), forming a complex we term DDR. We also found that DRD1 copurified with Pol V subunits and that RDM1, like DRD1 [3] and DMS3 [7], is required for the production of Pol V-dependent transcripts. These results suggest that the DDR complex acts in RdDM at a step upstream of the recruitment or activation of Pol V. © 2010 Elsevier Ltd. All rights reserved.

  9. A Protein Complex Required for Polymerase V Transcripts and RNA- Directed DNA Methylation in Arabidopsis

    KAUST Repository

    Law, Julie A.

    2010-05-01

    DNA methylation is an epigenetic modification associated with gene silencing. In Arabidopsis, DNA methylation is established by DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2), which is targeted by small interfering RNAs through a pathway termed RNA-directed DNA methylation (RdDM) [1, 2]. Recently, RdDM was shown to require intergenic noncoding (IGN) transcripts that are dependent on the Pol V polymerase. These transcripts are proposed to function as scaffolds for the recruitment of downstream RdDM proteins, including DRM2, to loci that produce both siRNAs and IGN transcripts [3]. However, the mechanism(s) through which Pol V is targeted to specific genomic loci remains largely unknown. Through affinity purification of two known RdDM components, DEFECTIVE IN RNA-DIRECTED DNA METHYLATION 1 (DRD1) [4] and DEFECTIVE IN MERISTEM SILENCING 3 (DMS3) [5, 6], we found that they copurify with each other and with a novel protein, RNA-DIRECTED DNA METHYLATION 1 (RDM1), forming a complex we term DDR. We also found that DRD1 copurified with Pol V subunits and that RDM1, like DRD1 [3] and DMS3 [7], is required for the production of Pol V-dependent transcripts. These results suggest that the DDR complex acts in RdDM at a step upstream of the recruitment or activation of Pol V. © 2010 Elsevier Ltd. All rights reserved.

  10. Cooperative activation of cardiac transcription through myocardin bridging of paired MEF2 sites

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, Courtney M. [Univ. of California, San Francisco, CA (United States). Cardiovascular Research Inst.; Hu, Jianxin [Univ. of California, San Francisco, CA (United States). Cardiovascular Research Inst.; Thomas, Reuben [Univ. of California, San Francisco, CA (United States). Gladstone Inst.; Gainous, T. Blair [Univ. of California, San Francisco, CA (United States). Cardiovascular Research Inst.; Celona, Barbara [Univ. of California, San Francisco, CA (United States). Cardiovascular Research Inst.; Sinha, Tanvi [Univ. of California, San Francisco, CA (United States). Cardiovascular Research Inst.; Dickel, Diane E. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Genomics Division; Heidt, Analeah B. [Univ. of California, San Francisco, CA (United States). Cardiovascular Research Inst.; Xu, Shan-Mei [Univ. of California, San Francisco, CA (United States). Cardiovascular Research Inst.; Bruneau, Benoit G. [Univ. of California, San Francisco, CA (United States). Cardiovascular Research Inst.; Univ. of California, San Francisco, CA (United States). Gladstone Inst.; Pollard, Katherine S. [Univ. of California, San Francisco, CA (United States). Gladstone Inst.; Pennacchio, Len A. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Genomics Division; Black, Brian L. [Univ. of California, San Francisco, CA (United States). Cardiovascular Research Inst.; Univ. of California, San Francisco, CA (United States). Dept. of

    2017-03-28

    Enhancers frequently contain multiple binding sites for the same transcription factor. These homotypic binding sites often exhibit synergy, whereby the transcriptional output from two or more binding sites is greater than the sum of the contributions of the individual binding sites alone. Although this phenomenon is frequently observed, the mechanistic basis for homotypic binding site synergy is poorly understood. Here in this paper, we identify a bona fide cardiac-specific Prkaa2 enhancer that is synergistically activated by homotypic MEF2 binding sites. We show that two MEF2 sites in the enhancer function cooperatively due to bridging of the MEF2C-bound sites by the SAP domain-containing co-activator protein myocardin, and we show that paired sites buffer the enhancer from integration site-dependent effects on transcription in vivo. Paired MEF2 sites are prevalent in cardiac enhancers, suggesting that this might be a common mechanism underlying synergy in the control of cardiac gene expression in vivo.

  11. Activating RNAs associate with Mediator to enhance chromatin architecture and transcription.

    Science.gov (United States)

    Lai, Fan; Orom, Ulf A; Cesaroni, Matteo; Beringer, Malte; Taatjes, Dylan J; Blobel, Gerd A; Shiekhattar, Ramin

    2013-02-28

    Recent advances in genomic research have revealed the existence of a large number of transcripts devoid of protein-coding potential in multiple organisms. Although the functional role for long non-coding RNAs (lncRNAs) has been best defined in epigenetic phenomena such as X-chromosome inactivation and imprinting, different classes of lncRNAs may have varied biological functions. We and others have identified a class of lncRNAs, termed ncRNA-activating (ncRNA-a), that function to activate their neighbouring genes using a cis-mediated mechanism. To define the precise mode by which such enhancer-like RNAs function, we depleted factors with known roles in transcriptional activation and assessed their role in RNA-dependent activation. Here we report that depletion of the components of the co-activator complex, Mediator, specifically and potently diminished the ncRNA-induced activation of transcription in a heterologous reporter assay using human HEK293 cells. In vivo, Mediator is recruited to ncRNA-a target genes and regulates their expression. We show that ncRNA-a interact with Mediator to regulate its chromatin localization and kinase activity towards histone H3 serine 10. The Mediator complex harbouring disease- displays diminished ability to associate with activating ncRNAs. Chromosome conformation capture confirmed the presence of DNA looping between the ncRNA-a loci and its targets. Importantly, depletion of Mediator subunits or ncRNA-a reduced the chromatin looping between the two loci. Our results identify the human Mediator complex as the transducer of activating ncRNAs and highlight the importance of Mediator and activating ncRNA association in human disease.

  12. The coat protein of Alfalfa mosaic virus interacts and interferes with the transcriptional activity of the bHLH transcription factor ILR3 promoting salicylic acid-dependent defence signalling response.

    Science.gov (United States)

    Aparicio, Frederic; Pallás, Vicente

    2017-02-01

    During virus infection, specific viral component-host factor interaction elicits the transcriptional reprogramming of diverse cellular pathways. Alfalfa mosaic virus (AMV) can establish a compatible interaction in tobacco and Arabidopsis hosts. We show that the coat protein (CP) of AMV interacts directly with transcription factor (TF) ILR3 of both species. ILR3 is a basic helix-loop-helix (bHLH) family member of TFs, previously proposed to participate in diverse metabolic pathways. ILR3 has been shown to regulate NEET in Arabidopsis, a critical protein in plant development, senescence, iron metabolism and reactive oxygen species (ROS) homeostasis. We show that the AMV CP-ILR3 interaction causes a fraction of this TF to relocate from the nucleus to the nucleolus. ROS, pathogenesis-related protein 1 (PR1) mRNAs, salicylic acid (SA) and jasmonic acid (JA) contents are increased in healthy Arabidopsis loss-of-function ILR3 mutant (ilr3.2) plants, which implicates ILR3 in the regulation of plant defence responses. In AMV-infected wild-type (wt) plants, NEET expression is reduced slightly, but is induced significantly in ilr3.2 mutant plants. Furthermore, the accumulation of SA and JA is induced in Arabidopsis wt-infected plants. AMV infection in ilr3.2 plants increases JA by over 10-fold, and SA is reduced significantly, indicating an antagonist crosstalk effect. The accumulation levels of viral RNAs are decreased significantly in ilr3.2 mutants, but the virus can still systemically invade the plant. The AMV CP-ILR3 interaction may down-regulate a host factor, NEET, leading to the activation of plant hormone responses to obtain a hormonal equilibrium state, where infection remains at a level that does not affect plant viability. © 2016 BSPP AND JOHN WILEY & SONS LTD.

  13. Redefining the transcriptional regulatory dynamics of classically and alternatively activated macrophages by deepCAGE transcriptomics

    KAUST Repository

    Roy, S.

    2015-06-27

    Classically or alternatively activated macrophages (M1 and M2, respectively) play distinct and important roles for microbiocidal activity, regulation of inflammation and tissue homeostasis. Despite this, their transcriptional regulatory dynamics are poorly understood. Using promoter-level expression profiling by non-biased deepCAGE we have studied the transcriptional dynamics of classically and alternatively activated macrophages. Transcription factor (TF) binding motif activity analysis revealed four motifs, NFKB1_REL_RELA, IRF1,2, IRF7 and TBP that are commonly activated but have distinct activity dynamics in M1 and M2 activation. We observe matching changes in the expression profiles of the corresponding TFs and show that only a restricted set of TFs change expression. There is an overall drastic and transient up-regulation in M1 and a weaker and more sustainable up-regulation in M2. Novel TFs, such as Thap6, Maff, (M1) and Hivep1, Nfil3, Prdm1, (M2) among others, were suggested to be involved in the activation processes. Additionally, 52 (M1) and 67 (M2) novel differentially expressed genes and, for the first time, several differentially expressed long non-coding RNA (lncRNA) transcriptome markers were identified. In conclusion, the finding of novel motifs, TFs and protein-coding and lncRNA genes is an important step forward to fully understand the transcriptional machinery of macrophage activation.

  14. Neurotoxocarosis alters myelin protein gene transcription and expression.

    Science.gov (United States)

    Heuer, Lea; Beyerbach, Martin; Lühder, Fred; Beineke, Andreas; Strube, Christina

    2015-06-01

    Neurotoxocarosis is an infection of the central nervous system caused by migrating larvae of the common dog and cat roundworms (Toxocara canis and Toxocara cati), which are zoonotic agents. As these parasites are prevalent worldwide and neuropathological and molecular investigations on neurotoxocarosis are scare, this study aims to characterise nerve fibre demyelination associated with neurotoxocarosis on a molecular level. Transcription of eight myelin-associated genes (Cnp, Mag, Mbp, Mog, Mrf-1, Nogo-A, Plp1, Olig2) was determined in the mouse model during six time points of the chronic phase of infection using qRT-PCR. Expression of selected proteins was analysed by Western blotting or immunohistochemistry. Additionally, demyelination and neuronal damage were investigated histologically. Significant differences (p ≤ 0.05) between transcription rates of T. canis-infected and uninfected control mice were detected for all analysed genes while T. cati affected five of eight investigated genes. Interestingly, 2', 3 ´-cyclic nucleotide 3'-phosphodiesterase (Cnp) and myelin oligodendrocyte glycoprotein (Mog) were upregulated in both T. canis- and T. cati-infected mice preceding demyelination. Later, CNPase expression was additionally enhanced. As expected, myelin basic protein (Mbp) was downregulated in cerebra and cerebella of T. canis-infected mice when severe demyelination was present 120 days post infectionem (dpi). The transcriptional pattern observed in the present study appears to reflect direct traumatic and hypoxic effects of larval migration as well as secondary processes including host immune reactions, demyelination and attempts to remyelinate damaged areas.

  15. Peroxisome proliferator-activated receptor gamma recruits the positive transcription elongation factor b complex to activate transcription and promote adipogenesis

    DEFF Research Database (Denmark)

    Iankova, Irena; Petersen, Rasmus K; Annicotte, Jean-Sébastien

    2006-01-01

    Positive transcription elongation factor b (P-TEFb) phosphorylates the C-terminal domain of RNA polymerase II, facilitating transcriptional elongation. In addition to its participation in general transcription, P-TEFb is recruited to specific promoters by some transcription factors such as c......-Myc or MyoD. The P-TEFb complex is composed of a cyclin-dependent kinase (cdk9) subunit and a regulatory partner (cyclin T1, cyclin T2, or cyclin K). Because cdk9 has been shown to participate in differentiation processes, such as muscle cell differentiation, we studied a possible role of cdk9...... with and phosphorylation of peroxisome proliferator-activated receptor gamma (PPARgamma), which is the master regulator of this process, on the promoter of PPARgamma target genes. PPARgamma-cdk9 interaction results in increased transcriptional activity of PPARgamma and therefore increased adipogenesis....

  16. Ctr9, a Protein in the Transcription Complex Paf1, Regulates Dopamine Transporter Activity at the Plasma Membrane.

    Science.gov (United States)

    De Gois, Stéphanie; Slama, Patrick; Pietrancosta, Nicolas; Erdozain, Amaia M; Louis, Franck; Bouvrais-Veret, Caroline; Daviet, Laurent; Giros, Bruno

    2015-07-17

    Dopamine (DA) is a major regulator of sensorimotor and cognitive functions. The DA transporter (DAT) is the key protein that regulates the spatial and temporal activity of DA release into the synaptic cleft via the rapid reuptake of DA into presynaptic termini. Several lines of evidence have suggested that transporter-interacting proteins may play a role in DAT function and regulation. Here, we identified the tetratricopeptide repeat domain-containing protein Ctr9 as a novel DAT binding partner using a yeast two-hybrid system. We showed that Ctr9 is expressed in dopaminergic neurons and forms a stable complex with DAT in vivo via GST pulldown and co-immunoprecipitation assays. In mammalian cells co-expressing both proteins, Ctr9 partially colocalizes with DAT at the plasma membrane. This interaction between DAT and Ctr9 results in a dramatic enhancement of DAT-mediated DA uptake due to an increased number of DAT transporters at the plasma membrane. We determined that the binding of Ctr9 to DAT requires residues YKF in the first half of the DAT C terminus. In addition, we characterized Ctr9, providing new insight into this protein. Using three-dimensional modeling, we identified three novel tetratricopeptide repeat domains in the Ctr9 sequence, and based on deletion mutation experiments, we demonstrated the role of the SH2 domain of Ctr9 in nuclear localization. Our results demonstrate that Ctr9 localization is not restricted to the nucleus, as previously described for the transcription complex Paf1. Taken together, our data provide evidence that Ctr9 modulates DAT function by regulating its trafficking. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Identification of DNA-binding sites for the activator involved in late transcription of the temperate lactococcal phage TP901-1

    DEFF Research Database (Denmark)

    Pedersen, Margit; Kilstrup, Mogens; Hammer, Karin

    2006-01-01

    Alt, encoded by the lactococcal phage TP901-1, is needed for late transcription. We identify Alt as a DNA-binding protein, and footprint analysis shows that Alt binds to a region containing four imperfect direct repeats (ALT boxes) located -76 to -32 relative to the P-late transcriptional start...... site. The importance of the ALT boxes was confirmed by deletion of one or two ALT boxes and by introducing mutations in ALT boxes 1 and 4. Alt is proposed to act as a tetramer or higher multimer activating transcription of TP901-1 late genes by binding to the four ALT boxes, and bending of the DNA may...... be important for transcriptional activation of P-late. Furthermore, our results suggest that DNA replication may be required for late transcription in TP901-1. Additionally, we identify gp28 of the related lactococcal phage Tuc2009 as an activator and show that the activators required for late transcription...

  18. A transcription activator-like effector (TALE) induction system mediated by proteolysis.

    Science.gov (United States)

    Copeland, Matthew F; Politz, Mark C; Johnson, Charles B; Markley, Andrew L; Pfleger, Brian F

    2016-04-01

    Simple and predictable trans-acting regulatory tools are needed in the fields of synthetic biology and metabolic engineering to build complex genetic circuits and optimize the levels of native and heterologous gene products. Transcription activator-like effectors (TALEs) are bacterial virulence factors that have recently gained traction in biotechnology applications owing to their customizable DNA-binding specificity. In this work we expanded the versatility of these transcription factors to create an inducible TALE system by inserting tobacco-etch virus (TEV) protease recognition sites into the TALE backbone. The resulting engineered TALEs maintain transcriptional repression of their target genes in Escherichia coli, but are degraded after induction of the TEV protease, thereby promoting expression of the previously repressed target gene of interest. This TALE-TEV technology enables both repression and induction of plasmid or chromosomal target genes in a manner analogous to traditional repressor proteins but with the added flexibility of being operator-agnostic.

  19. Protein Phosphatase 1-Dependent Transcriptional Programs for Long-Term Memory and Plasticity

    Science.gov (United States)

    Graff, Johannes; Koshibu, Kyoko; Jouvenceau, Anne; Dutar, Patrick; Mansuy, Isabelle M.

    2010-01-01

    Gene transcription is essential for the establishment and the maintenance of long-term memory (LTM) and for long-lasting forms of synaptic plasticity. The molecular mechanisms that control gene transcription in neuronal cells are complex and recruit multiple signaling pathways in the cytoplasm and the nucleus. Protein kinases (PKs) and…

  20. Rift valley fever virus nonstructural protein NSs promotes viral RNA replication and transcription in a minigenome system.

    Science.gov (United States)

    Ikegami, Tetsuro; Peters, C J; Makino, Shinji

    2005-05-01

    Rift Valley fever virus (RVFV), which belongs to the genus Phlebovirus, family Bunyaviridae, has a tripartite negative-strand genome (S, M, and L segments) and is an important mosquito-borne pathogen for domestic animals and humans. We established an RVFV T7 RNA polymerase-driven minigenome system in which T7 RNA polymerase from an expression plasmid drove expression of RNA transcripts for viral proteins and minigenome RNA transcripts carrying a reporter gene between both termini of the M RNA segment in 293T cells. Like other viruses of the Bunyaviridae family, replication and transcription of the RVFV minigenome required expression of viral N and L proteins. Unexpectedly, the coexpression of an RVFV nonstructural protein, NSs, with N and L proteins resulted in a significant enhancement of minigenome RNA replication. Coexpression of NSs protein with N and L proteins also enhanced minigenome mRNA transcription in the cells expressing viral-sense minigenome RNA transcripts. NSs protein expression increased the RNA replication of minigenomes that originated from S and L RNA segments. Enhancement of minigenome RNA synthesis by NSs protein occurred in cells lacking alpha/beta interferon (IFN-alpha/beta) genes, indicating that the effect of NSs protein on minigenome RNA replication was unrelated to a putative NSs protein-induced inhibition of IFN-alpha/beta production. Our finding that RVFV NSs protein augmented minigenome RNA synthesis was in sharp contrast to reports that Bunyamwera virus (genus Bunyavirus) NSs protein inhibits viral minigenome RNA synthesis, suggesting that RVFV NSs protein and Bunyamwera virus NSs protein have distinctly different biological roles in viral RNA synthesis.

  1. Structure of noncoding RNA is a determinant of function of RNA binding proteins in transcriptional regulation

    Directory of Open Access Journals (Sweden)

    Oyoshi Takanori

    2012-01-01

    Full Text Available Abstract The majority of the noncoding regions of mammalian genomes have been found to be transcribed to generate noncoding RNAs (ncRNAs, resulting in intense interest in their biological roles. During the past decade, numerous ncRNAs and aptamers have been identified as regulators of transcription. 6S RNA, first described as a ncRNA in E. coli, mimics an open promoter structure, which has a large bulge with two hairpin/stalk structures that regulate transcription through interactions with RNA polymerase. B2 RNA, which has stem-loops and unstructured single-stranded regions, represses transcription of mRNA in response to various stresses, including heat shock in mouse cells. The interaction of TLS (translocated in liposarcoma with CBP/p300 was induced by ncRNAs that bind to TLS, and this in turn results in inhibition of CBP/p300 histone acetyltransferase (HAT activity in human cells. Transcription regulator EWS (Ewing's sarcoma, which is highly related to TLS, and TLS specifically bind to G-quadruplex structures in vitro. The carboxy terminus containing the Arg-Gly-Gly (RGG repeat domains in these proteins are necessary for cis-repression of transcription activation and HAT activity by the N-terminal glutamine-rich domain. Especially, the RGG domain in the carboxy terminus of EWS is important for the G-quadruplex specific binding. Together, these data suggest that functions of EWS and TLS are modulated by specific structures of ncRNAs.

  2. Chimeras taking shape: Potential functions of proteins encoded by chimeric RNA transcripts

    Science.gov (United States)

    Frenkel-Morgenstern, Milana; Lacroix, Vincent; Ezkurdia, Iakes; Levin, Yishai; Gabashvili, Alexandra; Prilusky, Jaime; del Pozo, Angela; Tress, Michael; Johnson, Rory; Guigo, Roderic; Valencia, Alfonso

    2012-01-01

    Chimeric RNAs comprise exons from two or more different genes and have the potential to encode novel proteins that alter cellular phenotypes. To date, numerous putative chimeric transcripts have been identified among the ESTs isolated from several organisms and using high throughput RNA sequencing. The few corresponding protein products that have been characterized mostly result from chromosomal translocations and are associated with cancer. Here, we systematically establish that some of the putative chimeric transcripts are genuinely expressed in human cells. Using high throughput RNA sequencing, mass spectrometry experimental data, and functional annotation, we studied 7424 putative human chimeric RNAs. We confirmed the expression of 175 chimeric RNAs in 16 human tissues, with an abundance varying from 0.06 to 17 RPKM (Reads Per Kilobase per Million mapped reads). We show that these chimeric RNAs are significantly more tissue-specific than non-chimeric transcripts. Moreover, we present evidence that chimeras tend to incorporate highly expressed genes. Despite the low expression level of most chimeric RNAs, we show that 12 novel chimeras are translated into proteins detectable in multiple shotgun mass spectrometry experiments. Furthermore, we confirm the expression of three novel chimeric proteins using targeted mass spectrometry. Finally, based on our functional annotation of exon organization and preserved domains, we discuss the potential features of chimeric proteins with illustrative examples and suggest that chimeras significantly exploit signal peptides and transmembrane domains, which can alter the cellular localization of cognate proteins. Taken together, these findings establish that some chimeric RNAs are translated into potentially functional proteins in humans. PMID:22588898

  3. Leaderless Transcripts and Small Proteins Are Common Features of the Mycobacterial Translational Landscape.

    Directory of Open Access Journals (Sweden)

    Scarlet S Shell

    2015-11-01

    Full Text Available RNA-seq technologies have provided significant insight into the transcription networks of mycobacteria. However, such studies provide no definitive information on the translational landscape. Here, we use a combination of high-throughput transcriptome and proteome-profiling approaches to more rigorously understand protein expression in two mycobacterial species. RNA-seq and ribosome profiling in Mycobacterium smegmatis, and transcription start site (TSS mapping and N-terminal peptide mass spectrometry in Mycobacterium tuberculosis, provide complementary, empirical datasets to examine the congruence of transcription and translation in the Mycobacterium genus. We find that nearly one-quarter of mycobacterial transcripts are leaderless, lacking a 5' untranslated region (UTR and Shine-Dalgarno ribosome-binding site. Our data indicate that leaderless translation is a major feature of mycobacterial genomes and is comparably robust to leadered initiation. Using translational reporters to systematically probe the cis-sequence requirements of leaderless translation initiation in mycobacteria, we find that an ATG or GTG at the mRNA 5' end is both necessary and sufficient. This criterion, together with our ribosome occupancy data, suggests that mycobacteria encode hundreds of small, unannotated proteins at the 5' ends of transcripts. The conservation of small proteins in both mycobacterial species tested suggests that some play important roles in mycobacterial physiology. Our translational-reporter system further indicates that mycobacterial leadered translation initiation requires a Shine Dalgarno site in the 5' UTR and that ATG, GTG, TTG, and ATT codons can robustly initiate translation. Our combined approaches provide the first comprehensive view of mycobacterial gene structures and their non-canonical mechanisms of protein expression.

  4. Regulatory mechanisms for 3'-end alternative splicing and polyadenylation of the Glial Fibrillary Acidic Protein, GFAP, transcript

    DEFF Research Database (Denmark)

    Blechingberg, Jenny; Lykke-Andersen, Søren; Jensen, Torben Heick

    2007-01-01

    (PTB) protein enhanced both exon 7a polyadenylation and exon 7a splicing. Finally, increasing transcription by the VP16 trans-activator did not affect the frequency of use of the exon 7a polyadenylation signal whereas the exon 7a splicing frequency was decreased. Our data suggest a model...

  5. Butyrate transcriptionally enhances peptide transporter PepT1 expression and activity.

    Directory of Open Access Journals (Sweden)

    Guillaume Dalmasso

    Full Text Available BACKGROUND: PepT1, an intestinal epithelial apical di/tripeptide transporter, is normally expressed in the small intestine and induced in colon during chronic inflammation. This study aimed at investigating PepT1 regulation by butyrate, a short-chain fatty acid produced by commensal bacteria and accumulated inside inflamed colonocyte. RESULTS: We found that butyrate treatment of human intestinal epithelial Caco2-BBE cells increased human PepT1 (hPepT1 promoter activity in a dose- and time-dependent manner, with maximal activity observed in cells treated with 5 mM butyrate for 24 h. Under this condition, hPepT1 promoter activity, mRNA and protein expression levels were increased as assessed by luciferase assay, real-time RT-PCR and Western blot, respectively. hPepT1 transport activity was accordingly increased by approximately 2.5-fold. Butyrate did not alter hPepT1 mRNA half-life indicating that butyrate acts at the transcriptional level. Molecular analyses revealed that Cdx2 is the most important transcription factor for butyrate-induced increase of hPepT1 expression and activity in Caco2-BBE cells. Butyrate-activated Cdx2 binding to hPepT1 promoter was confirmed by gel shift and chromatin immunoprecipitation. Moreover, Caco2-BBE cells overexpressing Cdx2 exhibited greater hPepT1 expression level than wild-type cells. Finally, treatment of mice with 5 mM butyrate added to drinking water for 24 h increased colonic PepT1 mRNA and protein expression levels, as well as enhanced PepT1 transport activity in colonic apical membranes vesicles. CONCLUSIONS: Collectively, our results demonstrate that butyrate increases PepT1 expression and activity in colonic epithelial cells, which provides a new understanding of PepT1 regulation during chronic inflammation.

  6. Transcriptional switches in the control of macronutrient metabolism.

    Science.gov (United States)

    Wise, Alan

    2008-06-01

    This review shows how some transcription factors respond to alterations in macronutrients. Carbohydrates induce enzymes for their metabolism and fatty acid synthesis. Fatty acids reduce carbohydrate processing, induce enzymes for their metabolism, and increase both gluconeogenesis and storage of fat. Fat stores help control carbohydrate uptake by other cells. The following main transcription factors are discussed: carbohydrate response element-binding protein; sterol regulatory element-binding protein-1c, cyclic AMP response element-binding protein, peroxisome proliferator-activated receptor-alpha, and peroxisome proliferator-activated receptor-gamma.

  7. The intracellular immune receptor Rx1 regulates the DNA-binding activity of a Golden2-like transcription factor.

    Science.gov (United States)

    Townsend, Philip D; Dixon, Christopher H; Slootweg, Erik J; Sukarta, Octavina C A; Yang, Ally W H; Hughes, Timothy R; Sharples, Gary J; Pålsson, Lars-Olof; Takken, Frank L W; Goverse, Aska; Cann, Martin J

    2018-03-02

    Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable the immune system to recognize and respond to pathogen attack. An early consequence of immune activation is transcriptional reprogramming, and some NLRs have been shown to act in the nucleus and interact with transcription factors. The Rx1 NLR protein of potato is further able to bind and distort double-stranded DNA. However, Rx1 host targets that support a role for Rx1 in transcriptional reprogramming at DNA are unknown. Here, we report a functional interaction between Rx1 and Nb Glk1, a Golden2-like transcription factor. Rx1 binds to Nb Glk1 in vitro and in planta. Nb Glk1 binds to known Golden2-like consensus DNA sequences. Rx1 reduces the binding affinity of Nb Glk1 for DNA in vitro. Nb Glk1 activates cellular responses to potato virus X, whereas Rx1 associates with Nb Glk1 and prevents its assembly on DNA in planta unless activated by PVX. This study provides new mechanistic insight into how an NLR can coordinate an immune signaling response at DNA following pathogen perceptions. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Human-Phosphate-Binding-Protein inhibits HIV-1 gene transcription and replication

    Directory of Open Access Journals (Sweden)

    Candolfi Ermanno

    2011-07-01

    Full Text Available Abstract The Human Phosphate-Binding protein (HPBP is a serendipitously discovered lipoprotein that binds phosphate with high affinity. HPBP belongs to the DING protein family, involved in various biological processes like cell cycle regulation. We report that HPBP inhibits HIV-1 gene transcription and replication in T cell line, primary peripherical blood lymphocytes and primary macrophages. We show that HPBP is efficient in naïve and HIV-1 AZT-resistant strains. Our results revealed HPBP as a new and potent anti HIV molecule that inhibits transcription of the virus, which has not yet been targeted by HAART and therefore opens new strategies in the treatment of HIV infection.

  9. Identification of Two Protein-Signaling States Delineating Transcriptionally Heterogeneous Human Medulloblastoma

    Directory of Open Access Journals (Sweden)

    Walderik W. Zomerman

    2018-03-01

    Full Text Available Summary: The brain cancer medulloblastoma consists of different transcriptional subgroups. To characterize medulloblastoma at the phosphoprotein-signaling level, we performed high-throughput peptide phosphorylation profiling on a large cohort of SHH (Sonic Hedgehog, group 3, and group 4 medulloblastomas. We identified two major protein-signaling profiles. One profile was associated with rapid death post-recurrence and resembled MYC-like signaling for which MYC lesions are sufficient but not necessary. The second profile showed enrichment for DNA damage, as well as apoptotic and neuronal signaling. Integrative analysis demonstrated that heterogeneous transcriptional input converges on these protein-signaling profiles: all SHH and a subset of group 3 patients exhibited the MYC-like protein-signaling profile; the majority of the other group 3 subset and group 4 patients displayed the DNA damage/apoptotic/neuronal signaling profile. Functional analysis of enriched pathways highlighted cell-cycle progression and protein synthesis as therapeutic targets for MYC-like medulloblastoma. : Using peptide phosphorylation profiling, Zomerman et al. identify two medulloblastoma phosphoprotein-signaling profiles that have prognostic value and are potentially targetable. They find that these profiles extend across transcriptome-based subgroup borders. This suggests that diverse genetic information converges on common protein-signaling pathways and highlights protein-signaling as a unique information layer. Keywords: medulloblastoma, protein-signaling, protein synthesis, MYC, TP53, proteome, phosphoproteome

  10. Activation of peroxisome proliferator-activated receptors (PPARs) by their ligands and protein kinase A activators

    Science.gov (United States)

    Lazennec, Gwendal; Canaple, Laurence; Saugy, Damien; Wahli, Walter

    2000-01-01

    The nuclear peroxisome proliferator-activated receptors (PPARs) α, β and γ activate the transcription of multiple genes involved in lipid metabolism. Several natural and synthetic ligands have been identified for each PPAR isotype but little is known about the phosphorylation state of these receptors. We show here that activators of protein kinase A (PKA) can enhance mouse PPAR activity in the absence and the presence of exogenous ligands in transient transfection experiments. The activation function 1 (AF-1) of PPARs was dispensable for transcriptional enhancement, whereas the activation function 2 (AF-2) was required for this effect. We also show that several domains of PPAR can be phosphorylated by PKA in vitro. Moreover, gel experiments suggest that PKA stabilizes binding of the liganded PPAR to DNA. PKA inhibitors decreased not only the kinase dependent induction of PPARs but also their ligand-dependent induction, suggesting that the ligands may also mobilize the PKA pathway to lead to maximal transcriptional induction by PPARs. Moreover, comparing PPARα KO with PPARα wild-type mice, we show that the expression of the ACO gene can be regulated by PKA-activated PPARα in liver. These data demonstrate that the PKA pathway is an important modulator of PPAR activity and we propose a model associating this pathway in the control of fatty acid β-oxidation under conditions of fasting, stress and exercise. PMID:11117527

  11. Transcriptional regulation of hepatic lipogenesis.

    Science.gov (United States)

    Wang, Yuhui; Viscarra, Jose; Kim, Sun-Joong; Sul, Hei Sook

    2015-11-01

    Fatty acid and fat synthesis in the liver is a highly regulated metabolic pathway that is important for very low-density lipoprotein (VLDL) production and thus energy distribution to other tissues. Having common features at their promoter regions, lipogenic genes are coordinately regulated at the transcriptional level. Transcription factors, such as upstream stimulatory factors (USFs), sterol regulatory element-binding protein 1C (SREBP1C), liver X receptors (LXRs) and carbohydrate-responsive element-binding protein (ChREBP) have crucial roles in this process. Recently, insights have been gained into the signalling pathways that regulate these transcription factors. After feeding, high blood glucose and insulin levels activate lipogenic genes through several pathways, including the DNA-dependent protein kinase (DNA-PK), atypical protein kinase C (aPKC) and AKT-mTOR pathways. These pathways control the post-translational modifications of transcription factors and co-regulators, such as phosphorylation, acetylation or ubiquitylation, that affect their function, stability and/or localization. Dysregulation of lipogenesis can contribute to hepatosteatosis, which is associated with obesity and insulin resistance.

  12. The DNA damage- and transcription-associated protein Paxip1 controls thymocyte development and emigration

    DEFF Research Database (Denmark)

    Callen, E.; Faryabi, R.B.; Daniel, Jeremy Austin

    2012-01-01

    Histone 3 lysine 4 trimethylation (H3K4me3) is associated with promoters of active genes and found at hot spots for DNA recombination. Here we have shown that PAXIP1 (also known as PTIP), a protein associated with MLL3 and MLL4 methyltransferase and the DNA damage response, regulates RAG......-mediated cleavage and repair during V(D)J recombination in CD4 CD8 DP thymocytes. Loss of PAXIP1 in developing thymocytes diminished Jα H3K4me3 and germline transcription, suppressed double strand break formation at 3' Jα segments, but resulted in accumulation of unresolved T cell receptor α-chain gene (Tcra......) breaks. Moreover, PAXIP1 was essential for release of mature single positive (SP) αβ T cells from the thymus through transcriptional activation of sphingosine-1-phosphate receptor S1pr1 as well as for natural killer T cell development. Thus, in addition to maintaining genome integrity during Tcra...

  13. Age-dependent regulation of ERF-VII transcription factor activity in Arabidopsis thaliana.

    Science.gov (United States)

    Giuntoli, Beatrice; Shukla, Vinay; Maggiorelli, Federica; Giorgi, Federico M; Lombardi, Lara; Perata, Pierdomenico; Licausi, Francesco

    2017-10-01

    The Group VII Ethylene Responsive Factors (ERFs-VII) RAP2.2 and RAP2.12 have been mainly characterized with regard to their contribution as activators of fermentation in plants. However, transcriptional changes measured in conditions that stabilize these transcription factors exceed the mere activation of this biochemical pathway, implying additional roles performed by the ERF-VIIs in other processes. We evaluated gene expression in transgenic Arabidopsis lines expressing a stabilized form of RAP2.12, or hampered in ERF-VII activity, and identified genes affected by this transcriptional regulator and its homologs, including some involved in oxidative stress response, which are not universally induced under anaerobic conditions. The contribution of the ERF-VIIs in regulating this set of genes in response to chemically induced or submergence-stimulated mitochondria malfunctioning was found to depend on the plant developmental stage. A similar age-dependent mechanism also restrained ERF-VII activity upon the core-hypoxic genes, independently of the N-end rule pathway, which is accounted for the control of the anaerobic response. To conclude, this study shed new light on a dual role of ERF-VII proteins under submergence: as positive regulators of the hypoxic response and as repressors of oxidative-stress related genes, depending on the developmental stage at which plants are challenged by stress conditions. © 2017 John Wiley & Sons Ltd.

  14. Protein intrinsic disorder in Arabidopsis NAC transcription factors

    DEFF Research Database (Denmark)

    O'Shea, Charlotte; Jensen, Mikael Kryger; Stender, Emil G.P.

    2015-01-01

    of differences in binding mechanisms. Although substitution of both hydrophobic and acidic residues of the ANAC046 MoRF region abolished binding, substitution of other residues, even with α-helix-breaking proline, was less disruptive. Together, the biophysical analyses suggest that RCD1-ANAC046 complex formation......Protein ID (intrinsic disorder) plays a significant, yet relatively unexplored role in transcription factors (TFs). In the present paper, analysis of the transcription regulatory domains (TRDs) of six phylogenetically representative, plant-specific NAC [no apical meristem, ATAF (Arabidopsis...

  15. Sialotranscriptomics of Rhipicephalus zambeziensis reveals intricate expression profiles of secretory proteins and suggests tight temporal transcriptional regulation during blood-feeding.

    Science.gov (United States)

    de Castro, Minique Hilda; de Klerk, Daniel; Pienaar, Ronel; Rees, D Jasper G; Mans, Ben J

    2017-08-10

    Ticks secrete a diverse mixture of secretory proteins into the host to evade its immune response and facilitate blood-feeding, making secretory proteins attractive targets for the production of recombinant anti-tick vaccines. The largely neglected tick species, Rhipicephalus zambeziensis, is an efficient vector of Theileria parva in southern Africa but its available sequence information is limited. Next generation sequencing has advanced sequence availability for ticks in recent years and has assisted the characterisation of secretory proteins. This study focused on the de novo assembly and annotation of the salivary gland transcriptome of R. zambeziensis and the temporal expression of secretory protein transcripts in female and male ticks, before the onset of feeding and during early and late feeding. The sialotranscriptome of R. zambeziensis yielded 23,631 transcripts from which 13,584 non-redundant proteins were predicted. Eighty-six percent of these contained a predicted start and stop codon and were estimated to be putatively full-length proteins. A fifth (2569) of the predicted proteins were annotated as putative secretory proteins and explained 52% of the expression in the transcriptome. Expression analyses revealed that 2832 transcripts were differentially expressed among feeding time points and 1209 between the tick sexes. The expression analyses further indicated that 57% of the annotated secretory protein transcripts were differentially expressed. Dynamic expression profiles of secretory protein transcripts were observed during feeding of female ticks. Whereby a number of transcripts were upregulated during early feeding, presumably for feeding site establishment and then during late feeding, 52% of these were downregulated, indicating that transcripts were required at specific feeding stages. This suggested that secretory proteins are under stringent transcriptional regulation that fine-tunes their expression in salivary glands during feeding. No open

  16. Alterations in leukocyte transcriptional control pathway activity associated with major depressive disorder and antidepressant treatment.

    Science.gov (United States)

    Mellon, S H; Wolkowitz, O M; Schonemann, M D; Epel, E S; Rosser, R; Burke, H B; Mahan, L; Reus, V I; Stamatiou, D; Liew, C-C; Cole, S W

    2016-05-24

    Major depressive disorder (MDD) is associated with a significantly elevated risk of developing serious medical illnesses such as cardiovascular disease, immune impairments, infection, dementia and premature death. Previous work has demonstrated immune dysregulation in subjects with MDD. Using genome-wide transcriptional profiling and promoter-based bioinformatic strategies, we assessed leukocyte transcription factor (TF) activity in leukocytes from 20 unmedicated MDD subjects versus 20 age-, sex- and ethnicity-matched healthy controls, before initiation of antidepressant therapy, and in 17 of the MDD subjects after 8 weeks of sertraline treatment. In leukocytes from unmedicated MDD subjects, bioinformatic analysis of transcription control pathway activity indicated an increased transcriptional activity of cAMP response element-binding/activating TF (CREB/ATF) and increased activity of TFs associated with cellular responses to oxidative stress (nuclear factor erythroid-derived 2-like 2, NFE2l2 or NRF2). Eight weeks of antidepressant therapy was associated with significant reductions in Hamilton Depression Rating Scale scores and reduced activity of NRF2, but not in CREB/ATF activity. Several other transcriptional regulation pathways, including the glucocorticoid receptor (GR), nuclear factor kappa-B cells (NF-κB), early growth response proteins 1-4 (EGR1-4) and interferon-responsive TFs, showed either no significant differences as a function of disease or treatment, or activities that were opposite to those previously hypothesized to be involved in the etiology of MDD or effective treatment. Our results suggest that CREB/ATF and NRF2 signaling may contribute to MDD by activating immune cell transcriptome dynamics that ultimately influence central nervous system (CNS) motivational and affective processes via circulating mediators.

  17. Acetylation Increases EWS-FLI1 DNA Binding and Transcriptional Activity

    International Nuclear Information System (INIS)

    Schlottmann, Silke; Erkizan, Hayriye V.; Barber-Rotenberg, Julie S.; Knights, Chad; Cheema, Amrita; Üren, Aykut; Avantaggiati, Maria L.; Toretsky, Jeffrey A.

    2012-01-01

    Ewing Sarcoma (ES) is associated with a balanced chromosomal translocation that in most cases leads to the expression of the oncogenic fusion protein and transcription factor EWS-FLI1. EWS-FLI1 has been shown to be crucial for ES cell survival and tumor growth. However, its regulation is still enigmatic. To date, no functionally significant post-translational modifications of EWS-FLI1 have been shown. Since ES are sensitive to histone deacetylase inhibitors (HDI), and these inhibitors are advancing in clinical trials, we sought to identify if EWS-FLI1 is directly acetylated. We convincingly show acetylation of the C-terminal FLI1 (FLI1-CTD) domain, which is the DNA binding domain of EWS-FLI1. In vitro acetylation studies showed that acetylated FLI1-CTD has higher DNA binding activity than the non-acetylated protein. Over-expression of PCAF or treatment with HDI increased the transcriptional activity of EWS-FLI1, when co-expressed in Cos7 cells. However, our data that evaluates the acetylation of full-length EWS-FLI1 in ES cells remains unclear, despite creating acetylation specific antibodies to four potential acetylation sites. We conclude that EWS-FLI1 may either gain access to chromatin as a result of histone acetylation or undergo regulation by direct acetylation. These data should be considered when patients are treated with HDAC inhibitors. Further investigation of this phenomenon will reveal if this potential acetylation has an impact on tumor response.

  18. Differential Expression of the Activator Protein 1 Transcription Factor Regulates Interleukin-1ß Induction of Interleukin 6 in the Developing Enterocyte.

    Directory of Open Access Journals (Sweden)

    Catherine M Cahill

    Full Text Available The innate immune response is characterized by activation of transcription factors, nuclear factor kappa B and activator protein-1 and their downstream targets, the pro-inflammatory cytokines including interleukin 1β and interleukin 6. Normal development of this response in the intestine is critical to survival of the human neonate and delays can cause the onset of devastating inflammatory diseases such as necrotizing enterocolitis. Previous studies have addressed the role of nuclear factor kappa B in the development of the innate immune response in the enterocyte, however despite its central role in the control of multiple pro-inflammatory cytokine genes, little is known on the role of Activator Protein 1 in this response in the enterocyte. Here we show that the canonical Activator Protein 1 members, cJun and cFos and their upstream kinases JNK and p38 play an essential role in the regulation of interleukin 6 in the immature enterocyte. Our data supports a model whereby the cFos/cJun heterodimer and the more potent cJun homodimer downstream of JNK are replaced by less efficient JunD containing dimers, contributing to the decreased responsiveness to interleukin 1β and decreased interleukin 6 secretion observed in the mature enterocyte. The tissue specific expression of JunB in colonocytes and colon derived tissues together with its ability to repress Interleukin-1β induction of an Interleukin-6 gene reporter in the NCM-460 colonocyte suggests that induction of JunB containing dimers may offer an attractive therapeutic strategy for the control of IL-6 secretion during inflammatory episodes in this area of the intestine.

  19. The human papillomavirus type 11 and 16 E6 proteins modulate the cell-cycle regulator and transcription cofactor TRIP-Br1

    International Nuclear Information System (INIS)

    Gupta, Sanjay; Takhar, Param Parkash S; Degenkolbe, Roland; Heng Koh, Choon; Zimmermann, Holger; Maolin Yang, Christopher; Guan Sim, Khe; I-Hong Hsu, Stephen; Bernard, Hans-Ulrich

    2003-01-01

    The genital human papillomaviruses (HPVs) are a taxonomic group including HPV types that preferentially cause genital and laryngeal warts ('low-risk types'), such as HPV-6 and HPV-11, or cancer of the cervix and its precursor lesions ('high-risk types'), such as HPV-16. The transforming processes induced by these viruses depend on the proteins E5, E6, and E7. Among these oncoproteins, the E6 protein stands out because it supports a particularly large number of functions and interactions with cellular proteins, some of which are specific for the carcinogenic HPVs, while others are shared among low- and high-risk HPVs. Here we report yeast two-hybrid screens with HPV-6 and -11 E6 proteins that identified TRIP-Br1 as a novel cellular target. TRIP-Br1 was recently detected by two research groups, which described two separate functions, namely that of a transcriptional integrator of the E2F1/DP1/RB cell-cycle regulatory pathway (and then named TRIP-Br1), and that of an antagonist of the cyclin-dependent kinase suppression of p16INK4a (and then named p34SEI-1). We observed that TRIP-Br1 interacts with low- and high-risk HPV E6 proteins in yeast, in vitro and in mammalian cell cultures. Transcription activation of a complex consisting of E2F1, DP1, and TRIP-Br1 was efficiently stimulated by both E6 proteins. TRIP-Br1 has an LLG E6 interaction motif, which contributed to the binding of E6 proteins. Apparently, E6 does not promote degradation of TRIP-Br1. Our observations imply that the cell-cycle promoting transcription factor E2F1/DP1 is dually targeted by HPV oncoproteins, namely (i) by interference of the E7 protein with repression by RB, and (ii) by the transcriptional cofactor function of the E6 protein. Our data reveal the natural context of the transcription activator function of E6, which has been predicted without knowledge of the E2F1/DP1/TRIP-Br/E6 complex by studying chimeric constructs, and add a function to the limited number of transforming properties shared

  20. Replicative Stress Induces Intragenic Transcription of the ASE1 Gene that Negatively Regulates Ase1 Activity

    OpenAIRE

    McKnight, Kelly; Liu, Hong; Wang, Yanchang

    2014-01-01

    Intragenic transcripts initiate within the coding region of a gene, thereby producing shorter mRNAs and proteins. Although intragenic transcripts are widely expressed [1], their role in the functional regulation of genes remains largely unknown. In budding yeast, DNA replication stress activates the S-phase checkpoint that stabilizes replication forks and arrests cells in S-phase with a short spindle [2-4]. When yeast cells were treated with hydroxyurea (HU) to block DNA synthesis and induce ...

  1. Residual DNA-bound proteins are a source of in vitro transcription inhibitor peptides

    International Nuclear Information System (INIS)

    Venanzi, F.M.

    1989-01-01

    Enzymatic breakdown of residual proteins occurs at mild alkaline pH (pH optimum 8.5) as monitored by using radioiodinated, purified genomic DNA from calf thymus. These DNA fibers also possess a differential ability to hydrolyze added exogenous small and linker histones. The results described argue strongly that a putative protease activity, co-purified with DNA, is the source of short chain peptides which inhibit transcription in vitro. Therefore, we propose that RNA repressor peptides must be of higher molecular weight than previously reported

  2. The splicing regulator PTBP1 controls the activity of the transcription factor Pbx1 during neuronal differentiation.

    Science.gov (United States)

    Linares, Anthony J; Lin, Chia-Ho; Damianov, Andrey; Adams, Katrina L; Novitch, Bennett G; Black, Douglas L

    2015-12-24

    The RNA-binding proteins PTBP1 and PTBP2 control programs of alternative splicing during neuronal development. PTBP2 was found to maintain embryonic splicing patterns of many synaptic and cytoskeletal proteins during differentiation of neuronal progenitor cells (NPCs) into early neurons. However, the role of the earlier PTBP1 program in embryonic stem cells (ESCs) and NPCs was not clear. We show that PTBP1 controls a program of neuronal gene expression that includes the transcription factor Pbx1. We identify exons specifically regulated by PTBP1 and not PTBP2 as mouse ESCs differentiate into NPCs. We find that PTBP1 represses Pbx1 exon 7 and the expression of the neuronal Pbx1a isoform in ESCs. Using CRISPR-Cas9 to delete regulatory elements for exon 7, we induce Pbx1a expression in ESCs, finding that this activates transcription of neuronal genes. Thus, PTBP1 controls the activity of Pbx1 to suppress its neuronal transcriptional program prior to induction of NPC development.

  3. Dissecting the expression relationships between RNA-binding proteins and their cognate targets in eukaryotic post-transcriptional regulatory networks

    Science.gov (United States)

    Nishtala, Sneha; Neelamraju, Yaseswini; Janga, Sarath Chandra

    2016-05-01

    RNA-binding proteins (RBPs) are pivotal in orchestrating several steps in the metabolism of RNA in eukaryotes thereby controlling an extensive network of RBP-RNA interactions. Here, we employed CLIP (cross-linking immunoprecipitation)-seq datasets for 60 human RBPs and RIP-ChIP (RNP immunoprecipitation-microarray) data for 69 yeast RBPs to construct a network of genome-wide RBP- target RNA interactions for each RBP. We show in humans that majority (~78%) of the RBPs are strongly associated with their target transcripts at transcript level while ~95% of the studied RBPs were also found to be strongly associated with expression levels of target transcripts when protein expression levels of RBPs were employed. At transcript level, RBP - RNA interaction data for the yeast genome, exhibited a strong association for 63% of the RBPs, confirming the association to be conserved across large phylogenetic distances. Analysis to uncover the features contributing to these associations revealed the number of target transcripts and length of the selected protein-coding transcript of an RBP at the transcript level while intensity of the CLIP signal, number of RNA-Binding domains, location of the binding site on the transcript, to be significant at the protein level. Our analysis will contribute to improved modelling and prediction of post-transcriptional networks.

  4. Analysis of transcript and protein overlap in a human osteosarcoma cell line

    Directory of Open Access Journals (Sweden)

    Emanuelsson Olof

    2010-12-01

    Full Text Available Abstract Background An interesting field of research in genomics and proteomics is to compare the overlap between the transcriptome and the proteome. Recently, the tools to analyse gene and protein expression on a whole-genome scale have been improved, including the availability of the new generation sequencing instruments and high-throughput antibody-based methods to analyze the presence and localization of proteins. In this study, we used massive transcriptome sequencing (RNA-seq to investigate the transcriptome of a human osteosarcoma cell line and compared the expression levels with in situ protein data obtained in-situ from antibody-based immunohistochemistry (IHC and immunofluorescence microscopy (IF. Results A large-scale analysis based on 2749 genes was performed, corresponding to approximately 13% of the protein coding genes in the human genome. We found the presence of both RNA and proteins to a large fraction of the analyzed genes with 60% of the analyzed human genes detected by all three methods. Only 34 genes (1.2% were not detected on the transcriptional or protein level with any method. Our data suggest that the majority of the human genes are expressed at detectable transcript or protein levels in this cell line. Since the reliability of antibodies depends on possible cross-reactivity, we compared the RNA and protein data using antibodies with different reliability scores based on various criteria, including Western blot analysis. Gene products detected in all three platforms generally have good antibody validation scores, while those detected only by antibodies, but not by RNA sequencing, generally consist of more low-scoring antibodies. Conclusion This suggests that some antibodies are staining the cells in an unspecific manner, and that assessment of transcript presence by RNA-seq can provide guidance for validation of the corresponding antibodies.

  5. Elk3 from hamster-a ternary complex factor with strong transcriptional repressor activity

    DEFF Research Database (Denmark)

    Hjortoe, G.M.; Weilguny, D.; Willumsen, Berthe Marie

    2005-01-01

    the transcription of genes that are activated during entry into G1. We have isolated the Cricetulus griseus Elk3 gene from the Chinese hamster ovary (CHO) cell line and investigated the transcriptional potential of this factor. Transient transfections revealed that, in addition to its regulation of the c......-fos promoter, Elk3 from CHO cells seems to inhibit other promoters controlling expression of proteins involved in G1/S phase progression; Cyclin D1 and DHFR. As has been described for the Elk3 homologs Net (Mouse) and Sap-2 (Human), the results of the present study further indicate that hamster Elk3...

  6. The central domain of yeast transcription factor Rpn4 facilitates degradation of reporter protein in human cells.

    Science.gov (United States)

    Morozov, A V; Spasskaya, D S; Karpov, D S; Karpov, V L

    2014-10-16

    Despite high interest in the cellular degradation machinery and protein degradation signals (degrons), few degrons with universal activity along species have been identified. It has been shown that fusion of a target protein with a degradation signal from mammalian ornithine decarboxylase (ODC) induces fast proteasomal degradation of the chimera in both mammalian and yeast cells. However, no degrons from yeast-encoded proteins capable to function in mammalian cells were identified so far. Here, we demonstrate that the yeast transcription factor Rpn4 undergoes fast proteasomal degradation and its central domain can destabilize green fluorescent protein and Alpha-fetoprotein in human HEK 293T cells. Furthermore, we confirm the activity of this degron in yeast. Thus, the Rpn4 central domain is an effective interspecies degradation signal. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  7. The cellular transcription factor CREB corresponds to activating transcription factor 47 (ATF-47) and forms complexes with a group of polypeptides related to ATF-43.

    Science.gov (United States)

    Hurst, H C; Masson, N; Jones, N C; Lee, K A

    1990-12-01

    Promoter elements containing the sequence motif CGTCA are important for a variety of inducible responses at the transcriptional level. Multiple cellular factors specifically bind to these elements and are encoded by a multigene family. Among these factors, polypeptides termed activating transcription factor 43 (ATF-43) and ATF-47 have been purified from HeLa cells and a factor referred to as cyclic AMP response element-binding protein (CREB) has been isolated from PC12 cells and rat brain. We demonstrated that CREB and ATF-47 are identical and that CREB and ATF-43 form protein-protein complexes. We also found that the cis requirements for stable DNA binding by ATF-43 and CREB are different. Using antibodies to ATF-43 we have identified a group of polypeptides (ATF-43) in the size range from 40 to 43 kDa. ATF-43 polypeptides are related by their reactivity with anti-ATF-43, DNA-binding specificity, complex formation with CREB, heat stability, and phosphorylation by protein kinase A. Certain cell types vary in their ATF-43 complement, suggesting that CREB activity is modulated in a cell-type-specific manner through interaction with ATF-43. ATF-43 polypeptides do not appear simply to correspond to the gene products of the ATF multigene family, suggesting that the size of the ATF family at the protein level is even larger than predicted from cDNA-cloning studies.

  8. [The role of Smads and related transcription factors in the signal transduction of bone morphogenetic protein inducing bone formation].

    Science.gov (United States)

    Xu, Xiao-liang; Dai, Ke-rong; Tang, Ting-ting

    2003-09-01

    To clarify the mechanisms of the signal transduction of bone morphogenetic proteins (BMPs) inducing bone formation and to provide theoretical basis for basic and applying research of BMPs. We looked up the literature of the role of Smads and related transcription factors in the signal transduction of BMPs inducing bone formation. The signal transduction processes of BMPs included: 1. BMPs combined with type II and type I receptors; 2. the type I receptor phosphorylated Smads; and 3. Smads entered the cell nucleus, interacted with transcription factors and influenced the transcription of related proteins. Smads could be divided into receptor-regulated Smads (R-Smads: Smad1, Smad2, Smad3, Smad5, Smad8 and Smad9), common-mediator Smad (co-Smad: Smad4), and inhibitory Smads (I-Smads: Smad6 and Smad7). Smad1, Smad5, Smad8, and probable Smad9 were involved in the signal transduction of BMPs. Multiple kinases, such as focal adhesion kinase (FAK), Ras-extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3K), and Akt serine/threonine kinase were related to Smads signal transduction. Smad1 and Smad5 related with transcription factors included core binding factor A1 (CBFA1), smad-interacting protein 1 (SIP1), ornithine decarboxylase antizyme (OAZ), activating protein-1 (AP-1), xenopus ventralizing homeobox protein-2 (Xvent-2), sandostatin (Ski), antiproliferative proteins (Tob), and homeodomain-containing transcriptian factor-8 (Hoxc-8), et al. CBFA1 could interact with Smad1, Smad2, Smad3, and Smad5, so it was involved in TGF-beta and BMP-2 signal transduction, and played an important role in the bone formation. Cleidocranial dysplasia (CCD) was thought to be caused by heterozygous mutations in CBFA1. The CBFA1 knockout mice showed no osteogenesis and had maturational disturbance of chondrocytes. Smads and related transcription factors, especially Smad1, Smad5, Smad8 and CBFA1, play an important role in the signal transduction of BMPs inducing bone

  9. Nucleolin modulates the subcellular localization of GDNF-inducible zinc finger protein 1 and its roles in transcription and cell proliferation

    International Nuclear Information System (INIS)

    Dambara, Atsushi; Morinaga, Takatoshi; Fukuda, Naoyuki; Yamakawa, Yoshinori; Kato, Takuya; Enomoto, Atsushi; Asai, Naoya; Murakumo, Yoshiki; Matsuo, Seiichi; Takahashi, Masahide

    2007-01-01

    GZF1 is a zinc finger protein induced by glial cell-line-derived neurotrophic factor (GDNF). It is a sequence-specific transcriptional repressor with a BTB/POZ (Broad complex, Tramtrack, Bric a brac/Poxvirus and zinc finger) domain and ten zinc finger motifs. In the present study, we used immunoprecipitation and mass spectrometry to identify nucleolin as a GZF1-binding protein. Deletion analysis revealed that zinc finger motifs 1-4 of GZF1 mediate its association with nucleolin. When zinc fingers 1-4 were deleted from GZF1 or nucleolin expression was knocked down by short interference RNA (siRNA), nuclear localization of GZF1 was impaired. These results suggest that nucleolin is involved in the proper subcellular distribution of GZF1. In addition, overexpression of nucleolin moderately inhibited the transcriptional repressive activity of GZF1 whereas knockdown of nucleolin expression by siRNA enhanced its activity. Thus, the repressive activity of GZF1 is modulated by the level at which nucleolin is expressed. Finally, we found that knockdown of GZF1 and nucleolin expression markedly impaired cell proliferation. These findings suggest that the physiological functions of GZF1 may be regulated by the protein's association with nucleolin

  10. The regulation of transactivator of transcription on the activity of DNA-PKcs promoter

    International Nuclear Information System (INIS)

    Yang Tianyi; Zhang Shimeng; Qin Xia; Li Bing; Liu Xiaodan; Zhou Pingkun

    2012-01-01

    Objective: To explore the influence of human immunodeficiency virus transactivator of transcription (TAT) on the promoter activity of DNA dependent protein kinase catalytic subunit (DNA-PKcs). Methods: The truncated promoters of DNA-PKcs were cloned by PCR from the template DNA from HeLa genomic DNA, and the pGL3-basic-DNA-PKcs promoter reporter plasmids were constructed. The activity of DNA-PKcs promoters was detected by dual-luciferase reporter assay system. A Lac-repressor and Lacoperator based green fluorescent protein imaging system was used to assay the chromatin remodeling activity. Results: A series of reporter plasmids harboring the truncated promoters of DNA-PKcs from -939 bp to -1 bp were constructed. The sequence of -64 bp to-1 bp was identified as a critical element for the activity of DNA-PKes promoter. TAT can suppress the activity of DNA-PKcs promoter. TAT participates in the regulation of the large scale chromatin relaxation. Ionizing radiation attenuates the activity of TAT played in the chromatin remodeling. Conclusion: TAT represses the promoter activity of DNA repair protein DNA-PKcs, and also play a role of large scale chromatin remodeling which can te attenuated by ionizing radiation. (authors)

  11. Eviction of linker histone H1 by NAP-family histone chaperones enhances activated transcription.

    Science.gov (United States)

    Zhang, Qian; Giebler, Holli A; Isaacson, Marisa K; Nyborg, Jennifer K

    2015-01-01

    In the Metazoan nucleus, core histones assemble the genomic DNA to form nucleosome arrays, which are further compacted into dense chromatin structures by the linker histone H1. The extraordinary density of chromatin creates an obstacle for accessing the genetic information. Regulation of chromatin dynamics is therefore critical to cellular homeostasis, and histone chaperones serve as prominent players in these processes. In the current study, we examined the role of specific histone chaperones in negotiating the inherently repressive chromatin structure during transcriptional activation. Using a model promoter, we demonstrate that the human nucleosome assembly protein family members hNap1 and SET/Taf1β stimulate transcription in vitro during pre-initiation complex formation, prior to elongation. This stimulatory effect is dependent upon the presence of activators, p300, and Acetyl-CoA. We show that transcription from our chromatin template is strongly repressed by H1, and that both histone chaperones enhance RNA synthesis by overcoming H1-induced repression. Importantly, both hNap1 and SET/Taf1β directly bind H1, and function to enhance transcription by evicting the linker histone from chromatin reconstituted with H1. In vivo studies demonstrate that SET/Taf1β, but not hNap1, strongly stimulates activated transcription from the chromosomally-integrated model promoter, consistent with the observation that SET/Taf1β is nuclear, whereas hNap1 is primarily cytoplasmic. Together, these observations indicate that SET/Taf1β may serve as a critical regulator of H1 dynamics and gene activation in vivo. These studies uncover a novel function for SET that mechanistically couples transcriptional derepression with H1 dynamics. Furthermore, they underscore the significance of chaperone-dependent H1 displacement as an essential early step in the transition of a promoter from a dense chromatin state into one that is permissive to transcription factor binding and robust

  12. Nuclear protein IκB-ζ inhibits the activity of STAT3

    International Nuclear Information System (INIS)

    Wu, Zhihao; Zhang, Xiaoai; Yang, Juntao; Wu, Guangzhou; Zhang, Ying; Yuan, Yanzhi; Jin, Chaozhi; Chang, Zhijie; Wang, Jian; Yang, Xiaoming; He, Fuchu

    2009-01-01

    STAT3 (Signal transducer and activator of transcription 3) is a key transcription factor of the JAK-STAT (Janus kinase/signal transducer and activator of transcription) pathway that regulates cell proliferation and apoptosis. Activation of STAT3 is under tight regulation, and yet the different signaling pathways and the mechanisms that regulate its activity remain to be elucidated. Using a yeast two-hybrid screening, we have identified a nuclear protein IκB-ζ that interacts in a novel way with STAT3. This physical interaction was further confirmed by co-immunoprecipitation assays. The interaction regions were mapped to the coiled-coil domain of STAT3 and the C-terminal of IκB-ζ. Overexpression of IκB-ζ inhibited the transcriptional activity of STAT3. It also suppressed cell growth and induced cell apoptosis in SRC-simulated cells, which is partially mediated by down-regulation of expression of a known STAT3 target gene, MCL1. Our results suggest that IκB-ζ is a negative regulator of STAT3, and demonstrate a novel mechanism in which a component of the NF-κB signaling pathway inhibits the activation of STAT3.

  13. HECT E3 Ubiquitin Ligase Itch Functions as a Novel Negative Regulator of Gli-Similar 3 (Glis3 Transcriptional Activity.

    Directory of Open Access Journals (Sweden)

    Gary T ZeRuth

    Full Text Available The transcription factor Gli-similar 3 (Glis3 plays a critical role in the generation of pancreatic ß cells and the regulation insulin gene transcription and has been implicated in the development of several pathologies, including type 1 and 2 diabetes and polycystic kidney disease. However, little is known about the proteins and posttranslational modifications that regulate or mediate Glis3 transcriptional activity. In this study, we identify by mass-spectrometry and yeast 2-hybrid analyses several proteins that interact with the N-terminal region of Glis3. These include the WW-domain-containing HECT E3 ubiquitin ligases, Itch, Smurf2, and Nedd4. The interaction between Glis3 and the HECT E3 ubiquitin ligases was verified by co-immunoprecipitation assays and mutation analysis. All three proteins interact through their WW-domains with a PPxY motif located in the Glis3 N-terminus. However, only Itch significantly contributed to Glis3 polyubiquitination and reduced Glis3 stability by enhancing its proteasomal degradation. Itch-mediated degradation of Glis3 required the PPxY motif-dependent interaction between Glis3 and the WW-domains of Itch as well as the presence of the Glis3 zinc finger domains. Transcription analyses demonstrated that Itch dramatically inhibited Glis3-mediated transactivation and endogenous Ins2 expression by increasing Glis3 protein turnover. Taken together, our study identifies Itch as a critical negative regulator of Glis3-mediated transcriptional activity. This regulation provides a novel mechanism to modulate Glis3-driven gene expression and suggests that it may play a role in a number of physiological processes controlled by Glis3, such as insulin transcription, as well as in Glis3-associated diseases.

  14. Development of Transcriptional Fusions to Assess Leptospira interrogans Promoter Activity

    Science.gov (United States)

    Cerqueira, Gustavo M.; Souza, Natalie M.; Araújo, Eduardo R.; Barros, Aline T.; Morais, Zenaide M.; Vasconcellos, Sílvio A.; Nascimento, Ana L. T. O.

    2011-01-01

    Background Leptospirosis is a zoonotic infectious disease that affects both humans and animals. The existing genetic tools for Leptospira spp. have improved our understanding of the biology of this spirochete as well as the interaction of pathogenic leptospires with the mammalian host. However, new tools are necessary to provide novel and useful information to the field. Methodology and Principal Findings A series of promoter-probe vectors carrying a reporter gene encoding green fluorescent protein (GFP) were constructed for use in L. biflexa. They were tested by constructing transcriptional fusions between the lipL41, Leptospiral Immunoglobulin-like A (ligA) and Sphingomielynase 2 (sph2) promoters from L. interrogans and the reporter gene. ligA and sph2 promoters were the most active, in comparison to the lipL41 promoter and the non-induced controls. The results obtained are in agreement with LigA expression from the L. interrogans Fiocruz L1-130 strain. Conclusions The novel vectors facilitated the in vitro evaluation of L. interrogans promoter activity under defined growth conditions which simulate the mammalian host environment. The fluorescence and rt-PCR data obtained closely reflected transcriptional regulation of the promoters, thus demonstrating the suitability of these vectors for assessing promoter activity in L. biflexa. PMID:21445252

  15. Development of transcriptional fusions to assess Leptospira interrogans promoter activity.

    Directory of Open Access Journals (Sweden)

    Gustavo M Cerqueira

    Full Text Available BACKGROUND: Leptospirosis is a zoonotic infectious disease that affects both humans and animals. The existing genetic tools for Leptospira spp. have improved our understanding of the biology of this spirochete as well as the interaction of pathogenic leptospires with the mammalian host. However, new tools are necessary to provide novel and useful information to the field. METHODOLOGY AND PRINCIPAL FINDINGS: A series of promoter-probe vectors carrying a reporter gene encoding green fluorescent protein (GFP were constructed for use in L. biflexa. They were tested by constructing transcriptional fusions between the lipL41, Leptospiral Immunoglobulin-like A (ligA and Sphingomyelinase 2 (sph2 promoters from L. interrogans and the reporter gene. ligA and sph2 promoters were the most active, in comparison to the lipL41 promoter and the non-induced controls. The results obtained are in agreement with LigA expression from the L. interrogans Fiocruz L1-130 strain. CONCLUSIONS: The novel vectors facilitated the in vitro evaluation of L. interrogans promoter activity under defined growth conditions which simulate the mammalian host environment. The fluorescence and rt-PCR data obtained closely reflected transcriptional regulation of the promoters, thus demonstrating the suitability of these vectors for assessing promoter activity in L. biflexa.

  16. Intracellular delivery of cell-penetrating peptide-transcriptional factor fusion protein and its role in selective osteogenesis

    Science.gov (United States)

    Suh, Jin Sook; Lee, Jue Yeon; Choi, Yoon Jung; You, Hyung Keun; Hong, Seong-Doo; Chung, Chong Pyoung; Park, Yoon Jeong

    2014-01-01

    Protein-transduction technology has been attempted to deliver macromolecular materials, including protein, nucleic acids, and polymeric drugs, for either diagnosis or therapeutic purposes. Herein, fusion protein composed of an arginine-rich cell-penetrating peptide, termed low-molecular-weight protamine (LMWP), and a transcriptional coactivator with a PDZ-binding motif (TAZ) protein was prepared and applied in combination with biomaterials to increase bone-forming capacity. TAZ has been recently identified as a specific osteogenic stimulating transcriptional coactivator in human mesenchymal stem cell (hMSC) differentiation, while simultaneously blocking adipogenic differentiation. However, TAZ by itself cannot penetrate the cells, and thus needs a transfection tool for translocalization. The LMWP-TAZ fusion proteins were efficiently translocalized into the cytosol of hMSCs. The hMSCs treated with cell-penetrating LMWP-TAZ exhibited increased expression of osteoblastic genes and protein, producing significantly higher quantities of mineralized matrix compared to free TAZ. In contrast, adipogenic differentiation of the hMSCs was blocked by treatment of LMWP-TAZ fusion protein, as reflected by reduced marker-protein expression, adipocyte fatty acid-binding protein 2, and peroxisome proliferator-activated receptor-γ messenger ribonucleic acid levels. LMWP-TAZ was applied in alginate gel for the purpose of localization and controlled release. The LMWP-TAZ fusion protein-loaded alginate gel matrix significantly increased bone formation in rabbit calvarial defects compared with alginate gel matrix mixed with free TAZ protein. The protein transduction of TAZ fused with cell-penetrating LMWP peptide was able selectively to stimulate osteogenesis in vitro and in vivo. Taken together, this fusion protein-transduction technology for osteogenic protein can thus be applied in combination with biomaterials for tissue regeneration and controlled release for tissue

  17. Putrescine biosynthesis in Lactococcus lactis is transcriptionally activated at acidic pH and counteracts acidification of the cytosol.

    Science.gov (United States)

    Del Rio, Beatriz; Linares, Daniel; Ladero, Victor; Redruello, Begoña; Fernandez, Maria; Martin, Maria Cruz; Alvarez, Miguel A

    2016-11-07

    Lactococcus lactis subsp. cremoris CECT 8666 is a lactic acid bacterium that synthesizes the biogenic amine putrescine from agmatine via the agmatine deiminase (AGDI) pathway. The AGDI genes cluster includes aguR. This encodes a transmembrane protein that functions as a one-component signal transduction system, the job of which is to sense the agmatine concentration of the medium and accordingly regulate the transcription of the catabolic operon aguBDAC. The latter encodes the proteins necessary for agmatine uptake and its conversion into putrescine. This work reports the effect of extracellular pH on putrescine biosynthesis and on the genetic regulation of the AGDI pathway. Increased putrescine biosynthesis was detected at acidic pH (pH5) compared to neutral pH. Acidic pH induced the transcription of the catabolic operon via the activation of the aguBDAC promoter PaguB. However, the external pH had no significant effect on the activity of the aguR promoter PaguR, or on the transcription of the aguR gene. The transcriptional activation of the AGDI pathway was also found to require a lower agmatine concentration at pH5 than at neutral pH. Finally, the following of the AGDI pathway counteracted the acidification of the cytoplasm under acidic external conditions, suggesting it to provide protection against acid stress. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Intracellular delivery of cell-penetrating peptide-transcriptional factor fusion protein and its role in selective osteogenesis

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    Suh JS

    2014-03-01

    Full Text Available Jin Sook Suh,1,* Jue Yeon Lee,2,* Yoon Jung Choi,1 Hyung Keun You,3 Seong-Doo Hong,4 Chong Pyoung Chung,2 Yoon Jeong Park1,2 1Dental Regenerative Biotechnology, Dental Research Institute, School of Dentistry, Seoul National University, Seoul, 2Central Research Institute, Nano Intelligent Biomedical Engineering Corporation (NIBEC, Seoul, 3Department of Periodontology, College of Dentistry, Wonkwang University, Iksan, 4Department of Oral Pathology, School of Dentistry, Seoul National University, Seoul, Republic of Korea *These authors contributed equally to this work Abstract: Protein-transduction technology has been attempted to deliver macromolecular materials, including protein, nucleic acids, and polymeric drugs, for either diagnosis or therapeutic purposes. Herein, fusion protein composed of an arginine-rich cell-penetrating peptide, termed low-molecular-weight protamine (LMWP, and a transcriptional coactivator with a PDZ-binding motif (TAZ protein was prepared and applied in combination with biomaterials to increase bone-forming capacity. TAZ has been recently identified as a specific osteogenic stimulating transcriptional coactivator in human mesenchymal stem cell (hMSC differentiation, while simultaneously blocking adipogenic differentiation. However, TAZ by itself cannot penetrate the cells, and thus needs a transfection tool for translocalization. The LMWP-TAZ fusion proteins were efficiently translocalized into the cytosol of hMSCs. The hMSCs treated with cell-penetrating LMWP-TAZ exhibited increased expression of osteoblastic genes and protein, producing significantly higher quantities of mineralized matrix compared to free TAZ. In contrast, adipogenic differentiation of the hMSCs was blocked by treatment of LMWP-TAZ fusion protein, as reflected by reduced marker-protein expression, adipocyte fatty acid-binding protein 2, and peroxisome proliferator-activated receptor-γ messenger ribonucleic acid levels. LMWP-TAZ was applied in

  19. Rift Valley fever virus NSs protein promotes post-transcriptional downregulation of protein kinase PKR and inhibits eIF2alpha phosphorylation.

    Science.gov (United States)

    Ikegami, Tetsuro; Narayanan, Krishna; Won, Sungyong; Kamitani, Wataru; Peters, C J; Makino, Shinji

    2009-02-01

    Rift Valley fever virus (RVFV) (genus Phlebovirus, family Bunyaviridae) is a negative-stranded RNA virus with a tripartite genome. RVFV is transmitted by mosquitoes and causes fever and severe hemorrhagic illness among humans, and fever and high rates of abortions in livestock. A nonstructural RVFV NSs protein inhibits the transcription of host mRNAs, including interferon-beta mRNA, and is a major virulence factor. The present study explored a novel function of the RVFV NSs protein by testing the replication of RVFV lacking the NSs gene in the presence of actinomycin D (ActD) or alpha-amanitin, both of which served as a surrogate of the host mRNA synthesis suppression function of the NSs. In the presence of the host-transcriptional inhibitors, the replication of RVFV lacking the NSs protein, but not that carrying NSs, induced double-stranded RNA-dependent protein kinase (PKR)-mediated eukaryotic initiation factor (eIF)2alpha phosphorylation, leading to the suppression of host and viral protein translation. RVFV NSs promoted post-transcriptional downregulation of PKR early in the course of the infection and suppressed the phosphorylated eIF2alpha accumulation. These data suggested that a combination of RVFV replication and NSs-induced host transcriptional suppression induces PKR-mediated eIF2alpha phosphorylation, while the NSs facilitates efficient viral translation by downregulating PKR and inhibiting PKR-mediated eIF2alpha phosphorylation. Thus, the two distinct functions of the NSs, i.e., the suppression of host transcription, including that of type I interferon mRNAs, and the downregulation of PKR, work together to prevent host innate antiviral functions, allowing efficient replication and survival of RVFV in infected mammalian hosts.

  20. Mechano-genetic DNA hydrogels as a simple, reconstituted model to probe the effect of active fluctuations on gene transcription

    Science.gov (United States)

    Nguyen, Dan; Saleh, Omar

    Active fluctuations - non-directed fluctuations attributable, not to thermal energy, but to non-equilibrium processes - are thought to influence biology by increasing the diffusive motion of biomolecules. Dense DNA regions within cells (i.e. chromatin) are expected to exhibit such phenomena, as they are cross-linked networks that continually experience propagating forces arising from dynamic cellular activity. Additional agitation within these gene-encoding DNA networks could have potential genetic consequences. By changing the local mobility of transcriptional machinery and regulatory proteins towards/from their binding sites, and thereby influencing transcription rates, active fluctuations could prove to be a physical means of modulating gene expression. To begin probing this effect, we construct genetic DNA hydrogels, as a simple, reconstituted model of chromatin, and quantify transcriptional output from these hydrogels in the presence/absence of active fluctuations.

  1. High mobility group protein DSP1 negatively regulates HSP70 transcription in Crassostrea hongkongensis

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    Miao, Zongyu; Xu, Delin; Cui, Miao; Zhang, Qizhong, E-mail: zhangqzdr@126.com

    2016-06-10

    HSP70 acts mostly as a molecular chaperone and plays important roles in facilitating the folding of nascent peptides as well as the refolding or degradation of the denatured proteins. Under stressed conditions, the expression level of HSP70 is upregulated significantly and rapidly, as is known to be achieved by various regulatory factors controlling the transcriptional level. In this study, a high mobility group protein DSP1 was identified by DNA-affinity purification from the nuclear extracts of Crassostrea hongkongensis using the ChHSP70 promoter as a bait. The specific interaction between the prokaryotically expressed ChDSP1 and the FITC-labeled ChHSP70 promoter was confirmed by EMSA analysis. ChDSP1 was shown to negatively regulate ChHSP70 promoter expression by Luciferase Reporter Assay in the heterologous HEK293T cells. Both ChHSP70 and ChDSP1 transcriptions were induced by either thermal or CdCl{sub 2} stress, while the accumulated expression peaks of ChDSP1 were always slightly delayed when compared with that of ChHSP70. This indicates that ChDSP1 is involved, very likely to exert its suppressive role, in the recovery of the ChHSP70 expression from the induced level to its original state. This study is the first to report negative regulator of HSP70 gene transcription, and provides novel insights into the mechanisms controlling heat shock protein expression. -- Highlights: •HMG protein ChDSP1 shows affinity to ChHSP70 promoter in Crassostrea hongkongensis. •ChDSP1 negatively regulates ChHSP70 transcription. •ChHSP70 and ChDSP1 transcriptions were coordinately induced by thermal/Cd stress. •ChDSP1 may contribute to the recovery of the induced ChHSP70 to its original state. •This is the first report regarding negative regulator of HSP70 transcription.

  2. Yeast Interacting Proteins Database: YNL216W, YLR453C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available YNL216W RAP1 DNA-binding protein involved in either activation or repression of transcription, depending...NA-binding protein involved in either activation or repression of transcription, depending on binding site c

  3. AMP-activated protein kinase (AMPK mediates nutrient regulation of thioredoxin-interacting protein (TXNIP in pancreatic beta-cells.

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    Maayan Shaked

    Full Text Available Thioredoxin-interacting protein (TXNIP regulates critical biological processes including inflammation, stress and apoptosis. TXNIP is upregulated by glucose and is a critical mediator of hyperglycemia-induced beta-cell apoptosis in diabetes. In contrast, the saturated long-chain fatty acid palmitate, although toxic to the beta-cell, inhibits TXNIP expression. The mechanisms involved in the opposing effects of glucose and fatty acids on TXNIP expression are unknown. We found that both palmitate and oleate inhibited TXNIP in a rat beta-cell line and islets. Palmitate inhibition of TXNIP was independent of fatty acid beta-oxidation or esterification. AMP-activated protein kinase (AMPK has an important role in cellular energy sensing and control of metabolic homeostasis; therefore we investigated its involvement in nutrient regulation of TXNIP. As expected, glucose inhibited whereas palmitate stimulated AMPK. Pharmacologic activators of AMPK mimicked fatty acids by inhibiting TXNIP. AMPK knockdown increased TXNIP expression in presence of high glucose with and without palmitate, indicating that nutrient (glucose and fatty acids effects on TXNIP are mediated in part via modulation of AMPK activity. TXNIP is transcriptionally regulated by carbohydrate response element-binding protein (ChREBP. Palmitate inhibited glucose-stimulated ChREBP nuclear entry and recruitment to the Txnip promoter, thereby inhibiting Txnip transcription. We conclude that AMPK is an important regulator of Txnip transcription via modulation of ChREBP activity. The divergent effects of glucose and fatty acids on TXNIP expression result in part from their opposing effects on AMPK activity. In light of the important role of TXNIP in beta-cell apoptosis, its inhibition by fatty acids can be regarded as an adaptive/protective response to glucolipotoxicity. The finding that AMPK mediates nutrient regulation of TXNIP may have important implications for the pathophysiology and treatment

  4. A point mutation in the DNA-binding domain of HPV-2 E2 protein increases its DNA-binding capacity and reverses its transcriptional regulatory activity on the viral early promoter

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    Gao Chen

    2012-02-01

    Full Text Available Abstract Background The human papillomavirus (HPV E2 protein is a multifunctional DNA-binding protein. The transcriptional activity of HPV E2 is mediated by binding to its specific binding sites in the upstream regulatory region of the HPV genomes. Previously we reported a HPV-2 variant from a verrucae vulgaris patient with huge extensive clustered cutaneous, which have five point mutations in its E2 ORF, L118S, S235P, Y287H, S293R and A338V. Under the control of HPV-2 LCR, co-expression of the mutated HPV E2 induced an increased activity on the viral early promoter. In the present study, a series of mammalian expression plasmids encoding E2 proteins with one to five amino acid (aa substitutions for these mutations were constructed and transfected into HeLa, C33A and SiHa cells. Results CAT expression assays indicated that the enhanced promoter activity was due to the co-expressions of the E2 constructs containing A338V mutation within the DNA-binding domain. Western blots analysis demonstrated that the transiently transfected E2 expressing plasmids, regardless of prototype or the A338V mutant, were continuously expressed in the cells. To study the effect of E2 mutations on its DNA-binding activity, a serial of recombinant E2 proteins with various lengths were expressed and purified. Electrophoresis mobility shift assays (EMSA showed that the binding affinity of E2 protein with A338V mutation to both an artificial probe with two E2 binding sites or HPV-2 and HPV-16 promoter-proximal LCR sequences were significantly stronger than that of the HPV-2 prototype E2. Furthermore, co-expression of the construct containing A338V mutant exhibited increased activities on heterologous HPV-16 early promoter P97 than that of prototype E2. Conclusions These results suggest that the mutation from Ala to Val at aa 338 is critical for E2 DNA-binding and its transcriptional regulation.

  5. The Staphylococcus aureus group II biotin protein ligase BirA is an effective regulator of biotin operon transcription and requires the DNA binding domain for full enzymatic activity.

    Science.gov (United States)

    Henke, Sarah K; Cronan, John E

    2016-11-01

    Group II biotin protein ligases (BPLs) are characterized by the presence of an N-terminal DNA binding domain that functions in transcriptional regulation of the genes of biotin biosynthesis and transport. The Staphylococcus aureus Group II BPL which is called BirA has been reported to bind an imperfect inverted repeat located upstream of the biotin synthesis operon. DNA binding by other Group II BPLs requires dimerization of the protein which is triggered by synthesis of biotinoyl-AMP (biotinoyl-adenylate), the intermediate in the ligation of biotin to its cognate target proteins. However, the S. aureus BirA was reported to dimerize and bind DNA in the absence of biotin or biotinoyl-AMP (Soares da Costa et al. (2014) Mol Microbiol 91: 110-120). These in vitro results argued that the protein would be unable to respond to the levels of biotin or acceptor proteins and thus would lack the regulatory properties of the other characterized BirA proteins. We tested the regulatory function of the protein using an in vivo model system and examined its DNA binding properties in vitro using electrophoretic mobility shift and fluorescence anisotropy analyses. We report that the S. aureus BirA is an effective regulator of biotin operon transcription and that the prior data can be attributed to artifacts of mobility shift analyses. We also report that deletion of the DNA binding domain of the S. aureus BirA results in loss of virtually all of its ligation activity. © 2016 John Wiley & Sons Ltd.

  6. The artificial zinc finger coding gene 'Jazz' binds the utrophin promoter and activates transcription.

    Science.gov (United States)

    Corbi, N; Libri, V; Fanciulli, M; Tinsley, J M; Davies, K E; Passananti, C

    2000-06-01

    Up-regulation of utrophin gene expression is recognized as a plausible therapeutic approach in the treatment of Duchenne muscular dystrophy (DMD). We have designed and engineered new zinc finger-based transcription factors capable of binding and activating transcription from the promoter of the dystrophin-related gene, utrophin. Using the recognition 'code' that proposes specific rules between zinc finger primary structure and potential DNA binding sites, we engineered a new gene named 'Jazz' that encodes for a three-zinc finger peptide. Jazz belongs to the Cys2-His2 zinc finger type and was engineered to target the nine base pair DNA sequence: 5'-GCT-GCT-GCG-3', present in the promoter region of both the human and mouse utrophin gene. The entire zinc finger alpha-helix region, containing the amino acid positions that are crucial for DNA binding, was specifically chosen on the basis of the contacts more frequently represented in the available list of the 'code'. Here we demonstrate that Jazz protein binds specifically to the double-stranded DNA target, with a dissociation constant of about 32 nM. Band shift and super-shift experiments confirmed the high affinity and specificity of Jazz protein for its DNA target. Moreover, we show that chimeric proteins, named Gal4-Jazz and Sp1-Jazz, are able to drive the transcription of a test gene from the human utrophin promoter.

  7. Identification of the promoter region required for human adiponectin gene transcription: Association with CCAAT/enhancer binding protein-β and tumor necrosis factor-α

    International Nuclear Information System (INIS)

    Kita, Atsushi; Yamasaki, Hironori; Kuwahara, Hironaga; Moriuchi, Akie; Fukushima, Keiko; Kobayashi, Masakazu; Fukushima, Tetsuya; Takahashi, Ryoko; Abiru, Norio; Uotani, Shigeo; Kawasaki, Eiji; Eguchi, Katsumi

    2005-01-01

    Adiponectin, an adipose tissue-specific plasma protein, is involved in insulin sensitizing and has anti-atherosclerotic properties. Plasma levels of adiponectin are decreased in obese individuals and patients with type 2 diabetes with insulin resistance. Tumor necrosis factor-α (TNF-α) decreases the expression of adiponectin in adipocytes. The aims of the present study were: (1) to identify the promoter region responsible for basal transcription of the human adiponectin gene, and (2) to investigate the mechanism by which adiponectin was regulated by TNF-α. The human adiponectin promoter (2.1 kb) was isolated and used for luciferase reporter analysis by transient transfection into 3T3-L1 adipocytes. Deletion analysis demonstrated that the promoter region from -676 to +41 was sufficient for basal transcriptional activity. Mutation analysis of putative response elements for sterol regulatory element binding protein (SREBP) (-431 to -423) and CCAAT/enhancer binding protein (C/EBP) (-230 to -224) showed that both elements were required for basal promoter activity. Adiponectin transcription was increased 3-fold in cells that over-expressed constitutively active C/EBP-β. Electrophoretic mobility shift assay, using nuclear extract from 3T3-L1 cells and the -258 to -199 region as a probe, demonstrated specific DNA-protein binding, which was abolished by TNF-α treatment. The present data indicate that the putative response elements for SREBP and C/EBP are required for human adiponectin promoter activity, and that suppression by TNF-α may, at least in part, be associated with inactivation of C/EBP-β

  8. Increased accumulation of hypoxia-inducible factor-1α with reduced transcriptional activity mediates the antitumor effect of triptolide

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    Li Zheng

    2010-10-01

    Full Text Available Abstract Background Hypoxia-inducible factor-1α (HIF-1α, a critical transcription factor to reduced O2 availability, has been demonstrated to be extensively involved in tumor survival, aggressive progression, drug resistance and angiogenesis. Thus it has been considered as a potential anticancer target. Triptolide is the main principle responsible for the biological activities of the Traditional Chinese Medicine tripterygium wilfordii Hook F. Triptolide possesses great chemotherapy potential for cancer with its broad-spectrum anticancer, antiangiogenesis, and drug-resistance circumvention activities. Numerous biological molecules inhibited by triptolide have been viewed as its possible targets. However, the anticancer action mechanisms of triptolide remains to be further investigated. Here we used human ovarian SKOV-3 cancer cells as a model to probe the effect of triptolide on HIF-1α. Results Triptolide was observed to inhibit the proliferation of SKOV-3 cells, and meanwhile, to enhance the accumulation of HIF-1α protein in SKOV-3, A549 and DU145 cells under different conditions. Triptolide did not change the kinetics or nuclear localization of HIF-1α protein or the 26 S proteasome activity in SKOV-3 cells. However, triptolide was found to increase the levels of HIF-1α mRNA. Unexpectedly, the HIF-1α protein induced by triptolide appeared to lose its transcriptional activity, as evidenced by the decreased mRNA levels of its target genes including VEGF, BNIP3 and CAIX. The results were further strengthened by the lowered secretion of VEGF protein, the reduced sprout outgrowth from the rat aorta rings and the inhibitory expression of the hypoxia responsive element-driven luciferase reporter gene. Moreover, the silencing of HIF-1α partially prevented the cytotoxicity and apoptosis triggered by triptolide. Conclusions The potent induction of HIF-1α protein involved in its cytotoxicity, together with the suppression of HIF-1 transcriptional

  9. PI3K/AKT signaling modulates transcriptional expression of EWS/FLI1 through specificity protein 1.

    Science.gov (United States)

    Giorgi, Chiara; Boro, Aleksandar; Rechfeld, Florian; Lopez-Garcia, Laura A; Gierisch, Maria E; Schäfer, Beat W; Niggli, Felix K

    2015-10-06

    Ewing sarcoma (ES) is the second most frequent bone cancer in childhood and is characterized by the presence of the balanced translocation t(11;22)(q24;q12) in more than 85% of cases, generating a dysregulated transcription factor EWS/FLI1. This fusion protein is an essential oncogenic component of ES development which is necessary for tumor cell maintenance and represents an attractive therapeutic target. To search for modulators of EWS/FLI1 activity we screened a library of 153 targeted compounds and identified inhibitors of the PI3K pathway to directly modulate EWS/FLI1 transcription. Surprisingly, treatment of four different ES cell lines with BEZ235 resulted in down regulation of EWS/FLI1 mRNA and protein by ~50% with subsequent modulation of target gene expression. Analysis of the EWS/FLI1 promoter region (-2239/+67) using various deletion constructs identified two 14 bp minimal elements as being important for EWS/FLI1 transcription. We identified SP1 as modulator of EWS/FLI1 gene expression and demonstrated direct binding to one of these regions in the EWS/FLI1 promoter by EMSA and ChIP experiments. These results provide the first insights on the transcriptional regulation of EWS/FLI1, an area that has not been investigated so far, and offer an additional molecular explanation for the known sensitivity of ES cell lines to PI3K inhibition.

  10. Brassinosteroid-Induced Transcriptional Repression and Dephosphorylation-Dependent Protein Degradation Negatively Regulate BIN2-Interacting AIF2 (a BR Signaling-Negative Regulator) bHLH Transcription Factor.

    Science.gov (United States)

    Kim, Yoon; Song, Ji-Hye; Park, Seon-U; Jeong, You-Seung; Kim, Soo-Hwan

    2017-02-01

    Brassinosteroids (BRs) are plant polyhydroxy-steroids that play important roles in plant growth and development via extensive signal integration through direct interactions between regulatory components of different signaling pathways. Recent studies have shown that diverse helix-loop-helix/basic helix-loop-helix (HLH/bHLH) family proteins are actively involved in control of BR signaling pathways and interact with other signaling pathways. In this study, we show that ATBS1-INTERACTING FACTOR 2 (AIF2), a nuclear-localized atypical bHLH transcription factor, specifically interacts with BRASSINOSTEROID-INSENSITIVE 2 (BIN2) among other BR signaling molecules. Overexpression of AIF2 down-regulated transcript expression of growth-promoting genes, thus resulting in retardation of growth. AIF2 renders plants hyposensitive to BR-induced root growth inhibition, but shows little effects on BR-promoted hypocotyl elongation. Notably, AIF2 was dephosphorylated by BR, and the dephosphorylated AIF2 was subject to proteasome-mediated degradation. AIF2 degradation was greatly induced by BR and ABA, but relatively slightly by other hormones such as auxin, gibberellin, cytokinin and ethylene. Moreover, AIF2 transcription was significantly suppressed by a BRI1/BZR1-mediated BR signaling pathway through a direct binding of BRASSINAZOLE RESISTANT 1 (BZR1) to the BR response element (BRRE) region of the AIF2 promoter. In conclusion, our study suggests that BIN2-driven AIF2 phosphorylation could augment the BIN2/AIF2-mediated negative circuit of BR signaling pathways, and the BR-induced transcriptional repression and protein degradation negatively regulate AIF2 transcription factor, reinforcing the BZR1/BES1-mediated positive BR signaling pathway. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  11. Transcriptional activation of ribosomal RNA genes during compensatory renal hypertrophy

    International Nuclear Information System (INIS)

    Ouellette, A.J.; Moonka, R.; Zelenetz, A.; Malt, R.A.

    1986-01-01

    The overall rate of rDNA transcription increases by 50% during the first 24 hours of compensatory renal hypertrophy in the mouse. To study mechanisms of ribosome accumulation after uninephrectomy, transcription rates were measured in isolated kidneys by transcriptional runoff. 32 P-labeled nascent transcripts were hybridized to blots containing linearized, denatured cloned rDNA, and hybridization was quantitated autoradiographically and by direct counting. Overall transcriptional activity of rDNA was increased by 30% above control levels at 6 hrs after nephrectomy and by 50% at 12, 18, and 24 hrs after operation. Hybridizing RNA was insensitive to inhibiby alpha-amanitin, and no hybridization was detected to vector DNA. Thus, accelerated rDNA transcription is one regulatory element in the accretion of ribosomes in renal growth, and the regulatory event is an early event. Mechanisms of activation may include enhanced transcription of active genes or induction of inactive DNA

  12. Acetic acid activates the AMP-activated protein kinase signaling pathway to regulate lipid metabolism in bovine hepatocytes.

    Directory of Open Access Journals (Sweden)

    Xinwei Li

    Full Text Available The effect of acetic acid on hepatic lipid metabolism in ruminants differs significantly from that in monogastric animals. Therefore, the aim of this study was to investigate the regulation mechanism of acetic acid on the hepatic lipid metabolism in dairy cows. The AMP-activated protein kinase (AMPK signaling pathway plays a key role in regulating hepatic lipid metabolism. In vitro, bovine hepatocytes were cultured and treated with different concentrations of sodium acetate (neutralized acetic acid and BML-275 (an AMPKα inhibitor. Acetic acid consumed a large amount of ATP, resulting in an increase in AMPKα phosphorylation. The increase in AMPKα phosphorylation increased the expression and transcriptional activity of peroxisome proliferator-activated receptor α, which upregulated the expression of lipid oxidation genes, thereby increasing lipid oxidation in bovine hepatocytes. Furthermore, elevated AMPKα phosphorylation reduced the expression and transcriptional activity of the sterol regulatory element-binding protein 1c and the carbohydrate responsive element-binding protein, which reduced the expression of lipogenic genes, thereby decreasing lipid biosynthesis in bovine hepatocytes. In addition, activated AMPKα inhibited the activity of acetyl-CoA carboxylase. Consequently, the triglyceride content in the acetate-treated hepatocytes was significantly decreased. These results indicate that acetic acid activates the AMPKα signaling pathway to increase lipid oxidation and decrease lipid synthesis in bovine hepatocytes, thereby reducing liver fat accumulation in dairy cows.

  13. An alternatively spliced heat shock transcription factor, OsHSFA2dI, functions in the heat stress-induced unfolded protein response in rice.

    Science.gov (United States)

    Cheng, Q; Zhou, Y; Liu, Z; Zhang, L; Song, G; Guo, Z; Wang, W; Qu, X; Zhu, Y; Yang, D

    2015-03-01

    As sessile organisms, plants have evolved a wide range of defence pathways to cope with environmental stress such as heat shock. However, the molecular mechanism of these defence pathways remains unclear in rice. In this study, we found that OsHSFA2d, a heat shock transcriptional factor, encodes two main splice variant proteins, OsHSFA2dI and OsHSFA2dII in rice. Under normal conditions, OsHSFA2dII is the dominant but transcriptionally inactive spliced form. However, when the plant suffers heat stress, OsHSFA2d is alternatively spliced into a transcriptionally active form, OsHSFA2dI, which participates in the heat stress response (HSR). Further study found that this alternative splicing was induced by heat shock rather than photoperiod. We found that OsHSFA2dI is localised to the nucleus, whereas OsHSFA2dII is localised to the nucleus and cytoplasm. Moreover, expression of the unfolded protein response (UNFOLDED PROTEIN RESPONSE) sensors, OsIRE1, OsbZIP39/OsbZIP60 and the UNFOLDED PROTEIN RESPONSE marker OsBiP1, was up-regulated. Interestingly, OsbZIP50 was also alternatively spliced under heat stress, indicating that UNFOLDED PROTEIN RESPONSE signalling pathways were activated by heat stress to re-establish cellular protein homeostasis. We further demonstrated that OsHSFA2dI participated in the unfolded protein response by regulating expression of OsBiP1. © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands.

  14. Coordinate Regulation of Yeast Sterol Regulatory Element-binding Protein (SREBP) and Mga2 Transcription Factors.

    Science.gov (United States)

    Burr, Risa; Stewart, Emerson V; Espenshade, Peter J

    2017-03-31

    The Mga2 and Sre1 transcription factors regulate oxygen-responsive lipid homeostasis in the fission yeast Schizosaccharomyces pombe in a manner analogous to the mammalian sterol regulatory element-binding protein (SREBP)-1 and SREBP-2 transcription factors. Mga2 and SREBP-1 regulate triacylglycerol and glycerophospholipid synthesis, whereas Sre1 and SREBP-2 regulate sterol synthesis. In mammals, a shared activation mechanism allows for coordinate regulation of SREBP-1 and SREBP-2. In contrast, distinct pathways activate fission yeast Mga2 and Sre1. Therefore, it is unclear whether and how these two related pathways are coordinated to maintain lipid balance in fission yeast. Previously, we showed that Sre1 cleavage is defective in the absence of mga2 Here, we report that this defect is due to deficient unsaturated fatty acid synthesis, resulting in aberrant membrane transport. This defect is recapitulated by treatment with the fatty acid synthase inhibitor cerulenin and is rescued by addition of exogenous unsaturated fatty acids. Furthermore, sterol synthesis inhibition blocks Mga2 pathway activation. Together, these data demonstrate that Sre1 and Mga2 are each regulated by the lipid product of the other transcription factor pathway, providing a source of coordination for these two branches of lipid synthesis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. The silkworm Bombyx mori cuticular protein CPR55 gene is regulated by the transcription factor βFTZ-F1

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    Md. Saheb Ali

    2016-01-01

    Full Text Available The insect cuticle is composed of various proteins and formed during the moult under a complex biological process that depends on the cross talk between hormone levels and gene expression. In the present study, we aimed to clarify the ecdysone-dependent temporal regulation mechanisms of cuticular proteins expression and the underlying control of Bombyx mori metamorphosis. The expression of CPR55 was observed from the W3 early stage and peaked at pupation when the ecdysteroid titre declined. CPR55 was induced by the ecdysone pulse, and their expression peaked at 24 h after transfer to a hormone free medium. Transcripts of CPR55 were neither observed after the 20E pulse treatment in the presence of cycloheximide nor after the addition of 20E in V4 wing discs. We analysed the upstream region of the CPR55 gene using a transient reporter assay with a gene gun system which identified only one βFTZ-F1 binding site important for cis-acting elements for the transcription activation of the luciferase reporter gene by an ecdysone pulse. Site-directed mutagenesis of this element in the context of the 589-bp promoter fragment drastically decreased the reporter activity. The nuclear protein bound to βFTZ-F1 sites was identified by an electrophoretic mobility shift assay suggesting that CPR55 expression was regulated by βFTZ-F1 through the ecdysone pulse. The results confirmed that transcription factor, BmβFTZ-F1, binds to the cis-regulatory elements in the promoter of the gene coding for cuticle protein, CPR55, and regulates its expression during B. mori metamorphosis.

  16. Rift Valley fever virus NSs protein promotes post-transcriptional downregulation of protein kinase PKR and inhibits eIF2alpha phosphorylation.

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    Tetsuro Ikegami

    2009-02-01

    Full Text Available Rift Valley fever virus (RVFV (genus Phlebovirus, family Bunyaviridae is a negative-stranded RNA virus with a tripartite genome. RVFV is transmitted by mosquitoes and causes fever and severe hemorrhagic illness among humans, and fever and high rates of abortions in livestock. A nonstructural RVFV NSs protein inhibits the transcription of host mRNAs, including interferon-beta mRNA, and is a major virulence factor. The present study explored a novel function of the RVFV NSs protein by testing the replication of RVFV lacking the NSs gene in the presence of actinomycin D (ActD or alpha-amanitin, both of which served as a surrogate of the host mRNA synthesis suppression function of the NSs. In the presence of the host-transcriptional inhibitors, the replication of RVFV lacking the NSs protein, but not that carrying NSs, induced double-stranded RNA-dependent protein kinase (PKR-mediated eukaryotic initiation factor (eIF2alpha phosphorylation, leading to the suppression of host and viral protein translation. RVFV NSs promoted post-transcriptional downregulation of PKR early in the course of the infection and suppressed the phosphorylated eIF2alpha accumulation. These data suggested that a combination of RVFV replication and NSs-induced host transcriptional suppression induces PKR-mediated eIF2alpha phosphorylation, while the NSs facilitates efficient viral translation by downregulating PKR and inhibiting PKR-mediated eIF2alpha phosphorylation. Thus, the two distinct functions of the NSs, i.e., the suppression of host transcription, including that of type I interferon mRNAs, and the downregulation of PKR, work together to prevent host innate antiviral functions, allowing efficient replication and survival of RVFV in infected mammalian hosts.

  17. A temporal gate for viral enhancers to co-opt Toll-like-receptor transcriptional activation pathways upon acute infection.

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    Kai A Kropp

    2015-04-01

    Full Text Available Viral engagement with macrophages activates Toll-Like-Receptors (TLRs and viruses must contend with the ensuing inflammatory responses to successfully complete their replication cycle. To date, known counter-strategies involve the use of viral-encoded proteins that often employ mimicry mechanisms to block or redirect the host response to benefit the virus. Whether viral regulatory DNA sequences provide an opportunistic strategy by which viral enhancer elements functionally mimic innate immune enhancers is unknown. Here we find that host innate immune genes and the prototypical viral enhancer of cytomegalovirus (CMV have comparable expression kinetics, and positively respond to common TLR agonists. In macrophages but not fibroblasts we show that activation of NFκB at immediate-early times of infection is independent of virion-associated protein, M45. We find upon virus infection or transfection of viral genomic DNA the TLR-agonist treatment results in significant enhancement of the virus transcription-replication cycle. In macrophage time-course infection experiments we demonstrate that TLR-agonist stimulation of the viral enhancer and replication cycle is strictly delimited by a temporal gate with a determined half-maximal time for enhancer-activation of 6 h; after which TLR-activation blocks the viral transcription-replication cycle. By performing a systematic siRNA screen of 149 innate immune regulatory factors we identify not only anticipated anti-viral and pro-viral contributions but also new factors involved in the CMV transcription-replication cycle. We identify a central convergent NFκB-SP1-RXR-IRF axis downstream of TLR-signalling. Activation of the RXR component potentiated direct and indirect TLR-induced activation of CMV transcription-replication cycle; whereas chromatin binding experiments using wild-type and enhancer-deletion virus revealed IRF3 and 5 as new pro-viral host transcription factor interactions with the CMV enhancer in

  18. Molecular Characterization of the Schistosoma mansoni Zinc Finger Protein SmZF1 as a Transcription Factor

    Science.gov (United States)

    D'Astolfo, Diego S.; Cardoso, Fernanda C.; Rajão, Matheus A.; Mourão, Marina M.; Gava, Elisandra; Oliveira, Sérgio C.; Macedo, Andréa M.; Machado, Carlos R.; Pena, Sérgio D. J.; Kitten, Gregory T.; Franco, Glória R.

    2009-01-01

    Background During its development, the parasite Schistosoma mansoni is exposed to different environments and undergoes many morphological and physiological transformations as a result of profound changes in gene expression. Characterization of proteins involved in the regulation of these processes is of importance for the understanding of schistosome biology. Proteins containing zinc finger motifs usually participate in regulatory processes and are considered the major class of transcription factors in eukaryotes. It has already been shown, by EMSA (Eletrophoretic Mobility Shift Assay), that SmZF1, a S. mansoni zinc finger (ZF) protein, specifically binds both DNA and RNA oligonucleotides. This suggests that this protein might act as a transcription factor in the parasite. Methodology/Principal Findings In this study we extended the characterization of SmZF1 by determining its subcellular localization and by verifying its ability to regulate gene transcription. We performed immunohistochemistry assays using adult male and female worms, cercariae and schistosomula to analyze the distribution pattern of SmZF1 and verified that the protein is mainly detected in the cells nuclei of all tested life cycle stages except for adult female worms. Also, SmZF1 was heterologously expressed in mammalian COS-7 cells to produce the recombinant protein YFP-SmZF1, which was mainly detected in the nucleus of the cells by confocal microscopy and Western blot assays. To evaluate the ability of this protein to regulate gene transcription, cells expressing YFP-SmZF1 were tested in a luciferase reporter system. In this system, the luciferase gene is downstream of a minimal promoter, upstream of which a DNA region containing four copies of the SmZF1 putative best binding site (D1-3DNA) was inserted. SmZF1 increased the reporter gene transcription by two fold (p≤0.003) only when its specific binding site was present. Conclusion Taken together, these results strongly support the hypothesis

  19. CIKS, a connection to Ikappa B kinase and stress-activated protein kinase.

    Science.gov (United States)

    Leonardi, A; Chariot, A; Claudio, E; Cunningham, K; Siebenlist, U

    2000-09-12

    Pathogens, inflammatory signals, and stress cause acute transcriptional responses in cells. The induced expression of genes in response to these signals invariably involves transcription factors of the NF-kappaB and AP-1/ATF families. Activation of NF-kappaB factors is thought to be mediated primarily via IkappaB kinases (IKK), whereas that of AP-1/ATF can be mediated by stress-activated protein kinases (SAPKs; also named Jun kinases or JNKs). IKKalpha and IKKbeta are two catalytic subunits of a core IKK complex that also contains the regulatory subunit NEMO (NF-kappaB essential modulator)/IKKgamma. The latter protein is essential for activation of the IKKs, but its mechanism of action is not known. Here we describe the molecular cloning of CIKS (connection to IKK and SAPK/JNK), a previously unknown protein that directly interacts with NEMO/IKKgamma in cells. When ectopically expressed, CIKS stimulates IKK and SAPK/JNK kinases and it transactivates an NF-kappaB-dependent reporter. Activation of NF-kappaB is prevented in the presence of kinase-deficient, interfering mutants of the IKKs. CIKS may help to connect upstream signaling events to IKK and SAPK/JNK modules. CIKS could coordinate the activation of two stress-induced signaling pathways, functions reminiscent of those noted for tumor necrosis factor receptor-associated factor adaptor proteins.

  20. CIKS, a connection to IκB kinase and stress-activated protein kinase

    Science.gov (United States)

    Leonardi, Antonio; Chariot, Alain; Claudio, Estefania; Cunningham, Kirk; Siebenlist, Ulrich

    2000-01-01

    Pathogens, inflammatory signals, and stress cause acute transcriptional responses in cells. The induced expression of genes in response to these signals invariably involves transcription factors of the NF-κB and AP-1/ATF families. Activation of NF-κB factors is thought to be mediated primarily via IκB kinases (IKK), whereas that of AP-1/ATF can be mediated by stress-activated protein kinases (SAPKs; also named Jun kinases or JNKs). IKKα and IKKβ are two catalytic subunits of a core IKK complex that also contains the regulatory subunit NEMO (NF-κB essential modulator)/IKKγ. The latter protein is essential for activation of the IKKs, but its mechanism of action is not known. Here we describe the molecular cloning of CIKS (connection to IKK and SAPK/JNK), a previously unknown protein that directly interacts with NEMO/IKKγ in cells. When ectopically expressed, CIKS stimulates IKK and SAPK/JNK kinases and it transactivates an NF-κB-dependent reporter. Activation of NF-κB is prevented in the presence of kinase-deficient, interfering mutants of the IKKs. CIKS may help to connect upstream signaling events to IKK and SAPK/JNK modules. CIKS could coordinate the activation of two stress-induced signaling pathways, functions reminiscent of those noted for tumor necrosis factor receptor-associated factor adaptor proteins. PMID:10962033

  1. The homeodomain-leucine zipper (HD-Zip) class I transcription factors ATHB7 and ATHB12 modulate abscisic acid signalling by regulating protein phosphatase 2C and abscisic acid receptor gene activities.

    Science.gov (United States)

    Valdés, Ana Elisa; Overnäs, Elin; Johansson, Henrik; Rada-Iglesias, Alvaro; Engström, Peter

    2012-11-01

    Plants perceiving drought activate multiple responses to improve survival, including large-scale alterations in gene expression. This article reports on the roles in the drought response of two Arabidopsis thaliana homeodomain-leucine zipper class I genes; ATHB7 and ATHB12, both strongly induced by water-deficit and abscisic acid (ABA). ABA-mediated transcriptional regulation of both genes is shown to depend on the activity of protein phosphatases type 2C (PP2C). ATHB7 and ATHB12 are, thus, targets of the ABA signalling mechanism defined by the PP2Cs and the PYR/PYL family of ABA receptors, with which the PP2C proteins interact. Our results from chromatin immunoprecipitation and gene expression analyses demonstrate that ATHB7 and ATHB12 act as positive transcriptional regulators of PP2C genes, and thereby as negative regulators of abscisic acid signalling. In support of this notion, our results also show that ATHB7 and ATHB12 act to repress the transcription of genes encoding the ABA receptors PYL5 and PYL8 in response to an ABA stimulus. In summary, we demonstrate that ATHB7 and ATHB12 have essential functions in the primary response to drought, as mediators of a negative feedback effect on ABA signalling in the plant response to water deficit.

  2. p38 mitogen-activated protein kinase up-regulates NF-κB transcriptional activation through RelA phosphorylation during stretch-induced myogenesis

    International Nuclear Information System (INIS)

    Ji, Guoping; Liu, Dongxu; Liu, Jing; Gao, Hui; Yuan, Xiao; Shen, Gang

    2010-01-01

    p38 MAPK and nuclear factor-B (NF-B) signaling pathways play an indispensable role in the control of skeletal myogenesis. The specific contribution of these signaling pathways to the response of myoblast to the mechanical stimulation and the molecular mechanisms underlying this response remain unresolved. Using an established in vitro model, we now show that p38 MAP kinase activity regulates the transcriptional activation of NF-κB in response to mechanical stimulation of myoblasts. Furthermore, SB203580 blocked stretch-induced NF-κB activation during myogenesis, not through down-regulation of degradation of IκB-α, and consequent translocation of the p65 subunit of NF-κB to the nucleus. It is likely that stretch-induced NF-κB activation by phosphorylation of p65 NF-κB. Moreover, depletion of p38α using siRNA significantly reduces stretch-induced phosphorylation of RelA and NF-κB activity. These results provides the first evidence of a cross-talk between p38 MAPK and NF-κB signaling pathways during stretch-induced myogenesis, with phosphorylation of RelA being one of the effectors of this promyogenic mechanism. The α isoform of p38MAP kinase regulates the transcriptional activation of NF-κB following stimulation with cyclic stretch.

  3. p38 mitogen-activated protein kinase up-regulates NF-{kappa}B transcriptional activation through RelA phosphorylation during stretch-induced myogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Ji, Guoping [Department of Orthodontics, College of Stomatology, Ninth People' s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Research Institute of Stomatology, Shanghai 200011 (China); Liu, Dongxu [Department of Orthodontics, College of Stomatology, Shandong University, Jinan, Shandong Province 250012 (China); Liu, Jing [Department of Orthodontics, The Affiliated Qingdao Municipal Hospital, Qingdao University, Qingdao, Shandong Province 266075 (China); Gao, Hui [Department of Orthodontics, Tianjin Stomatological Hospital, Tianjin 300041 (China); Yuan, Xiao, E-mail: yuanxiaoqd@163.com [Department of Orthodontics, The Affiliated Qingdao Municipal Hospital, Qingdao University, Qingdao, Shandong Province 266075 (China); Shen, Gang, E-mail: ganshen2007@163.com [Department of Orthodontics, College of Stomatology, Ninth People' s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Research Institute of Stomatology, Shanghai 200011 (China)

    2010-01-01

    p38 MAPK and nuclear factor-B (NF-B) signaling pathways play an indispensable role in the control of skeletal myogenesis. The specific contribution of these signaling pathways to the response of myoblast to the mechanical stimulation and the molecular mechanisms underlying this response remain unresolved. Using an established in vitro model, we now show that p38 MAP kinase activity regulates the transcriptional activation of NF-{kappa}B in response to mechanical stimulation of myoblasts. Furthermore, SB203580 blocked stretch-induced NF-{kappa}B activation during myogenesis, not through down-regulation of degradation of I{kappa}B-{alpha}, and consequent translocation of the p65 subunit of NF-{kappa}B to the nucleus. It is likely that stretch-induced NF-{kappa}B activation by phosphorylation of p65 NF-{kappa}B. Moreover, depletion of p38{alpha} using siRNA significantly reduces stretch-induced phosphorylation of RelA and NF-{kappa}B activity. These results provides the first evidence of a cross-talk between p38 MAPK and NF-{kappa}B signaling pathways during stretch-induced myogenesis, with phosphorylation of RelA being one of the effectors of this promyogenic mechanism. The {alpha} isoform of p38MAP kinase regulates the transcriptional activation of NF-{kappa}B following stimulation with cyclic stretch.

  4. Promoter proximal polyadenylation sites reduce transcription activity

    DEFF Research Database (Denmark)

    Andersen, Pia Kjølhede; Lykke-Andersen, Søren; Jensen, Torben Heick

    2012-01-01

    Gene expression relies on the functional communication between mRNA processing and transcription. We previously described the negative impact of a point-mutated splice donor (SD) site on transcription. Here we demonstrate that this mutation activates an upstream cryptic polyadenylation (CpA) site......, which in turn causes reduced transcription. Functional depletion of U1 snRNP in the context of the wild-type SD triggers the same CpA event accompanied by decreased RNA levels. Thus, in accordance with recent findings, U1 snRNP can shield premature pA sites. The negative impact of unshielded pA sites...... on transcription requires promoter proximity, as demonstrated using artificial constructs and supported by a genome-wide data set. Importantly, transcription down-regulation can be recapitulated in a gene context devoid of splice sites by placing a functional bona fide pA site/transcription terminator within ∼500...

  5. Deep Proteomics of Mouse Skeletal Muscle Enables Quantitation of Protein Isoforms, Metabolic Pathways, and Transcription Factors*

    Science.gov (United States)

    Deshmukh, Atul S.; Murgia, Marta; Nagaraj, Nagarjuna; Treebak, Jonas T.; Cox, Jürgen; Mann, Matthias

    2015-01-01

    Skeletal muscle constitutes 40% of individual body mass and plays vital roles in locomotion and whole-body metabolism. Proteomics of skeletal muscle is challenging because of highly abundant contractile proteins that interfere with detection of regulatory proteins. Using a state-of-the art MS workflow and a strategy to map identifications from the C2C12 cell line model to tissues, we identified a total of 10,218 proteins, including skeletal muscle specific transcription factors like myod1 and myogenin and circadian clock proteins. We obtain absolute abundances for proteins expressed in a muscle cell line and skeletal muscle, which should serve as a valuable resource. Quantitation of protein isoforms of glucose uptake signaling pathways and in glucose and lipid metabolic pathways provides a detailed metabolic map of the cell line compared with tissue. This revealed unexpectedly complex regulation of AMP-activated protein kinase and insulin signaling in muscle tissue at the level of enzyme isoforms. PMID:25616865

  6. Adiponectin promotes hyaluronan synthesis along with increases in hyaluronan synthase 2 transcripts through an AMP-activated protein kinase/peroxisome proliferator-activated receptor-α-dependent pathway in human dermal fibroblasts

    International Nuclear Information System (INIS)

    Yamane, Takumi; Kobayashi-Hattori, Kazuo; Oishi, Yuichi

    2011-01-01

    Highlights: ► Adiponectin promotes hyaluronan synthesis along with an increase in HAS2 transcripts. ► Adiponectin also increases the phosphorylation of AMPK. ► A pharmacological activator of AMPK increases mRNA levels of PPARα and HAS2. ► Adiponectin-induced HAS2 mRNA expression is blocked by a PPARα antagonist. ► Adiponectin promotes hyaluronan synthesis via an AMPK/PPARα-dependent pathway. -- Abstract: Although adipocytokines affect the functions of skin, little information is available on the effect of adiponectin on the skin. In this study, we investigated the effect of adiponectin on hyaluronan synthesis and its regulatory mechanisms in human dermal fibroblasts. Adiponectin promoted hyaluronan synthesis along with an increase in the mRNA levels of hyaluronan synthase 2 (HAS2), which plays a primary role in hyaluronan synthesis. Adiponectin also increased the phosphorylation of AMP-activated protein kinase (AMPK). A pharmacological activator of AMPK, 5-aminoimidazole-4-carboxamide-1β-ribofuranoside (AICAR), increased mRNA levels of peroxisome proliferator-activated receptor-α (PPARα), which enhances the expression of HAS2 mRNA. In addition, AICAR increased the mRNA levels of HAS2. Adiponectin-induced HAS2 mRNA expression was blocked by GW6471, a PPARα antagonist, in a concentration-dependent manner. These results show that adiponectin promotes hyaluronan synthesis along with increases in HAS2 transcripts through an AMPK/PPARα-dependent pathway in human dermal fibroblasts. Thus, our study suggests that adiponectin may be beneficial for retaining moisture in the skin, anti-inflammatory activity, and the treatment of a variety of cutaneous diseases.

  7. MicroRNA-214 Suppresses Gluconeogenesis by Targeting Activating Transcriptional Factor 4*

    Science.gov (United States)

    Li, Kai; Zhang, Jin; Yu, Junjie; Liu, Bin; Guo, Yajie; Deng, Jiali; Chen, Shanghai; Wang, Chunxia; Guo, Feifan

    2015-01-01

    Although the gluconeogenesis pathway is already a target for the treatment of type 2 diabetes, the potential role of microRNAs (miRNAs) in gluconeogenesis remains unclear. Here, we investigated the physiological functions of miR-214 in gluconeogenesis. The expression of miR-214 was suppressed by glucagon via protein kinase A signaling in primary hepatocytes, and miR-214 was down-regulated in the livers of fasted, high fat diet-induced diabetic and leptin receptor-mutated (db/db) mice. The overexpression of miR-214 in primary hepatocytes suppressed glucose production, and silencing miR-214 reversed this effect. Gluconeogenesis was suppressed in the livers of mice injected with an adenovirus expressing miR-214 (Ad-miR-214). Additionally, Ad-miR-214 alleviated high fat diet-induced elevation of gluconeogenesis and hyperglycemia. Furthermore, we found that activating transcription factor 4 (ATF4), a reported target of miR-214, can reverse the suppressive effect of miR-214 on gluconeogenesis in primary hepatocytes, and this suppressive effect was blocked in liver-specific ATF4 knock-out mice. ATF4 regulated gluconeogenesis via affecting forkhead box protein O1 (FOXO1) transcriptional activity. Finally, liver-specific miR-214 transgenic mice exhibited suppressed gluconeogenesis and reduced expression of ATF4, phosphoenolpyruvate carboxykinase, and glucose-6-phosphatase in liver. Taken together, our results suggest that the miR-214-ATF4 axis is a novel pathway for the regulation of hepatic gluconeogenesis. PMID:25657009

  8. Transcriptional activation of LON Gene by a new form of mitochondrial stress: A role for the nuclear respiratory factor 2 in StAR overload response (SOR).

    Science.gov (United States)

    Bahat, Assaf; Perlberg, Shira; Melamed-Book, Naomi; Isaac, Sara; Eden, Amir; Lauria, Ines; Langer, Thomas; Orly, Joseph

    2015-06-15

    High output of steroid hormone synthesis in steroidogenic cells of the adrenal cortex and the gonads requires the expression of the steroidogenic acute regulatory protein (StAR) that facilitates cholesterol mobilization to the mitochondrial inner membrane where the CYP11A1/P450scc enzyme complex converts the sterol to the first steroid. Earlier studies have shown that StAR is active while pausing on the cytosolic face of the outer mitochondrial membrane while subsequent import of the protein into the matrix terminates the cholesterol mobilization activity. Consequently, during repeated activity cycles, high level of post-active StAR accumulates in the mitochondrial matrix. To prevent functional damage due to such protein overload effect, StAR is degraded by a sequence of three to four ATP-dependent proteases of the mitochondria protein quality control system, including LON and the m-AAA membranous proteases AFG3L2 and SPG7/paraplegin. Furthermore, StAR expression in both peri-ovulatory ovarian cells, or under ectopic expression in cell line models, results in up to 3-fold enrichment of the mitochondrial proteases and their transcripts. We named this novel form of mitochondrial stress as StAR overload response (SOR). To better understand the SOR mechanism at the transcriptional level we analyzed first the unexplored properties of the proximal promoter of the LON gene. Our findings suggest that the human nuclear respiratory factor 2 (NRF-2), also known as GA binding protein (GABP), is responsible for 88% of the proximal promoter activity, including the observed increase of transcription in the presence of StAR. Further studies are expected to reveal if common transcriptional determinants coordinate the SOR induced transcription of all the genes encoding the SOR proteases. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  9. A Function for the hnRNP A1/A2 Proteins in Transcription Elongation.

    Science.gov (United States)

    Lemieux, Bruno; Blanchette, Marco; Monette, Anne; Mouland, Andrew J; Wellinger, Raymund J; Chabot, Benoit

    2015-01-01

    The hnRNP A1 and A2 proteins regulate processes such as alternative pre-mRNA splicing and mRNA stability. Here, we report that a reduction in the levels of hnRNP A1 and A2 by RNA interference or their cytoplasmic retention by osmotic stress drastically increases the transcription of a reporter gene. Based on previous work, we propose that this effect may be linked to a decrease in the activity of the transcription elongation factor P-TEFb. Consistent with this hypothesis, the transcription of the reporter gene was stimulated when the catalytic component of P-TEFb, CDK9, was inhibited with DRB. While low levels of A1/A2 stimulated the association of RNA polymerase II with the reporter gene, they also increased the association of CDK9 with the repressor 7SK RNA, and compromised the recovery of promoter-distal transcription on the Kitlg gene after the release of pausing. Transcriptome analysis revealed that more than 50% of the genes whose expression was affected by the siRNA-mediated depletion of A1/A2 were also affected by DRB. RNA polymerase II-chromatin immunoprecipitation assays on DRB-treated and A1/A2-depleted cells identified a common set of repressed genes displaying increased occupancy of polymerases at promoter-proximal locations, consistent with pausing. Overall, our results suggest that lowering the levels of hnRNP A1/A2 elicits defective transcription elongation on a fraction of P-TEFb-dependent genes, hence favoring the transcription of P-TEFb-independent genes.

  10. A Function for the hnRNP A1/A2 Proteins in Transcription Elongation.

    Directory of Open Access Journals (Sweden)

    Bruno Lemieux

    Full Text Available The hnRNP A1 and A2 proteins regulate processes such as alternative pre-mRNA splicing and mRNA stability. Here, we report that a reduction in the levels of hnRNP A1 and A2 by RNA interference or their cytoplasmic retention by osmotic stress drastically increases the transcription of a reporter gene. Based on previous work, we propose that this effect may be linked to a decrease in the activity of the transcription elongation factor P-TEFb. Consistent with this hypothesis, the transcription of the reporter gene was stimulated when the catalytic component of P-TEFb, CDK9, was inhibited with DRB. While low levels of A1/A2 stimulated the association of RNA polymerase II with the reporter gene, they also increased the association of CDK9 with the repressor 7SK RNA, and compromised the recovery of promoter-distal transcription on the Kitlg gene after the release of pausing. Transcriptome analysis revealed that more than 50% of the genes whose expression was affected by the siRNA-mediated depletion of A1/A2 were also affected by DRB. RNA polymerase II-chromatin immunoprecipitation assays on DRB-treated and A1/A2-depleted cells identified a common set of repressed genes displaying increased occupancy of polymerases at promoter-proximal locations, consistent with pausing. Overall, our results suggest that lowering the levels of hnRNP A1/A2 elicits defective transcription elongation on a fraction of P-TEFb-dependent genes, hence favoring the transcription of P-TEFb-independent genes.

  11. The Development of Protein Microarrays and Their Applications in DNA-Protein and Protein-Protein Interaction Analyses of Arabidopsis Transcription Factors

    Science.gov (United States)

    Gong, Wei; He, Kun; Covington, Mike; Dinesh-Kumar, S. P.; Snyder, Michael; Harmer, Stacey L.; Zhu, Yu-Xian; Deng, Xing Wang

    2009-01-01

    We used our collection of Arabidopsis transcription factor (TF) ORFeome clones to construct protein microarrays containing as many as 802 TF proteins. These protein microarrays were used for both protein-DNA and protein-protein interaction analyses. For protein-DNA interaction studies, we examined AP2/ERF family TFs and their cognate cis-elements. By careful comparison of the DNA-binding specificity of 13 TFs on the protein microarray with previous non-microarray data, we showed that protein microarrays provide an efficient and high throughput tool for genome-wide analysis of TF-DNA interactions. This microarray protein-DNA interaction analysis allowed us to derive a comprehensive view of DNA-binding profiles of AP2/ERF family proteins in Arabidopsis. It also revealed four TFs that bound the EE (evening element) and had the expected phased gene expression under clock-regulation, thus providing a basis for further functional analysis of their roles in clock regulation of gene expression. We also developed procedures for detecting protein interactions using this TF protein microarray and discovered four novel partners that interact with HY5, which can be validated by yeast two-hybrid assays. Thus, plant TF protein microarrays offer an attractive high-throughput alternative to traditional techniques for TF functional characterization on a global scale. PMID:19802365

  12. The Varicella-Zoster Virus Immediate-Early 63 protein affects chromatin controlled gene transcription in a cell-type dependent manner

    Directory of Open Access Journals (Sweden)

    Bontems Sébastien

    2007-10-01

    Full Text Available Abstract Background Varicella Zoster Virus Immediate Early 63 protein (IE63 has been shown to be essential for VZV replication, and critical for latency establishment. The activity of the protein as a transcriptional regulator is not fully clear yet. Using transient transfection assays, IE63 has been shown to repress viral and cellular promoters containing typical TATA boxes by interacting with general transcription factors. Results In this paper, IE63 regulation properties on endogenous gene expression were evaluated using an oligonucleotide-based micro-array approach. We found that IE63 modulates the transcription of only a few genes in HeLa cells including genes implicated in transcription or immunity. Furthermore, we showed that this effect is mediated by a modification of RNA POL II binding on the promoters tested and that IE63 phosphorylation was essential for these effects. In MeWo cells, the number of genes whose transcription was modified by IE63 was somewhat higher, including genes implicated in signal transduction, transcription, immunity, and heat-shock signalling. While IE63 did not modify the basal expression of several NF-κB dependent genes such as IL-8, ICAM-1, and IκBα, it modulates transcription of these genes upon TNFα induction. This effect was obviously correlated with the amount of p65 binding to the promoter of these genes and with histone H3 acetylation and HDAC-3 removal. Conclusion While IE63 only affected transcription of a small number of cellular genes, it interfered with the TNF-inducibility of several NF-κB dependent genes by the accelerated resynthesis of the inhibitor IκBα.

  13. Role of protein phosphatase 1 in dephosphorylation of Ebola virus VP30 protein and its targeting for the inhibition of viral transcription.

    Science.gov (United States)

    Ilinykh, Philipp A; Tigabu, Bersabeh; Ivanov, Andrey; Ammosova, Tatiana; Obukhov, Yuri; Garron, Tania; Kumari, Namita; Kovalskyy, Dmytro; Platonov, Maxim O; Naumchik, Vasiliy S; Freiberg, Alexander N; Nekhai, Sergei; Bukreyev, Alexander

    2014-08-15

    The filovirus Ebola (EBOV) causes the most severe hemorrhagic fever known. The EBOV RNA-dependent polymerase complex includes a filovirus-specific VP30, which is critical for the transcriptional but not replication activity of EBOV polymerase; to support transcription, VP30 must be in a dephosphorylated form. Here we show that EBOV VP30 is phosphorylated not only at the N-terminal serine clusters identified previously but also at the threonine residues at positions 143 and 146. We also show that host cell protein phosphatase 1 (PP1) controls VP30 dephosphorylation because expression of a PP1-binding peptide cdNIPP1 increased VP30 phosphorylation. Moreover, targeting PP1 mRNA by shRNA resulted in the overexpression of SIPP1, a cytoplasm-shuttling regulatory subunit of PP1, and increased EBOV transcription, suggesting that cytoplasmic accumulation of PP1 induces EBOV transcription. Furthermore, we developed a small molecule compound, 1E7-03, that targeted a non-catalytic site of PP1 and increased VP30 dephosphorylation. The compound inhibited the transcription but increased replication of the viral genome and completely suppressed replication of EBOV in cultured cells. Finally, mutations of Thr(143) and Thr(146) of VP30 significantly inhibited EBOV transcription and strongly induced VP30 phosphorylation in the N-terminal Ser residues 29-46, suggesting a novel mechanism of regulation of VP30 phosphorylation. Our findings suggest that targeting PP1 with small molecules is a feasible approach to achieve dysregulation of the EBOV polymerase activity. This novel approach may be used for the development of antivirals against EBOV and other filovirus species. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Coordination of genomic structure and transcription by the main bacterial nucleoid-associated protein HU

    Science.gov (United States)

    Berger, Michael; Farcas, Anca; Geertz, Marcel; Zhelyazkova, Petya; Brix, Klaudia; Travers, Andrew; Muskhelishvili, Georgi

    2010-01-01

    The histone-like protein HU is a highly abundant DNA architectural protein that is involved in compacting the DNA of the bacterial nucleoid and in regulating the main DNA transactions, including gene transcription. However, the coordination of the genomic structure and function by HU is poorly understood. Here, we address this question by comparing transcript patterns and spatial distributions of RNA polymerase in Escherichia coli wild-type and hupA/B mutant cells. We demonstrate that, in mutant cells, upregulated genes are preferentially clustered in a large chromosomal domain comprising the ribosomal RNA operons organized on both sides of OriC. Furthermore, we show that, in parallel to this transcription asymmetry, mutant cells are also impaired in forming the transcription foci—spatially confined aggregations of RNA polymerase molecules transcribing strong ribosomal RNA operons. Our data thus implicate HU in coordinating the global genomic structure and function by regulating the spatial distribution of RNA polymerase in the nucleoid. PMID:20010798

  15. Binding of TFIIIC to sine elements controls the relocation of activity-dependent neuronal genes to transcription factories.

    Directory of Open Access Journals (Sweden)

    Luca Crepaldi

    Full Text Available In neurons, the timely and accurate expression of genes in response to synaptic activity relies on the interplay between epigenetic modifications of histones, recruitment of regulatory proteins to chromatin and changes to nuclear structure. To identify genes and regulatory elements responsive to synaptic activation in vivo, we performed a genome-wide ChIPseq analysis of acetylated histone H3 using somatosensory cortex of mice exposed to novel enriched environmental (NEE conditions. We discovered that Short Interspersed Elements (SINEs located distal to promoters of activity-dependent genes became acetylated following exposure to NEE and were bound by the general transcription factor TFIIIC. Importantly, under depolarizing conditions, inducible genes relocated to transcription factories (TFs, and this event was controlled by TFIIIC. Silencing of the TFIIIC subunit Gtf3c5 in non-stimulated neurons induced uncontrolled relocation to TFs and transcription of activity-dependent genes. Remarkably, in cortical neurons, silencing of Gtf3c5 mimicked the effects of chronic depolarization, inducing a dramatic increase of both dendritic length and branching. These findings reveal a novel and essential regulatory function of both SINEs and TFIIIC in mediating gene relocation and transcription. They also suggest that TFIIIC may regulate the rearrangement of nuclear architecture, allowing the coordinated expression of activity-dependent neuronal genes.

  16. Binding of TFIIIC to sine elements controls the relocation of activity-dependent neuronal genes to transcription factories.

    Science.gov (United States)

    Crepaldi, Luca; Policarpi, Cristina; Coatti, Alessandro; Sherlock, William T; Jongbloets, Bart C; Down, Thomas A; Riccio, Antonella

    2013-01-01

    In neurons, the timely and accurate expression of genes in response to synaptic activity relies on the interplay between epigenetic modifications of histones, recruitment of regulatory proteins to chromatin and changes to nuclear structure. To identify genes and regulatory elements responsive to synaptic activation in vivo, we performed a genome-wide ChIPseq analysis of acetylated histone H3 using somatosensory cortex of mice exposed to novel enriched environmental (NEE) conditions. We discovered that Short Interspersed Elements (SINEs) located distal to promoters of activity-dependent genes became acetylated following exposure to NEE and were bound by the general transcription factor TFIIIC. Importantly, under depolarizing conditions, inducible genes relocated to transcription factories (TFs), and this event was controlled by TFIIIC. Silencing of the TFIIIC subunit Gtf3c5 in non-stimulated neurons induced uncontrolled relocation to TFs and transcription of activity-dependent genes. Remarkably, in cortical neurons, silencing of Gtf3c5 mimicked the effects of chronic depolarization, inducing a dramatic increase of both dendritic length and branching. These findings reveal a novel and essential regulatory function of both SINEs and TFIIIC in mediating gene relocation and transcription. They also suggest that TFIIIC may regulate the rearrangement of nuclear architecture, allowing the coordinated expression of activity-dependent neuronal genes.

  17. Transcription initiation complex structures elucidate DNA opening.

    Science.gov (United States)

    Plaschka, C; Hantsche, M; Dienemann, C; Burzinski, C; Plitzko, J; Cramer, P

    2016-05-19

    Transcription of eukaryotic protein-coding genes begins with assembly of the RNA polymerase (Pol) II initiation complex and promoter DNA opening. Here we report cryo-electron microscopy (cryo-EM) structures of yeast initiation complexes containing closed and open DNA at resolutions of 8.8 Å and 3.6 Å, respectively. DNA is positioned and retained over the Pol II cleft by a network of interactions between the TATA-box-binding protein TBP and transcription factors TFIIA, TFIIB, TFIIE, and TFIIF. DNA opening occurs around the tip of the Pol II clamp and the TFIIE 'extended winged helix' domain, and can occur in the absence of TFIIH. Loading of the DNA template strand into the active centre may be facilitated by movements of obstructing protein elements triggered by allosteric binding of the TFIIE 'E-ribbon' domain. The results suggest a unified model for transcription initiation with a key event, the trapping of open promoter DNA by extended protein-protein and protein-DNA contacts.

  18. mTORC1 Balances Cellular Amino Acid Supply with Demand for Protein Synthesis through Post-transcriptional Control of ATF4

    Directory of Open Access Journals (Sweden)

    Yeonwoo Park

    2017-05-01

    Full Text Available The mammalian target of rapamycin complex 1 (mTORC1 is a master regulator of cell growth that is commonly deregulated in human diseases. Here we find that mTORC1 controls a transcriptional program encoding amino acid transporters and metabolic enzymes through a mechanism also used to regulate protein synthesis. Bioinformatic analysis of mTORC1-responsive mRNAs identified a promoter element recognized by activating transcription factor 4 (ATF4, a key effector of the integrated stress response. ATF4 translation is normally induced by the phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α through a mechanism that requires upstream open reading frames (uORFs in the ATF4 5′ UTR. mTORC1 also controls ATF4 translation through uORFs, but independently of changes in eIF2α phosphorylation. mTORC1 instead employs the 4E-binding protein (4E-BP family of translation repressors. These results link mTORC1-regulated demand for protein synthesis with an ATF4-regulated transcriptional program that controls the supply of amino acids to the translation machinery.

  19. C/EBPβ (CCAAT/enhancer-binding protein β) mediates progesterone production through transcriptional regulation in co-operation with SF-1 (steroidogenic factor-1).

    Science.gov (United States)

    Mizutani, Tetsuya; Ju, Yunfeng; Imamichi, Yoshitaka; Osaki, Tsukasa; Yazawa, Takashi; Kawabe, Shinya; Ishikane, Shin; Matsumura, Takehiro; Kanno, Masafumi; Kamiki, Yasue; Kimura, Kohei; Minamino, Naoto; Miyamoto, Kaoru

    2014-06-15

    The transcription factor SF-1 (steroidogenic factor-1) is a master regulator of steroidogenesis. Previously, we have found that SF-1 induces the differentiation of mesenchymal stem cells into steroidogenic cells. To elucidate the molecular mechanisms of SF-1-mediated functions, we attempted to identify protein components of the SF-1 nuclear protein complex in differentiated cells. SF-1 immunoaffinity chromatography followed by MS/MS analysis was performed, and 24 proteins were identified. Among these proteins, we focused on C/EBPβ (CCAAT/enhancer-binding protein β), which is an essential transcription factor for ovulation and luteinization, as the transcriptional mechanisms of C/EBPβ working together with SF-1 are poorly understood. C/EBPβ knockdown attenuated cAMP-induced progesterone production in granulosa tumour-derived KGN cells by altering STAR (steroidogenic acute regulatory protein), CYP11A1 (cytochrome P450, family 11, subfamily A, polypeptide 1) and HSD3B2 (hydroxy-δ-5-steroid dehydrogenase, 3β- and steroid δ-isomerase 2) expression. EMSA and ChIP assays revealed novel C/EBPβ-binding sites in the upstream regions of the HSD3B2 and CYP11A1 genes. These interactions were enhanced by cAMP stimulation. Luciferase assays showed that C/EBPβ-responsive regions were found in each promoter and C/EBPβ is involved in the cAMP-induced transcriptional activity of these genes together with SF-1. These results indicate that C/EBPβ is an important mediator of progesterone production by working together with SF-1, especially under tropic hormone-stimulated conditions.

  20. Ethanol-induced transcriptional activation of programmed cell death 4 (Pdcd4 is mediated by GSK-3β signaling in rat cortical neuroblasts.

    Directory of Open Access Journals (Sweden)

    Amanjot Kaur Riar

    Full Text Available Ingestion of ethanol (ETOH during pregnancy induces grave abnormalities in developing fetal brain. We have previously reported that ETOH induces programmed cell death 4 (PDCD4, a critical regulator of cell growth, in cultured fetal cerebral cortical neurons (PCNs and in the cerebral cortex in vivo and affect protein synthesis as observed in Fetal Alcohol Spectrum Disorder (FASD. However, the mechanism which activates PDCD4 in neuronal systems is unclear and understanding this regulation may provide a counteractive strategy to correct the protein synthesis associated developmental changes seen in FASD. The present study investigates the molecular mechanism by which ethanol regulates PDCD4 in cortical neuroblasts, the immediate precursor of neurons. ETOH treatment significantly increased PDCD4 protein and transcript expression in spontaneously immortalized rat brain neuroblasts. Since PDCD4 is regulated at both the post-translational and post-transcriptional level, we assessed ETOH's effect on PDCD4 protein and mRNA stability. Chase experiments demonstrated that ETOH does not significantly impact either PDCD4 protein or mRNA stabilization. PDCD4 promoter-reporter assays confirmed that PDCD4 is transcriptionally regulated by ETOH in neuroblasts. Given a critical role of glycogen synthase kinase 3β (GSK-3β signaling in regulating protein synthesis and neurotoxic mechanisms, we investigated the involvement of GSK-3β and showed that multifunctional GSK-3β was significantly activated in response to ETOH in neuroblasts. In addition, we found that ETOH-induced activation of PDCD4 was inhibited by pharmacologic blockade of GSK-3β using inhibitors, lithium chloride (LiCl and SB-216763 or siRNA mediated silencing of GSK-3β. These results suggest that ethanol transcriptionally upregulates PDCD4 by enhancing GSK-3β signaling in cortical neuroblasts. Further, we demonstrate that canonical Wnt-3a/GSK-3β signaling is involved in regulating PDCD4 protein

  1. RNA-guided transcriptional activation via CRISPR/dCas9 mimics overexpression phenotypes in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Jong-Jin Park

    Full Text Available Clustered regularly interspaced short palindromic repeats (CRISPR and the CRISPR associated protein 9 (Cas9 system allows effective gene modification through RNA-guided DNA targeting. The Cas9 has undergone a series of functional alterations from the original active endonuclease to partially or completely deactivated Cas9. The catalytically deactivated Cas9 (dCas9 offers a platform to regulate transcriptional expression with the addition of activator or repressor domains. We redesigned a CRISPR/Cas9 activation system by adding the p65 transactivating subunit of NF-kappa B and a heat-shock factor 1 (HSF activation domain to dCas9 bound with the VP64 (tetramer of VP16 activation domain for application in plants. The redesigned CRISPR/Cas9 activation system was tested in Arabidopsis to increase endogenous transcriptional levels of production of anthocyanin pigment 1 (PAP1 and Arabidopsis thaliana vacuolar H+-pyrophosphatase (AVP1. The expression of PAP1 was increased two- to three-fold and the activated plants exhibited purple leaves similar to that of PAP1 overexpressors. The AVP1 gene expression was increased two- to five-fold in transgenic plants. In comparison to the wild type, AVP1 activated plants had increased leaf numbers, larger single-leaf areas and improved tolerance to drought stress. The AVP1 activated plants showed similar phenotypes to AVP1 overexpressors. Therefore, the redesigned CRISPR/Cas9 activation system containing modified p65-HSF provides a simple approach for producing activated plants by upregulating endogenous transcriptional levels.

  2. dFOXO Activates Large and Small Heat Shock Protein Genes in Response to Oxidative Stress to Maintain Proteostasis in Drosophila.

    Science.gov (United States)

    Donovan, Marissa R; Marr, Michael T

    2016-09-02

    Maintaining protein homeostasis is critical for survival at the cellular and organismal level (Morimoto, R. I. (2011) Cold Spring Harb. Symp. Quant. Biol. 76, 91-99). Cells express a family of molecular chaperones, the heat shock proteins, during times of oxidative stress to protect against proteotoxicity. We have identified a second stress responsive transcription factor, dFOXO, that works alongside the heat shock transcription factor to activate transcription of both the small heat shock protein and the large heat shock protein genes. This expression likely protects cells from protein misfolding associated with oxidative stress. Here we identify the regions of the Hsp70 promoter essential for FOXO-dependent transcription using in vitro methods and find a physiological role for FOXO-dependent expression of heat shock proteins in vivo. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. RNA-guided transcriptional regulation in planta via synthetic dCas9-based transcription factors

    KAUST Repository

    Piatek, Agnieszka Anna

    2014-11-14

    Targeted genomic regulation is a powerful approach to accelerate trait discovery and development in agricultural biotechnology. Bacteria and archaea use clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) regulatory systems for adaptive molecular immunity against foreign nucleic acids introduced by invading phages and conjugative plasmids. The type II CRISPR/Cas system has been adapted for genome editing in many cell types and organisms. A recent study used the catalytically inactive Cas9 (dCas9) protein combined with guide-RNAs (gRNAs) as a DNA-targeting platform to modulate gene expression in bacterial, yeast, and human cells. Here, we modified this DNA-targeting platform for targeted transcriptional regulation in planta by developing chimeric dCas9-based transcriptional activators and repressors. To generate transcriptional activators, we fused the dCas9 C-terminus with the activation domains of EDLL and TAL effectors. To generate a transcriptional repressor, we fused the dCas9 C-terminus with the SRDX repression domain. Our data demonstrate that dCas9 fusion with the EDLL activation domain (dCas9:EDLL) and the TAL activation domain (dCas9:TAD), guided by gRNAs complementary to selected promoter elements, induce strong transcriptional activation on Bs3

  4. RNA-guided transcriptional regulation in planta via synthetic dCas9-based transcription factors

    KAUST Repository

    Piatek, Agnieszka Anna; Ali, Zahir; Baazim, Hatoon; Li, Lixin; Abulfaraj, Aala A.; Alshareef, Sahar; Aouida, Mustapha; Mahfouz, Magdy M.

    2014-01-01

    Targeted genomic regulation is a powerful approach to accelerate trait discovery and development in agricultural biotechnology. Bacteria and archaea use clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) regulatory systems for adaptive molecular immunity against foreign nucleic acids introduced by invading phages and conjugative plasmids. The type II CRISPR/Cas system has been adapted for genome editing in many cell types and organisms. A recent study used the catalytically inactive Cas9 (dCas9) protein combined with guide-RNAs (gRNAs) as a DNA-targeting platform to modulate gene expression in bacterial, yeast, and human cells. Here, we modified this DNA-targeting platform for targeted transcriptional regulation in planta by developing chimeric dCas9-based transcriptional activators and repressors. To generate transcriptional activators, we fused the dCas9 C-terminus with the activation domains of EDLL and TAL effectors. To generate a transcriptional repressor, we fused the dCas9 C-terminus with the SRDX repression domain. Our data demonstrate that dCas9 fusion with the EDLL activation domain (dCas9:EDLL) and the TAL activation domain (dCas9:TAD), guided by gRNAs complementary to selected promoter elements, induce strong transcriptional activation on Bs3

  5. Divergent Evolution of the Transcriptional Network Controlled by Snf1-Interacting Protein Sip4 in Budding Yeasts.

    Directory of Open Access Journals (Sweden)

    Constance Mehlgarten

    Full Text Available Cellular responses to starvation are of ancient origin since nutrient limitation has always been a common challenge to the stability of living systems. Hence, signaling molecules involved in sensing or transducing information about limiting metabolites are highly conserved, whereas transcription factors and the genes they regulate have diverged. In eukaryotes the AMP-activated protein kinase (AMPK functions as a central regulator of cellular energy homeostasis. The yeast AMPK ortholog SNF1 controls the transcriptional network that counteracts carbon starvation conditions by regulating a set of transcription factors. Among those Cat8 and Sip4 have overlapping DNA-binding specificity for so-called carbon source responsive elements and induce target genes upon SNF1 activation. To analyze the evolution of the Cat8-Sip4 controlled transcriptional network we have compared the response to carbon limitation of Saccharomyces cerevisiae to that of Kluyveromyces lactis. In high glucose, S. cerevisiae displays tumor cell-like aerobic fermentation and repression of respiration (Crabtree-positive while K. lactis has a respiratory-fermentative life-style, respiration being regulated by oxygen availability (Crabtree-negative, which is typical for many yeasts and for differentiated higher cells. We demonstrate divergent evolution of the Cat8-Sip4 network and present evidence that a role of Sip4 in controlling anabolic metabolism has been lost in the Saccharomyces lineage. We find that in K. lactis, but not in S. cerevisiae, the Sip4 protein plays an essential role in C2 carbon assimilation including induction of the glyoxylate cycle and the carnitine shuttle genes. Induction of KlSIP4 gene expression by KlCat8 is essential under these growth conditions and a primary function of KlCat8. Both KlCat8 and KlSip4 are involved in the regulation of lactose metabolism in K. lactis. In chromatin-immunoprecipitation experiments we demonstrate binding of both, KlSip4 and

  6. Transcription and expression of Plasmodium falciparum histidine-rich proteins in different stages and strains: implications for rapid diagnostic tests.

    Directory of Open Access Journals (Sweden)

    Joanne Baker

    Full Text Available BACKGROUND: Although rapid diagnostic tests (RDTs for Plasmodium falciparum infection that target histidine rich protein 2 (PfHRP2 are generally sensitive, their performance has been reported to be variable. One possible explanation for variable test performance is differences in expression level of PfHRP in different parasite isolates. METHODS: Total RNA and protein were extracted from synchronised cultures of 7 P. falciparum lines over 5 time points of the life cycle, and from synchronised ring stages of 10 falciparum lines. Using quantitative real-time polymerase chain reaction, Western blot analysis and ELISA we investigated variations in the transcription and protein levels of pfhrp2, pfhrp3 and PfHRP respectively in the different parasite lines, over the parasite intraerythrocytic life cycle. RESULTS: Transcription of pfhrp2 and pfhrp3 in different parasite lines over the parasite life cycle was observed to vary relative to the control parasite K1. In some parasite lines very low transcription of these genes was observed. The peak transcription was observed in ring-stage parasites. Pfhrp2 transcription was observed to be consistently higher than pfhrp3 transcription within parasite lines. The intraerythrocytic lifecycle stage at which the peak level of protein was present varied across strains. Total protein levels were more constant relative to total mRNA transcription, however a maximum 24 fold difference in expression at ring-stage parasites relative to the K1 strain was observed. CONCLUSIONS: The levels of transcription of pfhrp2 and pfhrp3, and protein expression of PfHRP varied between different P. falciparum strains. This variation may impact on the detection sensitivity of PfHRP2-detecting RDTs.

  7. Nuclear translocation of doublecortin-like protein kinase and phosphorylation of a transcription factor JDP2

    Energy Technology Data Exchange (ETDEWEB)

    Nagamine, Tadashi; Nomada, Shohgo; Onouchi, Takashi; Kameshita, Isamu; Sueyoshi, Noriyuki, E-mail: sueyoshi@ag.kagawa-u.ac.jp

    2014-03-28

    Highlights: • Doublecortin-like protein kinase (DCLK) is a microtubule-associated protein kinase. • In living cells, DCLK was cleaved into two functional fragments. • zDCLK(kinase) was translocated into the nucleus by osmotic stresses. • Jun dimerization protein 2 (JDP2) was identified as zDCLK(kinase)-binding protein. • JDP2 was efficiently phosphorylated by zDCLK(kinase) only when histone was present. - Abstract: Doublecortin-like protein kinase (DCLK) is a microtubule-associated protein kinase predominantly expressed in brain. In a previous paper, we reported that zebrafish DCLK2 (zDCLK) was cleaved into two functional fragments; the N-terminal zDCLK(DC + SP) with microtubule-binding activity and the C-terminal zDCLK(kinase) with a Ser/Thr protein kinase activity. In this study, we demonstrated that zDCLK(kinase) was widely distributed in the cytoplasm and translocated into the nucleus when the cells were treated under hyperosmotic conditions with NaCl or mannitol. By two-hybrid screening using the C-terminal domain of DCLK, Jun dimerization protein 2 (JDP2), a nuclear transcription factor, was identified as zDCLK(kinase)-binding protein. Furthermore, JDP2 served as an efficient substrate for zDCLK(kinase) only when histone was present. These results suggest that the kinase fragment of DCLK is translocated into the nucleus upon hyperosmotic stresses and that the kinase efficiently phosphorylates JDP2, a possible target in the nucleus, with the aid of histones.

  8. Transcriptional Regulation During Zygotic Genome Activation in Zebrafish and Other Anamniote Embryos.

    Science.gov (United States)

    Wragg, J; Müller, F

    2016-01-01

    Embryo development commences with the fusion of two terminally differentiated haploid gametes into the totipotent fertilized egg, which through a series of major cellular and molecular transitions generate a pluripotent cell mass. The activation of the zygotic genome occurs during the so-called maternal to zygotic transition and prepares the embryo for zygotic takeover from maternal factors, in the control of the development of cellular lineages during differentiation. Recent advances in next generation sequencing technologies have allowed the dissection of the genomic and epigenomic processes mediating this transition. These processes include reorganization of the chromatin structure to a transcriptionally permissive state, changes in composition and function of structural and regulatory DNA-binding proteins, and changeover of the transcriptome as it is overhauled from that deposited by the mother in the oocyte to a zygotically transcribed complement. Zygotic genome activation in zebrafish occurs 10 cell cycles after fertilization and provides an ideal experimental platform for elucidating the temporal sequence and dynamics of establishment of a transcriptionally active chromatin state and helps in identifying the determinants of transcription activation at polymerase II transcribed gene promoters. The relatively large number of pluripotent cells generated by the fast cell divisions before zygotic transcription provides sufficient biomass for next generation sequencing technology approaches to establish the temporal dynamics of events and suggest causative relationship between them. However, genomic and genetic technologies need to be improved further to capture the earliest events in development, where cell number is a limiting factor. These technologies need to be complemented with precise, inducible genetic interference studies using the latest genome editing tools to reveal the function of candidate determinants and to confirm the predictions made by classic

  9. The metabolic activator FOXO1 binds hepatitis B virus DNA and activates its transcription

    International Nuclear Information System (INIS)

    Shlomai, Amir; Shaul, Yosef

    2009-01-01

    Hepatitis B virus (HBV) is a small DNA virus that targets the liver and infects humans worldwide. Recently we have shown that the metabolic regulator PGC-1α coactivates HBV transcription thereby rendering the virus susceptible to fluctuations in the nutritional status of the liver. PGC-1α coactivation of HBV is mediated through the liver-enriched nuclear receptor HNF4α and through another yet unknown transcription factor(s). Here we show that the forkhead transcription factor FOXO1, a known target for PGC-1α coactivation and a central mediator of glucose metabolism in the liver, binds HBV core promoter and activates its transcription. This activation is further enhanced in the presence of PGC-1α, implying that FOXO1 is a target for PGC-1α coactivation of HBV transcription. Thus, our results identify another key metabolic regulator as an activator of HBV transcription, thereby supporting the principle that HBV gene expression is regulated in a similar way to key hepatic metabolic genes.

  10. The transcriptional repressor protein NsrR senses nitric oxide directly via a [2Fe-2S] cluster.

    Directory of Open Access Journals (Sweden)

    Nicholas P Tucker

    Full Text Available The regulatory protein NsrR, a member of the Rrf2 family of transcription repressors, is specifically dedicated to sensing nitric oxide (NO in a variety of pathogenic and non-pathogenic bacteria. It has been proposed that NO directly modulates NsrR activity by interacting with a predicted [Fe-S] cluster in the NsrR protein, but no experimental evidence has been published to support this hypothesis. Here we report the purification of NsrR from the obligate aerobe Streptomyces coelicolor. We demonstrate using UV-visible, near UV CD and EPR spectroscopy that the protein contains an NO-sensitive [2Fe-2S] cluster when purified from E. coli. Upon exposure of NsrR to NO, the cluster is nitrosylated, which results in the loss of DNA binding activity as detected by bandshift assays. Removal of the [2Fe-2S] cluster to generate apo-NsrR also resulted in loss of DNA binding activity. This is the first demonstration that NsrR contains an NO-sensitive [2Fe-2S] cluster that is required for DNA binding activity.

  11. The metazoan Mediator co-activator complex as an integrative hub for transcriptional regulation

    OpenAIRE

    Malik, Sohail; Roeder, Robert G.

    2010-01-01

    The Mediator is an evolutionarily conserved, multiprotein complex that is a key regulator of protein-coding genes. In metazoan cells, multiple pathways that are responsible for homeostasis, cell growth and differentiation converge on the Mediator through transcriptional activators and repressors that target one or more of the almost 30 subunits of this complex. Besides interacting directly with RNA polymerase II, Mediator has multiple functions and can interact with and coordinate the action ...

  12. cAMP-response Element-binding Protein (CREB) and NF-κB Transcription Factors Are Activated during Prolonged Hypoxia and Cooperatively Regulate the Induction of Matrix Metalloproteinase MMP1*

    Science.gov (United States)

    Nakayama, Koh

    2013-01-01

    Responses to low levels of oxygen (hypoxia) are essential to maintain homeostasis. During the hypoxic response, gene expression is altered by various transcription factors. The transcription factor, hypoxia-inducible factor (HIF), plays a central role in the hypoxic response. The α subunit of HIF, which is actively degraded during normoxia, becomes stabilized during hypoxia, which leads to HIF activation. A microarray analysis of HeLa cells showed that expression of matrix metalloproteinase 1 (MMP1) was markedly induced during prolonged hypoxia. CREB and NF-κB binding sites were identified in the MMP1 promoter region between 1945 and 1896 nucleotides upstream of the transcription start site. Assays with luciferase reporters demonstrated that HIF activity was induced during the early phase of hypoxia, whereas CREB and NF-κB were activated during the later (prolonged) phase. Depletion of CREB and/or NF-κB reduced MMP1 induction during prolonged hypoxia both at the mRNA and protein levels. A chromatin immunoprecipitation assay demonstrated binding of CREB and NF-κB to the MMP1 promoter. Finally, cell migration and invasion on a collagen matrix and pulmonary metastasis in nude mice were inhibited after depletion of CREB and NF-κB in MDA-MB-231 cells. Taken together, these results suggest that the cooperative action of CREB and NF-κB plays an important role to induce MMP1 expression during prolonged hypoxia and regulates cell migration and invasion in cancer cells. PMID:23775082

  13. Heterodimerization of the transcription factors E2F-1 and DP-1 is required for binding to the adenovirus E4 (ORF6/7) protein

    DEFF Research Database (Denmark)

    Helin, K; Harlow, E

    1994-01-01

    Adenovirus infection leads to E1A-dependent activation of the transcription factor E2F. E2F has recently been identified in complexes with cellular proteins such as the retinoblastoma protein (pRB) and the two pRB family members p107 and p130. E1A dissociates E2F from these cellular proteins...

  14. Changes in pH and NADPH regulate the DNA binding activity of neuronal PAS domain protein 2, a mammalian circadian transcription factor.

    Science.gov (United States)

    Yoshii, Katsuhiro; Tajima, Fumihisa; Ishijima, Sumio; Sagami, Ikuko

    2015-01-20

    Neuronal PAS domain protein 2 (NPAS2) is a core clock transcription factor that forms a heterodimer with BMAL1 to bind the E-box in the promoter of clock genes and is regulated by various environmental stimuli such as heme, carbon monoxide, and NAD(P)H. In this study, we investigated the effects of pH and NADPH on the DNA binding activity of NPAS2. In an electrophoretic mobility shift (EMS) assay, the pH of the reaction mixture affected the DNA binding activity of the NPAS2/BMAL1 heterodimer but not that of the BMAL1/BMAL1 homodimer. A change in pH from 7.0 to 7.5 resulted in a 1.7-fold increase in activity in the absence of NADPH, and NADPH additively enhanced the activity up to 2.7-fold at pH 7.5. The experiments using truncated mutants revealed that N-terminal amino acids 1-61 of NPAS2 were sufficient to sense the change in both pH and NADPH. We further analyzed the kinetics of formation and DNA binding of the NPAS2/BMAL1 heterodimer at various pH values. In the absence of NADPH, a change in pH from 6.5 to 8.0 decreased the KD(app) value of the E-box from 125 to 22 nM, with an 8-fold increase in the maximal level of DNA binding for the NPAS2/BMAL1 heterodimer. The addition of NADPH resulted in a further decrease in KD(app) to 9 nM at pH 8.0. Furthermore, NPAS2-dependent transcriptional activity in a luciferase assay using NIH3T3 cells also increased with the pH of the culture medium. These results suggest that NPAS2 has a role as a pH and metabolite sensor in regulating circadian rhythms.

  15. Zinc coordination is required for and regulates transcription activation by Epstein-Barr nuclear antigen 1.

    Directory of Open Access Journals (Sweden)

    Siddhesh Aras

    2009-06-01

    Full Text Available Epstein-Barr Nuclear Antigen 1 (EBNA1 is essential for Epstein-Barr virus to immortalize naïve B-cells. Upon binding a cluster of 20 cognate binding-sites termed the family of repeats, EBNA1 transactivates promoters for EBV genes that are required for immortalization. A small domain, termed UR1, that is 25 amino-acids in length, has been identified previously as essential for EBNA1 to activate transcription. In this study, we have elucidated how UR1 contributes to EBNA1's ability to transactivate. We show that zinc is necessary for EBNA1 to activate transcription, and that UR1 coordinates zinc through a pair of essential cysteines contained within it. UR1 dimerizes upon coordinating zinc, indicating that EBNA1 contains a second dimerization interface in its amino-terminus. There is a strong correlation between UR1-mediated dimerization and EBNA1's ability to transactivate cooperatively. Point mutants of EBNA1 that disrupt zinc coordination also prevent self-association, and do not activate transcription cooperatively. Further, we demonstrate that UR1 acts as a molecular sensor that regulates the ability of EBNA1 to activate transcription in response to changes in redox and oxygen partial pressure (pO(2. Mild oxidative stress mimicking such environmental changes decreases EBNA1-dependent transcription in a lymphoblastoid cell-line. Coincident with a reduction in EBNA1-dependent transcription, reductions are observed in EBNA2 and LMP1 protein levels. Although these changes do not affect LCL survival, treated cells accumulate in G0/G1. These findings are discussed in the context of EBV latency in body compartments that differ strikingly in their pO(2 and redox potential.

  16. Bacterial Genome Editing Strategy for Control of Transcription and Protein Stability

    DEFF Research Database (Denmark)

    Lauritsen, Ida; Martinez, Virginia; Ronda, Carlotta

    2018-01-01

    In molecular biology and cell factory engineering, tools that enable control of protein production and stability are highly important. Here, we describe protocols for tagging genes in Escherichia coli allowing for inducible degradation and transcriptional control of any soluble protein of interest....... The underlying molecular biology is based on the two cross-kingdom tools CRISPRi and the N-end rule for protein degradation. Genome editing is performed with the CRMAGE technology and randomization of the translational initiation region minimizes the polar effects of tag insertion. The approach has previously...... been applied for targeting proteins originating from essential operon-located genes and has potential to serve as a universal synthetic biology tool....

  17. The activation of peroxisome proliferator-activated receptor γ is regulated by Krüppel-like transcription factors 6 & 9 under steatotic conditions

    Energy Technology Data Exchange (ETDEWEB)

    Escalona-Nandez, Ivonne; Guerrero-Escalera, Dafne; Estanes-Hernández, Alma [Departamento de Gastroenterología, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Vasco de Quiroga 15 Sección XVI, Tlalpan, 14000, México, D.F. (Mexico); Ortíz-Ortega, Victor; Tovar, Armando R. [Departamento de Fisiología de la Nutrición, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Vasco de Quiroga 15 Sección XVI, Tlalpan, 14000, México, D.F. (Mexico); Pérez-Monter, Carlos, E-mail: carlos.perezm@incmnsz.mx [Departamento de Gastroenterología, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Vasco de Quiroga 15 Sección XVI, Tlalpan, 14000, México, D.F. (Mexico)

    2015-03-20

    Liver steatosis is characterised by lipid droplet deposition in hepatocytes that can leads to an inflammatory and fibrotic phenotype. Peroxisome proliferator-activated receptors (PPARs) play key roles in energetic homeostasis by regulating lipid metabolism in hepatic tissue. In adipose tissue PPARγ regulates the adipocyte differentiation by promoting the expression of lipid-associated genes. Within the liver PPARγ is up-regulated under steatotic conditions; however, which transcription factors participate in its expression is not completely understood. Krüppel-like transcription factors (KLFs) regulate various cellular mechanisms, such as cell proliferation and differentiation. KLFs are key components of adipogenesis by regulating the expression of PPARγ and other proteins such as the C-terminal enhancer binding protein (C/EBP). Here, we demonstrate that the transcript levels of Klf6, Klf9 and Pparγ are increased in response to a steatotic insult in vitro. Chromatin immunoprecipitation (ChIp) experiments showed that klf6 and klf9 are actively recruited to the Pparγ promoter region under these conditions. Accordingly, the loss-of-function experiments reduced cytoplasmic triglyceride accumulation. Here, we demonstrated that KLF6 and KLF9 proteins directly regulate PPARγ expression under steatotic conditions. - Highlights: • Palmitic acid promotes expression of KlF6 & KLF9 in HepG2 cells. • KLF6 and KLF9 promote the expression of PPARγ in response to palmitic acid. • Binding of KLF6 and KLF9 to the PPARγ promoter promotes steatosis in HepG2 cells. • KLF6 and KLF9 loss-of function diminishes the steatosis in HepG2 cells.

  18. Tip60 degradation by adenovirus relieves transcriptional repression of viral transcriptional activator EIA.

    Science.gov (United States)

    Gupta, A; Jha, S; Engel, D A; Ornelles, D A; Dutta, A

    2013-10-17

    Adenoviruses are linear double-stranded DNA viruses that infect human and rodent cell lines, occasionally transform them and cause tumors in animal models. The host cell challenges the virus in multifaceted ways to restrain viral gene expression and DNA replication, and sometimes even eliminates the infected cells by programmed cell death. To combat these challenges, adenoviruses abrogate the cellular DNA damage response pathway. Tip60 is a lysine acetyltransferase that acetylates histones and other proteins to regulate gene expression, DNA damage response, apoptosis and cell cycle regulation. Tip60 is a bona fide tumor suppressor as mice that are haploid for Tip60 are predisposed to tumors. We have discovered that Tip60 is degraded by adenovirus oncoproteins EIB55K and E4orf6 by a proteasome-mediated pathway. Tip60 binds to the immediate early adenovirus promoter and suppresses adenovirus EIA gene expression, which is a master regulator of adenovirus transcription, at least partly through retention of the virally encoded repressor pVII on this promoter. Thus, degradation of Tip60 by the adenoviral early proteins is important for efficient viral early gene transcription and for changes in expression of cellular genes.

  19. The transcriptional activator GAL4-VP16 regulates the intra ...

    Indian Academy of Sciences (India)

    Activator also reduced the TBP dimer levels both in vitro and in vivo, suggesting the dimer may be a direct target of transcriptional activators. The transcriptional activator facilitated the dimer to monomer transition and activated monomers further to help TBP bind even the weaker TATA boxes stably. The overall stimulatory ...

  20. Zinc finger proteins and other transcription regulators as response proteins in benzo[a]pyrene exposed cells

    International Nuclear Information System (INIS)

    Gao Zhihua; Jin Jinghua; Yang Jun; Yu Yingnian

    2004-01-01

    Proteomic analysis, which combines two-dimensional electrophoresis (2-DE) and mass spectrometry (MS), is an important approach to screen proteins responsive to specific stimuli. Benzo[a]pyrene (B[a]P), a prototype of polycyclic hydrocarbons (PAHs), is a potent procarcinogen generated from the combustion of fossil fuel and cigarette smoke. To further probe the molecular mechanism of mutagenesis and carcinogenesis, and to find potential molecular markers involved in cellular responses to B[a]P exposure, we performed proteomic analysis of whole cellular proteins in human amnion epithelial cells after B[a]P-treatment. Image visualization and statistical analysis indicated that more than 40 proteins showed significant changes following B[a]P-treatment (P<0.05). Among them, 20 proteins existed only in the control groups, while six were only present in B[a]P-treated cells. In addition, the expression of 10 proteins increased whereas 11 decreased after B[a]P-treatment. These proteins were subjected to in-gel tryptic digestion followed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) analysis. Using peptide mass fingerprinting (PMF) to search the nrNCBI database, we identified 22 proteins. Most of these proteins have unknown functions and have not been previously connected to a response to B[a]P exposure. To further annotate the characteristics of these proteins, GOblet analysis was carried out and results indicated that they were involved in multiple biological processes including regulation of transcription, cell proliferation, cell aging and other processes. However, expression changes were noted in a number of transcription regulators, including eight zinc finger proteins as well as SNF2L1 (SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 1), which is closely linked to the chromatin remodeling process. These data may provide new clues to further understand the implication of

  1. Conserved TRAM Domain Functions as an Archaeal Cold Shock Protein via RNA Chaperone Activity

    Directory of Open Access Journals (Sweden)

    Bo Zhang

    2017-08-01

    Full Text Available Cold shock proteins (Csps enable organisms to acclimate to and survive in cold environments and the bacterial CspA family exerts the cold protection via its RNA chaperone activity. However, most Archaea do not contain orthologs to the bacterial csp. TRAM, a conserved domain among RNA modification proteins ubiquitously distributed in organisms, occurs as an individual protein in most archaeal phyla and has a structural similarity to Csp proteins, yet its biological functions remain unknown. Through physiological and biochemical studies on four TRAM proteins from a cold adaptive archaeon Methanolobus psychrophilus R15, this work demonstrated that TRAM is an archaeal Csp and exhibits RNA chaperone activity. Three TRAM encoding genes (Mpsy_0643, Mpsy_3043, and Mpsy_3066 exhibited remarkable cold-shock induced transcription and were preferentially translated at lower temperature (18°C, while the fourth (Mpsy_2002 was constitutively expressed. They were all able to complement the cspABGE mutant of Escherichia coli BX04 that does not grow in cold temperatures and showed transcriptional antitermination. TRAM3066 (gene product of Mpsy_3066 and TRAM2002 (gene product of Mpsy_2002 displayed sequence-non-specific RNA but not DNA binding activity, and TRAM3066 assisted RNases in degradation of structured RNA, thus validating the RNA chaperone activity of TRAMs. Given the chaperone activity, TRAM is predicted to function beyond a Csp.

  2. Identification of an estrogen receptor α non covalent ubiquitin-binding surface: role in 17β-estradiol-induced transcriptional activity.

    Science.gov (United States)

    Pesiri, Valeria; La Rosa, Piergiorgio; Stano, Pasquale; Acconcia, Filippo

    2013-06-15

    Ubiquitin (Ub)-binding domains (UBDs) located in Ub receptors decode the ubiquitination signal by non-covalently engaging the Ub modification on their binding partners and transduce the Ub signalling through Ub-based molecular interactions. In this way, inducible protein ubiquitination regulates diverse biological processes. The estrogen receptor alpha (ERα) is a ligand-activated transcription factor that mediates the pleiotropic effects of the sex hormone 17β-estradiol (E2). Fine regulation of E2 pleiotropic actions depends on E2-dependent ERα association with a plethora of binding partners and/or on the E2 modulation of receptor ubiquitination. Indeed, E2-induced ERα polyubiquitination triggers receptor degradation and transcriptional activity, and E2-dependent reduction in ERα monoubiquitination is crucial for E2 signalling. Monoubiquitinated proteins often contain UBDs, but whether non-covalent Ub-ERα binding could occur and play a role in E2-ERα signalling is unknown. Here, we report an Ub-binding surface within the ERα ligand binding domain that directs in vitro the receptor interaction with both ubiquitinated proteins and recombinant Ub chains. Mutational analysis reveals that ERα residues leucine 429 and alanine 430 are involved in Ub binding. Moreover, impairment of ERα association to ubiquitinated species strongly affects E2-induced ERα transcriptional activity. Considering the importance of UBDs in the Ub-based signalling network and the central role of different ERα binding partners in the modulation of E2-dependent effects, our discoveries provide novel insights into ERα activity that could also be relevant for ERα-dependent diseases.

  3. Activating transcription factor 6 polymorphisms and haplotypes are associated with impaired glucose homeostasis and type 2 diabetes in dutch Caucasians

    NARCIS (Netherlands)

    Meex, Steven J. R.; van Greevenbroek, Marleen M. J.; Ayoubi, Torik A.; Vlietinck, Robert; van Vliet-Ostaptchouk, Jana V.; Hofker, Martin H.; Vermeulen, Vicky M. M. -J.; Schalkwijk, Casper G.; Feskens, Edith J. M.; Boer, Jolanda M. A.; Stehouwer, Coen D. A.; van der Kallen, Carla J. H.; de Bruin, Tjerk W. A.

    Context: Activating transcription factor 6 (ATF6) is critical for initiation and full activation of the unfolded protein response. An association between genetic variation in ATF6 and type 2 diabetes (DM2) was recently reported in Pima Indians. Objectives: To investigate the broader significance of

  4. Genomewide analyses define different modes of transcriptional regulation by peroxisome proliferator-activated receptor-β/δ (PPARβ/δ.

    Directory of Open Access Journals (Sweden)

    Till Adhikary

    Full Text Available Peroxisome proliferator-activated receptors (PPARs are nuclear receptors with essential functions in lipid, glucose and energy homeostasis, cell differentiation, inflammation and metabolic disorders, and represent important drug targets. PPARs heterodimerize with retinoid X receptors (RXRs and can form transcriptional activator or repressor complexes at specific DNA elements (PPREs. It is believed that the decision between repression and activation is generally governed by a ligand-mediated switch. We have performed genomewide analyses of agonist-treated and PPARβ/δ-depleted human myofibroblasts to test this hypothesis and to identify global principles of PPARβ/δ-mediated gene regulation. Chromatin immunoprecipitation sequencing (ChIP-Seq of PPARβ/δ, H3K4me3 and RNA polymerase II enrichment sites combined with transcriptional profiling enabled the definition of 112 bona fide PPARβ/δ target genes showing either of three distinct types of transcriptional response: (I ligand-independent repression by PPARβ/δ; (II ligand-induced activation and/or derepression by PPARβ/δ; and (III ligand-independent activation by PPARβ/δ. These data identify PPRE-mediated repression as a major mechanism of transcriptional regulation by PPARβ/δ, but, unexpectedly, also show that only a subset of repressed genes are activated by a ligand-mediated switch. Our results also suggest that the type of transcriptional response by a given target gene is connected to the structure of its associated PPRE(s and the biological function of its encoded protein. These observations have important implications for understanding the regulatory PPAR network and PPARβ/δ ligand-based drugs.

  5. Suitability of Yin Yang 1 transcript and protein levels for biomarker studies in B cell non-Hodgkin lymphoma.

    Science.gov (United States)

    Arribas Arranz, Jéssica; Winter, Dalia Nilufar; Drexler, Hans Günter; Eberth, Sonja

    2018-01-01

    Yin Yang 1 (YY1) is a transcription factor that plays an important role during all stages of B cell differentiation. Several studies reported upregulation of YY1 in B cell derived lymphoma, indicating that it might act as an oncogene. Furthermore, aberrant YY1 expression has been associated with survival in some entities of B cell non-Hodgkin lymphoma (B-NHL), suggesting that YY1 could be a valuable biomarker in B-NHL. However, studies are controversial and methodologically disparate, partially because some studies are based on transcript levels while others rely on YY1 protein data. Therefore, we aimed to investigate the dependence of YY1 protein levels on YY1 transcription. A panel of human cell lines representing different B-NHL subtypes was used to test for the correlation of YY1 mRNA and protein levels which were determined by quantitative PCR and immunoblotting. To analyze YY1 mRNA and YY1 protein stability cells were treated with actinomycin-D and cycloheximide, respectively. siRNAs were transfected to knockdown YY1 . Kaplan-Meier survival analyses were performed with data from published patient cohorts. Pearson's correlation analyses were assessed and statistical power was examined by Student's t-test. In the analyzed panel of B-NHL cell lines YY1 transcript levels do not correlate with their cellular protein amounts. YY1 protein levels were unaffected by transient block of transcription or by targeting YY1 mRNA using siRNA. Additionally, global inhibition of translation up to 48 h did not alter protein levels of YY1, indicating that YY1 is a highly stable protein in B-NHL. Furthermore, in a retrospective analysis of two different B-NHL cohorts, YY1 transcript levels had no impact on patients' survival probabilities. Our results point out the necessity to focus on YY1 protein expression to understand the potential role of YY1 as an oncogene and to unravel its suitability as clinical biomarker in B-NHL.

  6. Identification of NR4A2 as a transcriptional activator of IL-8 expression in human inflammatory arthritis.

    LENUS (Irish Health Repository)

    Aherne, Carol M

    2009-10-01

    Expression of the orphan nuclear receptor NR4A2 is controlled by pro-inflammatory mediators, suggesting that NR4A2 may contribute to pathological processes in the inflammatory lesion. This study identifies the chemoattractant protein, interleukin 8 (IL-8\\/CXCL8), as a molecular target of NR4A2 in human inflammatory arthritis and examines the mechanism through which NR4A2 modulates IL-8 expression. In TNF-alpha-activated human synoviocyte cells, enhanced expression of IL-8 mRNA and protein correspond to temporal changes in NR4A2 transcription and nuclear distribution. Ectopic expression of NR4A2 leads to robust changes in endogenous IL-8 mRNA levels and co-treatment with TNF-alpha results in significant (p<0.001) secretion of IL-8 protein. Transcriptional effects of NR4A2 on the human IL-8 promoter are enhanced in the presence of TNF-alpha, suggesting molecular crosstalk between TNF-alpha signalling and NR4A2. A dominant negative IkappaB kinase antagonizes the combined effects of NR4A2 and TNF-alpha on IL-8 promoter activity. Co-expression of NR4A2 and the p65 subunit of NF-kappaB enhances IL-8 transcription and functional studies indicate that transactivation occurs independently of NR4A2 binding to DNA or heterodimerization with additional nuclear receptors. The IL-8 minimal promoter region is sufficient to support NR4A2 and NF-kappaB\\/p65 co-operative activity and NR4A2 can interact with NF-kappaB\\/p65 on a 39bp sequence within this region. In patients treated with methotrexate for active inflammatory arthritis, a reduction in NR4A2 synovial tissue levels correlate significantly (n=10, r=0.73, p=0.002) with changes in IL-8 expression. Collectively, these data delineate an important role for NR4A2 in modulating IL-8 expression and reveal novel transcriptional responses to TNF-alpha in human inflammatory joint disease.

  7. SUMOylation regulates the transcriptional repression activity of FOG-2 and its association with GATA-4.

    Science.gov (United States)

    Perdomo, José; Jiang, Xing-Mai; Carter, Daniel R; Khachigian, Levon M; Chong, Beng H

    2012-01-01

    Friend of GATA 2 (FOG-2), a co-factor of several GATA transcription factors (GATA-4, -5 and 6), is a critical regulator of coronary vessel formation and heart morphogenesis. Here we demonstrate that FOG-2 is SUMOylated and that this modification modulates its transcriptional activity. FOG-2 SUMOylation occurs at four lysine residues (K324, 471, 915, 955) [corrected]. Three of these residues are part of the characteristic SUMO consensus site (ψKXE), while K955 is found in the less frequent TKXE motif. Absence of SUMOylation did not affect FOG-2's nuclear localization. However, mutation of the FOG-2 SUMOylation sites, or de-SUMOylation, with SENP-1 or SENP-8 resulted in stronger transcriptional repression activity in both heterologous cells and cardiomyocytes. Conversely, increased FOG-2 SUMOylation by overexpression of SUMO-1 or expression of a SUMO-1-FOG-2 fusion protein rendered FOG-2 incapable of repressing GATA-4-mediated activation of the B-type natriuretic peptide (BNP) promoter. Moreover, we demonstrate both increased interaction between a FOG-2 SUMO mutant and GATA-4 and enhanced SUMOylation of wild-type FOG-2 by co-expression of GATA-4. These data suggest a new dynamics in which GATA-4 may alter the activity of FOG-2 by influencing its SUMOylation status.

  8. Novel mechanism of gene regulation: the protein Rv1222 of Mycobacterium tuberculosis inhibits transcription by anchoring the RNA polymerase onto DNA.

    Science.gov (United States)

    Rudra, Paulami; Prajapati, Ranjit Kumar; Banerjee, Rajdeep; Sengupta, Shreya; Mukhopadhyay, Jayanta

    2015-07-13

    We propose a novel mechanism of gene regulation in Mycobacterium tuberculosis where the protein Rv1222 inhibits transcription by anchoring RNA polymerase (RNAP) onto DNA. In contrast to our existing knowledge that transcriptional repressors function either by binding to DNA at specific sequences or by binding to RNAP, we show that Rv1222-mediated transcription inhibition requires simultaneous binding of the protein to both RNAP and DNA. We demonstrate that the positively charged C-terminus tail of Rv1222 is responsible for anchoring RNAP on DNA, hence the protein slows down the movement of RNAP along the DNA during transcription elongation. The interaction between Rv1222 and DNA is electrostatic, thus the protein could inhibit transcription from any gene. As Rv1222 slows down the RNA synthesis, upon expression of the protein in Mycobacterium smegmatis or Escherichia coli, the growth rate of the bacteria is severely impaired. The protein does not possess any significant affinity for DNA polymerase, thus, is unable to inhibit DNA synthesis. The proposed mechanism by which Rv1222 inhibits transcription reveals a new repertoire of prokaryotic gene regulation. © Crown copyright 2015.

  9. MicroRNA-214 suppresses gluconeogenesis by targeting activating transcriptional factor 4.

    Science.gov (United States)

    Li, Kai; Zhang, Jin; Yu, Junjie; Liu, Bin; Guo, Yajie; Deng, Jiali; Chen, Shanghai; Wang, Chunxia; Guo, Feifan

    2015-03-27

    Although the gluconeogenesis pathway is already a target for the treatment of type 2 diabetes, the potential role of microRNAs (miRNAs) in gluconeogenesis remains unclear. Here, we investigated the physiological functions of miR-214 in gluconeogenesis. The expression of miR-214 was suppressed by glucagon via protein kinase A signaling in primary hepatocytes, and miR-214 was down-regulated in the livers of fasted, high fat diet-induced diabetic and leptin receptor-mutated (db/db) mice. The overexpression of miR-214 in primary hepatocytes suppressed glucose production, and silencing miR-214 reversed this effect. Gluconeogenesis was suppressed in the livers of mice injected with an adenovirus expressing miR-214 (Ad-miR-214). Additionally, Ad-miR-214 alleviated high fat diet-induced elevation of gluconeogenesis and hyperglycemia. Furthermore, we found that activating transcription factor 4 (ATF4), a reported target of miR-214, can reverse the suppressive effect of miR-214 on gluconeogenesis in primary hepatocytes, and this suppressive effect was blocked in liver-specific ATF4 knock-out mice. ATF4 regulated gluconeogenesis via affecting forkhead box protein O1 (FOXO1) transcriptional activity. Finally, liver-specific miR-214 transgenic mice exhibited suppressed gluconeogenesis and reduced expression of ATF4, phosphoenolpyruvate carboxykinase, and glucose-6-phosphatase in liver. Taken together, our results suggest that the miR-214-ATF4 axis is a novel pathway for the regulation of hepatic gluconeogenesis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Targeted genome regulation via synthetic programmable transcriptional regulators

    KAUST Repository

    Piatek, Agnieszka Anna

    2016-04-19

    Regulation of gene transcription controls cellular functions and coordinates responses to developmental, physiological and environmental cues. Precise and efficient molecular tools are needed to characterize the functions of single and multiple genes in linear and interacting pathways in a native context. Modular DNA-binding domains from zinc fingers (ZFs) and transcriptional activator-like proteins (TALE) are amenable to bioengineering to bind DNA target sequences of interest. As a result, ZF and TALE proteins were used to develop synthetic programmable transcription factors. However, these systems are limited by the requirement to re-engineer proteins for each new target sequence. The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR associated 9 (Cas9) genome editing tool was recently repurposed for targeted transcriptional regulation by inactivation of the nuclease activity of Cas9. Due to the facile engineering, simplicity, precision and amenability to library construction, the CRISPR/Cas9 system is poised to revolutionize the functional genomics field across diverse eukaryotic species. In this review, we discuss the development of synthetic customizable transcriptional regulators and provide insights into their current and potential applications, with special emphasis on plant systems, in characterization of gene functions, elucidation of molecular mechanisms and their biotechnological applications. © 2016 Informa UK Limited, trading as Taylor & Francis Group

  11. Amino-terminal residues of ΔNp63, mutated in ectodermal dysplasia, are required for its transcriptional activity.

    Science.gov (United States)

    Lena, Anna Maria; Duca, Sara; Novelli, Flavia; Melino, Sonia; Annicchiarico-Petruzzelli, Margherita; Melino, Gerry; Candi, Eleonora

    2015-11-13

    p63, a member of the p53 family, is a crucial transcription factor for epithelial development and skin homeostasis. Heterozygous mutations in TP63 gene have been associated with human ectodermal dysplasia disorders. Most of these TP63 mutations are missense mutations causing amino acidic substitutions at p63 DNA binding or SAM domains that reduce or abolish the transcriptional activity of mutants p63. A significant number of mutants, however, resides in part of the p63 protein that apparently do not affect DNA binding and/or transcriptional activity, such as the N-terminal domain. Here, we characterize five p63 mutations at the 5' end of TP63 gene aiming to understand the pathogenesis of the diseases and to uncover the role of ΔNp63α N-terminus residues in determining its transactivation potential. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Glucose-6-phosphate mediates activation of the carbohydrate responsive binding protein (ChREBP)

    International Nuclear Information System (INIS)

    Li, Ming V.; Chen, Weiqin; Harmancey, Romain N.; Nuotio-Antar, Alli M.; Imamura, Minako; Saha, Pradip; Taegtmeyer, Heinrich; Chan, Lawrence

    2010-01-01

    Carbohydrate response element binding protein (ChREBP) is a Mondo family transcription factor that activates a number of glycolytic and lipogenic genes in response to glucose stimulation. We have previously reported that high glucose can activate the transcriptional activity of ChREBP independent of the protein phosphatase 2A (PP2A)-mediated increase in nuclear entry and DNA binding. Here, we found that formation of glucose-6-phosphate (G-6-P) is essential for glucose activation of ChREBP. The glucose response of GAL4-ChREBP is attenuated by D-mannoheptulose, a potent hexokinase inhibitor, as well as over-expression of glucose-6-phosphatase (G6Pase); kinetics of activation of GAL4-ChREBP can be modified by exogenously expressed GCK. Further metabolism of G-6-P through the two major glucose metabolic pathways, glycolysis and pentose-phosphate pathway, is not required for activation of ChREBP; over-expression of glucose-6-phosphate dehydrogenase (G6PD) diminishes, whereas RNAi knockdown of the enzyme enhances, the glucose response of GAL4-ChREBP, respectively. Moreover, the glucose analogue 2-deoxyglucose (2-DG), which is phosphorylated by hexokinase, but not further metabolized, effectively upregulates the transcription activity of ChREBP. In addition, over-expression of phosphofructokinase (PFK) 1 and 2, synergistically diminishes the glucose response of GAL4-ChREBP. These multiple lines of evidence support the conclusion that G-6-P mediates the activation of ChREBP.

  13. Kaposi's sarcoma-associated herpesvirus-encoded LANA associates with glucocorticoid receptor and enhances its transcriptional activities

    International Nuclear Information System (INIS)

    Togi, Sumihito; Nakasuji, Misa; Muromoto, Ryuta; Ikeda, Osamu; Okabe, Kanako; Kitai, Yuichi; Kon, Shigeyuki; Oritani, Kenji; Matsuda, Tadashi

    2015-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded latency-associated nuclear antigen (LANA), which interacts with cellular proteins, plays a central role in modification of viral and/or cellular gene expression. Here, we show that LANA associates with glucocorticoid receptor (GR), and that LANA enhances the transcriptional activity of GR. Co-immunoprecipitation revealed a physical interaction between LANA and GR in transiently transfected 293T and HeLa cells. In human B-lymphoma cells, LANA overexpression enhanced GR activity and cell growth suppression following glucocorticoid stimulation. Furthermore, confocal microscopy showed that activated GR was bound to LANA and accumulated in the nucleus, leading to an increase in binding of activated GR to the glucocorticoid response element of target genes. Taken together, KSHV-derived LANA acts as a transcriptional co-activator of GR. Our results might suggest a careful use of glucocorticoids in the treatment of patients with KSHV-related malignancies such as Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman disease. - Highlights: • KSHV-LANA enhances the transcriptional activity of GR in 293T and HeLa cells. • KSHV-LANA physically associates with GR. • KSHV-LANA enhances GR activation and cell growth suppression in human B-lymphocytes. • KSHV-LANA influences the nuclear retention and DNA binding activity of GR

  14. Kaposi's sarcoma-associated herpesvirus-encoded LANA associates with glucocorticoid receptor and enhances its transcriptional activities

    Energy Technology Data Exchange (ETDEWEB)

    Togi, Sumihito; Nakasuji, Misa; Muromoto, Ryuta; Ikeda, Osamu; Okabe, Kanako; Kitai, Yuichi; Kon, Shigeyuki [Department of Immunology, Graduate School of Pharmaceutical Sciences Hokkaido University, Sapporo 060-0812 (Japan); Oritani, Kenji [Department of Hematology and Oncology, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan); Matsuda, Tadashi, E-mail: tmatsuda@pharm.hokudai.ac.jp [Department of Immunology, Graduate School of Pharmaceutical Sciences Hokkaido University, Sapporo 060-0812 (Japan)

    2015-07-31

    Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded latency-associated nuclear antigen (LANA), which interacts with cellular proteins, plays a central role in modification of viral and/or cellular gene expression. Here, we show that LANA associates with glucocorticoid receptor (GR), and that LANA enhances the transcriptional activity of GR. Co-immunoprecipitation revealed a physical interaction between LANA and GR in transiently transfected 293T and HeLa cells. In human B-lymphoma cells, LANA overexpression enhanced GR activity and cell growth suppression following glucocorticoid stimulation. Furthermore, confocal microscopy showed that activated GR was bound to LANA and accumulated in the nucleus, leading to an increase in binding of activated GR to the glucocorticoid response element of target genes. Taken together, KSHV-derived LANA acts as a transcriptional co-activator of GR. Our results might suggest a careful use of glucocorticoids in the treatment of patients with KSHV-related malignancies such as Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman disease. - Highlights: • KSHV-LANA enhances the transcriptional activity of GR in 293T and HeLa cells. • KSHV-LANA physically associates with GR. • KSHV-LANA enhances GR activation and cell growth suppression in human B-lymphocytes. • KSHV-LANA influences the nuclear retention and DNA binding activity of GR.

  15. Resveratrol induces growth arrest and apoptosis through activation of FOXO transcription factors in prostate cancer cells.

    Directory of Open Access Journals (Sweden)

    Qinghe Chen

    2010-12-01

    Full Text Available Resveratrol, a naturally occurring phytopolyphenol compound, has attracted extensive interest in recent years because of its diverse pharmacological characteristics. Although resveratrol possesses chemopreventive properties against several cancers, the molecular mechanisms by which it inhibits cell growth and induces apoptosis have not been clearly understood. The present study was carried out to examine whether PI3K/AKT/FOXO pathway mediates the biological effects of resveratrol.Resveratrol inhibited the phosphorylation of PI3K, AKT and mTOR. Resveratrol, PI3K inhibitors (LY294002 and Wortmannin and AKT inhibitor alone slightly induced apoptosis in LNCaP cells. These inhibitors further enhanced the apoptosis-inducing potential of resveratrol. Overexpression of wild-type PTEN slightly induced apoptosis. Wild type PTEN and PTEN-G129E enhanced resveratrol-induced apoptosis, whereas PTEN-G129R had no effect on proapoptotic effects of resveratrol. Furthermore, apoptosis-inducing potential of resveratrol was enhanced by dominant negative AKT, and inhibited by wild-type AKT and constitutively active AKT. Resveratrol has no effect on the expression of FKHR, FKHRL1 and AFX genes. The inhibition of FOXO phosphorylation by resveratrol resulted in its nuclear translocation, DNA binding and transcriptional activity. The inhibition of PI3K/AKT pathway induced FOXO transcriptional activity resulting in induction of Bim, TRAIL, p27/KIP1, DR4 and DR5, and inhibition of cyclin D1. Similarly, resveratrol-induced FOXO transcriptional activity was further enhanced when activation of PI3K/AKT pathway was blocked. Over-expression of phosphorylation deficient mutants of FOXO proteins (FOXO1-TM, FOXO3A-TM and FOXO4-TM induced FOXO transcriptional activity, which was further enhanced by resveratrol. Inhibition of FOXO transcription factors by shRNA blocked resveratrol-induced upregulation of Bim, TRAIL, DR4, DR5, p27/KIP1 and apoptosis, and inhibition of cyclin D1 by

  16. Deep proteomics of mouse skeletal muscle enables quantitation of protein isoforms, metabolic pathways, and transcription factors.

    Science.gov (United States)

    Deshmukh, Atul S; Murgia, Marta; Nagaraj, Nagarjuna; Treebak, Jonas T; Cox, Jürgen; Mann, Matthias

    2015-04-01

    Skeletal muscle constitutes 40% of individual body mass and plays vital roles in locomotion and whole-body metabolism. Proteomics of skeletal muscle is challenging because of highly abundant contractile proteins that interfere with detection of regulatory proteins. Using a state-of-the art MS workflow and a strategy to map identifications from the C2C12 cell line model to tissues, we identified a total of 10,218 proteins, including skeletal muscle specific transcription factors like myod1 and myogenin and circadian clock proteins. We obtain absolute abundances for proteins expressed in a muscle cell line and skeletal muscle, which should serve as a valuable resource. Quantitation of protein isoforms of glucose uptake signaling pathways and in glucose and lipid metabolic pathways provides a detailed metabolic map of the cell line compared with tissue. This revealed unexpectedly complex regulation of AMP-activated protein kinase and insulin signaling in muscle tissue at the level of enzyme isoforms. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Downregulation of RND3/RhoE in glioblastoma patients promotes tumorigenesis through augmentation of notch transcriptional complex activity

    International Nuclear Information System (INIS)

    Liu, Baohui; Lin, Xi; Yang, Xiangsheng; Dong, Huimin; Yue, Xiaojing; Andrade, Kelsey C; Guo, Zhentao; Yang, Jian; Wu, Liquan; Zhu, Xiaonan; Zhang, Shenqi; Tian, Daofeng; Wang, Junmin; Cai, Qiang; Chen, Qizuan; Mao, Shanping; Chen, Qianxue; Chang, Jiang

    2015-01-01

    Activation of Notch signaling contributes to glioblastoma multiform (GBM) tumorigenesis. However, the molecular mechanism that promotes the Notch signaling augmentation during GBM genesis remains largely unknown. Identification of new factors that regulate Notch signaling is critical for tumor treatment. The expression levels of RND3 and its clinical implication were analyzed in GBM patients. Identification of RND3 as a novel factor in GBM genesis was demonstrated in vitro by cell experiments and in vivo by a GBM xenograft model. We found that RND3 expression was significantly decreased in human glioblastoma. The levels of RND3 expression were inversely correlated with Notch activity, tumor size, and tumor cell proliferation, and positively correlated with patient survival time. We demonstrated that RND3 functioned as an endogenous repressor of the Notch transcriptional complex. RND3 physically interacted with NICD, CSL, and MAML1, the Notch transcriptional complex factors, promoted NICD ubiquitination, and facilitated the degradation of these cofactor proteins. We further revealed that RND3 facilitated the binding of NICD to FBW7, a ubiquitin ligase, and consequently enhanced NICD protein degradation. Therefore, Notch transcriptional activity was inhibited. Forced expression of RND3 repressed Notch signaling, which led to the inhibition of glioblastoma cell proliferation in vitro and tumor growth in the xenograft mice in vivo. Downregulation of RND3, however, enhanced Notch signaling activity, and subsequently promoted glioma cell proliferation. Inhibition of Notch activity abolished RND3 deficiency-mediated GBM cell proliferation. We conclude that downregulation of RND3 is responsible for the enhancement of Notch activity that promotes glioblastoma genesis

  18. Characterization of a putative cis-regulatory element that controls transcriptional activity of the pig uroplakin II gene promoter

    International Nuclear Information System (INIS)

    Kwon, Deug-Nam; Park, Mi-Ryung; Park, Jong-Yi; Cho, Ssang-Goo; Park, Chankyu; Oh, Jae-Wook; Song, Hyuk; Kim, Jae-Hwan; Kim, Jin-Hoi

    2011-01-01

    Highlights: → The sequences of -604 to -84 bp of the pUPII promoter contained the region of a putative negative cis-regulatory element. → The core promoter was located in the 5F-1. → Transcription factor HNF4 can directly bind in the pUPII core promoter region, which plays a critical role in controlling promoter activity. → These features of the pUPII promoter are fundamental to development of a target-specific vector. -- Abstract: Uroplakin II (UPII) is a one of the integral membrane proteins synthesized as a major differentiation product of mammalian urothelium. UPII gene expression is bladder specific and differentiation dependent, but little is known about its transcription response elements and molecular mechanism. To identify the cis-regulatory elements in the pig UPII (pUPII) gene promoter region, we constructed pUPII 5' upstream region deletion mutants and demonstrated that each of the deletion mutants participates in controlling the expression of the pUPII gene in human bladder carcinoma RT4 cells. We also identified a new core promoter region and putative negative cis-regulatory element within a minimal promoter region. In addition, we showed that hepatocyte nuclear factor 4 (HNF4) can directly bind in the pUPII core promoter (5F-1) region, which plays a critical role in controlling promoter activity. Transient cotransfection experiments showed that HNF4 positively regulates pUPII gene promoter activity. Thus, the binding element and its binding protein, HNF4 transcription factor, may be involved in the mechanism that specifically regulates pUPII gene transcription.

  19. Rift Valley fever virus NSs inhibits host transcription independently of the degradation of dsRNA-dependent Protein Kinase PKR

    OpenAIRE

    Kalveram, Birte; Lihoradova, Olga; Indran, Sabarish V.; Lokugamage, Nandadeva; Head, Jennifer A.; Ikegami, Tetsuro

    2012-01-01

    Rift Valley fever virus (RVFV) encodes one major virulence factor, the NSs protein. NSs suppresses host general transcription, including interferon (IFN)-β mRNA synthesis, and promotes degradation of the dsRNA-dependent protein kinase (PKR). We generated a novel RVFV mutant (rMP12-NSsR173A) specifically lacking the function to promote PKR degradation. rMP12-NSsR173A infection induces early phosphorylation of eIF2α through PKR activation, while retaining the function to inhibit host general tr...

  20. Feast/famine regulatory proteins (FFRPs): Escherichia coli Lrp, AsnC and related archaeal transcription factors.

    Science.gov (United States)

    Yokoyama, Katsushi; Ishijima, Sanae A; Clowney, Lester; Koike, Hideaki; Aramaki, Hironori; Tanaka, Chikako; Makino, Kozo; Suzuki, Masashi

    2006-01-01

    Feast/famine regulatory proteins comprise a diverse family of transcription factors, which have been referred to in various individual identifications, including Escherichia coli leucine-responsive regulatory protein and asparagine synthase C gene product. A full length feast/famine regulatory protein consists of the N-terminal DNA-binding domain and the C-domain, which is involved in dimerization and further assembly, thereby producing, for example, a disc or a chromatin-like cylinder. Various ligands of the size of amino acids bind at the interface between feast/famine regulatory protein dimers, thereby altering their assembly forms. Also, the combination of feast/famine regulatory protein subunits forming the same assembly is altered. In this way, a small number of feast/famine regulatory proteins are able to regulate a large number of genes in response to various environmental changes. Because feast/famine regulatory proteins are shared by archaea and eubacteria, the genome-wide regulation by feast/famine regulatory proteins is traceable back to their common ancestor, being the prototype of highly differentiated transcription regulatory mechanisms found in organisms nowadays.

  1. Transcription and DNA Damage: Holding Hands or Crossing Swords?

    Science.gov (United States)

    D'Alessandro, Giuseppina; d'Adda di Fagagna, Fabrizio

    2017-10-27

    Transcription has classically been considered a potential threat to genome integrity. Collision between transcription and DNA replication machinery, and retention of DNA:RNA hybrids, may result in genome instability. On the other hand, it has been proposed that active genes repair faster and preferentially via homologous recombination. Moreover, while canonical transcription is inhibited in the proximity of DNA double-strand breaks, a growing body of evidence supports active non-canonical transcription at DNA damage sites. Small non-coding RNAs accumulate at DNA double-strand break sites in mammals and other organisms, and are involved in DNA damage signaling and repair. Furthermore, RNA binding proteins are recruited to DNA damage sites and participate in the DNA damage response. Here, we discuss the impact of transcription on genome stability, the role of RNA binding proteins at DNA damage sites, and the function of small non-coding RNAs generated upon damage in the signaling and repair of DNA lesions. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. The RNA binding protein HuR does not interact directly with HIV-1 reverse transcriptase and does not affect reverse transcription in vitro

    Directory of Open Access Journals (Sweden)

    Gronenborn Angela M

    2010-05-01

    Full Text Available Abstract Background Lemay et al recently reported that the RNA binding protein HuR directly interacts with the ribonuclease H (RNase H domain of HIV-1 reverse transcriptase (RT and influences the efficiency of viral reverse transcription (Lemay et al., 2008, Retrovirology 5:47. HuR is a member of the embryonic lethal abnormal vision protein family and contains 3 RNA recognition motifs (RRMs that bind AU-rich elements (AREs. To define the structural determinants of the HuR-RT interaction and to elucidate the mechanism(s by which HuR influences HIV-1 reverse transcription activity in vitro, we cloned and purified full-length HuR as well as three additional protein constructs that contained the N-terminal and internal RRMs, the internal and C-terminal RRMs, or the C-terminal RRM only. Results All four HuR proteins were purified and characterized by biophysical methods. They are well structured and exist as monomers in solution. No direct protein-protein interaction between HuR and HIV-1 RT was detected using NMR titrations with 15N labeled HuR variants or the 15N labeled RNase H domain of HIV-1 RT. Furthermore, HuR did not significantly affect the kinetics of HIV-1 reverse transcription in vitro, even on RNA templates that contain AREs. Conclusions Our results suggest that HuR does not impact HIV-1 replication through a direct protein-protein interaction with the viral RT.

  3. Diurnal rhythms in neurexins transcripts and inhibitory/excitatory synapse scaffold proteins in the biological clock.

    Directory of Open Access Journals (Sweden)

    Mika Shapiro-Reznik

    Full Text Available The neurexin genes (NRXN1/2/3 encode two families (α and β of highly polymorphic presynaptic proteins that are involved in excitatory/inhibitory synaptic balance. Recent studies indicate that neuronal activation and memory formation affect NRXN1/2/3α expression and alternative splicing at splice sites 3 and 4 (SS#3/SS#4. Neurons in the biological clock residing in the suprachiasmatic nuclei of the hypothalamus (SCN act as self-sustained oscillators, generating rhythms in gene expression and electrical activity, to entrain circadian bodily rhythms to the 24 hours day/night cycles. Cell autonomous oscillations in NRXN1/2/3α expression and SS#3/SS#4 exons splicing and their links to rhythms in excitatory/inhibitory synaptic balance in the circadian clock were explored. NRXN1/2/3α expression and SS#3/SS#4 splicing, levels of neurexin-2α and the synaptic scaffolding proteins PSD-95 and gephyrin (representing excitatory and inhibitory synapses, respectively were studied in mRNA and protein extracts obtained from SCN of C3H/J mice at different times of the 24 hours day/night cycle. Further studies explored the circadian oscillations in these components and causality relationships in immortalized rat SCN2.2 cells. Diurnal rhythms in mNRXN1α and mNRXN2α transcription, SS#3/SS#4 exon-inclusion and PSD-95 gephyrin and neurexin-2α levels were found in the SCN in vivo. No such rhythms were found with mNRXN3α. SCN2.2 cells also exhibited autonomous circadian rhythms in rNRXN1/2 expression SS#3/SS#4 exon inclusion and PSD-95, gephyrin and neurexin-2α levels. rNRXN3α and rNRXN1/2β were not expressed. Causal relationships were demonstrated, by use of specific siRNAs, between rNRXN2α SS#3 exon included transcripts and gephyrin levels in the SCN2.2 cells. These results show for the first time dynamic, cell autonomous, diurnal rhythms in expression and splicing of NRXN1/2 and subsequent effects on the expression of neurexin-2α and postsynaptic

  4. Chromosomal loop/nuclear matrix organization of transcriptionally active and inactive RNA polymerases in HeLa nuclei.

    Science.gov (United States)

    Roberge, M; Dahmus, M E; Bradbury, E M

    1988-06-05

    The relative distribution of transcriptionally active and inactive RNA polymerases I and II between the nuclear matrix/scaffold and chromosomal loops of HeLa cells was determined. Total RNA polymerase was assessed by immunoblotting and transcribing RNA polymerase by a photoaffinity labeling technique in isolated nuclei. Nuclear matrix/scaffold was isolated by three methods using high-salt, intermediate-salt or low-salt extraction. The distribution of RNA polymerases I and II were very similar within each of the methods, but considerable differences in distributions were found between the different preparation methods. Either intermediate-salt or high-salt treatment of DNase I-digested nuclei showed significant association of RNA polymerases with the nuclear matrix. However, intermediate-salt followed by high-salt treatment released all transcribing and non-transcribing RNA polymerases. Nuclear scaffolds isolated with lithium diiodosalicylate (low-salt) contained very little of the RNA polymerases. This treatment, however, caused the dissociation of RNA polymerase II transcription complexes. These results show unambiguously that RNA polymerases, both in their active and inactive forms, are not nuclear matrix proteins. The data support models in which the transcriptional machinery moves around DNA loops during transcription.

  5. A transcript cleavage factor of Mycobacterium tuberculosis important for its survival.

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    Arnab China

    Full Text Available After initiation of transcription, a number of proteins participate during elongation and termination modifying the properties of the RNA polymerase (RNAP. Gre factors are one such group conserved across bacteria. They regulate transcription by projecting their N-terminal coiled-coil domain into the active center of RNAP through the secondary channel and stimulating hydrolysis of the newly synthesized RNA in backtracked elongation complexes. Rv1080c is a putative gre factor (MtbGre in the genome of Mycobacterium tuberculosis. The protein enhanced the efficiency of promoter clearance by lowering abortive transcription and also rescued arrested and paused elongation complexes on the GC rich mycobacterial template. Although MtbGre is similar in domain organization and shares key residues for catalysis and RNAP interaction with the Gre factors of Escherichia coli, it could not complement an E. coli gre deficient strain. Moreover, MtbGre failed to rescue E. coli RNAP stalled elongation complexes, indicating the importance of specific protein-protein interactions for transcript cleavage. Decrease in the level of MtbGre reduced the bacterial survival by several fold indicating its essential role in mycobacteria. Another Gre homolog, Rv3788 was not functional in transcript cleavage activity indicating that a single Gre is sufficient for efficient transcription of the M. tuberculosis genome.

  6. Strong negative self regulation of Prokaryotic transcription factors increases the intrinsic noise of protein expression

    Directory of Open Access Journals (Sweden)

    Jenkins Dafyd J

    2008-01-01

    Full Text Available Abstract Background Many prokaryotic transcription factors repress their own transcription. It is often asserted that such regulation enables a cell to homeostatically maintain protein abundance. We explore the role of negative self regulation of transcription in regulating the variability of protein abundance using a variety of stochastic modeling techniques. Results We undertake a novel analysis of a classic model for negative self regulation. We demonstrate that, with standard approximations, protein variance relative to its mean should be independent of repressor strength in a physiological range. Consequently, in that range, the coefficient of variation would increase with repressor strength. However, stochastic computer simulations demonstrate that there is a greater increase in noise associated with strong repressors than predicted by theory. The discrepancies between the mathematical analysis and computer simulations arise because with strong repressors the approximation that leads to Michaelis-Menten-like hyperbolic repression terms ceases to be valid. Because we observe that strong negative feedback increases variability and so is unlikely to be a mechanism for noise control, we suggest instead that negative feedback is evolutionarily favoured because it allows the cell to minimize mRNA usage. To test this, we used in silico evolution to demonstrate that while negative feedback can achieve only a modest improvement in protein noise reduction compared with the unregulated system, it can achieve good improvement in protein response times and very substantial improvement in reducing mRNA levels. Conclusion Strong negative self regulation of transcription may not always be a mechanism for homeostatic control of protein abundance, but instead might be evolutionarily favoured as a mechanism to limit the use of mRNA. The use of hyperbolic terms derived from quasi-steady-state approximation should also be avoided in the analysis of stochastic

  7. DELLA-induced early transcriptional changes during etiolated development in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Javier Gallego-Bartolomé

    Full Text Available The hormones gibberellins (GAs control a wide variety of processes in plants, including stress and developmental responses. This task largely relies on the activity of the DELLA proteins, nuclear-localized transcriptional regulators that do not seem to have DNA binding capacity. The identification of early target genes of DELLA action is key not only to understand how GAs regulate physiological responses, but also to get clues about the molecular mechanisms by which DELLAs regulate gene expression. Here, we have investigated the global, early transcriptional response triggered by the Arabidopsis DELLA protein GAI during skotomorphogenesis, a developmental program tightly regulated by GAs. Our results show that the induction of GAI activity has an almost immediate effect on gene expression. Although this transcriptional regulation is largely mediated by the PIFs and HY5 transcription factors based on target meta-analysis, additional evidence points to other transcription factors that would be directly involved in DELLA regulation of gene expression. First, we have identified cis elements recognized by Dofs and type-B ARRs among the sequences enriched in the promoters of GAI targets; and second, an enrichment in additional cis elements appeared when this analysis was extended to a dataset of early targets of the DELLA protein RGA: CArG boxes, bound by MADS-box proteins, and the E-box CACATG that links the activity of DELLAs to circadian transcriptional regulation. Finally, Gene Ontology analysis highlights the impact of DELLA regulation upon the homeostasis of the GA, auxin, and ethylene pathways, as well as upon pre-existing transcriptional networks.

  8. A Small Ras-like protein Ray/Rab1c modulates the p53-regulating activity of PRPK

    International Nuclear Information System (INIS)

    Abe, Yasuhito; Takeuchi, Takashi; Imai, Yoshinori; Murase, Ryuichi; Kamei, Yoshiaki; Fujibuchi, Taketsugu; Matsumoto, Suguru; Ueda, Norifumi; Ogasawara, Masahito; Shigemoto, Kazuhiro; Kito, Katsumi

    2006-01-01

    PRPK phosphorylates serine-15 residue of p53 and enhances transcriptional activity. PRPK possesses a bipartite nuclear localization signal and localizes in nucleus when over-expressed in cells. However, intrinsic PRPK localizes mainly in the cytosol in situ. While studying the mechanisms in the distribution of intrinsic PRPK, we identified a PRPK binding protein, an ubiquitously expressed Small Ras-like GTPase, Rab1c, also named Ray or Rab35. The over-expressed Ray was distributed in the nucleus, cytosol, and cell membrane. Both Ray wild type and GTP-restrictively binding mutant Ray-Q67L, but not guanine nucleotide unstable binding mutant Ray-N120I, partially distributed the over-expressed PRPK to the cytosol and also suppressed the PRPK-induced p53-transcriptional activity profoundly. A Small Ras-like GTPase protein Ray was thus indicated to modulate p53 transcriptional activity of PRPK

  9. Dataset of transcriptional landscape of B cell early activation

    Directory of Open Access Journals (Sweden)

    Alexander S. Garruss

    2015-09-01

    Full Text Available Signaling via B cell receptors (BCR and Toll-like receptors (TLRs result in activation of B cells with distinct physiological outcomes, but transcriptional regulatory mechanisms that drive activation and distinguish these pathways remain unknown. At early time points after BCR and TLR ligand exposure, 0.5 and 2 h, RNA-seq was performed allowing observations on rapid transcriptional changes. At 2 h, ChIP-seq was performed to allow observations on important regulatory mechanisms potentially driving transcriptional change. The dataset includes RNA-seq, ChIP-seq of control (Input, RNA Pol II, H3K4me3, H3K27me3, and a separate RNA-seq for miRNA expression, which can be found at Gene Expression Omnibus Dataset GSE61608. Here, we provide details on the experimental and analysis methods used to obtain and analyze this dataset and to examine the transcriptional landscape of B cell early activation.

  10. Human I-mfa domain proteins specifically interact with KSHV LANA and affect its regulation of Wnt signaling-dependent transcription

    Energy Technology Data Exchange (ETDEWEB)

    Kusano, Shuichi, E-mail: skusano@m2.kufm.kagoshima-u.ac.jp [Division of Persistent and Oncogenic Viruses, Center for Chronic Viral Diseases, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Eizuru, Yoshito [Division of Persistent and Oncogenic Viruses, Center for Chronic Viral Diseases, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan)

    2010-06-04

    Kaposi's sarcoma-associated herpes virus (KSHV)-encoded latency-associated nuclear antigen (LANA) protein has been reported to interact with glycogen synthase kinase 3{beta} (GSK-3{beta}) and to negatively regulate its activity, leading to stimulation of GSK-3{beta}-dependent {beta}-catenin degradation. We show here that the I-mfa domain proteins, HIC (human I-mfa domain-containing protein) and I-mfa (inhibitor of MyoD family a), interacted in vivo with LANA through their C-terminal I-mfa domains. This interaction affected the intracellular localization of HIC, inhibited the LANA-dependent transactivation of a {beta}-catenin-regulated reporter construct, and decreased the level of the LANA.GSK-3{beta} complex. These data reveal for the first time that I-mfa domain proteins interact with LANA and negatively regulate LANA-mediated activation of Wnt signaling-dependent transcription by inhibiting the formation of the LANA.GSK-3{beta} complex.

  11. Maternal protein restriction induced-hypertension is associated to oxidative disruption at transcriptional and functional levels in the medulla oblongata.

    Science.gov (United States)

    de Brito Alves, José L; de Oliveira, Jéssica M D; Ferreira, Diorginis J S; Barros, Monique A de V; Nogueira, Viviane O; Alves, Débora S; Vidal, Hubert; Leandro, Carol G; Lagranha, Cláudia J; Pirola, Luciano; da Costa-Silva, João H

    2016-12-01

    Maternal protein restriction during pregnancy and lactation predisposes the adult offspring to sympathetic overactivity and arterial hypertension. Although the underlying mechanisms are poorly understood, dysregulation of the oxidative balance has been proposed as a putative trigger of neural-induced hypertension. The aim of the study was to evaluate the association between the oxidative status at transcriptional and functional levels in the medulla oblongata and maternal protein restriction induced-hypertension. Wistar rat dams were fed a control (normal protein; 17% protein) or a low protein ((Lp); 8% protein) diet during pregnancy and lactation, and male offspring was studied at 90 days of age. Direct measurements of baseline arterial blood pressure (ABP) and heart rate (HR) were recorded in awakened offspring. In addition, quantitative RT-PCR was used to assess the mRNA expression of superoxide dismutase 1 (SOD1) and 2 (SOD2), catalase (CAT), glutathione peroxidase (GPx), Glutamatergic receptors (Grin1, Gria1 and Grm1) and GABA(A)-receptor-associated protein like 1 (Gabarapl1). Malondialdehyde (MDA) levels, CAT and SOD activities were examined in ventral and dorsal medulla. Lp rats exhibited higher ABP. The mRNA expression levels of SOD2, GPx and Gabarapl1 were down regulated in medullary tissue of Lp rats (Pmedulla. Taken together, our data suggest that maternal protein restriction induced-hypertension is associated with medullary oxidative dysfunction at transcriptional level and with impaired antioxidant capacity in the ventral medulla. © 2016 John Wiley & Sons Australia, Ltd.

  12. In Vitro Anticancer Activity of Phlorofucofuroeckol A via Upregulation of Activating Transcription Factor 3 against Human Colorectal Cancer Cells

    Directory of Open Access Journals (Sweden)

    Hyun Ji Eo

    2016-03-01

    Full Text Available Phlorofucofuroeckol A (PFF-A, one of the phlorotannins found in brown algae, has been reported to exert anti-cancer property. However, the molecular mechanism for the anti-cancer effect of PFF-A has not been known. Activating transcription factor 3 (ATF3 has been reported to be associated with apoptosis in colorectal cancer. The present study was performed to investigate the molecular mechanism by which PFF-A stimulates ATF3 expression and apoptosis in human colorectal cancer cells. PFF-A decreased cell viability through apoptosis of human colorectal cancer cells. PFF-A increased ATF3 expression through regulating transcriptional activity. The responsible cis-element for ATF3 transcriptional activation by PFF-A was cAMP response element binding protein (CREB, located between positions −147 and −85 of the ATF3 promoter. Inhibition of p38, c-Jun N-terminal kinases (JNK, glycogen synthase kinase (GSK 3β, and IκB kinase (IKK-α blocked PFF-A-mediated ATF3 expression. ATF3 knockdown by ATF3 siRNA attenuated the cleavage of poly (ADP-ribose polymerase (PARP by PFF-A, while ATF3 overexpression increased PFF-A-mediated cleaved PARP. These results suggest that PFF-A may exert anti-cancer property through inducing apoptosis via the ATF3-mediated pathway in human colorectal cancer cells.

  13. The T alpha 2 nuclear protein binding site from the human T cell receptor alpha enhancer functions as both a T cell-specific transcriptional activator and repressor

    OpenAIRE

    1990-01-01

    T cell-specific expression of the human T cell receptor alpha (TCR- alpha) gene is regulated by the interaction of variable region promoter elements with a transcriptional enhancer that is located 4.5 kb 3' of the TCR-alpha constant region (C alpha) gene segment. The minimal TCR- alpha enhancer is composed of two nuclear protein binding sites, T alpha 1 and T alpha 2, that are both required for the T cell-specific activity of the enhancer. The T alpha 1 binding site contains a consensus cAMP ...

  14. Common and distinct DNA-binding and regulatory activities of the BEN-solo transcription factor family.

    Science.gov (United States)

    Dai, Qi; Ren, Aiming; Westholm, Jakub O; Duan, Hong; Patel, Dinshaw J; Lai, Eric C

    2015-01-01

    Recently, the BEN (BANP, E5R, and NAC1) domain was recognized as a new class of conserved DNA-binding domain. The fly genome encodes three proteins that bear only a single BEN domain ("BEN-solo" factors); namely, Insensitive (Insv), Bsg25A (Elba1), and CG9883 (Elba2). Insv homodimers preferentially bind CCAATTGG palindromes throughout the genome to mediate transcriptional repression, whereas Bsg25A and Elba2 heterotrimerize with their obligate adaptor, Elba3 (i.e., the ELBA complex), to recognize a CCAATAAG motif in the Fab-7 insulator. While these data suggest distinct DNA-binding properties of BEN-solo proteins, we performed reporter assays that indicate that both Bsg25A and Elba2 can individually recognize Insv consensus sites efficiently. We confirmed this by solving the structure of Bsg25A complexed to the Insv site, which showed that key aspects of the BEN:DNA recognition strategy are similar between these proteins. We next show that both Insv and ELBA proteins are competent to mediate transcriptional repression via Insv consensus sequences but that the ELBA complex appears to be selective for the ELBA site. Reciprocally, genome-wide analysis reveals that Insv exhibits significant cobinding to class I insulator elements, indicating that it may also contribute to insulator function. Indeed, we observed abundant Insv binding within the Hox complexes with substantial overlaps with class I insulators, many of which bear Insv consensus sites. Moreover, Insv coimmunoprecipitates with the class I insulator factor CP190. Finally, we observed that Insv harbors exclusive activity among fly BEN-solo factors with respect to regulation of Notch-mediated cell fate choices in the peripheral nervous system. This in vivo activity is recapitulated by BEND6, a mammalian BEN-solo factor that conserves the Notch corepressor function of Insv but not its capacity to bind Insv consensus sites. Altogether, our data define an array of common and distinct biochemical and functional

  15. Gcn4-Mediator Specificity Is Mediated by a Large and Dynamic Fuzzy Protein-Protein Complex

    Directory of Open Access Journals (Sweden)

    Lisa M. Tuttle

    2018-03-01

    Full Text Available Summary: Transcription activation domains (ADs are inherently disordered proteins that often target multiple coactivator complexes, but the specificity of these interactions is not understood. Efficient transcription activation by yeast Gcn4 requires its tandem ADs and four activator-binding domains (ABDs on its target, the Mediator subunit Med15. Multiple ABDs are a common feature of coactivator complexes. We find that the large Gcn4-Med15 complex is heterogeneous and contains nearly all possible AD-ABD interactions. Gcn4-Med15 forms via a dynamic fuzzy protein-protein interface, where ADs bind the ABDs in multiple orientations via hydrophobic regions that gain helicity. This combinatorial mechanism allows individual low-affinity and specificity interactions to generate a biologically functional, specific, and higher affinity complex despite lacking a defined protein-protein interface. This binding strategy is likely representative of many activators that target multiple coactivators, as it allows great flexibility in combinations of activators that can cooperate to regulate genes with variable coactivator requirements. : Tuttle et al. report a “fuzzy free-for-all” interaction mechanism that explains how seemingly unrelated transcription activators converge on a limited number of coactivator targets. The mechanism provides a rationale for the observation that individually weak and low-specificity interactions can combine to produce biologically critical function without requiring highly ordered structure. Keywords: transcription activation, intrinsically disordered proteins, fuzzy binding

  16. Iro/IRX transcription factors negatively regulate Dpp/TGF-β pathway activity during intestinal tumorigenesis.

    Science.gov (United States)

    Martorell, Òscar; Barriga, Francisco M; Merlos-Suárez, Anna; Stephan-Otto Attolini, Camille; Casanova, Jordi; Batlle, Eduard; Sancho, Elena; Casali, Andreu

    2014-11-01

    Activating mutations in Wnt and EGFR/Ras signaling pathways are common in colorectal cancer (CRC). Remarkably, clonal co-activation of these pathways in the adult Drosophila midgut induces "tumor-like" overgrowths. Here, we show that, in these clones and in CRC cell lines, Dpp/TGF-β acts as a tumor suppressor. Moreover, we discover that the Iroquois/IRX-family-protein Mirror downregulates the transcription of core components of the Dpp pathway, reducing its tumor suppressor activity. We also show that this genetic interaction is conserved in human CRC cells, where the Iro/IRX proteins IRX3 and IRX5 diminish the response to TGF-β. IRX3 and IRX5 are upregulated in human adenomas, and their levels correlate inversely with the gene expression signature of response to TGF-β. In addition, Irx5 expression confers a growth advantage in the presence of TGF-β, but is selected against in its absence. Together, our results identify a set of Iro/IRX proteins as conserved negative regulators of Dpp/TGF-β activity. We propose that during the characteristic adenoma-to-carcinoma transition of human CRC, the activity of IRX proteins could reduce the sensitivity to the cytostatic effect of TGF-β, conferring a growth advantage to tumor cells prior to the acquisition of mutations in TGF-β pathway components. © 2014 The Authors.

  17. TRANSCRIPTIONAL INHIBITION OF INTERLEUKIN-12 PROMOTER ACTIVITY IN LEISHMANIA SPP.-INFECTED MACROPHAGES

    Science.gov (United States)

    Jayakumar, Asha; Widenmaier, Robyn; Ma, Xiaojing; McDowell, Mary Ann

    2009-01-01

    To establish and persist within a host, Leishmania spp. parasites delay the onset of cell-mediated immunity by suppressing interleukin-12 (IL-12) production from host macrophages. Although it is established that Leishmania spp.-infected macrophages have impaired IL-12 production, the mechanisms that account for this suppression remain to be completely elucidated. Using a luciferase reporter assay assessing IL-12 transcription, we report here that Leishmania major, Leishmania donovani, and Leishmania chagasi inhibit IL-12 transcription in response to interferon-gamma, lipopolysaccharide, and CD40 ligand and that Leishmania spp. lipophosphoglycan, phosphoglycans, and major surface protein are not necessary for inhibition. In addition, all the Leishmania spp. strains and life-cycle stages tested inhibited IL-12 promoter activity. Our data further reveal that autocrine-acting host factors play no role in the inhibitory response and that phagocytosis signaling is necessary for inhibition of IL-12. PMID:18372625

  18. First Exon Length Controls Active Chromatin Signatures and Transcription

    Directory of Open Access Journals (Sweden)

    Nicole I. Bieberstein

    2012-07-01

    Full Text Available Here, we explore the role of splicing in transcription, employing both genome-wide analysis of human ChIP-seq data and experimental manipulation of exon-intron organization in transgenic cell lines. We show that the activating histone modifications H3K4me3 and H3K9ac map specifically to first exon-intron boundaries. This is surprising, because these marks help recruit general transcription factors (GTFs to promoters. In genes with long first exons, promoter-proximal levels of H3K4me3 and H3K9ac are greatly reduced; consequently, GTFs and RNA polymerase II are low at transcription start sites (TSSs and exhibit a second, promoter-distal peak from which transcription also initiates. In contrast, short first exons lead to increased H3K4me3 and H3K9ac at promoters, higher expression levels, accuracy in TSS usage, and a lower frequency of antisense transcription. Therefore, first exon length is predictive for gene activity. Finally, splicing inhibition and intron deletion reduce H3K4me3 levels and transcriptional output. Thus, gene architecture and splicing determines transcription quantity and quality as well as chromatin signatures.

  19. The transcriptional landscape

    DEFF Research Database (Denmark)

    Nielsen, Henrik

    2011-01-01

    The application of new and less biased methods to study the transcriptional output from genomes, such as tiling arrays and deep sequencing, has revealed that most of the genome is transcribed and that there is substantial overlap of transcripts derived from the two strands of DNA. In protein coding...... regions, the map of transcripts is very complex due to small transcripts from the flanking ends of the transcription unit, the use of multiple start and stop sites for the main transcript, production of multiple functional RNA molecules from the same primary transcript, and RNA molecules made...... by independent transcription from within the unit. In genomic regions separating those that encode proteins or highly abundant RNA molecules with known function, transcripts are generally of low abundance and short-lived. In most of these cases, it is unclear to what extent a function is related to transcription...

  20. Use of prokaryotic transcriptional activators as metabolite biosensors in eukaryotic cells

    DEFF Research Database (Denmark)

    2018-01-01

    The present invention relates to the use of transcriptional activators from prokaryotic organisms for use in eukaryotic cells, such as yeast as sensors of intracellular and extracellular accumulation of a ligand or metabolite specifically activating this transcriptional activator in a eukaryot...

  1. The regulation of mitochondrial transcription factor A (Tfam) expression during skeletal muscle cell differentiation.

    Science.gov (United States)

    Collu-Marchese, Melania; Shuen, Michael; Pauly, Marion; Saleem, Ayesha; Hood, David A

    2015-05-19

    The ATP demand required for muscle development is accommodated by elevations in mitochondrial biogenesis, through the co-ordinated activities of the nuclear and mitochondrial genomes. The most important transcriptional activator of the mitochondrial genome is mitochondrial transcription factor A (Tfam); however, the regulation of Tfam expression during muscle differentiation is not known. Thus, we measured Tfam mRNA levels, mRNA stability, protein expression and localization and Tfam transcription during the progression of muscle differentiation. Parallel 2-fold increases in Tfam protein and mRNA were observed, corresponding with 2-3-fold increases in mitochondrial content. Transcriptional activity of a 2051 bp promoter increased during this differentiation period and this was accompanied by a 3-fold greater Tfam mRNA stabilization. Interestingly, truncations of the promoter at 1706 bp, 978 bp and 393 bp promoter all exhibited 2-3-fold higher transcriptional activity than the 2051 bp construct, indicating the presence of negative regulatory elements within the distal 350 bp of the promoter. Activation of AMP kinase augmented Tfam transcription within the proximal promoter, suggesting the presence of binding sites for transcription factors that are responsive to cellular energy state. During differentiation, the accumulating Tfam protein was progressively distributed to the mitochondrial matrix where it augmented the expression of mtDNA and COX (cytochrome c oxidase) subunit I, an mtDNA gene product. Our data suggest that, during muscle differentiation, Tfam protein levels are regulated by the availability of Tfam mRNA, which is controlled by both transcription and mRNA stability. Changes in energy state and Tfam localization also affect Tfam expression and action in differentiating myotubes. © 2015 Authors.

  2. RNA-guided transcriptional regulation

    Science.gov (United States)

    Church, George M.; Mali, Prashant G.; Esvelt, Kevin M.

    2016-02-23

    Methods of modulating expression of a target nucleic acid in a cell are provided including introducing into the cell a first foreign nucleic acid encoding one or more RNAs complementary to DNA, wherein the DNA includes the target nucleic acid, introducing into the cell a second foreign nucleic acid encoding a nuclease-null Cas9 protein that binds to the DNA and is guided by the one or more RNAs, introducing into the cell a third foreign nucleic acid encoding a transcriptional regulator protein or domain, wherein the one or more RNAs, the nuclease-null Cas9 protein, and the transcriptional regulator protein or domain are expressed, wherein the one or more RNAs, the nuclease-null Cas9 protein and the transcriptional regulator protein or domain co-localize to the DNA and wherein the transcriptional regulator protein or domain regulates expression of the target nucleic acid.

  3. In Situ Tagged nsp15 Reveals Interactions with Coronavirus Replication/Transcription Complex-Associated Proteins

    Directory of Open Access Journals (Sweden)

    Jeremiah Athmer

    2017-01-01

    Full Text Available Coronavirus (CoV replication and transcription are carried out in close proximity to restructured endoplasmic reticulum (ER membranes in replication/transcription complexes (RTC. Many of the CoV nonstructural proteins (nsps are required for RTC function; however, not all of their functions are known. nsp15 contains an endoribonuclease domain that is conserved in the CoV family. While the enzymatic activity and crystal structure of nsp15 are well defined, its role in replication remains elusive. nsp15 localizes to sites of RNA replication, but whether it acts independently or requires additional interactions for its function remains unknown. To begin to address these questions, we created an in situ tagged form of nsp15 using the prototypic CoV, mouse hepatitis virus (MHV. In MHV, nsp15 contains the genomic RNA packaging signal (P/S, a 95-bp RNA stem-loop structure that is not required for viral replication or nsp15 function. Utilizing this knowledge, we constructed an internal hemagglutinin (HA tag that replaced the P/S. We found that nsp15-HA was localized to discrete perinuclear puncta and strongly colocalized with nsp8 and nsp12, both well-defined members of the RTC, but not the membrane (M protein, involved in virus assembly. Finally, we found that nsp15 interacted with RTC-associated proteins nsp8 and nsp12 during infection, and this interaction was RNA independent. From this, we conclude that nsp15 localizes and interacts with CoV proteins in the RTC, suggesting it plays a direct or indirect role in virus replication. Furthermore, the use of in situ epitope tags could be used to determine novel nsp-nsp interactions in coronaviruses.

  4. A Synthetic Biology Framework for Programming Eukaryotic Transcription Functions

    Science.gov (United States)

    Khalil, Ahmad S.; Lu, Timothy K.; Bashor, Caleb J.; Ramirez, Cherie L.; Pyenson, Nora C.; Joung, J. Keith; Collins, James J.

    2013-01-01

    SUMMARY Eukaryotic transcription factors (TFs) perform complex and combinatorial functions within transcriptional networks. Here, we present a synthetic framework for systematically constructing eukaryotic transcription functions using artificial zinc fingers, modular DNA-binding domains found within many eukaryotic TFs. Utilizing this platform, we construct a library of orthogonal synthetic transcription factors (sTFs) and use these to wire synthetic transcriptional circuits in yeast. We engineer complex functions, such as tunable output strength and transcriptional cooperativity, by rationally adjusting a decomposed set of key component properties, e.g., DNA specificity, affinity, promoter design, protein-protein interactions. We show that subtle perturbations to these properties can transform an individual sTF between distinct roles (activator, cooperative factor, inhibitory factor) within a transcriptional complex, thus drastically altering the signal processing behavior of multi-input systems. This platform provides new genetic components for synthetic biology and enables bottom-up approaches to understanding the design principles of eukaryotic transcriptional complexes and networks. PMID:22863014

  5. [CNC proteins in physiology and pathology].

    Science.gov (United States)

    Gęgotek, Agnieszka; Skrzydlewska, Elżbieta

    2015-07-06

    CNC proteins consist of Bach1, Bach2 and 4 homologous transcription factors: Nrf1, Nrf2, Nrf3 and p45NF-E2. Transcription factors belonging to this group of proteins play a crucial role in protection of cells against oxidative stress. Under physiological conditions, they remain in the cytoplasm in the inactive form or are degraded. However, in oxidative stress conditions, they are translocated to the nucleus, and bind to DNA in the ARE sequence. Consequently, there is transcription of genes encoding cytoprotective proteins, such as phase II enzymes, or low molecular weight antioxidant proteins (i.e., thioredoxin, ferritin, metallothionein) responsible for protecting cells from reactive oxygen species (ROS) action. The activity of transcriptional proteins depends directly on the redox state of the cell. ROS as second messenger signals, control inhibitors of cytoplasmic CNC proteins or potentiate the activity of kinases (MAPK, PKC, PI3K, PERK), leading to phosphorylation of transcription factors. This is conducive to translocation of these molecules into the nucleus and to formation of complexes that initiate the gene expression. Disorders of regulation of the activity of transcription factors belonging to the CNC proteins caused by gene mutations, epigenetic modifications or increased activity of p62, p21, or k-Ras, B-Raf and c-Myc oncogenes, induce changes in the level of ARE-dependent gene expression, which can lead even to the development of carcinogenesis. On the other hand, Nrf transcription factors, inducing the expression of antioxidants and enzymes responsible for the detoxification of xenobiotics, can be considered as a potential target of the action of chemopreventive factors in anticancer therapy.

  6. CNC proteins in physiology and pathology

    Directory of Open Access Journals (Sweden)

    Agnieszka Gęgotek

    2015-07-01

    Full Text Available CNC proteins consist of Bach1, Bach2 and 4 homologous transcription factors: Nrf1, Nrf2, Nrf3 and p45NF-E2. Transcription factors belonging to this group of proteins play a crucial role in protection of cells against oxidative stress. Under physiological conditions, they remain in the cytoplasm in the inactive form or are degraded. However, in oxidative stress conditions, they are translocated to the nucleus, and bind to DNA in the ARE sequence. Consequently, there is transcription of genes encoding cytoprotective proteins, such as phase II enzymes, or low molecular weight antioxidant proteins (i.e., thioredoxin, ferritin, metallothionein responsible for protecting cells from reactive oxygen species (ROS action. The activity of transcriptional proteins depends directly on the redox state of the cell. ROS as second messenger signals, control inhibitors of cytoplasmic CNC proteins or potentiate the activity of kinases (MAPK, PKC, PI3K, PERK, leading to phosphorylation of transcription factors. This is conducive to translocation of these molecules into the nucleus and to formation of complexes that initiate the gene expression. Disorders of regulation of the activity of transcription factors belonging to the CNC proteins caused by gene mutations, epigenetic modifications or increased activity of p62, p21, or k-Ras, B-Raf and c-Myc oncogenes, induce changes in the level of ARE-dependent gene expression, which can lead even to the development of carcinogenesis. On the other hand, Nrf transcription factors, inducing the expression of antioxidants and enzymes responsible for the detoxification of xenobiotics, can be considered as a potential target of the action of chemopreventive factors in anticancer therapy.

  7. Histone acetyltransferase (HAT) activity of p300 modulates human T lymphotropic virus type 1 p30II-mediated repression of LTR transcriptional activity

    International Nuclear Information System (INIS)

    Michael, Bindhu; Nair, Amrithraj M.; Datta, Antara; Hiraragi, Hajime; Ratner, Lee; Lairmore, Michael D.

    2006-01-01

    Human T-lymphotropic virus type-1 (HTLV-1) is a deltaretrovirus that causes adult T cell leukemia/lymphoma, and is implicated in a variety of lymphocyte-mediated inflammatory disorders. HTLV-1 provirus has regulatory and accessory genes in four pX open reading frames. HTLV-1 pX ORF-II encodes two proteins, p13 II and p30 II , which are incompletely defined in virus replication or pathogenesis. We have demonstrated that pX ORF-II mutations block virus replication in vivo and that ORF-II encoded p30 II , a nuclear-localizing protein that binds with CREB-binding protein (CBP)/p300, represses CREB and Tax responsive element (TRE)-mediated transcription. Herein, we have identified p30 II motifs important for p300 binding and in regulating TRE-mediated transcription in the absence and presence of HTLV-1 provirus. Within amino acids 100-179 of p30 II , a region important for repression of LTR-mediated transcription, we identified a single lysine residue at amino acid 106 (K3) that significantly modulates the ability of p30 II to repress TRE-mediated transcription. Exogenous p300, in a dose-responsive manner, reverses p30 II -dependent repression of TRE-mediated transcription, in the absence or presence of the provirus, In contrast to wild type p300, p300 HAT mutants (defective in histone acetyltransferase activity) only partially rescued p30 II -mediated LTR repression. Deacetylation by histone deacetylase-1 (HDAC-1) enhanced p30 II -mediated LTR repression, while inhibition of deacetylation by trichostatin A decreases p30 II -mediated LTR repression. Collectively, our data indicate that HTLV-1 p30 II modulates viral gene expression in a cooperative manner with p300-mediated acetylation

  8. Interference with Activator Protein-2 transcription factors leads to induction of apoptosis and an increase in chemo- and radiation- sensitivity in breast cancer cells

    LENUS (Irish Health Repository)

    Thewes, Verena

    2010-05-11

    Abstract Background Activator Protein-2 (AP-2) transcription factors are critically involved in a variety of fundamental cellular processes such as proliferation, differentiation and apoptosis and have also been implicated in carcinogenesis. Expression of the family members AP-2α and AP-2γ is particularly well documented in malignancies of the female breast. Despite increasing evaluation of single AP-2 isoforms in mammary tumors the functional role of concerted expression of multiple AP-2 isoforms in breast cancer remains to be elucidated. AP-2 proteins can form homo- or heterodimers, and there is growing evidence that the net effect whether a cell will proliferate, undergo apoptosis or differentiate is partly dependent on the balance between different AP-2 isoforms. Methods We simultaneously interfered with all AP-2 isoforms expressed in ErbB-2-positive murine N202.1A breast cancer cells by conditionally over-expressing a dominant-negative AP-2 mutant. Results We show that interference with AP-2 protein function lead to reduced cell number, induced apoptosis and increased chemo- and radiation-sensitivity. Analysis of global gene expression changes upon interference with AP-2 proteins identified 139 modulated genes (90 up-regulated, 49 down-regulated) compared with control cells. Gene Ontology (GO) investigations for these genes revealed Cell Death and Cell Adhesion and Migration as the main functional categories including 25 and 12 genes, respectively. By using information obtained from Ingenuity Pathway Analysis Systems we were able to present proven or potential connections between AP-2 regulated genes involved in cell death and response to chemo- and radiation therapy, (i.e. Ctgf, Nrp1, Tnfaip3, Gsta3) and AP-2 and other main apoptosis players and to create a unique network. Conclusions Expression of AP-2 transcription factors in breast cancer cells supports proliferation and contributes to chemo- and radiation-resistance of tumor cells by impairing the

  9. Interference with Activator Protein-2 transcription factors leads to induction of apoptosis and an increase in chemo- and radiation-sensitivity in breast cancer cells

    International Nuclear Information System (INIS)

    Thewes, Verena; Orso, Francesca; Jäger, Richard; Eckert, Dawid; Schäfer, Sabine; Kirfel, Gregor; Garbe, Stephan; Taverna, Daniela; Schorle, Hubert

    2010-01-01

    Activator Protein-2 (AP-2) transcription factors are critically involved in a variety of fundamental cellular processes such as proliferation, differentiation and apoptosis and have also been implicated in carcinogenesis. Expression of the family members AP-2α and AP-2γ is particularly well documented in malignancies of the female breast. Despite increasing evaluation of single AP-2 isoforms in mammary tumors the functional role of concerted expression of multiple AP-2 isoforms in breast cancer remains to be elucidated. AP-2 proteins can form homo- or heterodimers, and there is growing evidence that the net effect whether a cell will proliferate, undergo apoptosis or differentiate is partly dependent on the balance between different AP-2 isoforms. We simultaneously interfered with all AP-2 isoforms expressed in ErbB-2-positive murine N202.1A breast cancer cells by conditionally over-expressing a dominant-negative AP-2 mutant. We show that interference with AP-2 protein function lead to reduced cell number, induced apoptosis and increased chemo- and radiation-sensitivity. Analysis of global gene expression changes upon interference with AP-2 proteins identified 139 modulated genes (90 up-regulated, 49 down-regulated) compared with control cells. Gene Ontology (GO) investigations for these genes revealed Cell Death and Cell Adhesion and Migration as the main functional categories including 25 and 12 genes, respectively. By using information obtained from Ingenuity Pathway Analysis Systems we were able to present proven or potential connections between AP-2 regulated genes involved in cell death and response to chemo- and radiation therapy, (i.e. Ctgf, Nrp1, Tnfaip3, Gsta3) and AP-2 and other main apoptosis players and to create a unique network. Expression of AP-2 transcription factors in breast cancer cells supports proliferation and contributes to chemo- and radiation-resistance of tumor cells by impairing the ability to induce apoptosis. Therefore, interference

  10. c-Jun binds the N terminus of human TAF(II)250 to derepress RNA polymerase II transcription in vitro.

    Science.gov (United States)

    Lively, T N; Ferguson, H A; Galasinski, S K; Seto, A G; Goodrich, J A

    2001-07-06

    c-Jun is an oncoprotein that activates transcription of many genes involved in cell growth and proliferation. We studied the mechanism of transcriptional activation by human c-Jun in a human RNA polymerase II transcription system composed of highly purified recombinant and native transcription factors. Transcriptional activation by c-Jun depends on the TATA-binding protein (TBP)-associated factor (TAF) subunits of transcription factor IID (TFIID). Protein-protein interaction assays revealed that c-Jun binds with high specificity to the largest subunit of human TFIID, TAF(II)250. The region of TAF(II)250 bound by c-Jun lies in the N-terminal 163 amino acids. This same region of TAF(II)250 binds to TBP and represses its interaction with TATA boxes, thereby decreasing DNA binding by TFIID. We hypothesized that c-Jun is capable of derepressing the effect of the TAF(II)250 N terminus on TFIID-driven transcription. In support of this hypothesis, we found that c-Jun increased levels of TFIID-driven transcription in vitro when added at high concentrations to a DNA template lacking activator protein 1 (AP-1) sites. Moreover, c-Jun blocked the repression of TBP DNA binding caused by the N terminus of TAF(II)250. In addition to revealing a mechanism by which c-Jun activates transcription, our studies provide the first evidence that an activator can bind directly to the N terminus of TAF(II)250 to derepress RNA polymerase II transcription in vitro.

  11. SUMOylation regulates the transcriptional repression activity of FOG-2 and its association with GATA-4.

    Directory of Open Access Journals (Sweden)

    José Perdomo

    Full Text Available Friend of GATA 2 (FOG-2, a co-factor of several GATA transcription factors (GATA-4, -5 and 6, is a critical regulator of coronary vessel formation and heart morphogenesis. Here we demonstrate that FOG-2 is SUMOylated and that this modification modulates its transcriptional activity. FOG-2 SUMOylation occurs at four lysine residues (K324, 471, 915, 955 [corrected]. Three of these residues are part of the characteristic SUMO consensus site (ψKXE, while K955 is found in the less frequent TKXE motif. Absence of SUMOylation did not affect FOG-2's nuclear localization. However, mutation of the FOG-2 SUMOylation sites, or de-SUMOylation, with SENP-1 or SENP-8 resulted in stronger transcriptional repression activity in both heterologous cells and cardiomyocytes. Conversely, increased FOG-2 SUMOylation by overexpression of SUMO-1 or expression of a SUMO-1-FOG-2 fusion protein rendered FOG-2 incapable of repressing GATA-4-mediated activation of the B-type natriuretic peptide (BNP promoter. Moreover, we demonstrate both increased interaction between a FOG-2 SUMO mutant and GATA-4 and enhanced SUMOylation of wild-type FOG-2 by co-expression of GATA-4. These data suggest a new dynamics in which GATA-4 may alter the activity of FOG-2 by influencing its SUMOylation status.

  12. A transcription factor active on the epidermal growth factor receptor gene

    International Nuclear Information System (INIS)

    Kageyama, R.; Merlino, G.T.; Pastan, I.

    1988-01-01

    The authors have developed an in vitro transcription system for the epidermal growth factor receptor (EGFR) oncogene by using nuclear extracts of A431 human epidermoid carcinoma cells, which overproduce EGFR. They found that a nuclear factor, termed EGFR-specific transcription factor (ETF), specifically stimulated EGFR transcription by 5- to 10-fold. In this report, ETF, purified by using sequence-specific oligonucleotide affinity chromatography, is shown by renaturing material eluted from a NaDodSO 4 /polyacrylamide gel to be a protein with a molecular mass of 120 kDa. ETF binds to the promoter region, as measured by DNase I footprinting and gel-mobility-shift assays, and specifically stimulates the transcription of the EGFR gene in a reconstituted in vitro transcription system. These results suggest that ETF could play a role in the overexpression of the cellular oncogene EGFR

  13. Intracellular protein delivery activity of peptides derived from insulin-like growth factor binding proteins 3 and 5

    International Nuclear Information System (INIS)

    Goda, Natsuko; Tenno, Takeshi; Inomata, Kosuke; Shirakawa, Masahiro; Tanaka, Toshiki; Hiroaki, Hidekazu

    2008-01-01

    Insulin-like growth factor binding proteins (IGFBPs) have various IGF-independent cellular activities, including receptor-independent cellular uptake followed by transcriptional regulation, although mechanisms of cellular entry remain unclear. Herein, we focused on their receptor-independent cellular entry mechanism in terms of protein transduction domain (PTD) activity, which is an emerging technique useful for clinical applications. The peptides of 18 amino acid residues derived from IGFBP-3 and IGFBP-5, which involve heparin-binding regions, mediated cellular delivery of an exogenous protein into NIH3T3 and HeLa cells. Relative protein delivery activities of IGFBP-3/5-derived peptides were approximately 20-150% compared to that of the HIV-Tat peptide, a potent PTD. Heparin inhibited the uptake of the fusion proteins with IGFBP-3 and IGFBP-5, indicating that the delivery pathway is heparin-dependent endocytosis, similar to that of HIV-Tat. The delivery of GST fused to HIV-Tat was competed by either IGFBP-3 or IGFBP-5-derived synthetic peptides. Therefore, the entry pathways of the three PTDs are shared. Our data has shown a new approach for designing protein delivery systems using IGFBP-3/5 derived peptides based on the molecular mechanisms of IGF-independent activities of IGFBPs

  14. ZFP226 is a novel artificial transcription factor for selective activation of tumor suppressor KIBRA.

    Science.gov (United States)

    Schelleckes, Katrin; Schmitz, Boris; Lenders, Malte; Mewes, Mirja; Brand, Stefan-Martin; Brand, Eva

    2018-03-09

    KIBRA has been suggested as a key regulator of the hippo pathway, regulating organ size, cell contact inhibition as well as tissue regeneration and tumorigenesis. Recently, alterations of KIBRA expression caused by promotor methylation have been reported for several types of cancer. Our current study aimed to design an artificial transcription factor capable of re-activating expression of the tumor suppressor KIBRA and the hippo pathway. We engineered a new gene named 'ZFP226' encoding for a ~23 kDa fusion protein. ZFP226 belongs to the Cys2-His2 zinc finger type and recognizes a nine base-pair DNA sequence 5'-GGC-GGC-GGC-3' in the KIBRA core promoter P1a. ZFP226 showed nuclear localization in human immortalized kidney epithelial cells and activated the KIBRA core promoter (p < 0.001) resulting in significantly increased KIBRA mRNA and protein levels (p < 0.001). Furthermore, ZFP226 led to activation of hippo signaling marked by elevated YAP and LATS phosphorylation. In Annexin V flow cytometry assays ZFP226 overexpression showed strong pro-apoptotic capacity on MCF-7 breast cancer cells (p < 0.01 early-, p < 0.001 late-apoptotic cells). We conclude that the artificial transcription factor ZFP226 can be used for target KIBRA and hippo pathway activation. This novel molecule may represent a molecular tool for the development of future applications in cancer treatment.

  15. c-Myc Antagonises the Transcriptional Activity of the Androgen Receptor in Prostate Cancer Affecting Key Gene Networks.

    Science.gov (United States)

    Barfeld, Stefan J; Urbanucci, Alfonso; Itkonen, Harri M; Fazli, Ladan; Hicks, Jessica L; Thiede, Bernd; Rennie, Paul S; Yegnasubramanian, Srinivasan; DeMarzo, Angelo M; Mills, Ian G

    2017-04-01

    Prostate cancer (PCa) is the most common non-cutaneous cancer in men. The androgen receptor (AR), a ligand-activated transcription factor, constitutes the main drug target for advanced cases of the disease. However, a variety of other transcription factors and signaling networks have been shown to be altered in patients and to influence AR activity. Amongst these, the oncogenic transcription factor c-Myc has been studied extensively in multiple malignancies and elevated protein levels of c-Myc are commonly observed in PCa. Its impact on AR activity, however, remains elusive. In this study, we assessed the impact of c-Myc overexpression on AR activity and transcriptional output in a PCa cell line model and validated the antagonistic effect of c-MYC on AR-targets in patient samples. We found that c-Myc overexpression partially reprogrammed AR chromatin occupancy and was associated with altered histone marks distribution, most notably H3K4me1 and H3K27me3. We found c-Myc and the AR co-occupy a substantial number of binding sites and these exhibited enhancer-like characteristics. Interestingly, c-Myc overexpression antagonised clinically relevant AR target genes. Therefore, as an example, we validated the antagonistic relationship between c-Myc and two AR target genes, KLK3 (alias PSA, prostate specific antigen), and Glycine N-Methyltransferase (GNMT), in patient samples. Our findings provide unbiased evidence that MYC overexpression deregulates the AR transcriptional program, which is thought to be a driving force in PCa. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

  16. c-Myc Antagonises the Transcriptional Activity of the Androgen Receptor in Prostate Cancer Affecting Key Gene Networks

    Directory of Open Access Journals (Sweden)

    Stefan J. Barfeld

    2017-04-01

    Full Text Available Prostate cancer (PCa is the most common non-cutaneous cancer in men. The androgen receptor (AR, a ligand-activated transcription factor, constitutes the main drug target for advanced cases of the disease. However, a variety of other transcription factors and signaling networks have been shown to be altered in patients and to influence AR activity. Amongst these, the oncogenic transcription factor c-Myc has been studied extensively in multiple malignancies and elevated protein levels of c-Myc are commonly observed in PCa. Its impact on AR activity, however, remains elusive. In this study, we assessed the impact of c-Myc overexpression on AR activity and transcriptional output in a PCa cell line model and validated the antagonistic effect of c-MYC on AR-targets in patient samples. We found that c-Myc overexpression partially reprogrammed AR chromatin occupancy and was associated with altered histone marks distribution, most notably H3K4me1 and H3K27me3. We found c-Myc and the AR co-occupy a substantial number of binding sites and these exhibited enhancer-like characteristics. Interestingly, c-Myc overexpression antagonised clinically relevant AR target genes. Therefore, as an example, we validated the antagonistic relationship between c-Myc and two AR target genes, KLK3 (alias PSA, prostate specific antigen, and Glycine N-Methyltransferase (GNMT, in patient samples. Our findings provide unbiased evidence that MYC overexpression deregulates the AR transcriptional program, which is thought to be a driving force in PCa.

  17. CCS mRNA transcripts and serum CCS protein as copper marker in adults suffering inflammatory processes.

    Science.gov (United States)

    Araya, Magdalena; Gutiérrez, Ricardo; Arredondo, Miguel

    2014-08-01

    The chaperone to Zn-Cu superoxide dismutase (CCS) has been postulated as a candidate copper indicator, changing in a consistent manner in induced and recovered copper deficiency, in experimental cell and animal models. In real life people have various conditions that may modify molecules acting as acute phase proteins, such as serum ceruloplasmin and copper concentration and could alter CCS responses. With the hypothesis that CCS mRNA transcripts and protein would be different in individuals suffering inflammatory processes in comparison to healthy individuals, we assessed adult individuals who, although not ill had conditions known to induce variable degrees of inflammation. Screening of 600 adults resulted in two study groups, formed on the basis of their clinical history and levels of serum C reactive protein (CRP): Group 1 (n = 61, mean (range) CRP = 0.9 (0.3-2.0 mg/dL) and Group 2 (n = 150, mean (range) CRP = 6.1 (4.3-8.7 mg/dL). Results showed that mRNA transcripts relative abundance was not different for CCS, MTIIA, TNF-alpha and Cu-Zn-SOD by group (p > 0.05, one way Anova), nor between sexes (p > 0.05, one way Anova). Distribution of CCS mRNA transcripts and CCS protein in serum did not show any differences or trends. Results disproved our hypothesis that CCS abundance of transcripts and CCS protein would be different in individuals suffering inflammatory processes, adding further support to the idea that CCS may be a copper marker.

  18. Heterochromatin protein 1 (HP1a positively regulates euchromatic gene expression through RNA transcript association and interaction with hnRNPs in Drosophila.

    Directory of Open Access Journals (Sweden)

    Lucia Piacentini

    2009-10-01

    Full Text Available Heterochromatin Protein 1 (HP1a is a well-known conserved protein involved in heterochromatin formation and gene silencing in different species including humans. A general model has been proposed for heterochromatin formation and epigenetic gene silencing in different species that implies an essential role for HP1a. According to the model, histone methyltransferase enzymes (HMTases methylate the histone H3 at lysine 9 (H3K9me, creating selective binding sites for itself and the chromodomain of HP1a. This complex is thought to form a higher order chromatin state that represses gene activity. It has also been found that HP1a plays a role in telomere capping. Surprisingly, recent studies have shown that HP1a is present at many euchromatic sites along polytene chromosomes of Drosophila melanogaster, including the developmental and heat-shock-induced puffs, and that this protein can be removed from these sites by in vivo RNase treatment, thus suggesting an association of HP1a with the transcripts of many active genes. To test this suggestion, we performed an extensive screening by RIP-chip assay (RNA-immunoprecipitation on microarrays, and we found that HP1a is associated with transcripts of more than one hundred euchromatic genes. An expression analysis in HP1a mutants shows that HP1a is required for positive regulation of these genes. Cytogenetic and molecular assays show that HP1a also interacts with the well known proteins DDP1, HRB87F, and PEP, which belong to different classes of heterogeneous nuclear ribonucleoproteins (hnRNPs involved in RNA processing. Surprisingly, we found that all these hnRNP proteins also bind heterochromatin and are dominant suppressors of position effect variegation. Together, our data show novel and unexpected functions for HP1a and hnRNPs proteins. All these proteins are in fact involved both in RNA transcript processing and in heterochromatin formation. This suggests that, in general, similar epigenetic mechanisms

  19. Coordinating repair of oxidative DNA damage with transcription and replication

    International Nuclear Information System (INIS)

    Cooper, P.K.

    2003-01-01

    Transcription-coupled repair (TCR) preferentially removes DNA lesions from template strands of active genes. Defects in TCR, which acts both on lesions removed by nucleotide excision repair (NER) and on oxidative lesions removed by base excision repair (BER), underlie the fatal developmental disorder Cockayne syndrome. Although its detailed mechanism remains unknown, TCR involves recognition of a stalled RNA polymerase (RNAP), removal or remodeling of RNAP to allow access to the lesion, and recruitment of repair enzymes. At a minimum, these early steps require a non-enzymatic function of the multifunctional repair protein XPG, the CSB protein with ATP-dependent chromatin remodeling activity, and the TFIIH complex (including the XPB and XPD helicases) that is also required for basal transcription initiation and NER. XPG exists in the cell in a complex with TFIIH, and in vitro evidence has suggested that it interacts with CSB. To address the mechanism of TCR, we are characterizing protein-DNA and protein-protein interactions of XPG. We show that XPG preferentially binds to double-stranded DNA containing bubbles resembling in size the unpaired regions associated with transcription. Two distinct domains of XPG are required for the observed strong binding specificity and stability. XPG both interacts directly with CSB and synergistically binds with it to bubble DNA, and it strongly stimulates the bubble DNA-dependent ATPase activity of CSB. Significantly for TCR, XPG also interacts directly with RNAP II, binds both the protein and nucleic acid components (the R-loop) of a stalled RNA polymerase, and forms a ternary complex with CSB and the stalled RNAP. These results are consistent with the model that XPG and CSB jointly interact with the DNA/chromatin structure in the vicinity of the stalled transcriptional apparatus and with the transcriptional machinery itself to remodel the chromatin and either move or remodel the blocked RNA polymerase to expose the lesion

  20. AMP-activated protein kinase phosphorylates CtBP1 and down-regulates its activity

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jae-Hwan; Choi, Soo-Youn; Kang, Byung-Hee; Lee, Soon-Min [National Creative Research Center for Epigenome Reprogramming Network, Departments of Biomedical Sciences and Biochemistry and Molecular Biology, Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of); Park, Hyung Soon; Kang, Gum-Yong; Bang, Joo Young [Center for Biomedical Mass Spectrometry, Diatech Korea Co., Ltd., Seoul (Korea, Republic of); Cho, Eun-Jung [National Research Laboratory for Chromatin Dynamics, College of Pharmacy, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of); Youn, Hong-Duk, E-mail: hdyoun@snu.ac.kr [National Creative Research Center for Epigenome Reprogramming Network, Departments of Biomedical Sciences and Biochemistry and Molecular Biology, Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of); WCU Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence and Technology, Seoul National University, Seoul (Korea, Republic of)

    2013-02-01

    Highlights: ► AMPK phosphorylates CtBP1 on serine 158. ► AMPK-mediated phosphorylation of CtBP1 causes the ubiquitination and nuclear export of CtBP1. ► AMPK downregulates the CtBP1-mediated repression of Bax transcription. -- Abstract: CtBP is a transcriptional repressor which plays a significant role in the regulation of cell proliferation and tumor progression. It was reported that glucose withdrawal causes induction of Bax due to the dissociation of CtBP from the Bax promoter. However, the precise mechanism involved in the regulation of CtBP still remains unclear. In this study, we found that an activated AMP-activated protein kinase (AMPK) phosphorylates CtBP1 on Ser-158 upon metabolic stresses. Moreover, AMPK-mediated phosphorylation of CtBP1 (S158) attenuates the repressive function of CtBP1. We also confirmed that triggering activation of AMPK by various factors resulted in an increase of Bax gene expression. These findings provide connections of AMPK with CtBP1-mediated regulation of Bax expression for cell death under metabolic stresses.

  1. Cold shock protein YB-1 is involved in hypoxia-dependent gene transcription

    International Nuclear Information System (INIS)

    Rauen, Thomas; Frye, Bjoern C.; Wang, Jialin; Raffetseder, Ute; Alidousty, Christina; En-Nia, Abdelaziz; Floege, Jürgen; Mertens, Peter R.

    2016-01-01

    Hypoxia-dependent gene regulation is largely orchestrated by hypoxia-inducible factors (HIFs), which associate with defined nucleotide sequences of hypoxia-responsive elements (HREs). Comparison of the regulatory HRE within the 3′ enhancer of the human erythropoietin (EPO) gene with known binding motifs for cold shock protein Y-box (YB) protein-1 yielded strong similarities within the Y-box element and 3′ adjacent sequences. DNA binding assays confirmed YB-1 binding to both, single- and double-stranded HRE templates. Under hypoxia, we observed nuclear shuttling of YB-1 and co-immunoprecipitation assays demonstrated that YB-1 and HIF-1α physically interact with each other. Cellular YB-1 depletion using siRNA significantly induced hypoxia-dependent EPO production at both, promoter and mRNA level. Vice versa, overexpressed YB-1 significantly reduced EPO-HRE-dependent gene transcription, whereas this effect was minor under normoxia. HIF-1α overexpression induced hypoxia-dependent gene transcription through the same element and accordingly, co-expression with YB-1 reduced HIF-1α-mediated EPO induction under hypoxic conditions. Taken together, we identified YB-1 as a novel binding factor for HREs that participates in fine-tuning of the hypoxia transcriptome. - Highlights: • Hypoxia drives nuclear translocation of cold shock protein YB-1. • YB-1 physically interacts with hypoxia-inducible factor (HIF)-1α. • YB-1 binds to the hypoxia-responsive element (HRE) within the erythropoietin (EPO) 3′ enhancer. • YB-1 trans-regulates transcription of hypoxia-dependent genes such as EPO and VEGF.

  2. Eukaryotic Initiation Factor 4H Is under Transcriptional Control of p65/NF-κB

    Science.gov (United States)

    Fiume, Giuseppe; Rossi, Annalisa; de Laurentiis, Annamaria; Falcone, Cristina; Pisano, Antonio; Vecchio, Eleonora; Pontoriero, Marilena; Scala, Iris; Scialdone, Annarita; Masci, Francesca Fasanella; Mimmi, Selena; Palmieri, Camillo; Scala, Giuseppe; Quinto, Ileana

    2013-01-01

    Protein synthesis is mainly regulated at the initiation step, allowing the fast, reversible and spatial control of gene expression. Initiation of protein synthesis requires at least 13 translation initiation factors to assemble the 80S ribosomal initiation complex. Loss of translation control may result in cell malignant transformation. Here, we asked whether translational initiation factors could be regulated by NF-κB transcription factor, a major regulator of genes involved in cell proliferation, survival, and inflammatory response. We show that the p65 subunit of NF-κB activates the transcription of eIF4H gene, which is the regulatory subunit of eIF4A, the most relevant RNA helicase in translation initiation. The p65-dependent transcriptional activation of eIF4H increased the eIF4H protein content augmenting the rate of global protein synthesis. In this context, our results provide novel insights into protein synthesis regulation in response to NF-κB activation signalling, suggesting a transcription-translation coupled mechanism of control. PMID:23776612

  3. Deduction of upstream sequences of Xanthomonas campestris flagellar genes responding to transcription activation by FleQ

    International Nuclear Information System (INIS)

    Hu, R.-M.; Yang, T.-C.; Yang, S.-H.; Tseng, Y.-H.

    2005-01-01

    Xanthomonas campestris pv. campestris (Xcc), a close relative to Pseudomonas aeruginosa, is the pathogen causing black rot in cruciferous plants. In P. aeruginosa, FleQ serves as a cognate activator of σ 54 in transcription from several σ 54 -dependent promoters of flagellar genes. These P. aeruginosa promoters have been analyzed for FleQ-binding sequences; however, no consensus was deduced. Xcc, although lacks fleSR, has a fleQ homologue residing among over 40 contiguously clustered flagellar genes. A fleQ mutant, Xc17fleQ, constructed by insertional mutation is deficient in FleQ protein, non-flagellated, and immobile. Transcriptional fusion assays on six putative σ 54 -dependent promoters of the flagellar genes, fliE, fliQ, fliL, flgG, flgB, and flhF, indicated that each of them is also FleQ dependent. Each of these promoters has a sequence with weak consensus to 5'-gaaacCCgccgCcgctTt-3', immediately upstream of the predicted σ 54 -binding site, with an imperfect inverted repeat containing a GC-rich center flanked by several A and T at 5'- and 3'-ends, respectively. Replacing this region in fliE promoter with a HindIII recognition sequence abolished the transcription, indicating that this region responds to transcription activation by FleQ

  4. Promoter polymorphisms in genes involved in porcine myogenesis influence their transcriptional activity.

    Science.gov (United States)

    Bongiorni, Silvia; Tilesi, Francesca; Bicorgna, Silvia; Iacoponi, Francesca; Willems, Daniela; Gargani, Maria; D'Andrea, MariaSilvia; Pilla, Fabio; Valentini, Alessio

    2014-11-07

    Success of meat production and selection for improvement of meat quality is among the primary aims in animal production. Meat quality traits are economically important in swine; however, the underlying genetic nature is very complex. Therefore, an improved pork production strongly depends on identifying and studying how genetic variations contribute to modulate gene expression. Promoters are key regions in gene modulation as they harbour several binding motifs to transcription regulatory factors. Therefore, polymorphisms in these regions are likely to deeply affect RNA levels and consequently protein synthesis. In this study, we report the identification of single nucleotide polymorphisms (SNPs) in promoter regions of candidate genes involved in development, cellular differentiation and muscle growth in Sus scrofa. We identified SNPs in the promoter regions of genes belonging to the Myogenic Regulatory Factors (MRF) gene family (the Myogenic Differentiation gene, MYOD1) and to Growth and Differentiation Factors (GDF) gene family (Myostatin gene, MSTN, GDF8), in Casertana and Large White breeds. The purpose of this study was to investigate if polymorphisms in the promoters could affect the transcriptional activity of these genes. With this aim, we evaluated in vitro the functional activity of the luciferase reporter gene luc2 activity, driven by two constructs carrying different promoter haplotypes. We tested the effects of the G302A (U12574) transition on the promoter efficiency in MYOD1 gene. We ascertained a difference in transcription efficiency for the two variants. A stronger activity of the A-carrying construct is more evident in C2C12. The luciferase expression driven by the MYOD1-A allelic variant displayed a 3.8-fold increased transcriptional activity. We investigated the activity of two haplotype variants (AY527152) in the promoter of GDF8 gene. The haploptype-1 (A435-A447-A879) up-regulated the expression of the reporter gene by a two-fold increase, and

  5. Keap1 silencing boosts lipopolysaccharide-induced transcription of interleukin 6 via activation of nuclear factor κB in macrophages

    International Nuclear Information System (INIS)

    Lv, Peng; Xue, Peng; Dong, Jian; Peng, Hui; Clewell, Rebecca; Wang, Aiping; Wang, Yue; Peng, Shuangqing; Qu, Weidong; Zhang, Qiang; Andersen, Melvin E.; Pi, Jingbo

    2013-01-01

    Interleukin-6 (IL6) is a multifunctional cytokine that regulates immune and inflammatory responses. Multiple transcription factors, including nuclear factor κB (NF-κB) and nuclear factor E2-related factor 2 (Nrf2), regulate IL6 transcription. Kelch-like ECH-associated protein 1 (Keap1) is a substrate adaptor protein for the Cullin 3-dependent E3 ubiquitin ligase complex, which regulates the degradation of many proteins, including Nrf2 and IκB kinase β (IKKβ). Here, we found that stable knockdown of Keap1 (Keap1-KD) in RAW 264.7 (RAW) mouse macrophages and human monocyte THP-1 cells significantly increased expression of Il6, and Nrf2-target genes, under basal and lipopolysaccharide (LPS, 0.001–0.1 μg/ml)-challenged conditions. However, Nrf2 activation alone, by tert-butylhydroquinone treatment of RAW cells, did not increase expression of Il6. Compared to cells transduced with scrambled non-target negative control shRNA, Keap1-KD RAW cells showed enhanced protein levels of IKKβ and increased expression and phosphorylation of NF-κB p65 under non-stressed and LPS-treated conditions. Because the expression of Il6 in Keap1-KD RAW cells was significantly attenuated by silencing of Ikkβ, but not Nrf2, it appears that stabilized IKKβ is responsible for the enhanced transactivation of Il6 in Keap1-KD cells. This study demonstrated that silencing of Keap1 in macrophages boosts LPS-induced transcription of Il6 via NF-κB activation. Given the importance of IL6 in the inflammatory response, the Keap1–IKKβ–NF-κB pathway may be a novel target for treatment and prevention of inflammation and associated disorders. - Highlights: • Knockdown of Keap1 increases expression of Il6 in macrophages. • Silencing of Keap1 results in protein accumulation of IKKβ and NF-κB p65. • Induction of Il6 resulting from Keap1 silencing is attributed to NF-κB activation

  6. Keap1 silencing boosts lipopolysaccharide-induced transcription of interleukin 6 via activation of nuclear factor κB in macrophages

    Energy Technology Data Exchange (ETDEWEB)

    Lv, Peng [Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing (China); Institute for Chemical Safety Sciences, The Hamner Institutes for Health Sciences, 6 Davis Drive, Research Triangle Park, NC 27709 (United States); Xue, Peng; Dong, Jian [Institute for Chemical Safety Sciences, The Hamner Institutes for Health Sciences, 6 Davis Drive, Research Triangle Park, NC 27709 (United States); Peng, Hui [Institute for Chemical Safety Sciences, The Hamner Institutes for Health Sciences, 6 Davis Drive, Research Triangle Park, NC 27709 (United States); Evaluation and Research Center for Toxicology, Institute of Disease Control and Prevention, Academy of Military Medical Sciences (China); Clewell, Rebecca [Institute for Chemical Safety Sciences, The Hamner Institutes for Health Sciences, 6 Davis Drive, Research Triangle Park, NC 27709 (United States); Wang, Aiping [Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing (China); Wang, Yue [Institute for Medical Device Standardization Administration, National Institutes for Food and Drug Control, Beijing (China); Peng, Shuangqing [Evaluation and Research Center for Toxicology, Institute of Disease Control and Prevention, Academy of Military Medical Sciences (China); Qu, Weidong [Key Laboratory of the Public Health Safety, Ministry of Education, School of Public Health, Fudan University, Shanghai (China); Zhang, Qiang; Andersen, Melvin E. [Institute for Chemical Safety Sciences, The Hamner Institutes for Health Sciences, 6 Davis Drive, Research Triangle Park, NC 27709 (United States); Pi, Jingbo, E-mail: jpi@thehamner.org [Institute for Chemical Safety Sciences, The Hamner Institutes for Health Sciences, 6 Davis Drive, Research Triangle Park, NC 27709 (United States)

    2013-11-01

    Interleukin-6 (IL6) is a multifunctional cytokine that regulates immune and inflammatory responses. Multiple transcription factors, including nuclear factor κB (NF-κB) and nuclear factor E2-related factor 2 (Nrf2), regulate IL6 transcription. Kelch-like ECH-associated protein 1 (Keap1) is a substrate adaptor protein for the Cullin 3-dependent E3 ubiquitin ligase complex, which regulates the degradation of many proteins, including Nrf2 and IκB kinase β (IKKβ). Here, we found that stable knockdown of Keap1 (Keap1-KD) in RAW 264.7 (RAW) mouse macrophages and human monocyte THP-1 cells significantly increased expression of Il6, and Nrf2-target genes, under basal and lipopolysaccharide (LPS, 0.001–0.1 μg/ml)-challenged conditions. However, Nrf2 activation alone, by tert-butylhydroquinone treatment of RAW cells, did not increase expression of Il6. Compared to cells transduced with scrambled non-target negative control shRNA, Keap1-KD RAW cells showed enhanced protein levels of IKKβ and increased expression and phosphorylation of NF-κB p65 under non-stressed and LPS-treated conditions. Because the expression of Il6 in Keap1-KD RAW cells was significantly attenuated by silencing of Ikkβ, but not Nrf2, it appears that stabilized IKKβ is responsible for the enhanced transactivation of Il6 in Keap1-KD cells. This study demonstrated that silencing of Keap1 in macrophages boosts LPS-induced transcription of Il6 via NF-κB activation. Given the importance of IL6 in the inflammatory response, the Keap1–IKKβ–NF-κB pathway may be a novel target for treatment and prevention of inflammation and associated disorders. - Highlights: • Knockdown of Keap1 increases expression of Il6 in macrophages. • Silencing of Keap1 results in protein accumulation of IKKβ and NF-κB p65. • Induction of Il6 resulting from Keap1 silencing is attributed to NF-κB activation.

  7. Transcriptional regulation of the HMGA1 gene by octamer-binding proteins Oct-1 and Oct-2.

    Directory of Open Access Journals (Sweden)

    Eusebio Chiefari

    Full Text Available The High-Mobility Group AT-Hook 1 (HMGA1 protein is an architectural transcription factor that binds to AT-rich sequences in the promoter region of DNA and functions as a specific cofactor for gene activation. Previously, we demonstrated that HMGA1 is a key regulator of the insulin receptor (INSR gene and an important downstream target of the INSR signaling cascade. Moreover, from a pathogenic point of view, overexpression of HMGA1 has been associated with human cancer, whereas functional variants of the HMGA1 gene have been recently linked to type 2 diabetes mellitus and metabolic syndrome. However, despite of this biological and pathological relevance, the mechanisms that control HMGA1 gene expression remain unknown. In this study, to define the molecular mechanism(s that regulate HMGA1 gene expression, the HMGA1 gene promoter was investigated by transient transfection of different cell lines, either before or after DNA and siRNA cotransfections. An octamer motif was identified as an important element of transcriptional regulation of this gene, the interaction of which with the octamer transcription factors Oct-1 and Oct-2 is crucial in modulating HMGA1 gene and protein expression. Additionally, we demonstrate that HMGA1 binds its own promoter and contributes to its transactivation by Oct-2 (but not Oct-1, supporting its role in an auto-regulatory circuit. Overall, our results provide insight into the transcriptional regulation of the HMGA1 gene, revealing a differential control exerted by both Oct-1 and Oct-2. Furthermore, they consistently support the hypothesis that a putative defect in Oct-1 and/or Oct-2, by affecting HMGA1 expression, may cause INSR dysfunction, leading to defects of the INSR signaling pathway.

  8. The roles of the catalytic and noncatalytic activities of Rpd3L and Rpd3S in the regulation of gene transcription in yeast.

    Directory of Open Access Journals (Sweden)

    Daniella Yeheskely-Hayon

    Full Text Available In budding yeasts, the histone deacetylase Rpd3 resides in two different complexes called Rpd3L (large and Rpd3S (small that exert opposing effects on the transcription of meiosis-specific genes. By introducing mutations that disrupt the integrity and function of either Rpd3L or Rpd3S, we show here that Rpd3 function is determined by its association with either of these complexes. Specifically, the catalytic activity of Rpd3S activates the transcription of the two major positive regulators of meiosis, IME1 and IME2, under all growth conditions and activates the transcription of NDT80 only during vegetative growth. In contrast, the effects of Rpd3L depends on nutrients; it represses or activates transcription in the presence or absence of a nitrogen source, respectively. Further, we show that transcriptional activation does not correlate with histone H4 deacetylation, suggesting an effect on a nonhistone protein. Comparison of rpd3-null and catalytic-site point mutants revealed an inhibitory activity that is independent of either the catalytic activity of Rpd3 or the integrity of Rpd3L and Rpd3S.

  9. The Roles of the Catalytic and Noncatalytic Activities of Rpd3L and Rpd3S in the Regulation of Gene Transcription in Yeast

    Science.gov (United States)

    Yeheskely-Hayon, Daniella; Kotler, Anat; Stark, Michal; Hashimshony, Tamar; Sagee, Shira; Kassir, Yona

    2013-01-01

    In budding yeasts, the histone deacetylase Rpd3 resides in two different complexes called Rpd3L (large) and Rpd3S (small) that exert opposing effects on the transcription of meiosis-specific genes. By introducing mutations that disrupt the integrity and function of either Rpd3L or Rpd3S, we show here that Rpd3 function is determined by its association with either of these complexes. Specifically, the catalytic activity of Rpd3S activates the transcription of the two major positive regulators of meiosis, IME1 and IME2, under all growth conditions and activates the transcription of NDT80 only during vegetative growth. In contrast, the effects of Rpd3L depends on nutrients; it represses or activates transcription in the presence or absence of a nitrogen source, respectively. Further, we show that transcriptional activation does not correlate with histone H4 deacetylation, suggesting an effect on a nonhistone protein. Comparison of rpd3-null and catalytic-site point mutants revealed an inhibitory activity that is independent of either the catalytic activity of Rpd3 or the integrity of Rpd3L and Rpd3S. PMID:24358376

  10. Regulation of TCF ETS-domain transcription factors by helix-loop-helix motifs.

    Science.gov (United States)

    Stinson, Julie; Inoue, Toshiaki; Yates, Paula; Clancy, Anne; Norton, John D; Sharrocks, Andrew D

    2003-08-15

    DNA binding by the ternary complex factor (TCF) subfamily of ETS-domain transcription factors is tightly regulated by intramolecular and intermolecular interactions. The helix-loop-helix (HLH)-containing Id proteins are trans-acting negative regulators of DNA binding by the TCFs. In the TCF, SAP-2/Net/ERP, intramolecular inhibition of DNA binding is promoted by the cis-acting NID region that also contains an HLH-like motif. The NID also acts as a transcriptional repression domain. Here, we have studied the role of HLH motifs in regulating DNA binding and transcription by the TCF protein SAP-1 and how Cdk-mediated phosphorylation affects the inhibitory activity of the Id proteins towards the TCFs. We demonstrate that the NID region of SAP-1 is an autoinhibitory motif that acts to inhibit DNA binding and also functions as a transcription repression domain. This region can be functionally replaced by fusion of Id proteins to SAP-1, whereby the Id moiety then acts to repress DNA binding in cis. Phosphorylation of the Ids by cyclin-Cdk complexes results in reduction in protein-protein interactions between the Ids and TCFs and relief of their DNA-binding inhibitory activity. In revealing distinct mechanisms through which HLH motifs modulate the activity of TCFs, our results therefore provide further insight into the role of HLH motifs in regulating TCF function and how the inhibitory properties of the trans-acting Id HLH proteins are themselves regulated by phosphorylation.

  11. Melatonin regulates CRE-dependent gene transcription underlying osteoblast proliferation by activating Src and PKA in parallel.

    Science.gov (United States)

    Tao, Lin; Zhu, Yue

    2018-01-01

    Several studies have indicated a relationship between melatonin and idiopathic scoliosis, including our previous work which demonstrated that melatonin can inhibit osteoblast proliferation; however, the mechanism remains unclear. Here, we utilized a MTT assay to show that melatonin significantly reduces osteoblast proliferation in a concentration-and time-dependent manner. Through a combination of techniques, including real-time PCR, MTT assays, immunofluorescence, and luciferase assays, we confirmed that melatonin-induced changes in phosphorylated cAMP response element-binding protein (CREB) reduced transcriptional activity in a melatonin receptor-dependent manner. Surprisingly, treatment of osteoblasts with the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) inhibitor PD98059 up-regulated other cascades upstream of CREB. We next treated cells with PKA and Src inhibitors and observed that melatonin can also activate the protein kinase A (PKA) and Src pathways. To examine whether Src is upstream from the cAMP-PKA pathway, we measured cAMP levels in response to melatonin with and without a Src inhibitor (PP2) and found that PP2 had no additional effect. Therefore, the transcription-dependent mechanisms involved in CREB phosphorylation, along with melatonin, activated Src via a parallel signaling pathway that was separate from that of PKA. Finally, we transfected osteoblasts with lentiviral CREB short hairpin (sh) RNAs and found a decrease in the expression of proliferating cell nuclear antigen (PCNA) and osteoblast proliferation. These results suggest that CREB and PCNA are downstream targets of melatonin signaling, and that the down-regulation of CREB, which is regulated via PKA and Src pathways, contributes to the melatonin-induced inhibition of osteoblast proliferation.

  12. Transcriptional control of the tissue-specific, developmentally regulated osteocalcin gene requires a binding motif for the Msx family of homeodomain proteins.

    Science.gov (United States)

    Hoffmann, H M; Catron, K M; van Wijnen, A J; McCabe, L R; Lian, J B; Stein, G S; Stein, J L

    1994-12-20

    The OC box of the rat osteocalcin promoter (nt -99 to -76) is the principal proximal regulatory element contributing to both tissue-specific and developmental control of osteocalcin gene expression. The central motif of the OC box includes a perfect consensus DNA binding site for certain homeodomain proteins. Homeodomain proteins are transcription factors that direct proper development by regulating specific temporal and spatial patterns of gene expression. We therefore addressed the role of the homeodomain binding motif in the activity of the OC promoter. In this study, by the combined application of mutagenesis and site-specific protein recognition analysis, we examined interactions of ROS 17/2.8 osteosarcoma cell nuclear proteins and purified Msx-1 homeodomain protein with the OC box. We detected a series of related specific protein-DNA interactions, a subset of which were inhibited by antibodies directed against the Msx-1 homeodomain but which also recognize the Msx-2 homeodomain. Our results show that the sequence requirements for binding the Msx-1 or Msx-2 homeodomain closely parallel those necessary for osteocalcin gene promoter activity in vivo. This functional relationship was demonstrated by transient expression in ROS 17/2.8 osteosarcoma cells of a series of osteocalcin promoter (nt -1097 to +24)-reporter gene constructs containing mutations within and flanking the homeodomain binding site of the OC box. Northern blot analysis of several bone-related cell types showed that all of the cells expressed msx-1, whereas msx-2 expression was restricted to cells transcribing osteocalcin. Taken together, our results suggest a role for Msx-1 and -2 or related homeodomain proteins in transcription of the osteocalcin gene.

  13. Extended region of nodulation genes in Rhizobium meliloti 1021. II. Nucleotide sequence, transcription start sites and protein products

    International Nuclear Information System (INIS)

    Fisher, R.F.; Swanson, J.A.; Mulligan, J.T.; Long, S.R.

    1987-01-01

    The authors have established the DNA sequence and analyzed the transcription and translation products of a series of putative nodulation (nod) genes in Rhizobium meliloti strain 1021. Four loci have been designated nodF, nodE, nodG and nodH. The correlation of transposon insertion positions with phenotypes and open reading frames was confirmed by sequencing the insertion junctions of the transposons. The protein products of these nod genes were visualized by in vitro expression of cloned DNA segments in a R. meliloti transcription-translation system. In addition, the sequence for nodG was substantiated by creating translational fusions in all three reading frames at several points in the sequence; the resulting fusions were expressed in vitro in both E. coli and R. meliloti transcription-translation systems. A DNA segment bearing several open reading frames downstream of nodG corresponds to the putative nod gene mutated in strain nod-216. The transcription start sites of nodF and nodH were mapped by primer extension of RNA from cells induced with the plant flavone, luteolin. Initiation of transcription occurs approximately 25 bp downstream from the conserved sequence designated the nod box, suggesting that this conserved sequence acts as an upstream regulator of inducible nod gene expression. Its distance from the transcription start site is more suggestive of an activator binding site rather than an RNA polymerase binding site

  14. Specificity versus redundancy in the RAP2.4 transcription factor family of Arabidopsis thaliana: transcriptional regulation of genes for chloroplast peroxidases.

    Science.gov (United States)

    Rudnik, Radoslaw; Bulcha, Jote Tafese; Reifschneider, Elena; Ellersiek, Ulrike; Baier, Margarete

    2017-08-23

    The Arabidopsis ERFIb / RAP2.4 transcription factor family consists of eight members with highly conserved DNA binding domains. Selected members have been characterized individually, but a systematic comparison is pending. The redox-sensitive transcription factor RAP2.4a mediates chloroplast-to-nucleus redox signaling and controls induction of the three most prominent chloroplast peroxidases, namely 2-Cys peroxiredoxin A (2CPA) and thylakoid- and stromal ascorbate peroxidase (tAPx and sAPx). To test the specificity and redundancy of RAP2.4 transcription factors in the regulation of genes for chloroplast peroxidases, we compared the DNA-binding sites of the transcription factors in tertiary structure models, analyzed transcription factor and target gene regulation by qRT-PCR in RAP2.4, 2-Cys peroxiredoxin and ascorbate peroxidase T-DNA insertion lines and RAP2.4 overexpressing lines of Arabidopsis thaliana and performed promoter binding studies. All RAP2.4 proteins bound the tAPx promoter, but only the four RAP2.4 proteins with identical DNA contact sites, namely RAP2.4a, RAP2.4b, RAP2.4d and RAP2.4h, interacted stably with the redox-sensitive part of the 2CPA promoter. Gene expression analysis in RAP2.4 knockout lines revealed that RAP2.4a is the only one supporting 2CPA and chloroplast APx expression. Rap2.4h binds to the same promoter region as Rap2.4a and antagonizes 2CPA expression. Like the other six RAP2.4 proteins, Rap2.4 h promotes APx mRNA accumulation. Chloroplast ROS signals induced RAP2.4b and RAP2.4d expression, but these two transcription factor genes are (in contrast to RAP2.4a) insensitive to low 2CP availability, and their expression decreased in APx knockout lines. RAP2.4e and RAP2.4f gradually responded to chloroplast APx availability and activated specifically APx expression. These transcription factors bound, like RAP2.4c and RAP2.4g, the tAPx promoter, but hardly the 2CPA promoter. The RAP2.4 transcription factors form an environmentally and

  15. Transcriptional mutagenesis: causes and involvement in tumor development

    Science.gov (United States)

    Brégeon, Damien; Doetsch, Paul W.

    2013-01-01

    The majority of normal cells in a human do not multiply continuously but are quiescent and devote most of their energy to gene transcription. When DNA damages in the transcribed strand of an active gene are bypassed by an RNA polymerase, they can miscode at the damaged site and produce mutant transcripts. This process known as transcriptional mutagenesis can lead to the production of mutant proteins that could be important in tumor development. PMID:21346784

  16. Eukaryote-like Ser/Thr protein kinase PrkA modulates sporulation via regulating the transcriptional factor σ(K) in Bacillus subtilis.

    Science.gov (United States)

    Yan, Jinyuan; Zou, Wei; Fang, Juan; Huang, Xiaowei; Gao, Feng; He, Zeying; Zhang, Keqin; Zhao, Ninghui

    2015-01-01

    Protein kinase A (PrkA), also known as AMP-activated protein kinase, functions as a serine/threonine protein kinase (STPK), has been shown to be involved in a variety of important biologic processes, including pathogenesis of many important diseases in mammals. However, the biological functions of PrkA are less known in prokaryote cells. Here, we explored the function of PrkA as well as its underlying molecular mechanisms using the model bacterium Bacillus subtilis168. When PrkA is inhibited by 9-β-D-arabinofuranosyladenine (ara-A) in the wild type strain or deleted in the ΔprkA mutant strain, we observed sporulation defects in B. subtilis 168, suggesting that PrkA functions as a sporulation-related protein. Transcriptional analysis using the lacZ reporter gene demonstrated that deletion of prkA significantly reduced the expression of the transcriptional factor σ(K) and its downstream genes. Complementation of sigK gene in prkA knockout mutant partially rescued the phenotype of ΔprkA, further supporting the hypothesis that the decreased σ(K) expression should be one of the reasons for the sporulation defect resulting from prkA disruption. Finally, our data confirmed that Hpr (ScoC) negatively controlled the expression of transcriptional factor σ(K), and thus PrkA accelerated sporulation and the expression of σ(K) by suppression of Hpr (ScoC). Taken together, our study discovered a novel function of the eukaryotic-like STPK PrkA in spore development as well as its underlying molecular mechanism in B. subtilis.

  17. Activator Protein-1: redox switch controlling structure and DNA-binding

    Energy Technology Data Exchange (ETDEWEB)

    Yin, Zhou; Machius, Mischa; Nestler, Eric J.; Rudenko, Gabby (Texas-MED); (Icahn)

    2017-09-07

    The transcription factor, activator protein-1 (AP-1), binds to cognate DNA under redox control; yet, the underlying mechanism has remained enigmatic. A series of crystal structures of the AP-1 FosB/JunD bZIP domains reveal ordered DNA-binding regions in both FosB and JunD even in absence DNA. However, while JunD is competent to bind DNA, the FosB bZIP domain must undergo a large conformational rearrangement that is controlled by a ‘redox switch’ centered on an inter-molecular disulfide bond. Solution studies confirm that FosB/JunD cannot undergo structural transition and bind DNA when the redox-switch is in the ‘OFF’ state, and show that the mid-point redox potential of the redox switch affords it sensitivity to cellular redox homeostasis. The molecular and structural studies presented here thus reveal the mechanism underlying redox-regulation of AP-1 Fos/Jun transcription factors and provide structural insight for therapeutic interventions targeting AP-1 proteins.

  18. A Mini Zinc-Finger Protein (MIF from Gerbera hybrida Activates the GASA Protein Family Gene, GEG, to Inhibit Ray Petal Elongation

    Directory of Open Access Journals (Sweden)

    Meixiang Han

    2017-09-01

    Full Text Available Petal appearance is an important horticultural trail that is generally used to evaluate the ornamental value of plants. However, knowledge of the molecular regulation of petal growth is mostly derived from analyses of Arabidopsis thaliana, and relatively little is known about this process in ornamental plants. Previously, GEG (Gerbera hybrida homolog of the gibberellin [GA]–stimulated transcript 1 [GAST1] from tomato, a gene from the GA stimulated Arabidopsis (GASA family, was reported to be an inhibitor of ray petal growth in the ornamental species, G. hybrida. To explore the molecular regulatory mechanism of GEG in petal growth inhibition, a mini zinc-finger protein (MIF was identified using yeast one-hybrid (Y1H screen. The direct binding of GhMIF to the GEG promoter was verified by using an electrophoretic mobility shift assay and a dual-luciferase assay. A yeast two-hybrid (Y2H revealed that GhMIF acts as a transcriptional activator. Transient transformation assay indicated that GhMIF is involved in inhibiting ray petal elongation by activating the expression of GEG. Spatiotemporal expression analyses and hormone treatment assay showed that the expression of GhMIF and GEG is coordinated during petal development. Taken together, these results suggest that GhMIF acts as a direct transcriptional activator of GEG, a gene from the GASA protein family to regulate the petal elongation.

  19. Transcriptional regulation of genes related to progesterone production.

    Science.gov (United States)

    Mizutani, Tetsuya; Ishikane, Shin; Kawabe, Shinya; Umezawa, Akihiro; Miyamoto, Kaoru

    2015-01-01

    Steroid hormones are synthesized from cholesterol in various tissues, mainly in the adrenal glands and gonads. Because these lipid-soluble steroid hormones immediately diffuse through the cells in which they are produced, their secretion directly reflects the activity of the genes related to their production. Progesterone is important not only for luteinization and maintenance of pregnancy, but also as a substrate for most other steroids. Steroidogenic acute regulatory protein (STAR), cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), and 3β-hydroxysteroid dehydrogenase/Δ(5)-Δ(4) isomerase (3β-HSD) are well-known proteins essential for progesterone production. In addition to them, glutathione S-transferase A1-1 and A3-3 are shown to exert Δ(5)-Δ(4) isomerization activity to produce progesterone in a cooperative fashion with 3β-HSD. 5-Aminolevulinic acid synthase 1, ferredoxin 1, and ferredoxin reductase also play a role in steroidogenesis as accessory factors. Members of the nuclear receptor 5A (NR5A) family (steroidogenic factor 1 and liver receptor homolog 1) play a crucial role in the transcriptional regulation of these genes. The NR5A family activates these genes by binding to NR5A responsive elements present within their promoter regions, as well as to the elements far from their promoters. In addition, various NR5A-interacting proteins including peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), nuclear receptor subfamily 0, group B, member 1 (DAX-1), and CCAAT/enhancer-binding proteins (C/EBP) are involved in the transcription of NR5A target genes and regulate the transcription either positively or negatively under both basal and tropic hormone-stimulated conditions. In this review, we describe the transcriptional regulation of genes related to progesterone production.

  20. Karyopherin alpha2 is essential for rRNA transcription and protein synthesis in proliferative keratinocytes.

    Directory of Open Access Journals (Sweden)

    Noriko Umegaki-Arao

    Full Text Available Karyopherin proteins mediate nucleocytoplasmic trafficking and are critical for protein and RNA subcellular localization. Recent studies suggest KPNA2 expression is induced in tumor cells and is strongly associated with prognosis, although the precise roles and mechanisms of KPNA2 overexpression in proliferative disorders have not been defined. We found that KPNA2 expression is induced in various proliferative disorders of the skin such as psoriasis, Bowen's disease, actinic keratosis, squamous cell carcinoma, Paget's disease, Merkel cell carcinoma, and mycosis fungoides. siRNA-mediated KPNA suppression revealed that KPNA2 is essential for significant suppression of HaCaT proliferation under starvation conditions. Ribosomal RNA transcription and protein synthesis were suppressed by starvation combined with knockdown of KPNA (including KPNA2 expression. KPNA2 localized to the nucleolus and interacted with proteins associated with mRNA processing, ribonucleoprotein complex biogenesis, chromatin modification, and transcription, as demonstrated by tandem affinity purification and mass spectrometry. KPNA2 may be an important promoter of ribosomal RNA and protein synthesis in tumor cells.

  1. Small-Molecule Inhibitors of the SOX18 Transcription Factor.

    Science.gov (United States)

    Fontaine, Frank; Overman, Jeroen; Moustaqil, Mehdi; Mamidyala, Sreeman; Salim, Angela; Narasimhan, Kamesh; Prokoph, Nina; Robertson, Avril A B; Lua, Linda; Alexandrov, Kirill; Koopman, Peter; Capon, Robert J; Sierecki, Emma; Gambin, Yann; Jauch, Ralf; Cooper, Matthew A; Zuegg, Johannes; Francois, Mathias

    2017-03-16

    Pharmacological modulation of transcription factors (TFs) has only met little success over the past four decades. This is mostly due to standard drug discovery approaches centered on blocking protein/DNA binding or interfering with post-translational modifications. Recent advances in the field of TF biology have revealed a central role of protein-protein interaction in their mode of action. In an attempt to modulate the activity of SOX18 TF, a known regulator of vascular growth in development and disease, we screened a marine extract library for potential small-molecule inhibitors. We identified two compounds, which inspired a series of synthetic SOX18 inhibitors, able to interfere with the SOX18 HMG DNA-binding domain, and to disrupt HMG-dependent protein-protein interaction with RBPJ. These compounds also perturbed SOX18 transcriptional activity in a cell-based reporter gene system. This approach may prove useful in developing a new class of anti-angiogenic compounds based on the inhibition of TF activity. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Mediator, SWI/SNF and SAGA complexes regulate Yap8-dependent transcriptional activation of ACR2 in response to arsenate.

    Science.gov (United States)

    Menezes, Regina Andrade; Pimentel, Catarina; Silva, Ana Rita Courelas; Amaral, Catarina; Merhej, Jawad; Devaux, Frédéric; Rodrigues-Pousada, Claudina

    2017-04-01

    Response to arsenic stress in Saccharomyces cerevisiae is orchestrated by the regulatory protein Yap8, which mediates transcriptional activation of ACR2 and ACR3. This study contributes to the state of art knowledge of the molecular mechanisms underlying yeast stress response to arsenate as it provides the genetic and biochemical evidences that Yap8, through cysteine residues 132, 137, and 274, is the sensor of presence of arsenate in the cytosol. Moreover, it is here reported for the first time the essential role of the Mediator complex in the transcriptional activation of ACR2 by Yap8. Based on our data, we propose an order-of-function map to recapitulate the sequence of events taking place in cells injured with arsenate. Modification of the sulfhydryl state of these cysteines converts Yap8 in its activated form, triggering the recruitment of the Mediator complex to the ACR2/ACR3 promoter, through the interaction with the tail subunit Med2. The Mediator complex then transfers the regulatory signals conveyed by Yap8 to the core transcriptional machinery, which culminates with TBP occupancy, ACR2 upregulation and cell adaptation to arsenate stress. Additional co-factors are required for the transcriptional activation of ACR2 by Yap8, particularly the nucleosome remodeling activity of SWI/SNF and SAGA complexes. Copyright © 2017. Published by Elsevier B.V.

  3. Transcriptional profiling of protein expression related genes of Pichia pastoris under simulated microgravity.

    Directory of Open Access Journals (Sweden)

    Feng Qi

    Full Text Available The physiological responses and transcription profiling of Pichia pastoris GS115 to simulated microgravity (SMG were substantially changed compared with normal gravity (NG control. We previously reported that the recombinant P. pastoris grew faster under SMG than NG during methanol induction phase and the efficiencies of recombinant enzyme production and secretion were enhanced under SMG, which was considered as the consequence of changed transcriptional levels of some key genes. In this work, transcriptiome profiling of P. pastoris cultured under SMG and NG conditions at exponential and stationary phases were determined using next-generation sequencing (NGS technologies. Four categories of 141 genes function as methanol utilization, protein chaperone, RNA polymerase and protein transportation or secretion classified according to Gene Ontology (GO were chosen to be analyzed on the basis of NGS results. And 80 significantly changed genes were weighted and estimated by Cluster 3.0. It was found that most genes of methanol metabolism (85% of 20 genes and protein transportation or secretion (82.2% of 45 genes were significantly up-regulated under SMG. Furthermore the quantity and fold change of up-regulated genes in exponential phase of each category were higher than those of stationary phase. The results indicate that the up-regulated genes of methanol metabolism and protein transportation or secretion mainly contribute to enhanced production and secretion of the recombinant protein under SMG.

  4. Engineering prokaryotic transcriptional activators as metabolite biosensors in yeast

    DEFF Research Database (Denmark)

    Skjødt, Mette Louise; Snoek, Tim; Kildegaard, Kanchana Rueksomtawin

    2016-01-01

    ,cis-muconic acid at different levels, and found that reporter gene output correlated with production. The transplantation of prokaryotic transcriptional activators into the eukaryotic chassis illustrates the potential of a hitherto untapped biosensor resource useful for biotechnological applications....... real-time monitoring of production has attracted attention. Here we applied systematic engineering of multiple parameters to search for a general biosensor design in the budding yeast Saccharomyces cerevisiae based on small-molecule binding transcriptional activators from the prokaryote superfamily...

  5. Activation of Akt is essential for the propagation of mitochondrial respiratory stress signaling and activation of the transcriptional coactivator heterogeneous ribonucleoprotein A2.

    Science.gov (United States)

    Guha, Manti; Fang, Ji-Kang; Monks, Robert; Birnbaum, Morris J; Avadhani, Narayan G

    2010-10-15

    Mitochondrial respiratory stress (also called mitochondrial retrograde signaling) activates a Ca(2+)/calcineurin-mediated signal that culminates in transcription activation/repression of a large number of nuclear genes. This signal is propagated through activation of the regulatory proteins NFκB c-Rel/p50, C/EBPδ, CREB, and NFAT. Additionally, the heterogeneous ribonucleoprotein A2 (hnRNPA2) functions as a coactivator in up-regulating the transcription of Cathepsin L, RyR1, and Glut-4, the target genes of stress signaling. Activation of IGF1R, which causes a metabolic switch to glycolysis, cell invasiveness, and resistance to apoptosis, is a phenotypic hallmark of C2C12 myoblasts subjected to mitochondrial stress. In this study, we report that mitochondrial stress leads to increased expression, activation, and nuclear localization of Akt1. Mitochondrial respiratory stress also activates Akt1-gene expression, which involves hnRNPA2 as a coactivator, indicating a complex interdependency of these two factors. Using Akt1(-/-) mouse embryonic fibroblasts and Akt1 mRNA-silenced C2C12 cells, we show that Akt1-mediated phosphorylation is crucial for the activation and recruitment of hnRNPA2 to the enhanceosome complex. Akt1 mRNA silencing in mtDNA-depleted cells resulted in reversal of the invasive phenotype, accompanied by sensitivity to apoptotic stimuli. These results show that Akt1 is an important regulator of the nuclear transcriptional response to mitochondrial stress.

  6. Structural Insight on the Mechanism of Regulation of the MarR Family of Proteins: High-Resolution Crystal Structure of a Transcriptional Repressor from Methanobacterium thermoautotrophicum

    Energy Technology Data Exchange (ETDEWEB)

    Saridakis, Vivian; Shahinas, Dea; Xu, Xiaohui; Christendat, Dinesh (York); (Toronto); (CG)

    2008-03-31

    Transcriptional regulators belonging to the MarR family are characterized by a winged-helix DNA binding domain. These transcriptional regulators regulate the efflux and influx of phenolic agents in bacteria and archaea. In Escherichia coli, MarR regulates the multiple antibiotic resistance operon and its inactivation produces a multiple antibiotic resistance phenotype. In some organisms, active efflux of drug compounds will produce a drug resistance phenotype, whereas in other organisms, active influx of chlorinated hydrocarbons results in their rapid degradation. Although proteins in the MarR family are regulators of important biological processes, their mechanism of action is not well understood and structural information about how phenolic agents regulate the activity of these proteins is lacking. This article presents the three-dimensional structure of a protein of the MarR family, MTH313, in its apo form and in complex with salicylate, a known inactivator. A comparison of these two structures indicates that the mechanism of regulation involves a large conformational change in the DNA binding lobe. Electrophoretic mobility shift assay and biophysical analyses further suggest that salicylate inactivates MTH313 and prevents it from binding to its promoter region.

  7. Lycopene activates antioxidant enzymes and nuclear transcription factor systems in heat-stressed broilers.

    Science.gov (United States)

    Sahin, K; Orhan, C; Tuzcu, M; Sahin, N; Hayirli, A; Bilgili, S; Kucuk, O

    2016-05-01

    This study was conducted to evaluate the effects of dietary lycopene supplementation on growth performance, antioxidant status, and muscle nuclear transcription factor [Kelch like-ECH-associated protein 1 (Keap1) and (erythroid-derived 2)-like 2 (Nrf2)] expressions in broiler chickens exposed to heat stress (HS). A total of 180 one-day-old male broiler chicks (Ross 308) were assigned randomly to one of 2×3 factorially arranged treatments: two housing temperatures (22°C for 24 h/d; thermoneutral, TN or 34°C for 8 h/d HS) and three dietary lycopene levels (0, 200, or 400 mg/kg). Each treatment consisted of three replicates of 10 birds. Birds were reared to 42 d of age. Heat stress caused reductions in feed intake and weight gain by 12.2 and 20.7% and increased feed efficiency by 10.8% (Plycopene level improved performance in both environments. Birds reared under the HS environment had lower serum and muscle lycopene concentration (0.34 vs. 0.50 μg/mL and 2.80 vs. 2.13 μg/g), activities of superoxide dismutase (151 vs. 126 U/mL and 131 vs. 155 U/mg protein), glutathione peroxidase (184 vs. 154 U/mL and 1.39 vs. 1.74 U/mg protein), and higher malondialdehyde (MDA) concentration (0.53 vs. 0.83 μg/mL and 0.78 vs. 0.45 μg/ mg protein) than birds reared under the TN environment. Changes in levels of lycopene and MDA and activities of enzymes in serum and muscle varied by the environmental temperature as dietary lycopene level increased. Moreover, increasing dietary lycopene level suppressed muscle Keap1 expression and enhanced muscle Nrf2 expression, which had increased by 150% and decreased by 40%, respectively in response to HS. In conclusion, lycopene supplementation alleviates adverse effects of HS on performance through modulating expressions of stress-related nuclear transcription factors. © 2016 Poultry Science Association Inc.

  8. E-cadherin is transcriptionally activated via suppression of ZEB1 transcriptional repressor by small RNA-mediated gene silencing.

    Directory of Open Access Journals (Sweden)

    Minami Mazda

    Full Text Available RNA activation has been reported to be induced by small interfering RNAs (siRNAs that act on the promoters of several genes containing E-cadherin. In this study, we present an alternative mechanism of E-cadherin activation in human PC-3 cells by siRNAs previously reported to possess perfect-complementary sequences to E-cadherin promoter. We found that activation of E-cadherin can be also induced via suppression of ZEB1, which is a transcriptional repressor of E-cadherin, by seed-dependent silencing mechanism of these siRNAs. The functional seed-complementary sites of the siRNAs were found in the coding region in addition to the 3' untranslated region of ZEB1 mRNA. Promoter analyses indicated that E-boxes, which are ZEB1-binding sites, in the upstream promoter region are indispensable for E-cadherin transcription by the siRNAs. Thus, the results caution against ignoring siRNA seed-dependent silencing effects in genome-wide transcriptional regulation. In addition, members of miR-302/372/373/520 family, which have the same seed sequences with one of the siRNAs containing perfect-complementarity to E-cadherin promoter, are also found to activate E-cadherin transcription. Thus, E-cadherin could be upregulated by the suppression of ZEB1 transcriptional repressor by miRNAs in vivo.

  9. Analysis of a Plant Transcriptional Regulatory Network Using Transient Expression Systems.

    Science.gov (United States)

    Díaz-Triviño, Sara; Long, Yuchen; Scheres, Ben; Blilou, Ikram

    2017-01-01

    In plant biology, transient expression systems have become valuable approaches used routinely to rapidly study protein expression, subcellular localization, protein-protein interactions, and transcriptional activity prior to in vivo studies. When studying transcriptional regulation, luciferase reporter assays offer a sensitive readout for assaying promoter behavior in response to different regulators or environmental contexts and to confirm and assess the functional relevance of predicted binding sites in target promoters. This chapter aims to provide detailed methods for using luciferase reporter system as a rapid, efficient, and versatile assay to analyze transcriptional regulation of target genes by transcriptional regulators. We describe a series of optimized transient expression systems consisting of Arabidopsis thaliana protoplasts, infiltrated Nicotiana benthamiana leaves, and human HeLa cells to study the transcriptional regulations of two well-characterized transcriptional regulators SCARECROW (SCR) and SHORT-ROOT (SHR) on one of their targets, CYCLIN D6 (CYCD6).Here, we illustrate similarities and differences in outcomes when using different systems. The plant-based systems revealed that the SCR-SHR complex enhances CYCD6 transcription, while analysis in HeLa cells showed that the complex is not sufficient to strongly induce CYCD6 transcription, suggesting that additional, plant-specific regulators are required for full activation. These results highlight the importance of the system and suggest that including heterologous systems, such as HeLa cells, can provide a more comprehensive analysis of a complex gene regulatory network.

  10. A critical role for topoisomerase IIb and DNA double strand breaks in transcription.

    Science.gov (United States)

    Calderwood, Stuart K

    2016-05-26

    Recent studies have indicated a novel role for topoisomerase IIb in transcription. Transcription of heat shock genes, serum-induced immediate early genes and nuclear receptor-activated genes, each required DNA double strands generated by topoisomerase IIb. Such strand breaks seemed both necessary and sufficient for transcriptional activation. In addition, such transcription was associated with initiation of the DNA damage response pathways, including the activation of the enzymes: ataxia-telangiectasia mutated (ATM), DNA-dependent protein kinase and poly (ADP ribose) polymerase 1. DNA damage response signaling was involved both in transcription and in repair of DNA breaks generated by topoisomerase IIb.

  11. Human GW182 Paralogs Are the Central Organizers for RNA-Mediated Control of Transcription.

    Science.gov (United States)

    Hicks, Jessica A; Li, Liande; Matsui, Masayuki; Chu, Yongjun; Volkov, Oleg; Johnson, Krystal C; Corey, David R

    2017-08-15

    In the cytoplasm, small RNAs can control mammalian translation by regulating the stability of mRNA. In the nucleus, small RNAs can also control transcription and splicing. The mechanisms for RNA-mediated nuclear regulation are not understood and remain controversial, hindering the effective application of nuclear RNAi and investigation of its natural regulatory roles. Here, we reveal that the human GW182 paralogs TNRC6A/B/C are central organizing factors critical to RNA-mediated transcriptional activation. Mass spectrometry of purified nuclear lysates followed by experimental validation demonstrates that TNRC6A interacts with proteins involved in protein degradation, RNAi, the CCR4-NOT complex, the mediator complex, and histone-modifying complexes. Functional analysis implicates TNRC6A, NAT10, MED14, and WDR5 in RNA-mediated transcriptional activation. These findings describe protein complexes capable of bridging RNA-mediated sequence-specific recognition of noncoding RNA transcripts with the regulation of gene transcription. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  12. Nerve growth factor enhances the CRE-dependent transcriptional activity activated by nobiletin in PC12 cells.

    Science.gov (United States)

    Takito, Jiro; Kimura, Junko; Kajima, Koji; Uozumi, Nobuyuki; Watanabe, Makoto; Yokosuka, Akihito; Mimaki, Yoshihiro; Nakamura, Masanori; Ohizumi, Yasushi

    2016-07-01

    Prevention and treatment of Alzheimer disease are urgent problems for elderly people in developed countries. We previously reported that nobiletin, a poly-methoxylated flavone from the citrus peel, improved the symptoms in various types of animal models of memory loss and activated the cAMP responsive element (CRE)-dependent transcription in PC12 cells. Nobiletin activated the cAMP/PKA/MEK/Erk/MAPK signaling pathway without using the TrkA signaling activated by nerve growth factor (NGF). Here, we examined the effect of combination of nobiletin and NGF on the CRE-dependent transcription in PC12 cells. Although NGF alone had little effect on the CRE-dependent transcription, NGF markedly enhanced the CRE-dependent transcription induced by nobiletin. The NGF-induced enhancement was neutralized by a TrkA antagonist, K252a. This effect of NGF was effective on the early signaling event elicited by nobiletin. These results suggested that there was crosstalk between NGF and nobiletin signaling in activating the CRE-dependent transcription in PC12 cells.

  13. CSL protein regulates transcription of genes required to prevent catastrophic mitosis in fission yeast.

    Science.gov (United States)

    Převorovský, Martin; Oravcová, Martina; Zach, Róbert; Jordáková, Anna; Bähler, Jürg; Půta, František; Folk, Petr

    2016-11-16

    For every eukaryotic cell to grow and divide, intricately coordinated action of numerous proteins is required to ensure proper cell-cycle progression. The fission yeast Schizosaccharomyces pombe has been instrumental in elucidating the fundamental principles of cell-cycle control. Mutations in S. pombe 'cut' (cell untimely torn) genes cause failed coordination between cell and nuclear division, resulting in catastrophic mitosis. Deletion of cbf11, a fission yeast CSL transcription factor gene, triggers a 'cut' phenotype, but the precise role of Cbf11 in promoting mitotic fidelity is not known. We report that Cbf11 directly activates the transcription of the acetyl-coenzyme A carboxylase gene cut6, and the biotin uptake/biosynthesis genes vht1 and bio2, with the former 2 implicated in mitotic fidelity. Cbf11 binds to a canonical, metazoan-like CSL response element (GTGGGAA) in the cut6 promoter. Expression of Cbf11 target genes shows apparent oscillations during the cell cycle using temperature-sensitive cdc25-22 and cdc10-M17 block-release experiments, but not with other synchronization methods. The penetrance of catastrophic mitosis in cbf11 and cut6 mutants is nutrient-dependent. We also show that drastic decrease in biotin availability arrests cell proliferation but does not cause mitotic defects. Taken together, our results raise the possibility that CSL proteins play conserved roles in regulating cell-cycle progression, and they could guide experiments into mitotic CSL functions in mammals.

  14. Genome-wide mRNA processing in methanogenic archaea reveals post-transcriptional regulation of ribosomal protein synthesis.

    Science.gov (United States)

    Qi, Lei; Yue, Lei; Feng, Deqin; Qi, Fengxia; Li, Jie; Dong, Xiuzhu

    2017-07-07

    Unlike stable RNAs that require processing for maturation, prokaryotic cellular mRNAs generally follow an 'all-or-none' pattern. Herein, we used a 5΄ monophosphate transcript sequencing (5΄P-seq) that specifically captured the 5΄-end of processed transcripts and mapped the genome-wide RNA processing sites (PSSs) in a methanogenic archaeon. Following statistical analysis and stringent filtration, we identified 1429 PSSs, among which 23.5% and 5.4% were located in 5΄ untranslated region (uPSS) and intergenic region (iPSS), respectively. A predominant uridine downstream PSSs served as a processing signature. Remarkably, 5΄P-seq detected overrepresented uPSS and iPSS in the polycistronic operons encoding ribosomal proteins, and the majority upstream and proximal ribosome binding sites, suggesting a regulatory role of processing on translation initiation. The processed transcripts showed increased stability and translation efficiency. Particularly, processing within the tricistronic transcript of rplA-rplJ-rplL enhanced the translation of rplL, which can provide a driving force for the 1:4 stoichiometry of L10 to L12 in the ribosome. Growth-associated mRNA processing intensities were also correlated with the cellular ribosomal protein levels, thereby suggesting that mRNA processing is involved in tuning growth-dependent ribosome synthesis. In conclusion, our findings suggest that mRNA processing-mediated post-transcriptional regulation is a potential mechanism of ribosomal protein synthesis and stoichiometry. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  15. Step out of the groove : epigenetic gene control systems and engineered transcription factors

    NARCIS (Netherlands)

    Verschure, P.J.; Visser, A.E.; Rots, M.G.

    2006-01-01

    At the linear DNA level, gene activity is believed to be driven by binding of transcription factors, which subsequently recruit the RNA polymerase to the gene promoter region. However, it has become clear that transcriptional activation involves large complexes of many different proteins, which not

  16. Pumilio and nanos RNA-binding proteins counterbalance the transcriptional consequences of RB1 inactivation.

    Science.gov (United States)

    Miles, Wayne O; Dyson, Nicholas J

    2014-01-01

    The ability of the retinoblastoma protein (RB) tumor suppressor to repress transcription stimulated by the E2 promoter binding factors (E2F) is integral to its biological functions. Our recent report described a conserved feedback mechanism mediated by the RNA-binding proteins Pumilio and Nanos that increases in importance following RB loss and helps cells to tolerate deregulated E2F.

  17. Transcriptionally Active Heterochromatin in Rye B Chromosomes[W

    Science.gov (United States)

    Carchilan, Mariana; Delgado, Margarida; Ribeiro, Teresa; Costa-Nunes, Pedro; Caperta, Ana; Morais-Cecílio, Leonor; Jones, R. Neil; Viegas, Wanda; Houben, Andreas

    2007-01-01

    B chromosomes (Bs) are dispensable components of the genomes of numerous species. Thus far, there is a lack of evidence for any transcripts of Bs in plants, with the exception of some rDNA sequences. Here, we show that the Giemsa banding-positive heterochromatic subterminal domain of rye (Secale cereale) Bs undergoes decondensation during interphase. Contrary to the heterochromatic regions of A chromosomes, this domain is simultaneously marked by trimethylated H3K4 and by trimethylated H3K27, an unusual combination of apparently conflicting histone modifications. Notably, both types of B-specific high copy repeat families (E3900 and D1100) of the subterminal domain are transcriptionally active, although with different tissue type–dependent activity. No small RNAs were detected specifically for the presence of Bs. The lack of any significant open reading frame and the highly heterogeneous size of mainly polyadenylated transcripts indicate that the noncoding RNA may function as structural or catalytic RNA. PMID:17586652

  18. Evaluation of SMN protein, transcript, and copy number in the biomarkers for spinal muscular atrophy (BforSMA clinical study.

    Directory of Open Access Journals (Sweden)

    Thomas O Crawford

    Full Text Available The universal presence of a gene (SMN2 nearly identical to the mutated SMN1 gene responsible for Spinal Muscular Atrophy (SMA has proved an enticing incentive to therapeutics development. Early disappointments from putative SMN-enhancing agent clinical trials have increased interest in improving the assessment of SMN expression in blood as an early "biomarker" of treatment effect.A cross-sectional, single visit, multi-center design assessed SMN transcript and protein in 108 SMA and 22 age and gender-matched healthy control subjects, while motor function was assessed by the Modified Hammersmith Functional Motor Scale (MHFMS. Enrollment selectively targeted a broad range of SMA subjects that would permit maximum power to distinguish the relative influence of SMN2 copy number, SMA type, present motor function, and age.SMN2 copy number and levels of full-length SMN2 transcripts correlated with SMA type, and like SMN protein levels, were lower in SMA subjects compared to controls. No measure of SMN expression correlated strongly with MHFMS. A key finding is that SMN2 copy number, levels of transcript and protein showed no correlation with each other.This is a prospective study that uses the most advanced techniques of SMN transcript and protein measurement in a large selectively-recruited cohort of individuals with SMA. There is a relationship between measures of SMN expression in blood and SMA type, but not a strong correlation to motor function as measured by the MHFMS. Low SMN transcript and protein levels in the SMA subjects relative to controls suggest that these measures of SMN in accessible tissues may be amenable to an "early look" for target engagement in clinical trials of putative SMN-enhancing agents. Full length SMN transcript abundance may provide insight into the molecular mechanism of phenotypic variation as a function of SMN2 copy number.Clinicaltrials.gov NCT00756821.

  19. Transcriptional decomposition reveals active chromatin architectures and cell specific regulatory interactions

    DEFF Research Database (Denmark)

    Rennie, Sarah; Dalby, Maria; van Duin, Lucas

    2018-01-01

    Transcriptional regulation is tightly coupled with chromosomal positioning and three-dimensional chromatin architecture. However, it is unclear what proportion of transcriptional activity is reflecting such organisation, how much can be informed by RNA expression alone and how this impacts disease...... proportion of total levels and is highly informative of topological associating domain activities and organisation, revealing boundaries and chromatin compartments. Furthermore, expression data alone accurately predict individual enhancer-promoter interactions, drawing features from expression strength...... between transcription and chromatin architecture....

  20. Proteasome activity or expression is not altered by activation of the heat shock transcription factor Hsf1 in cultured fibroblasts or myoblasts.

    Science.gov (United States)

    Taylor, David M; Kabashi, Edor; Agar, Jeffrey N; Minotti, Sandra; Durham, Heather D

    2005-01-01

    Heat shock proteins (Hsps) with chaperoning function work together with the ubiquitin-proteasome pathway to prevent the accumulation of misfolded, potentially toxic proteins, as well as to control catabolism of the bulk of cytoplasmic, cellular protein. There is evidence for the involvement of both systems in neurodegenerative disease, and a therapeutic target is the heat shock transcription factor, Hsf1, which mediates upregulation of Hsps in response to cellular stress. The mechanisms regulating expression of proteasomal proteins in mammalian cells are less well defined. To assess any direct effect of Hsf1 on expression of proteasomal subunits and activity in mammalian cells, a plasmid encoding a constitutively active form of Hsf1 (Hsf1act) was expressed in mouse embryonic fibroblasts lacking Hsf1 and in cultured human myoblasts. Plasmid encoding an inactivatible form of Hsf1 (Hsf1inact) served as control. In cultures transfected with plasmid hsf1act, robust expression of the major stress-inducible Hsp, Hsp70, occurred but not in cultures transfected with hsf1inact. No significant changes in the level of expression of representative proteasomal proteins (structural [20Salpha], a nonpeptidase beta subunit [20Sbeta3], or 2 regulatory subunits [19S subunit 6b, 11 Salpha]) or in chymotrypsin-, trypsin-, and caspaselike activities of the proteasome were measured. Thus, stress-induced or pharmacological activation of Hsf1 in mammalian cells would upregulate Hsps but not directly affect expression or activity of proteasomes.

  1. MrkH, a novel c-di-GMP-dependent transcriptional activator, controls Klebsiella pneumoniae biofilm formation by regulating type 3 fimbriae expression.

    Directory of Open Access Journals (Sweden)

    Jonathan J Wilksch

    2011-08-01

    Full Text Available Klebsiella pneumoniae causes significant morbidity and mortality worldwide, particularly amongst hospitalized individuals. The principle mechanism for pathogenesis in hospital environments involves the formation of biofilms, primarily on implanted medical devices. In this study, we constructed a transposon mutant library in a clinical isolate, K. pneumoniae AJ218, to identify the genes and pathways implicated in biofilm formation. Three mutants severely defective in biofilm formation contained insertions within the mrkABCDF genes encoding the main structural subunit and assembly machinery for type 3 fimbriae. Two other mutants carried insertions within the yfiN and mrkJ genes, which encode GGDEF domain- and EAL domain-containing c-di-GMP turnover enzymes, respectively. The remaining two isolates contained insertions that inactivated the mrkH and mrkI genes, which encode for novel proteins with a c-di-GMP-binding PilZ domain and a LuxR-type transcriptional regulator, respectively. Biochemical and functional assays indicated that the effects of these factors on biofilm formation accompany concomitant changes in type 3 fimbriae expression. We mapped the transcriptional start site of mrkA, demonstrated that MrkH directly activates transcription of the mrkA promoter and showed that MrkH binds strongly to the mrkA regulatory region only in the presence of c-di-GMP. Furthermore, a point mutation in the putative c-di-GMP-binding domain of MrkH completely abolished its function as a transcriptional activator. In vivo analysis of the yfiN and mrkJ genes strongly indicated their c-di-GMP-specific function as diguanylate cyclase and phosphodiesterase, respectively. In addition, in vitro assays showed that purified MrkJ protein has strong c-di-GMP phosphodiesterase activity. These results demonstrate for the first time that c-di-GMP can function as an effector to stimulate the activity of a transcriptional activator, and explain how type 3 fimbriae

  2. USP10 Antagonizes c-Myc Transcriptional Activation through SIRT6 Stabilization to Suppress Tumor Formation

    Directory of Open Access Journals (Sweden)

    Zhenghong Lin

    2013-12-01

    Full Text Available The reduced protein expression of SIRT6 tumor suppressor is involved in tumorigenesis. The molecular mechanisms underlying SIRT6 protein downregulation in human cancers remain unknown. Using a proteomic approach, we have identified the ubiquitin-specific peptidase USP10, another tumor suppressor, as one of the SIRT6-interacting proteins. USP10 suppresses SIRT6 ubiquitination to protect SIRT6 from proteasomal degradation. USP10 antagonizes the transcriptional activity of the c-Myc oncogene through SIRT6, as well as p53, to inhibit cell-cycle progression, cancer cell growth, and tumor formation. To support this conclusion, we detected significant reductions in both USP10 and SIRT6 protein expression in human colon cancers. Our study discovered crosstalk between two tumor-suppressive genes in regulating cell-cycle progression and proliferation and showed that dysregulated USP10 function promotes tumorigenesis through SIRT6 degradation.

  3. Human Mediator Enhances Activator-Facilitated Recruitment of RNA Polymerase II and Promoter Recognition by TATA-Binding Protein (TBP) Independently of TBP-Associated Factors

    OpenAIRE

    Wu, Shwu-Yuan; Zhou, Tianyuan; Chiang, Cheng-Ming

    2003-01-01

    Mediator is a general cofactor implicated in the functions of many transcriptional activators. Although Mediator with different protein compositions has been isolated, it remains unclear how Mediator facilitates activator-dependent transcription, independent of its general stimulation of basal transcription. To define the mechanisms of Mediator function, we isolated two forms of human Mediator complexes (Mediator-P.5 and Mediator-P.85) and demonstrated that Mediator-P.5 clearly functions by e...

  4. Inhibitory effects of curcumin and capsaicin on phorbol ester-induced activation of eukaryotic transcription factors, NF-kappaB and AP-1.

    Science.gov (United States)

    Surh, Y J; Han, S S; Keum, Y S; Seo, H J; Lee, S S

    2000-01-01

    Recently, considerable attention has been focused on identifying dietary and medicinal phytochemicals that can inhibit, retard or reverse the multi-stage carcinogenesis. Spices and herbs contain phenolic substances with potent antioxidative and chemopreventive properties. Curcumin, a yellow colouring agent from turmeric and capsaicin, a pungent principle of red pepper exhibit profound anticarcinogenic and antimutagenic activities. Two well-defined eukaryotic transcription factors, nuclear factor-kappa B (NF-kappaB) and activator protein 1 (AP-1) have been implicated in pathogenesis of many human diseases including cancer. These transcription factors are known to be activated by a wide array of external stimuli, such as tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), tumor necrosis factor, reactive oxygen species, bacterial lipopolysaccharide, and ultraviolet. In the present study, we found that topical application of TPA onto dorsal skin of female ICR mice resulted in marked activation of epidermal NF-kappaB and AP-1. Curcumin and capsaicin, when topically applied prior to TPA, significantly attenuated TPA-induced activation of each transcription factor in mouse skin. Likewise, both compounds inhibited NF-kappaB and AP-1 activation in cultured human promyelocytic leukemia (HL-60) cells stimulated with TPA. Based on these findings, it is likely that curcumin and capsaicin exert anti-tumor promotional effects through suppression of the tumor promoter-induced activation of transcription factors, NF-kappaB and AP-1.

  5. Multimode drug inducible CRISPR/Cas9 devices for transcriptional activation and genome editing

    Science.gov (United States)

    Lu, Jia; Zhao, Chen; Zhao, Yingze; Zhang, Jingfang; Zhang, Yue; Chen, Li; Han, Qiyuan; Ying, Yue; Peng, Shuai; Ai, Runna; Wang, Yu

    2018-01-01

    Abstract Precise investigation and manipulation of dynamic biological processes often requires molecular modulation in a controlled inducible manner. The clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) has emerged as a versatile tool for targeted gene editing and transcriptional programming. Here, we designed and vigorously optimized a series of Hybrid drug Inducible CRISPR/Cas9 Technologies (HIT) for transcriptional activation by grafting a mutated human estrogen receptor (ERT2) to multiple CRISPR/Cas9 systems, which renders them 4-hydroxytamoxifen (4-OHT) inducible for the access of genome. Further, extra functionality of simultaneous genome editing was achieved with one device we named HIT2. Optimized terminal devices herein delivered advantageous performances in comparison with several existing designs. They exerted selective, titratable, rapid and reversible response to drug induction. In addition, these designs were successfully adapted to an orthogonal Cas9. HIT systems developed in this study can be applied for controlled modulation of potentially any genomic loci in multiple modes. PMID:29237052

  6. Regulation of the activity of the dual-function DnaA protein in Caulobacter crescentus.

    Directory of Open Access Journals (Sweden)

    Carmen Fernandez-Fernandez

    Full Text Available DnaA is a conserved essential bacterial protein that acts as the initiator of chromosomal replication as well as a master transcriptional regulator in Caulobacter crescentus. Thus, the intracellular levels of active DnaA need to be tightly regulated during the cell cycle. Our previous work suggested that DnaA may be regulated at the level of its activity by the replisome-associated protein HdaA. Here, we describe the construction of a mutant DnaA protein [DnaA(R357A]. The R357 residue in the AAA+ domain of the C. crescentus DnaA protein is equivalent to the R334 residue of the E. coli DnaA protein, which is required for the Regulatory Inactivation of DnaA (RIDA. We found that the expression of the DnaA(R357A mutant protein in C. crescentus, but not the expression of the wild-type DnaA protein at similar levels, causes a severe phenotype of over-initiation of chromosomal replication and that it blocks cell division. Thus, the mutant DnaA(R357A protein is hyper-active to promote the initiation of DNA replication, compared to the wild-type DnaA protein. DnaA(R357A could not replace DnaA in vivo, indicating that the switch in DnaA activity once chromosomal replication has started may be an essential process in C. crescentus. We propose that the inactivation of DnaA is the main mechanism ensuring that chromosomal replication starts only once per cell cycle. We further observed that the R357A substitution in DnaA does not promote the activity of DnaA as a direct transcriptional activator of four important genes, encoding HdaA, the GcrA master cell cycle regulator, the FtsZ cell division protein and the MipZ spatial regulator of cell division. Thus, the AAA+ domain of DnaA may play a role in temporally regulating the bifunctionality of DnaA by reallocating DnaA molecules from initiating DNA replication to transcribing genes within the unique DnaA regulon of C. crescentus.

  7. Regulation of hepatic lipogenesis by the transcription complex Prep1-Pbx1

    OpenAIRE

    Cabaro, Serena

    2011-01-01

    Prep1 is an homeodomain transcription factor belonging to the TALE proteins, including also Pbx1, which plays an essential role in hematopoiesis, organogenesis and development. Prep1 forms transcriptionally active complexes with Pbx1 and regulates the activity of several genes. The Prep1 null mutation leads to embryonic death at a very early stage. Therefore, Prep1 hypomorphic (Prep1i/i) mice have been generated. Prep1 heterozygous (Prep1i/+) mice, which express only 55-57% of protein, have a...

  8. The same pocket in menin binds both MLL and JUND but has opposite effects on transcription

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Jing; Gurung, Buddha; Wan, Bingbing; Matkar, Smita; Veniaminova, Natalia A.; Wan, Ke; Merchant, Juanita L.; Hua, Xianxin; Lei, Ming (Michigan); (Michigan-Med); (UPENN-MED)

    2013-04-08

    Menin is a tumour suppressor protein whose loss or inactivation causes multiple endocrine neoplasia 1 (MEN1), a hereditary autosomal dominant tumour syndrome that is characterized by tumorigenesis in multiple endocrine organs. Menin interacts with many proteins and is involved in a variety of cellular processes. Menin binds the JUN family transcription factor JUND and inhibits its transcriptional activity. Several MEN1 missense mutations disrupt the menin-JUND interaction, suggesting a correlation between the tumour-suppressor function of menin and its suppression of JUND-activated transcription. Menin also interacts with mixed lineage leukaemia protein 1 (MLL1), a histone H3 lysine 4 methyltransferase, and functions as an oncogenic cofactor to upregulate gene transcription and promote MLL1-fusion-protein-induced leukaemogenesis. A recent report on the tethering of MLL1 to chromatin binding factor lens epithelium-derived growth factor (LEDGF) by menin indicates that menin is a molecular adaptor coordinating the functions of multiple proteins. Despite its importance, how menin interacts with many distinct partners and regulates their functions remains poorly understood. Here we present the crystal structures of human menin in its free form and in complexes with MLL1 or with JUND, or with an MLL1-LEDGF heterodimer. These structures show that menin contains a deep pocket that binds short peptides of MLL1 or JUND in the same manner, but that it can have opposite effects on transcription. The menin-JUND interaction blocks JUN N-terminal kinase (JNK)-mediated JUND phosphorylation and suppresses JUND-induced transcription. In contrast, menin promotes gene transcription by binding the transcription activator MLL1 through the peptide pocket while still interacting with the chromatin-anchoring protein LEDGF at a distinct surface formed by both menin and MLL1.

  9. Role of hypoxia-inducible factor-α in hepatitis-B-virus X protein-mediated MDR1 activation

    International Nuclear Information System (INIS)

    Han, Hyo-Kyung; Han, Chang Yeob; Cheon, Eun-Pa; Lee, Jaewon; Kang, Keon Wook

    2007-01-01

    The transition from chemotherapy-responsive cancer cells to chemotherapy-resistant cancer cells is mainly accompanied by the increased expression of multi-drug resistance 1 (MDR1). We found that hepatitis-B-virus X protein (HBx) increases the transcriptional activity and protein level of MDR1 in a hepatoma cell line, H4IIE. In addition, HBx overexpression made H4IIE cells more resistant to verapamil-uptake. HBx stabilized hypoxia-inducible factor-1α (HIF-1α) and induced the nuclear translocation of C/EBPβ. Reporter gene analyses showed that HBx increased the reporter activity in the cells transfected with the reporter containing MDR1 gene promoter. Moreover, the luciferase reporter gene activity was significantly inhibited by HIF-1α siRNA but not by overexpression of C/EBP dominant negative mutant. These results imply that HBx increases the MDR1 transporter activity through the transcriptional activation of the MDR1 gene with HIF-1α activation, and suggest HIF-1α for the therapeutic target of HBV-mediated chemoresistance

  10. The pat1 protein kinase controls transcription of the mating-type genes in fission yeast

    DEFF Research Database (Denmark)

    Nielsen, O; Egel, R; Nielsen, Olaf

    1990-01-01

    . This differentiation process is characterized by a transcriptional induction of the mating-type genes. Conjugation can also be induced in pat1-ts mutants by a shift to a semi-permissive temperature. The pat1 gene encodes a protein kinase, which also functions further downstream in the developmental pathway controlling...... of the mating-type genes in the zygote leads to complete loss of pat1 protein kinase activity causing entry into meiosis. Thus, pat1 can promote its own inactivation. We suggest a model according to which a stepwise inactivation of pat1 leads to sequential derepression of the processes of conjugation......The developmental programme of fission yeast brings about a transition from mitotic cell division to the dormant state of ascospores. In response to nitrogen starvation, two cells of opposite mating type conjugate to form a diploid zygote, which then undergoes meiosis and sporulation...

  11. Model of transcriptional activation by MarA in Escherichia coli.

    Science.gov (United States)

    Wall, Michael E; Markowitz, David A; Rosner, Judah L; Martin, Robert G

    2009-12-01

    The AraC family transcription factor MarA activates approximately 40 genes (the marA/soxS/rob regulon) of the Escherichia coli chromosome resulting in different levels of resistance to a wide array of antibiotics and to superoxides. Activation of marA/soxS/rob regulon promoters occurs in a well-defined order with respect to the level of MarA; however, the order of activation does not parallel the strength of MarA binding to promoter sequences. To understand this lack of correspondence, we developed a computational model of transcriptional activation in which a transcription factor either increases or decreases RNA polymerase binding, and either accelerates or retards post-binding events associated with transcription initiation. We used the model to analyze data characterizing MarA regulation of promoter activity. The model clearly explains the lack of correspondence between the order of activation and the MarA-DNA affinity and indicates that the order of activation can only be predicted using information about the strength of the full MarA-polymerase-DNA interaction. The analysis further suggests that MarA can activate without increasing polymerase binding and that activation can even involve a decrease in polymerase binding, which is opposite to the textbook model of activation by recruitment. These findings are consistent with published chromatin immunoprecipitation assays of interactions between polymerase and the E. coli chromosome. We find that activation involving decreased polymerase binding yields lower latency in gene regulation and therefore might confer a competitive advantage to cells. Our model yields insights into requirements for predicting the order of activation of a regulon and enables us to suggest that activation might involve a decrease in polymerase binding which we expect to be an important theme of gene regulation in E. coli and beyond.

  12. Model of transcriptional activation by MarA in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Michael E Wall

    2009-12-01

    Full Text Available The AraC family transcription factor MarA activates approximately 40 genes (the marA/soxS/rob regulon of the Escherichia coli chromosome resulting in different levels of resistance to a wide array of antibiotics and to superoxides. Activation of marA/soxS/rob regulon promoters occurs in a well-defined order with respect to the level of MarA; however, the order of activation does not parallel the strength of MarA binding to promoter sequences. To understand this lack of correspondence, we developed a computational model of transcriptional activation in which a transcription factor either increases or decreases RNA polymerase binding, and either accelerates or retards post-binding events associated with transcription initiation. We used the model to analyze data characterizing MarA regulation of promoter activity. The model clearly explains the lack of correspondence between the order of activation and the MarA-DNA affinity and indicates that the order of activation can only be predicted using information about the strength of the full MarA-polymerase-DNA interaction. The analysis further suggests that MarA can activate without increasing polymerase binding and that activation can even involve a decrease in polymerase binding, which is opposite to the textbook model of activation by recruitment. These findings are consistent with published chromatin immunoprecipitation assays of interactions between polymerase and the E. coli chromosome. We find that activation involving decreased polymerase binding yields lower latency in gene regulation and therefore might confer a competitive advantage to cells. Our model yields insights into requirements for predicting the order of activation of a regulon and enables us to suggest that activation might involve a decrease in polymerase binding which we expect to be an important theme of gene regulation in E. coli and beyond.

  13. 6-Mercaptopurine, an activator of Nur77, enhances transcriptional activity of HIF-1alpha resulting in new vessel formation.

    Science.gov (United States)

    Yoo, Y-G; Na, T-Y; Yang, W-K; Kim, H-J; Lee, I-K; Kong, G; Chung, J-H; Lee, M-O

    2007-05-31

    Hypoxia-inducible factor-1alpha (HIF-1alpha) plays a central role in oxygen homeostasis. Previously, we reported that the orphan nuclear receptor Nur77 functions in stabilizing HIF-1alpha. Here, we demonstrate that 6-mercaptopurine (6-MP), an activator of the NR4A family members, enhances transcriptional activity of HIF-1. 6-MP enhanced the protein-level of HIF-1alpha as well as vascular endothelial growth factor (VEGF) in a dose- and time-dependent manner. The induction of HIF-1alpha was abolished by the transfection of either a dominant-negative Nur77 mutant or si-Nur77, indicating a critical role of Nur77 in the 6-MP action. The HIF-1alpha protein level remained up to 60 min in the presence of 6-MP when de novo protein synthesis was blocked by cycloheximide, suggesting that 6-MP induces stabilization of the HIF-1alpha protein. The fact that 6-MP decreased the association of HIF-1alpha with von Hippel-Lindau protein and the acetylation of HIF-1alpha, may explain how 6-MP induced stability of HIF-1alpha. Further, 6-MP induced the transactivation function of HIF-1alpha by recruiting co-activator cyclic-AMP-response-element-binding protein. Finally, 6-MP enhanced the expression of HIF-1alpha and VEGF, and the formation of capillary tubes in human umbilical vascular endothelial cells. Together, our results provide a new insight for 6-MP action in the stabilization of HIF-1alpha and imply a potential application of 6-MP in hypoxia-associated human vascular diseases.

  14. Identification of nucleosome assembly protein 1 (NAP1) as an interacting partner of plant ribosomal protein S6 (RPS6) and a positive regulator of rDNA transcription

    Energy Technology Data Exchange (ETDEWEB)

    Son, Ora [Department of Biological Science, Sookmyung Women' s University, Seoul 140-742 (Korea, Republic of); Kim, Sunghan [Department of Biological Science, Sookmyung Women' s University, Seoul 140-742 (Korea, Republic of); Department of Plant Science, Plant Genomics and Breeding Institute, Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 151-921 (Korea, Republic of); Shin, Yun-jeong [Department of Biological Science, Sookmyung Women' s University, Seoul 140-742 (Korea, Republic of); Kim, Woo-Young [College of Pharmacy, Sookmyung Women' s University, Seoul 140-742 (Korea, Republic of); Koh, Hee-Jong, E-mail: heejkoh@snu.ac.kr [Department of Plant Science, Plant Genomics and Breeding Institute, Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 151-921 (Korea, Republic of); Cheon, Choong-Ill, E-mail: ccheon@sookmyung.ac.kr [Department of Biological Science, Sookmyung Women' s University, Seoul 140-742 (Korea, Republic of)

    2015-09-18

    The ribosomal protein S6 (RPS6) is a downstream component of the signaling mediated by the target of rapamycin (TOR) kinase that acts as a central regulator of the key metabolic processes, such as protein translation and ribosome biogenesis, in response to various environmental cues. In our previous study, we identified a novel role of plant RPS6, which negatively regulates rDNA transcription, forming a complex with a plant-specific histone deacetylase, AtHD2B. Here we report that the Arabidopsis RPS6 interacts additionally with a histone chaperone, nucleosome assembly protein 1(AtNAP1;1). The interaction does not appear to preclude the association of RPS6 with AtHD2B, as the AtNAP1 was also able to interact with AtHD2B as well as with an RPS6-AtHD2B fusion protein in the BiFC assay and pulldown experiment. Similar to a positive effect of the ribosomal S6 kinase 1 (AtS6K1) on rDNA transcription observed in this study, overexpression or down regulation of the AtNAP1;1 resulted in concomitant increase and decrease, respectively, in rDNA transcription suggesting a positive regulatory role played by AtNAP1 in plant rDNA transcription, possibly through derepression of the negative effect of the RPS6-AtHD2B complex. - Highlights: • Nucleosome assembly protein 1 (AtNAP1) interacts with RPS6 as well as with AtHD2B. • rDNA transcription is regulated S6K1. • Overexpression or down regulation of AtNAP1 results in concomitant increase or decrease in rDNA transcription.

  15. BTG2 is an LXXLL-dependent co-repressor for androgen receptor transcriptional activity

    International Nuclear Information System (INIS)

    Hu, Xu-Dong; Meng, Qing-Hui; Xu, Jia-Ying; Jiao, Yang; Ge, Chun-Min; Jacob, Asha; Wang, Ping; Rosen, Eliot M; Fan, Saijun

    2011-01-01

    Research highlights: → BTG2 associates with AR, androgen causes an increase of the interaction. → BTG2 as a co-repressor inhibits the AR-mediated transcription activity. → BTG2 inhibits the transcription activity and expression of PSA. → An intact 92 LxxLL 96 motif is essential and necessary for these activities of BTG2, while the 20 LxxLL 24 motif is not required. → Ectopic expression of BTG2 reduces proliferation of prostate cancer cells. -- Abstract: The tumor suppressor gene, BTG2 has been down-regulated in prostate cancer and the ectopic expression of this gene has been shown to inhibit prostate cancer cell growth. Sequence analysis revealed that the BTG2 protein contains two leucine-rich motifs ( 20 LxxLL 24 and 92 LxxLL 96 ), which are usually found in nuclear receptor co-factors. Based on this, we postulated that there will be an association between BTG2 and AR. In this study, we discovered that BTG2 directly bound to the androgen receptor (AR) in the absence of 5α-dihydrotestosterone (DHT), and in the presence of the androgen, this interaction was increased. BTG2 bearing the mutant 20 LxxLL 24 motif bound to AR equally efficient as the wild-type BTG2, while BTG2 bearing the mutant 92 LxxLL 96 motif failed to interact with AR. Functional studies indicated that ectopic expression of BTG2 caused a significant inhibition of AR-mediated transcriptional activity and a decreased growth of prostate cancer cells. Androgen-induced promoter activation and expression of prostate-specific antigen (PSA) are significantly attenuated by BTG2. The intact 92 LxxLL 96 motif is required for these activities. These findings, for the first time, demonstrate that BTG2 complexes with AR via an LxxLL-dependent mechanism and may play a role in prostate cancer via modulating the AR signaling pathway.

  16. BTG2 is an LXXLL-dependent co-repressor for androgen receptor transcriptional activity

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Xu-Dong [School of Radiation Medicine and Public Health, Medical College of Soochow University, Suzhou 215123 (China); Meng, Qing-Hui [Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20057 (United States); Xu, Jia-Ying; Jiao, Yang [School of Radiation Medicine and Public Health, Medical College of Soochow University, Suzhou 215123 (China); Ge, Chun-Min [Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20057 (United States); Jacob, Asha; Wang, Ping [North Shore University Hospital-Long Island Jewish Medical Center and The Feinstein Institute for Medical Research, Manhasset, NY 11030 (United States); Rosen, Eliot M [Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20057 (United States); Fan, Saijun, E-mail: sjfan@suda.edu.cn [School of Radiation Medicine and Public Health, Medical College of Soochow University, Suzhou 215123 (China)

    2011-01-28

    Research highlights: {yields} BTG2 associates with AR, androgen causes an increase of the interaction. {yields} BTG2 as a co-repressor inhibits the AR-mediated transcription activity. {yields} BTG2 inhibits the transcription activity and expression of PSA. {yields} An intact {sup 92}LxxLL{sup 96} motif is essential and necessary for these activities of BTG2, while the {sup 20}LxxLL{sup 24} motif is not required. {yields} Ectopic expression of BTG2 reduces proliferation of prostate cancer cells. -- Abstract: The tumor suppressor gene, BTG2 has been down-regulated in prostate cancer and the ectopic expression of this gene has been shown to inhibit prostate cancer cell growth. Sequence analysis revealed that the BTG2 protein contains two leucine-rich motifs ({sup 20}LxxLL{sup 24} and {sup 92}LxxLL{sup 96}), which are usually found in nuclear receptor co-factors. Based on this, we postulated that there will be an association between BTG2 and AR. In this study, we discovered that BTG2 directly bound to the androgen receptor (AR) in the absence of 5{alpha}-dihydrotestosterone (DHT), and in the presence of the androgen, this interaction was increased. BTG2 bearing the mutant {sup 20}LxxLL{sup 24} motif bound to AR equally efficient as the wild-type BTG2, while BTG2 bearing the mutant {sup 92}LxxLL{sup 96} motif failed to interact with AR. Functional studies indicated that ectopic expression of BTG2 caused a significant inhibition of AR-mediated transcriptional activity and a decreased growth of prostate cancer cells. Androgen-induced promoter activation and expression of prostate-specific antigen (PSA) are significantly attenuated by BTG2. The intact {sup 92}LxxLL{sup 96} motif is required for these activities. These findings, for the first time, demonstrate that BTG2 complexes with AR via an LxxLL-dependent mechanism and may play a role in prostate cancer via modulating the AR signaling pathway.

  17. Foot-and-mouth disease virus leader proteinase inhibits dsRNA-induced type I interferon transcription by decreasing interferon regulatory factor 3/7 in protein levels

    International Nuclear Information System (INIS)

    Wang, Dang; Fang, Liurong; Luo, Rui; Ye, Rui; Fang, Ying; Xie, Lilan; Chen, Huanchun; Xiao, Shaobo

    2010-01-01

    Research highlights: → FMDV L pro inhibits poly(I:C)-induced IFN-α1/β mRNA expression. → L pro inhibits MDA5-mediated activation of the IFN-α1/β promoter. → L pro significantly reduced the transcription of multiple IRF-responsive genes. → L pro inhibits IFN-α1/β promoter activation by decreasing IRF-3/7 in protein levels. → The ability to process eIF-4G of L pro is not necessary to inhibit IFN-α1/β activation. -- Abstract: The leader proteinase (L pro ) of foot-and-mouth disease virus (FMDV) has been identified as an interferon-β (IFN-β) antagonist that disrupts the integrity of transcription factor nuclear factor κB (NF-κB). In this study, we showed that the reduction of double stranded RNA (dsRNA)-induced IFN-α1/β expression caused by L pro was also associated with a decrease of interferon regulatory factor 3/7 (IRF-3/7) in protein levels, two critical transcription factors for activation of IFN-α/β. Furthermore, overexpression of L pro significantly reduced the transcription of multiple IRF-responsive genes including 2',5'-OAS, ISG54, IP-10, and RANTES. Screening L pro mutants indicated that the ability to process eIF-4G of L pro is not required for suppressing dsRNA-induced activation of the IFN-α1/β promoter and decreasing IRF-3/7 expression. Taken together, our results demonstrate that, in addition to disrupting NF-κB, L pro also decreases IRF-3/7 expression to suppress dsRNA-induced type I IFN production, suggesting multiple strategies used by FMDV to counteract the immune response to viral infection.

  18. Foot-and-mouth disease virus leader proteinase inhibits dsRNA-induced type I interferon transcription by decreasing interferon regulatory factor 3/7 in protein levels

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Dang; Fang, Liurong; Luo, Rui; Ye, Rui; Fang, Ying; Xie, Lilan; Chen, Huanchun [Division of Animal Infectious Diseases, State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070 (China); Xiao, Shaobo, E-mail: shaoboxiao@yahoo.com [Division of Animal Infectious Diseases, State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070 (China)

    2010-08-13

    Research highlights: {yields} FMDV L{sup pro} inhibits poly(I:C)-induced IFN-{alpha}1/{beta} mRNA expression. {yields} L{sup pro} inhibits MDA5-mediated activation of the IFN-{alpha}1/{beta} promoter. {yields} L{sup pro} significantly reduced the transcription of multiple IRF-responsive genes. {yields} L{sup pro} inhibits IFN-{alpha}1/{beta} promoter activation by decreasing IRF-3/7 in protein levels. {yields} The ability to process eIF-4G of L{sup pro} is not necessary to inhibit IFN-{alpha}1/{beta} activation. -- Abstract: The leader proteinase (L{sup pro}) of foot-and-mouth disease virus (FMDV) has been identified as an interferon-{beta} (IFN-{beta}) antagonist that disrupts the integrity of transcription factor nuclear factor {kappa}B (NF-{kappa}B). In this study, we showed that the reduction of double stranded RNA (dsRNA)-induced IFN-{alpha}1/{beta} expression caused by L{sup pro} was also associated with a decrease of interferon regulatory factor 3/7 (IRF-3/7) in protein levels, two critical transcription factors for activation of IFN-{alpha}/{beta}. Furthermore, overexpression of L{sup pro} significantly reduced the transcription of multiple IRF-responsive genes including 2',5'-OAS, ISG54, IP-10, and RANTES. Screening L{sup pro} mutants indicated that the ability to process eIF-4G of L{sup pro} is not required for suppressing dsRNA-induced activation of the IFN-{alpha}1/{beta} promoter and decreasing IRF-3/7 expression. Taken together, our results demonstrate that, in addition to disrupting NF-{kappa}B, L{sup pro} also decreases IRF-3/7 expression to suppress dsRNA-induced type I IFN production, suggesting multiple strategies used by FMDV to counteract the immune response to viral infection.

  19. Impaired PRC2 activity promotes transcriptional instability and favors breast tumorigenesis.

    Science.gov (United States)

    Wassef, Michel; Rodilla, Veronica; Teissandier, Aurélie; Zeitouni, Bruno; Gruel, Nadege; Sadacca, Benjamin; Irondelle, Marie; Charruel, Margaux; Ducos, Bertrand; Michaud, Audrey; Caron, Matthieu; Marangoni, Elisabetta; Chavrier, Philippe; Le Tourneau, Christophe; Kamal, Maud; Pasmant, Eric; Vidaud, Michel; Servant, Nicolas; Reyal, Fabien; Meseure, Dider; Vincent-Salomon, Anne; Fre, Silvia; Margueron, Raphaël

    2015-12-15

    Alterations of chromatin modifiers are frequent in cancer, but their functional consequences often remain unclear. Focusing on the Polycomb protein EZH2 that deposits the H3K27me3 (trimethylation of Lys27 of histone H3) mark, we showed that its high expression in solid tumors is a consequence, not a cause, of tumorigenesis. In mouse and human models, EZH2 is dispensable for prostate cancer development and restrains breast tumorigenesis. High EZH2 expression in tumors results from a tight coupling to proliferation to ensure H3K27me3 homeostasis. However, this process malfunctions in breast cancer. Low EZH2 expression relative to proliferation and mutations in Polycomb genes actually indicate poor prognosis and occur in metastases. We show that while altered EZH2 activity consistently modulates a subset of its target genes, it promotes a wider transcriptional instability. Importantly, transcriptional changes that are consequences of EZH2 loss are predominantly irreversible. Our study provides an unexpected understanding of EZH2's contribution to solid tumors with important therapeutic implications. © 2015 Wassef et al.; Published by Cold Spring Harbor Laboratory Press.

  20. CDK11p58 represses vitamin D receptor-mediated transcriptional activation through promoting its ubiquitin-proteasome degradation

    International Nuclear Information System (INIS)

    Chi, Yayun; Hong, Yi; Zong, Hongliang; Wang, Yanlin; Zou, Weiying; Yang, Junwu; Kong, Xiangfei; Yun, Xiaojing; Gu, Jianxin

    2009-01-01

    Vitamin D receptor (VDR) is a member of the nuclear receptor superfamily and regulates transcription of target genes. In this study, we identified CDK11 p58 as a novel protein involved in the regulation of VDR. CDK11 p58 , a member of the large family of p34cdc2-related kinases, is associated with cell cycle progression, tumorigenesis, and apoptotic signaling. Our study demonstrated that CDK11 p58 interacted with VDR and repressed VDR-dependent transcriptional activation. Furthermore, overexpression of CDK11 p58 decreased the stability of VDR through promoting its ubiquitin-proteasome-mediated degradation. Taken together, these results suggest that CDK11 p58 is involved in the negative regulation of VDR.

  1. The transcription factor ATF3 is upregulated during chondrocyte differentiation and represses cyclin D1 and A gene transcription

    Directory of Open Access Journals (Sweden)

    James Claudine G

    2006-09-01

    Full Text Available Abstract Background Coordinated chondrocyte proliferation and differentiation are required for normal endochondral bone growth. Transcription factors binding to the cyclicAMP response element (CRE are known to regulate these processes. One member of this family, Activating Tanscription Factor 3 (ATF3, is expressed during skeletogenesis and acts as a transcriptional repressor, but the function of this protein in chondrogenesis is unknown. Results Here we demonstrate that Atf3 mRNA levels increase during mouse chondrocyte differentiation in vitro and in vivo. In addition, Atf3 mRNA levels are increased in response to cytochalasin D treatment, an inducer of chondrocyte maturation. This is accompanied by increased Atf3 promoter activity in cytochalasin D-treated chondrocytes. We had shown earlier that transcription of the cell cycle genes cyclin D1 and cyclin A in chondrocytes is dependent on CREs. Here we demonstrate that overexpression of ATF3 in primary mouse chondrocytes results in reduced transcription of both genes, as well as decreased activity of a CRE reporter plasmid. Repression of cyclin A transcription by ATF3 required the CRE in the cyclin A promoter. In parallel, ATF3 overexpression reduces the activity of a SOX9-dependent promoter and increases the activity of a RUNX2-dependent promoter. Conclusion Our data suggest that transcriptional induction of the Atf3 gene in maturing chondrocytes results in down-regulation of cyclin D1 and cyclin A expression as well as activation of RUNX2-dependent transcription. Therefore, ATF3 induction appears to facilitate cell cycle exit and terminal differentiation of chondrocytes.

  2. AguR, a Transmembrane Transcription Activator of the Putrescine Biosynthesis Operon in Lactococcus lactis, Acts in Response to the Agmatine Concentration.

    Science.gov (United States)

    Linares, Daniel M; Del Rio, Beatriz; Redruello, Begoña; Ladero, Victor; Martin, M Cruz; de Jong, Anne; Kuipers, Oscar P; Fernandez, Maria; Alvarez, Miguel A

    2015-09-01

    Dairy industry fermentative processes mostly use Lactococcus lactis as a starter. However, some dairy L. lactis strains produce putrescine, a biogenic amine that raises food safety and spoilage concerns, via the agmatine deiminase (AGDI) pathway. The enzymatic activities responsible for putrescine biosynthesis in this bacterium are encoded by the AGDI gene cluster. The role of the catabolic genes aguB, aguD, aguA, and aguC has been studied, but knowledge regarding the role of aguR (the first gene in the cluster) remains limited. In the present work, aguR was found to be a very low level constitutively expressed gene that is essential for putrescine biosynthesis and is transcribed independently of the polycistronic mRNA encoding the catabolic genes (aguBDAC). In response to agmatine, AguR acts as a transcriptional activator of the aguB promoter (PaguB), which drives the transcription of the aguBDAC operon. Inverted sequences required for PaguB activity were identified by deletion analysis. Further work indicated that AguR is a transmembrane protein which might function as a one-component signal transduction system that senses the agmatine concentration of the medium and, accordingly, regulates the transcription of the aguBDAC operon through a C-terminal cytoplasmic DNA-binding domain typically found in LuxR-like proteins. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  3. Induction of the unfolded protein response by cigarette smoke is primarily an activating transcription factor 4-C/EBP homologous protein mediated process

    Directory of Open Access Journals (Sweden)

    Geraghty P

    2011-06-01

    Full Text Available Patrick Geraghty, Alison Wallace, Jeanine M D'ArmientoDepartment of Medicine, Divisions of Molecular and Pulmonary Medicine, Columbia University College of Physicians and Surgeons, New York, NY, USAPurpose: Cigarette smoke is the major risk factor associated with the development of chronic obstructive pulmonary disease (COPD. Recent studies propose a link between endoplasmic reticulum (ER stress and emphysema, demonstrated by increased ER stress markers under smoking conditions. Here, we investigate whether cigarette smoke-induced ER stress is cell specific and correlates with acute and chronic cigarette smoke exposure.Methods: Gene and protein expression changes in human primary lung cell cultures following cigarette smoke extract (CSE exposure were monitored by qPCR and Western blot analysis. Mice and guinea pigs were exposed to cigarette smoke and ER stress markers examined in whole lung homogenates. Inflammatory cells from the bronchoalveolar lavage fluid of 10 days smoke exposed mice were also examined.Results: Cigarette smoke induced a trend increase in the ER stress response through an activating transcription factor 4 (ATF4 mediated induction of C/EBP homologous protein (CHOP in primary small airway epithelial cells. Bronchial epithelial cells and macrophages responded similarly to CSE. Wild-type mice and guinea pigs exposed to acute levels of cigarette smoke exhibited increased levels of CHOP but not at significant levels. However, after long-term chronic cigarette smoke exposure, CHOP expression was reduced. Interestingly, inflammatory cells from smoke exposed mice had a significant increase in CHOP/ATF4 expression.Conclusion: A trend increase in CHOP levels appear in multiple human lung cell types following acute cigarette smoke exposure in vitro. In vivo, inflammatory cells, predominately macrophages, demonstrate significant cigarette smoke-induced ER stress. Early induction of CHOP in cigarette smoke may play a pivotal role in early

  4. Pharmacological targeting of the transcription factor SOX18 delays breast cancer in mice

    Science.gov (United States)

    Overman, Jeroen; Fontaine, Frank; Moustaqil, Mehdi; Mittal, Deepak; Sierecki, Emma; Sacilotto, Natalia; Zuegg, Johannes; Robertson, Avril AB; Holmes, Kelly; Salim, Angela A; Mamidyala, Sreeman; Butler, Mark S; Robinson, Ashley S; Lesieur, Emmanuelle; Johnston, Wayne; Alexandrov, Kirill; Black, Brian L; Hogan, Benjamin M; De Val, Sarah; Capon, Robert J; Carroll, Jason S; Bailey, Timothy L; Koopman, Peter; Jauch, Ralf; Smyth, Mark J; Cooper, Matthew A; Gambin, Yann; Francois, Mathias

    2017-01-01

    Pharmacological targeting of transcription factors holds great promise for the development of new therapeutics, but strategies based on blockade of DNA binding, nuclear shuttling, or individual protein partner recruitment have yielded limited success to date. Transcription factors typically engage in complex interaction networks, likely masking the effects of specifically inhibiting single protein-protein interactions. Here, we used a combination of genomic, proteomic and biophysical methods to discover a suite of protein-protein interactions involving the SOX18 transcription factor, a known regulator of vascular development and disease. We describe a small-molecule that is able to disrupt a discrete subset of SOX18-dependent interactions. This compound selectively suppressed SOX18 transcriptional outputs in vitro and interfered with vascular development in zebrafish larvae. In a mouse pre-clinical model of breast cancer, treatment with this inhibitor significantly improved survival by reducing tumour vascular density and metastatic spread. Our studies validate an interactome-based molecular strategy to interfere with transcription factor activity, for the development of novel disease therapeutics. DOI: http://dx.doi.org/10.7554/eLife.21221.001 PMID:28137359

  5. Transcriptionally active LTR retrotransposons in Eucalyptus genus are differentially expressed and insertionally polymorphic.

    Science.gov (United States)

    Marcon, Helena Sanches; Domingues, Douglas Silva; Silva, Juliana Costa; Borges, Rafael Junqueira; Matioli, Fábio Filippi; Fontes, Marcos Roberto de Mattos; Marino, Celso Luis

    2015-08-14

    In Eucalyptus genus, studies on genome composition and transposable elements (TEs) are particularly scarce. Nearly half of the recently released Eucalyptus grandis genome is composed by retrotransposons and this data provides an important opportunity to understand TE dynamics in Eucalyptus genome and transcriptome. We characterized nine families of transcriptionally active LTR retrotransposons from Copia and Gypsy superfamilies in Eucalyptus grandis genome and we depicted genomic distribution and copy number in two Eucalyptus species. We also evaluated genomic polymorphism and transcriptional profile in three organs of five Eucalyptus species. We observed contrasting genomic and transcriptional behavior in the same family among different species. RLC_egMax_1 was the most prevalent family and RLC_egAngela_1 was the family with the lowest copy number. Most families of both superfamilies have their insertions occurring Eucalyptus species. Using EST analysis and qRT-PCRs, we observed transcriptional activity in several tissues and in all evaluated species. In some families, osmotic stress increases transcript values. Our strategy was successful in isolating transcriptionally active retrotransposons in Eucalyptus, and each family has a particular genomic and transcriptional pattern. Overall, our results show that retrotransposon activity have differentially affected genome and transcriptome among Eucalyptus species.

  6. Alterations in transcription factor binding in radioresistant human melanoma cells after ionizing radiation

    International Nuclear Information System (INIS)

    Sahijdak, W.M.; Yang, Chin-Rang; Zuckerman, J.S.; Meyers, M.; Boothman, D.A.

    1994-01-01

    We analyzed alterations in transcription factor binding to specific, known promoter DNA consensus sequences between irradiated and unirradiated radioresistant human melanoma (U1-Mel) cells. The goal of this study was to begin to investigate which transcription factors and DNA-binding sites are responsible for the induction of specific transcripts and proteins after ionizing radiation. Transcription factor binding was observed using DNA band-shift assays and oligonucleotide competition analyses. Confluence-arrested U1-Mel cells were irradiated (4.5 Gy) and harvested at 4 h. Double-stranded oligonucleotides containing known DNA-binding consensus sites for specific transcription factors were used. Increased DNA binding activity after ionizing radiation was noted with oligonucleotides containing the CREB, NF-kB and Sp1 consensus sites. No changes in protein binding to AP-1, AP-2, AP-3, or CTF/NF1, GRE or Oct-1 consensus sequences were noted. X-ray activation of select transcription factors, which bind certain consensus sites in promoters, may cause specific induction or repression of gene transcription. 22 refs., 2 figs

  7. Functional and transcriptomic analysis of the key unfolded protein response transcription factor HacA in Aspergillus oryzae.

    Science.gov (United States)

    Zhou, Bin; Xie, Jingyi; Liu, Xiaokai; Wang, Bin; Pan, Li

    2016-11-15

    HacA is a conserved basic leucine zipper transcription factor that serves as the master transcriptional regulator in the unfolded protein response (UPR). To comprehensively evaluate the role of HacA in Aspergillus oryzae, a homokaryotic hacA disruption mutant (HacA-DE) and a strain that expressed a constitutively active form of HacA (HacA-CA) were successfully generated, and transcriptome analyses of these mutants were performed. Growth and phenotypic profiles demonstrated that hyphal growth and sporulation were impaired in the HacA-DE and HacA-CA strains that were grown on complete and minimal media, and the growth impairment was more pronounced for the HacA-CA strain. Compared with a wild-type (WT) strain, the transcriptome results indicated that differentially expressed genes in these mutants mainly fell into four categories: the protein secretory pathway, amino acid metabolism, lipid metabolism, and carbohydrate metabolism. Furthermore, we identified 80 and 36 genes of the secretory pathway whose expression significantly differed in the HacA-CA strain (compared with the WT and HacA-DE strains) and HacA-DE strain (compared with the WT strain), respectively, which mostly belonged to protein folding/UPR, glycosylation, and vesicle transport processes. Both the HacA-CA and HacA-DE strains exhibited reduced expression of extracellular enzymes, especially amylolytic enzymes, which resulted from the activation of the repression under secretion stress mechanism in response to endoplasmic reticulum stress. Collectively, our results suggest that the function of HacA is important not only for UPR induction, but also for growth and fungal physiology, as it serves to reduce secretion stress in A. oryzae. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Endoplasmic reticulum stress-responsive transcription factor ATF6α directs recruitment of the Mediator of RNA polymerase II transcription and multiple histone acetyltransferase complexes.

    Science.gov (United States)

    Sela, Dotan; Chen, Lu; Martin-Brown, Skylar; Washburn, Michael P; Florens, Laurence; Conaway, Joan Weliky; Conaway, Ronald C

    2012-06-29

    The basic leucine zipper transcription factor ATF6α functions as a master regulator of endoplasmic reticulum (ER) stress response genes. Previous studies have established that, in response to ER stress, ATF6α translocates to the nucleus and activates transcription of ER stress response genes upon binding sequence specifically to ER stress response enhancer elements in their promoters. In this study, we investigate the biochemical mechanism by which ATF6α activates transcription. By exploiting a combination of biochemical and multidimensional protein identification technology-based mass spectrometry approaches, we have obtained evidence that ATF6α functions at least in part by recruiting to the ER stress response enhancer elements of ER stress response genes a collection of RNA polymerase II coregulatory complexes, including the Mediator and multiple histone acetyltransferase complexes, among which are the Spt-Ada-Gcn5 acetyltransferase (SAGA) and Ada-Two-A-containing (ATAC) complexes. Our findings shed new light on the mechanism of action of ATF6α, and they outline a straightforward strategy for applying multidimensional protein identification technology mass spectrometry to determine which RNA polymerase II transcription factors and coregulators are recruited to promoters and other regulatory elements to control transcription.

  9. Cdk phosphorylation of the Ste11 transcription factor constrains differentiation-specific transcription to G1

    DEFF Research Database (Denmark)

    Kjaerulff, Søren; Andersen, Nicoline Resen; Borup, Mia Trolle

    2007-01-01

    Eukaryotic cells normally differentiate from G(1); here we investigate the mechanism preventing expression of differentiation-specific genes outside G(1). In fission yeast, induction of the transcription factor Ste11 triggers sexual differentiation. We find that Ste11 is only active in G(1) when...... Cdk activity is low. In the remaining part of the cell cycle, Ste11 becomes Cdk-phosphorylated at Thr 82 (T82), which inhibits its DNA-binding activity. Since the ste11 gene is autoregulated and the Ste11 protein is highly unstable, this Cdk switch rapidly extinguishes Ste11 activity when cells enter...... S phase. When we mutated T82 to aspartic acid, mimicking constant phosphorylation, cells no longer underwent differentiation. Conversely, changing T82 to alanine rendered Ste11-controlled transcription constitutive through the cell cycle, and allowed mating from S phase with increased frequency...

  10. Mechanical stress activates Smad pathway through PKCδ to enhance interleukin-11 gene transcription in osteoblasts.

    Directory of Open Access Journals (Sweden)

    Shinsuke Kido

    Full Text Available BACKGROUND: Mechanical stress rapidly induces ΔFosB expression in osteoblasts, which binds to interleukin (IL-11 gene promoter to enhance IL-11 expression, and IL-11 enhances osteoblast differentiation. Because bone morphogenetic proteins (BMPs also stimulate IL-11 expression in osteoblasts, there is a possibility that BMP-Smad signaling is involved in the enhancement of osteoblast differentiation by mechanical stress. The present study was undertaken to clarify whether mechanical stress affects BMP-Smad signaling, and if so, to elucidate the role of Smad signaling in mechanical stress-induced enhancement of IL-11 gene transcription. METHODOLOGY/PRINCIPAL FINDINGS: Mechanical loading by fluid shear stress (FSS induced phosphorylation of BMP-specific receptor-regulated Smads (BR-Smads, Smad1/5, in murine primary osteoblasts (mPOBs. FSS rapidly phosphorylated Y311 of protein kinase C (PKCδ, and phosphorylated PKCδ interacted with BR-Smads to phosphorylate BR-Smads. Transfection of PKCδ siRNA or Y311F mutant PKCδ abrogated BR-Smads phosphorylation and suppressed IL-11 gene transcription enhanced by FSS. Activated BR-Smads bound to the Smad-binding element (SBE of IL-11 gene promoter and formed complex with ΔFosB/JunD heterodimer via binding to the C-terminal region of JunD. Site-directed mutagenesis in the SBE and the AP-1 site revealed that both SBE and AP-1 sites were required for full activation of IL-11 gene promoter by FSS. CONCLUSIONS/SIGNIFICANCE: These results demonstrate that PKCδ-BR-Smads pathway plays an important role in the intracellular signaling in response to mechanical stress, and that a cross-talk between PKCδ-BR-Smads and ΔFosB/JunD pathways synergistically stimulates IL-11 gene transcription in response to mechanical stress.

  11. Evolutionary Analysis of DELLA-Associated Transcriptional Networks

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    Miguel A. Blázquez

    2017-04-01

    Full Text Available DELLA proteins are transcriptional regulators present in all land plants which have been shown to modulate the activity of over 100 transcription factors in Arabidopsis, involved in multiple physiological and developmental processes. It has been proposed that DELLAs transduce environmental information to pre-wired transcriptional circuits because their stability is regulated by gibberellins (GAs, whose homeostasis largely depends on environmental signals. The ability of GAs to promote DELLA degradation coincides with the origin of vascular plants, but the presence of DELLAs in other land plants poses at least two questions: what regulatory properties have DELLAs provided to the behavior of transcriptional networks in land plants, and how has the recruitment of DELLAs by GA signaling affected this regulation. To address these issues, we have constructed gene co-expression networks of four different organisms within the green lineage with different properties regarding DELLAs: Arabidopsis thaliana and Solanum lycopersicum (both with GA-regulated DELLA proteins, Physcomitrella patens (with GA-independent DELLA proteins and Chlamydomonas reinhardtii (a green alga without DELLA, and we have examined the relative evolution of the subnetworks containing the potential DELLA-dependent transcriptomes. Network analysis indicates a relative increase in parameters associated with the degree of interconnectivity in the DELLA-associated subnetworks of land plants, with a stronger effect in species with GA-regulated DELLA proteins. These results suggest that DELLAs may have played a role in the coordination of multiple transcriptional programs along evolution, and the function of DELLAs as regulatory ‘hubs’ became further consolidated after their recruitment by GA signaling in higher plants.

  12. A Dual Luciferase Reporter System for B. burgdorferi Measures Transcriptional Activity during Tick-Pathogen Interactions

    Directory of Open Access Journals (Sweden)

    Philip P. Adams

    2017-05-01

    Full Text Available Knowledge of the transcriptional responses of vector-borne pathogens at the vector-pathogen interface is critical for understanding disease transmission. Borrelia (Borreliella burgdorferi, the causative agent of Lyme disease in the United States, is transmitted by the bite of infected Ixodes sp. ticks. It is known that B. burgdorferi has altered patterns of gene expression during tick acquisition, persistence and transmission. Recently, we and others have discovered in vitro expression of RNAs found internal, overlapping, and antisense to annotated open reading frames in the B. burgdorferi genome. However, there is a lack of molecular genetic tools for B. burgdorferi for quantitative, strand-specific, comparative analysis of these transcripts in distinct environments such as the arthropod vector. To address this need, we have developed a dual luciferase reporter system to quantify B. burgdorferi promoter activities in a strand-specific manner. We demonstrate that constitutive expression of a B. burgdorferi codon-optimized Renilla reniformis luciferase gene (rlucBb allows normalization of the activity of a promoter of interest when fused to the B. burgdorferi codon-optimized Photinus pyralis luciferase gene (flucBb on the same plasmid. Using the well characterized, differentially regulated, promoters for flagellin (flaBp, outer surface protein A (ospAp and outer surface protein C (ospCp, we document the efficacy of the dual luciferase system for quantitation of promoter activities during in vitro growth and in infected ticks. Cumulatively, the dual luciferase method outlined herein is the first dual reporter system for B. burgdorferi, providing a novel and highly versatile approach for strand-specific molecular genetic analyses.

  13. Negative transcriptional regulation of mitochondrial transcription factor A (TFAM) by nuclear TFAM

    International Nuclear Information System (INIS)

    Lee, Eun Jin; Kang, Young Cheol; Park, Wook-Ha; Jeong, Jae Hoon; Pak, Youngmi Kim

    2014-01-01

    Highlights: • TFAM localizes in nuclei and mitochondria of neuronal cells. • Nuclear TFAM does not bind the Tfam promoter. • Nuclear TFAM reduced the Tfam promoter activity via suppressing NRF-1 activity. • A novel self-negative feedback regulation of Tfam gene expression is explored. • FAM may play different roles depending on its subcellular localizations. - Abstract: The nuclear DNA-encoded mitochondrial transcription factor A (TFAM) is synthesized in cytoplasm and transported into mitochondria. TFAM enhances both transcription and replication of mitochondrial DNA. It is unclear, however, whether TFAM plays a role in regulating nuclear gene expression. Here, we demonstrated that TFAM was localized to the nucleus and mitochondria by immunostaining, subcellular fractionation, and TFAM-green fluorescent protein hybrid protein studies. In HT22 hippocampal neuronal cells, human TFAM (hTFAM) overexpression suppressed human Tfam promoter-mediated luciferase activity in a dose-dependent manner. The mitochondria targeting sequence-deficient hTFAM also repressed Tfam promoter activity to the same degree as hTFAM. It indicated that nuclear hTFAM suppressed Tfam expression without modulating mitochondrial activity. The repression required for nuclear respiratory factor-1 (NRF-1), but hTFAM did not bind to the NRF-1 binding site of its promoter. TFAM was co-immunoprecipitated with NRF-1. Taken together, we suggest that nuclear TFAM down-regulate its own gene expression as a NRF-1 repressor, showing that TFAM may play different roles depending on its subcellular localizations

  14. Transcriptional robustness and protein interactions are associated in yeast

    Directory of Open Access Journals (Sweden)

    Conant Gavin C

    2011-05-01

    Full Text Available Abstract Background Robustness to insults, both external and internal, is a characteristic feature of life. One level of biological organization for which noise and robustness have been extensively studied is gene expression. Cells have a variety of mechanisms for buffering noise in gene expression, but it is not completely clear what rules govern whether or not a given gene uses such tools to maintain appropriate expression. Results Here, we show a general association between the degree to which yeast cells have evolved mechanisms to buffer changes in gene expression and whether they possess protein-protein interactions. We argue that this effect bears an affinity to epistasis, because yeast appears to have evolved regulatory mechanisms such that distant changes in gene copy number for a protein-protein interaction partner gene can alter a gene's expression. This association is not unexpected given recent work linking epistasis and the deleterious effects of changes in gene dosage (i.e., the dosage balance hypothesis. Using gene expression data from artificial aneuploid strains of bakers' yeast, we found that genes coding for proteins that physically interact with other proteins show less expression variation in response to aneuploidy than do other genes. This effect is even more pronounced for genes whose products interact with proteins encoded on aneuploid chromosomes. We further found that genes targeted by transcription factors encoded on aneuploid chromosomes were more likely to change in expression after aneuploidy. Conclusions We suggest that these observations can be best understood as resulting from the higher fitness cost of misexpression in epistatic genes and a commensurate greater regulatory control of them.

  15. The equine herpesvirus-1 IR3 gene that lies antisense to the sole immediate-early (IE) gene is trans-activated by the IE protein, and is poorly expressed to a protein

    International Nuclear Information System (INIS)

    Ahn, Byung Chul; Breitenbach, Jonathan E.; Kim, Seong K.; O'Callaghan, Dennis J.

    2007-01-01

    The unique IR3 gene of equine herpesvirus 1 (EHV-1) is expressed as a late 1.0-kb transcript. Previous studies confirmed the IR3 transcription initiation site and tentatively identified other cis-acting elements specific to IR3 such as a TATA box, a 443 base pair 5'untranslated region (UTR), a 285 base pair open reading frame (ORF), and a poly adenylation (A) signal [Holden, V.R., Harty, R.N., Yalamanchili, R.R., O'Callaghan, D.J., 1992. The IR3 gene of equine herpesvirus type 1: a unique gene regulated by sequences within the intron of the immediate-early gene. DNA Seq. 3, 143-152]. Transient transfection assays revealed that the IR3 promoter is strongly trans-activated by the IE protein (IEP) and that coexpression of the IEP with the early EICP0 and IR4 regulatory proteins results in maximal trans-activation of the IR3 promoter. Gel shift assays revealed that the IEP directly binds to the IR3 promoter region. Western blot analysis showed that the IR3 protein produced in E. coli was detected by antibodies to IR3 synthetic peptides; however, the IR3 protein was not detected in EHV-1 infected cell extracts by these same anti-IR3 antibodies, even though the IR3 transcript was detected by northern blot. These findings suggest that the IR3 may not be expressed to a protein. Expression of an IR3/GFP fusion gene was not observed, but expression of a GFP/IR3 fusion gene was detected by fluorescent microscopy. In further attempts to detect the IR3/GFP fusion protein using anti-GFP antibody, western blot analysis showed that the IR3/GFP fusion protein was not detected in vivo. Interestingly, a truncated form of the GFP/IR3 protein was synthesized from the GFP/IR3 fusion gene. However, GFP/IR3 and IR3/GFP fusion proteins of the predicted sizes were synthesized by in vitro coupled transcription and translation of the fusion genes, suggesting poor expression of the IR3 protein in vivo. The possible role of the IR3 transcript in EHV-1 infection is discussed

  16. Activator Protein-1: redox switch controlling structure and DNA-binding.

    Science.gov (United States)

    Yin, Zhou; Machius, Mischa; Nestler, Eric J; Rudenko, Gabby

    2017-11-02

    The transcription factor, activator protein-1 (AP-1), binds to cognate DNA under redox control; yet, the underlying mechanism has remained enigmatic. A series of crystal structures of the AP-1 FosB/JunD bZIP domains reveal ordered DNA-binding regions in both FosB and JunD even in absence DNA. However, while JunD is competent to bind DNA, the FosB bZIP domain must undergo a large conformational rearrangement that is controlled by a 'redox switch' centered on an inter-molecular disulfide bond. Solution studies confirm that FosB/JunD cannot undergo structural transition and bind DNA when the redox-switch is in the 'OFF' state, and show that the mid-point redox potential of the redox switch affords it sensitivity to cellular redox homeostasis. The molecular and structural studies presented here thus reveal the mechanism underlying redox-regulation of AP-1 Fos/Jun transcription factors and provide structural insight for therapeutic interventions targeting AP-1 proteins. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  17. Activation of the Arabidopsis membrane-bound transcription factor bZIP28 is mediated by site-2 protease, but not site-1 protease.

    Science.gov (United States)

    Iwata, Yuji; Ashida, Makoto; Hasegawa, Chisa; Tabara, Kazuki; Mishiba, Kei-Ichiro; Koizumi, Nozomu

    2017-08-01

    The unfolded protein response (UPR) is a homeostatic cellular response conserved in eukaryotic cells to alleviate the accumulation of unfolded proteins in the endoplasmic reticulum (ER). Arabidopsis bZIP28 is a membrane-bound transcription factor activated by proteolytic cleavage in response to ER stress, thereby releasing its cytosolic portion containing the bZIP domain from the membrane to translocate into the nucleus where it induces the transcription of genes encoding ER-resident molecular chaperones and folding enzymes. It has been widely recognized that the proteolytic activation of bZIP28 is mediated by the sequential cleavage of site-1 protease (S1P) and site-2 protease (S2P). In the present study we provide evidence that bZIP28 protein is cleaved by S2P, but not by S1P. We demonstrated that wild-type and s1p mutant plants produce the active, nuclear form of bZIP28 in response to the ER stress inducer tunicamycin. In contrast, tunicamycin-treated s2p mutants do not accumulate the active, nuclear form of bZIP28. Consistent with these observations, s2p mutants, but not s1p mutants, exhibited a defective transcriptional response of ER stress-responsive genes and significantly higher sensitivity to tunicamycin. Interestingly, s2p mutants accumulate two membrane-bound bZIP28 fragments with a shorter ER lumen-facing C-terminal domain. Importantly, the predicted cleavage sites are located far from the canonical S1P recognition motif previously described. We propose that ER stress-induced proteolytic activation of bZIP28 is mediated by the sequential actions of as-yet-unidentified protease(s) and S2P, and does not require S1P. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  18. Drosophila TDP-43 RNA-Binding Protein Facilitates Association of Sister Chromatid Cohesion Proteins with Genes, Enhancers and Polycomb Response Elements.

    Directory of Open Access Journals (Sweden)

    Amanda Swain

    2016-09-01

    Full Text Available The cohesin protein complex mediates sister chromatid cohesion and participates in transcriptional control of genes that regulate growth and development. Substantial reduction of cohesin activity alters transcription of many genes without disrupting chromosome segregation. Drosophila Nipped-B protein loads cohesin onto chromosomes, and together Nipped-B and cohesin occupy essentially all active transcriptional enhancers and a large fraction of active genes. It is unknown why some active genes bind high levels of cohesin and some do not. Here we show that the TBPH and Lark RNA-binding proteins influence association of Nipped-B and cohesin with genes and gene regulatory sequences. In vitro, TBPH and Lark proteins specifically bind RNAs produced by genes occupied by Nipped-B and cohesin. By genomic chromatin immunoprecipitation these RNA-binding proteins also bind to chromosomes at cohesin-binding genes, enhancers, and Polycomb response elements (PREs. RNAi depletion reveals that TBPH facilitates association of Nipped-B and cohesin with genes and regulatory sequences. Lark reduces binding of Nipped-B and cohesin at many promoters and aids their association with several large enhancers. Conversely, Nipped-B facilitates TBPH and Lark association with genes and regulatory sequences, and interacts with TBPH and Lark in affinity chromatography and immunoprecipitation experiments. Blocking transcription does not ablate binding of Nipped-B and the RNA-binding proteins to chromosomes, indicating transcription is not required to maintain binding once established. These findings demonstrate that RNA-binding proteins help govern association of sister chromatid cohesion proteins with genes and enhancers.

  19. Tuning of Recombinant Protein Expression in Escherichia coli by Manipulating Transcription, Translation Initiation Rates, and Incorporation of Noncanonical Amino Acids.

    Science.gov (United States)

    Schlesinger, Orr; Chemla, Yonatan; Heltberg, Mathias; Ozer, Eden; Marshall, Ryan; Noireaux, Vincent; Jensen, Mogens Høgh; Alfonta, Lital

    2017-06-16

    Protein synthesis in cells has been thoroughly investigated and characterized over the past 60 years. However, some fundamental issues remain unresolved, including the reasons for genetic code redundancy and codon bias. In this study, we changed the kinetics of the Eschrichia coli transcription and translation processes by mutating the promoter and ribosome binding domains and by using genetic code expansion. The results expose a counterintuitive phenomenon, whereby an increase in the initiation rates of transcription and translation lead to a decrease in protein expression. This effect can be rescued by introducing slow translating codons into the beginning of the gene, by shortening gene length or by reducing initiation rates. On the basis of the results, we developed a biophysical model, which suggests that the density of co-transcriptional-translation plays a role in bacterial protein synthesis. These findings indicate how cells use codon bias to tune translation speed and protein synthesis.

  20. Transcription factor activating protein 2 beta (TFAP2B) mediates noradrenergic neuronal differentiation in neuroblastoma.

    Science.gov (United States)

    Ikram, Fakhera; Ackermann, Sandra; Kahlert, Yvonne; Volland, Ruth; Roels, Frederik; Engesser, Anne; Hertwig, Falk; Kocak, Hayriye; Hero, Barbara; Dreidax, Daniel; Henrich, Kai-Oliver; Berthold, Frank; Nürnberg, Peter; Westermann, Frank; Fischer, Matthias

    2016-02-01

    Neuroblastoma is an embryonal pediatric tumor that originates from the developing sympathetic nervous system and shows a broad range of clinical behavior, ranging from fatal progression to differentiation into benign ganglioneuroma. In experimental neuroblastoma systems, retinoic acid (RA) effectively induces neuronal differentiation, and RA treatment has been therefore integrated in current therapies. However, the molecular mechanisms underlying differentiation are still poorly understood. We here investigated the role of transcription factor activating protein 2 beta (TFAP2B), a key factor in sympathetic nervous system development, in neuroblastoma pathogenesis and differentiation. Microarray analyses of primary neuroblastomas (n = 649) demonstrated that low TFAP2B expression was significantly associated with unfavorable prognostic markers as well as adverse patient outcome. We also found that low TFAP2B expression was strongly associated with CpG methylation of the TFAP2B locus in primary neuroblastomas (n = 105) and demethylation with 5-aza-2'-deoxycytidine resulted in induction of TFAP2B expression in vitro, suggesting that TFAP2B is silenced by genomic methylation. Tetracycline inducible re-expression of TFAP2B in IMR-32 and SH-EP neuroblastoma cells significantly impaired proliferation and cell cycle progression. In IMR-32 cells, TFAP2B induced neuronal differentiation, which was accompanied by up-regulation of the catecholamine biosynthesizing enzyme genes DBH and TH, and down-regulation of MYCN and REST, a master repressor of neuronal genes. By contrast, knockdown of TFAP2B by lentiviral transduction of shRNAs abrogated RA-induced neuronal differentiation of SH-SY5Y and SK-N-BE(2)c neuroblastoma cells almost completely. Taken together, our results suggest that TFAP2B is playing a vital role in retaining RA responsiveness and mediating noradrenergic neuronal differentiation in neuroblastoma. Copyright © 2015 Federation of European Biochemical Societies