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Sample records for conditionally activated transcription

  1. Induction of latent memory for conditioned food aversion and its transformation into "active" state depend on translation and transcription processes.

    Science.gov (United States)

    Solntseva, S V; Nikitin, V P

    2014-05-01

    Mechanisms of induction and retrieval of latent (hidden) memory for conditioned food aversion were investigated in snails. After initial training (single combination of a food stimulus with electric shock), aversive reactions to presentation of the conditioned food stimulus were not revealed. Repeated presentation of the stimuli in 12 days after the first combination was followed by the appearance of aversive food reactions that persisted for at least 14 days. Injections of inhibitors of protein (cycloheximide) or RNA (α-amanitin) synthesis immediately after the first or second combined presentation of the stimuli disturbed skill performance. We hypothesized that single combination of food and reinforcing stimuli led to translation- and transcription-dependent induction of latent conditioned food aversion memory. Transformation of this memory into an active state after repeated presentation of the stimulus combination also depends on the synthesis of new proteins and RNA.

  2. 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.

  3. Transcriptional and metabolic regulation of denitrification in Paracoccus denitrificans allows low but significant activity of nitrous oxide reductase under oxic conditions.

    Science.gov (United States)

    Qu, Zhi; Bakken, Lars R; Molstad, Lars; Frostegård, Åsa; Bergaust, Linda L

    2016-09-01

    Oxygen is known to repress denitrification at the transcriptional and metabolic levels. It has been a common notion that nitrous oxide reductase (N2 OR) is the most sensitive enzyme among the four N-oxide reductases involved in denitrification, potentially leading to increased N2 O production under suboxic or fluctuating oxygen conditions. We present detailed gas kinetics and transcription patterns from batch culture experiments with Paracoccus denitrificans, allowing in vivo estimation of e(-) -flow to O2 and N2 O under various O2 regimes. Transcription of nosZ took place concomitantly with that of narG under suboxic conditions, whereas transcription of nirS and norB was inhibited until O2 levels approached 0 μM in the liquid. Catalytically functional N2 OR was synthesized and active in aerobically raised cells transferred to vials with 7 vol% O2 in headspace, but N2 O reduction rates were 10 times higher when anaerobic pre-cultures were subjected to the same conditions. Upon oxygen exposure, there was an incomplete and transient inactivation of N2 OR that could be ascribed to its lower ability to compete for electrons compared with terminal oxidases. The demonstrated reduction of N2 O at high O2 partial pressure and low N2 O concentrations by a bacterium not known as a typical aerobic denitrifier may provide one clue to the understanding of why some soils appear to act as sinks rather than sources for atmospheric N2 O. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

  4. Histone variant H2A.Z antagonizes the positive effect of the transcriptional activator CPC1 to regulate catalase-3 expression under normal and oxidative stress conditions.

    Science.gov (United States)

    Dong, Qing; Wang, Yajun; Qi, Shaohua; Gai, Kexin; He, Qun; Wang, Ying

    2018-05-05

    In eukaryotes, deposition of the histone variant H2A.Z into nucleosomes through the chromatin remodeling complex, SWR1, is a crucial step in modulating gene transcription. Recently, H2A.Z has been shown to control the expression of responsive genes, but the underlying mechanism of how H2A.Z responds to physiological stimuli is not well understood. Here, we reveal that, in Neurospora crassa, H2A.Z is a negative regulator of catalase-3 gene, which is responsible for resistance to oxidative stress. H2A.Z represses cat-3 gene expression through direct incorporation at cat-3 locus in a SWR1 complex dependent pathway. Notably, loss of H2A.Z or SWR1 subunits leads to increased binding of a transcription factor, CPC1, at cat-3 locus. Additionally, introduction of plasmids containing gene encoding H2A.Z or SWR1 complex subunits into wild-type strains decreased CAT-3 expression, indicating that H2A.Z counteracts the positive effect of CPC1 to achieve low level cat-3 expression under non-inductive condition. Furthermore, upon oxidative stress, H2A.Z is rapidly evicted from cat-3 locus for the recruitment of CPC1, resulting in robust and full cat-3 gene expression in response to external stimuli. Collectively, this study strongly demonstrates that H2A.Z antagonizes the function of transcription factor to regulate responsive gene transcription under normal conditions and to poise for gene full activation under oxidative stress. Copyright © 2018. Published by Elsevier Inc.

  5. Allele-specific MMP-3 transcription under in vivo conditions

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    Chaoyong, Zhu [Atherosclerosis Research Unit, King Gustav V Research Institute, Department of Medicine, Karolinska Institute, Stockholm (Sweden); Odeberg, Jacob [Atherosclerosis Research Unit, King Gustav V Research Institute, Department of Medicine, Karolinska Institute, Stockholm (Sweden); Department of Biotechnology, AlbaNova University Center, Royal Institute of Technology, Stockholm (Sweden); Hamsten, Anders [Atherosclerosis Research Unit, King Gustav V Research Institute, Department of Medicine, Karolinska Institute, Stockholm (Sweden); Eriksson, Per [Atherosclerosis Research Unit, King Gustav V Research Institute, Department of Medicine, Karolinska Institute, Stockholm (Sweden)

    2006-09-29

    A common matrix metalloproteinases-3 (MMP-3) -1612 5A/6A promoter polymorphism is associated with risk for cardiovascular disease, rheumatoid arthritis, and other diseases. Here we used the haplotype chromatin immunoprecipitation method to study allele-specific MMP-3 expression under in vivo conditions in heterozygous THP-1 cells. Pyrosequencing was used to analyse the ratio of 5A-allele to 6A-allele after chromatin immunoprecipitation using an antibody against phosphorylated active RNA polymerase II. There was no allele-specific difference in transcriptional activity during basal conditions, i.e., in unstimulated monocytic THP-1 cells. However, after stimulation of MMP-3 expression by monocyte differentiation or incubation with IL-1{beta}, the haplotype containing the 5A-allele was associated with higher transcriptional activity compared with the 6A-containing haplotype. Electromobility shift assay demonstrated increased binding of nuclear proteins to the 5A-allele after monocyte differentiation. In conclusion, the common MMP-3 5A/6A promoter polymorphism appears to be functional only during specific environmental conditions involving inflammation.

  6. 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...

  7. 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.

  8. 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

  9. 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 ...

  10. 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

  11. Effect of growth conditions on expression of the acid phosphatase (cyx-appA) operon and the appY gene, which encodes a transcriptional activator of Escherichia coli

    DEFF Research Database (Denmark)

    Brøndsted, Lone; Atlung, Tove

    1996-01-01

    The expression and transcriptional regulation of the Escherichia coli cyx-appA operon and the appY gene has been investigated during different environmental conditions using single copy transcriptional lacZ fusions. The cyx-appA operon encodes acid phosphatase and a putative cytochrome oxidase...... of the cyx-appA operon. The nitrate repression was partially dependent on NarL. A high expression of the operon was obtained in glucose medium supplemented with formate, where E.coli obtains energy by fermentation. The formate induction was independent of the fhlA gene product. The results presented...... in this paper indicate a clear difference in the regulation of the cyx-appA operon compared to the cyd operon, encoding the cytochrome d oxidase complex. The results suggest that cytochrome x oxidase has a function at even more oxygen limiting conditions than cytochrome d oxidase. The expression of the app...

  12. Global transcriptional regulatory network for Escherichia coli robustly connects gene expression to transcription factor activities

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    Fang, Xin; Sastry, Anand; Mih, Nathan; Kim, Donghyuk; Tan, Justin; Lloyd, Colton J.; Gao, Ye; Yang, Laurence; Palsson, Bernhard O.

    2017-01-01

    Transcriptional regulatory networks (TRNs) have been studied intensely for >25 y. Yet, even for the Escherichia coli TRN—probably the best characterized TRN—several questions remain. Here, we address three questions: (i) How complete is our knowledge of the E. coli TRN; (ii) how well can we predict gene expression using this TRN; and (iii) how robust is our understanding of the TRN? First, we reconstructed a high-confidence TRN (hiTRN) consisting of 147 transcription factors (TFs) regulating 1,538 transcription units (TUs) encoding 1,764 genes. The 3,797 high-confidence regulatory interactions were collected from published, validated chromatin immunoprecipitation (ChIP) data and RegulonDB. For 21 different TF knockouts, up to 63% of the differentially expressed genes in the hiTRN were traced to the knocked-out TF through regulatory cascades. Second, we trained supervised machine learning algorithms to predict the expression of 1,364 TUs given TF activities using 441 samples. The algorithms accurately predicted condition-specific expression for 86% (1,174 of 1,364) of the TUs, while 193 TUs (14%) were predicted better than random TRNs. Third, we identified 10 regulatory modules whose definitions were robust against changes to the TRN or expression compendium. Using surrogate variable analysis, we also identified three unmodeled factors that systematically influenced gene expression. Our computational workflow comprehensively characterizes the predictive capabilities and systems-level functions of an organism’s TRN from disparate data types. PMID:28874552

  13. Cytokinins induce transcriptional reprograming and improve Arabidopsis plant performance under drought and salt stress conditions.

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    Natali Shirron

    2016-10-01

    Full Text Available In nature, annual plants respond to abiotic stresses by activating a specific genetic program leading to early flowering and accelerated senescence. Although, in nature, this phenomenon supports survival under unfavorable environmental conditions, it may have negative agro-economic impacts on crop productivity. Overcoming this genetic programing by cytokinins (CK has recently been shown in transgenic plants that overproduce CK. These transgenic plants displayed a significant increase in plant productivity under drought stress conditions. We investigated the role of CK in reverting the transcriptional program that is activated under abiotic stress conditions and allowing sustainable plant growth. We employed 2 complementary approaches: Ectopic overexpression of CK, and applying exogenous CK to detached Arabidopsis leaves. Transgenic Arabidopsis plants transformed with the isopentyltransferase (IPT gene under the regulation of the senescence associated receptor kinase (SARK promoter displayed a significant drought resistance. A transcriptomic analysis using RNA sequencing was performed to explore the response mechanisms under elevated CK levels during salinity stress. This analysis showed that under such stress, CK triggered transcriptional reprograming that resulted in attenuated stress-dependent inhibition of vegetative growth and delayed premature plant senescence. Our data suggest that elevated CK levels led to stress tolerance by retaining the expression of genes associated with plant growth and metabolism whose expression typically decreases under stress conditions. In conclusion, we hypothesize that CK allows sustainable plant growth under unfavorable environmental conditions by activating gene expression related to growth processes and by preventing the expression of genes related to the activation of premature senescence.

  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.

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    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. From DNA binding to transcriptional activation: Is the TALE complete?

    Science.gov (United States)

    Bobola, Nicoletta

    2017-09-04

    How transcription factors (TFs) control enhancer and promoter functions to effect changes in gene expression is an important question. In this issue, Hau et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201701154) show that the TALE TF MEIS recruits the histone modifier PARP1/ARTD1 at promoters to decompact chromatin and activate transcription. © 2017 Bobola.

  17. 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...

  18. 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....

  19. Dataset of transcriptional landscape of B cell early activation

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    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.

  20. 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.)

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

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    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.

  2. Transcription elongation factor GreA has functional chaperone activity.

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    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.

  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. Fungal mediator tail subunits contain classical transcriptional activation domains.

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    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.

  6. First Exon Length Controls Active Chromatin Signatures and Transcription

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    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.

  7. 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

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

    Directory of Open Access Journals (Sweden)

    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.

  9. Transcriptional profiles of Treponema denticola in response to environmental conditions.

    Directory of Open Access Journals (Sweden)

    Ian McHardy

    Full Text Available The periodontal pathogen T. denticola resides in a stressful environment rife with challenges, the human oral cavity. Knowledge of the stress response capabilities of this invasive spirochete is currently very limited. Whole genome expression profiles in response to different suspected stresses including heat shock, osmotic downshift, oxygen and blood exposure were examined. Most of the genes predicted to encode conserved heat shock proteins (HSPs were found to be induced under heat and oxygen stress. Several of these HSPs also seem to be important for survival in hypotonic solutions and blood. In addition to HSPs, differential regulation of many genes encoding metabolic proteins, hypothetical proteins, transcriptional regulators and transporters was observed in patterns that could betoken functional associations. In summary, stress responses in T. denticola exhibit many similarities to the corresponding stress responses in other organisms but also employ unique components including the induction of hypothetical proteins.

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

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    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

  11. 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

  12. Transcriptional activity of transposable elements in coelacanth.

    Science.gov (United States)

    Forconi, Mariko; Chalopin, Domitille; Barucca, Marco; Biscotti, Maria Assunta; De Moro, Gianluca; Galiana, Delphine; Gerdol, Marco; Pallavicini, Alberto; Canapa, Adriana; Olmo, Ettore; Volff, Jean-Nicolas

    2014-09-01

    The morphological stasis of coelacanths has long suggested a slow evolutionary rate. General genomic stasis might also imply a decrease of transposable elements activity. To evaluate the potential activity of transposable elements (TEs) in "living fossil" species, transcriptomic data of Latimeria chalumnae and its Indonesian congener Latimeria menadoensis were compared through the RNA-sequencing mapping procedures in three different organs (liver, testis, and muscle). The analysis of coelacanth transcriptomes highlights a significant percentage of transcribed TEs in both species. Major contributors are LINE retrotransposons, especially from the CR1 family. Furthermore, some particular elements such as a LF-SINE and a LINE2 sequences seem to be more expressed than other elements. The amount of TEs expressed in testis suggests possible transposition burst in incoming generations. Moreover, significant amount of TEs in liver and muscle transcriptomes were also observed. Analyses of elements displaying marked organ-specific expression gave us the opportunity to highlight exaptation cases, that is, the recruitment of TEs as new cellular genes, but also to identify a new Latimeria-specific family of Short Interspersed Nuclear Elements called CoeG-SINEs. Overall, transcriptome results do not seem to be in line with a slow-evolving genome with poor TE activity. © 2013 Wiley Periodicals, Inc.

  13. Exploring cellular memory molecules marking competent and active transcriptions

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    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.

  14. Transcriptional Profiling of Chromera velia Under Diverse Environmental Conditions

    KAUST Repository

    Tayyrov, Annageldi

    2014-05-01

    Since its description in 2008, Chromera velia has drawn profound interest as the closest free-­‐living photosynthetic relative of apicomplexan parasites that are significant pathogens, causing enormous health and economic problems. There-­‐ fore, this newly described species holds a great potential to understand evolu-­‐ tionary basis of how photosynthetic algae evolved into the fully pathogenic Apicomplexa and how their common ancestors may have lived before they evolved into obligate parasites. Hence, the aim of this work is to understand how C. velia function and respond to different environmental conditions. This study aims to reveal how C. velia is able to respond to environmental perturbations that are applied individually and simultaneously since, studying stress factors in separation fails to elucidate complex responses to multi stress factors and un-­‐ derstanding the systemic regulation of involved genes. To extract biologically significant information and to identify genes involved in various physiological processes under variety of environmental conditions (i.e. a combination of vary-­‐ ing temperatures, iron availability, and salinity in the growth medium) we pre-­‐ pared strand specific RNA-­‐seq libraries for 83 samples in diverse environmental conditions. Here, we report the set of significantly differentially expressed genes as a re-­‐ sponse to the each condition and their combinations. Several interesting up-­‐ regulated and down-­‐regulated genes were found and their functions and in-­‐ volved pathways were studied. We showed that the profound regulation of HSP20 proteins is significant under stress conditions and hypothesized that the-­‐ se proteins might be involved in their movements.

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

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    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.

  16. The transcriptionally active regions in the genome of Bacillus subtilis

    DEFF Research Database (Denmark)

    Rasmussen, Simon; Nielsen, Henrik Bjørn; Jarmer, Hanne Østergaard

    2009-01-01

    The majority of all genes have so far been identified and annotated systematically through in silico gene finding. Here we report the finding of 3662 strand-specific transcriptionally active regions (TARs) in the genome of Bacillus subtilis by the use of tiling arrays. We have measured the genome...

  17. Versatility of cooperative transcriptional activation: a thermodynamical modeling analysis for greater-than-additive and less-than-additive effects.

    Directory of Open Access Journals (Sweden)

    Till D Frank

    Full Text Available We derive a statistical model of transcriptional activation using equilibrium thermodynamics of chemical reactions. We examine to what extent this statistical model predicts synergy effects of cooperative activation of gene expression. We determine parameter domains in which greater-than-additive and less-than-additive effects are predicted for cooperative regulation by two activators. We show that the statistical approach can be used to identify different causes of synergistic greater-than-additive effects: nonlinearities of the thermostatistical transcriptional machinery and three-body interactions between RNA polymerase and two activators. In particular, our model-based analysis suggests that at low transcription factor concentrations cooperative activation cannot yield synergistic greater-than-additive effects, i.e., DNA transcription can only exhibit less-than-additive effects. Accordingly, transcriptional activity turns from synergistic greater-than-additive responses at relatively high transcription factor concentrations into less-than-additive responses at relatively low concentrations. In addition, two types of re-entrant phenomena are predicted. First, our analysis predicts that under particular circumstances transcriptional activity will feature a sequence of less-than-additive, greater-than-additive, and eventually less-than-additive effects when for fixed activator concentrations the regulatory impact of activators on the binding of RNA polymerase to the promoter increases from weak, to moderate, to strong. Second, for appropriate promoter conditions when activator concentrations are increased then the aforementioned re-entrant sequence of less-than-additive, greater-than-additive, and less-than-additive effects is predicted as well. Finally, our model-based analysis suggests that even for weak activators that individually induce only negligible increases in promoter activity, promoter activity can exhibit greater

  18. Global Transcriptional Responses to Osmotic, Oxidative, and Imipenem Stress Conditions in Pseudomonas putida.

    Science.gov (United States)

    Bojanovič, Klara; D'Arrigo, Isotta; Long, Katherine S

    2017-04-01

    Bacteria cope with and adapt to stress by modulating gene expression in response to specific environmental cues. In this study, the transcriptional response of Pseudomonas putida KT2440 to osmotic, oxidative, and imipenem stress conditions at two time points was investigated via identification of differentially expressed mRNAs and small RNAs (sRNAs). A total of 440 sRNA transcripts were detected, of which 10% correspond to previously annotated sRNAs, 40% to novel intergenic transcripts, and 50% to novel transcripts antisense to annotated genes. Each stress elicits a unique response as far as the extent and dynamics of the transcriptional changes. Nearly 200 protein-encoding genes exhibited significant changes in all stress types, implicating their participation in a general stress response. Almost half of the sRNA transcripts were differentially expressed under at least one condition, suggesting possible functional roles in the cellular response to stress conditions. The data show a larger fraction of differentially expressed sRNAs than of mRNAs with >5-fold expression changes. The work provides detailed insights into the mechanisms through which P. putida responds to different stress conditions and increases understanding of bacterial adaptation in natural and industrial settings. IMPORTANCE This study maps the complete transcriptional response of P. putida KT2440 to osmotic, oxidative, and imipenem stress conditions at short and long exposure times. Over 400 sRNA transcripts, consisting of both intergenic and antisense transcripts, were detected, increasing the number of identified sRNA transcripts in the strain by a factor of 10. Unique responses to each type of stress are documented, including both the extent and dynamics of the gene expression changes. The work adds rich detail to previous knowledge of stress response mechanisms due to the depth of the RNA sequencing data. Almost half of the sRNAs exhibit significant expression changes under at least one

  19. Regulation of the yeast metabolic cycle by transcription factors with periodic activities

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    Pellegrini Matteo

    2011-10-01

    Full Text Available Abstract Background When growing budding yeast under continuous, nutrient-limited conditions, over half of yeast genes exhibit periodic expression patterns. Periodicity can also be observed in respiration, in the timing of cell division, as well as in various metabolite levels. Knowing the transcription factors involved in the yeast metabolic cycle is helpful for determining the cascade of regulatory events that cause these patterns. Results Transcription factor activities were estimated by linear regression using time series and genome-wide transcription factor binding data. Time-translation matrices were estimated using least squares and were used to model the interactions between the most significant transcription factors. The top transcription factors have functions involving respiration, cell cycle events, amino acid metabolism and glycolysis. Key regulators of transitions between phases of the yeast metabolic cycle appear to be Hap1, Hap4, Gcn4, Msn4, Swi6 and Adr1. Conclusions Analysis of the phases at which transcription factor activities peak supports previous findings suggesting that the various cellular functions occur during specific phases of the yeast metabolic cycle.

  20. Neuronal activity-regulated gene transcription: how are distant synaptic signals conveyed to the nucleus?

    Science.gov (United States)

    Matamales, Miriam

    2012-12-19

    Synaptic activity can trigger gene expression programs that are required for the stable change of neuronal properties, a process that is essential for learning and memory. Currently, it is still unclear how the stimulation of dendritic synapses can be coupled to transcription in the nucleus in a timely way given that large distances can separate these two cellular compartments. Although several mechanisms have been proposed to explain long distance communication between synapses and the nucleus, the possible co-existence of these models and their relevance in physiological conditions remain elusive. One model suggests that synaptic activation triggers the translocation to the nucleus of certain transcription regulators localised at postsynaptic sites that function as synapto-nuclear messengers. Alternatively, it has been hypothesised that synaptic activity initiates propagating regenerative intracellular calcium waves that spread through dendrites into the nucleus where nuclear transcription machinery is thereby regulated. It has also been postulated that membrane depolarisation of voltage-gated calcium channels on the somatic membrane is sufficient to increase intracellular calcium concentration and activate transcription without the need for transported signals from distant synapses. Here I provide a critical overview of the suggested mechanisms for coupling synaptic stimulation to transcription, the underlying assumptions behind them and their plausible physiological significance.

  1. Oncogenes Activate an Autonomous Transcriptional Regulatory Circuit That Drives Glioblastoma

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    Dinesh K. Singh

    2017-01-01

    Full Text Available Efforts to identify and target glioblastoma (GBM drivers have primarily focused on receptor tyrosine kinases (RTKs. Clinical benefits, however, have been elusive. Here, we identify an SRY-related box 2 (SOX2 transcriptional regulatory network that is independent of upstream RTKs and capable of driving glioma-initiating cells. We identified oligodendrocyte lineage transcription factor 2 (OLIG2 and zinc-finger E-box binding homeobox 1 (ZEB1, which are frequently co-expressed irrespective of driver mutations, as potential SOX2 targets. In murine glioma models, we show that different combinations of tumor suppressor and oncogene mutations can activate Sox2, Olig2, and Zeb1 expression. We demonstrate that ectopic co-expression of the three transcription factors can transform tumor-suppressor-deficient astrocytes into glioma-initiating cells in the absence of an upstream RTK oncogene. Finally, we demonstrate that the transcriptional inhibitor mithramycin downregulates SOX2 and its target genes, resulting in markedly reduced proliferation of GBM cells in vivo.

  2. The effect of phenobarbital on the transcriptional activity of liver.

    OpenAIRE

    Hardwick, J P; Schwalm, F; Richardson, A

    1983-01-01

    The effect of phenobarbital on the transcriptional activity of liver was studied by measuring the synthesis of RNA by suspensions of hepatocytes isolated from rats treated with phenobarbital for various time periods. The absolute rates of RNA synthesis by isolated hepatocytes were determined by measuring the incorporation of [3H]orotic acid into RNA as UMP and the specific radioactivity of the UTP pool. The specific radioactivity of the UTP extracted from hepatocytes isolated from phenobarbit...

  3. 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.

  4. Global Transcriptional Responses to Osmotic, Oxidative, and Imipenem Stress Conditions in Pseudomonas putida

    DEFF Research Database (Denmark)

    Bojanovic, Klara; D'Arrigo, Isotta; Long, Katherine

    2017-01-01

    functional roles in the cellular response to stress conditions. The data show a larger fraction of differentially expressed sRNAs than of mRNAs with >5-fold expression changes. The work provides detailed insights into the mechanisms through which P. putida responds to different stress conditions...... intergenic and antisense transcripts, were detected, increasing the number of identified sRNA transcripts in the strain by a factor of 10. Unique responses to each type of stress are documented, including both the extent and dynamics of the gene expression changes. The work adds rich detail to previous......Bacteria cope with and adapt to stress by modulating gene expression in response to specific environmental cues. In this study, the transcriptional response of Pseudomonas putida KT2440 to osmotic, oxidative, and imipenem stress conditions at two time points was investigated via identification...

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. 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.

  10. 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.

  11. Dynamic Effects of Topoisomerase I Inhibition on R-Loops and Short Transcripts at Active Promoters.

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    Jessica Marinello

    Full Text Available Topoisomerase I-DNA-cleavage complexes (Top1cc stabilized by camptothecin (CPT have specific effects at transcriptional levels. We recently reported that Top1cc increase antisense transcript (aRNAs levels at divergent CpG-island promoters and, transiently, DNA/RNA hybrids (R-loop in nuclear and mitochondrial genomes of colon cancer HCT116 cells. However, the relationship between R-loops and aRNAs was not established. Here, we show that aRNAs can form R-loops in N-TERA-2 cells under physiological conditions, and that promoter-associated R-loops are somewhat increased and extended in length immediately upon cell exposure to CPT. In contrast, persistent Top1ccs reduce the majority of R-loops suggesting that CPT-accumulated aRNAs are not commonly involved in R-loops. The enhancement of aRNAs by Top1ccs is present both in human colon cancer HCT116 cells and WI38 fibroblasts suggesting a common response of cancer and normal cells. Although Top1ccs lead to DSB and DDR kinases activation, we do not detect a dependence of aRNA accumulation on ATM or DNA-PK activation. However, we showed that the cell response to persistent Top1ccs can involve an impairment of aRNA turnover rather than a higher synthesis rate. Finally, a genome-wide analysis shows that persistent Top1ccs also determine an accumulation of sense transcripts at 5'-end gene regions suggesting an increased occurrence of truncated transcripts. Taken together, the results indicate that Top1 may regulate transcription initiation by modulating RNA polymerase-generated negative supercoils, which can in turn favor R-loop formation at promoters, and that transcript accumulation at TSS is a response to persistent transcriptional stress by Top1 poisoning.

  12. 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.

  13. Environmental phthalate monoesters activate pregnane X receptor-mediated transcription

    International Nuclear Information System (INIS)

    Hurst, Christopher H.; Waxman, David J.

    2004-01-01

    Phthalate esters, widely used as plasticizers in the manufacture of products made of polyvinyl chloride, induce reproductive and developmental toxicities in rodents. The mechanism that underlies these effects of phthalate exposure, including the potential role of members of the nuclear receptor superfamily, is not known. The present study investigates the effects of phthalates on the pregnane X receptor (PXR), which mediates the induction of enzymes involved in steroid metabolism and xenobiotic detoxification. The ability of phthalate monoesters to activate PXR-mediated transcription was assayed in a HepG2 cell reporter assay following transfection with mouse PXR (mPXR), human PXR (hPXR), or the hPXR allelic variants V140M, D163G, and A370T. Mono-2-ethylhexyl phthalate (MEHP) increased the transcriptional activity of both mPXR and hPXR (5- and 15-fold, respectively) with EC 50 values of 7-8 μM. mPXR and hPXR were also activated by monobenzyl phthalate (MBzP, up to 5- to 6-fold) but were unresponsive to monomethyl phthalate and mono-n-butyl phthalate (M(n)BP) at the highest concentrations tested (300 μM). hPXR-V140M and hPXR-A370T exhibited patterns of phthalate responses similar to the wild-type receptor. By contrast, hPXR-D163G was unresponsive to all phthalate monoesters tested. Further studies revealed that hPXR-D163G did respond to rifampicin, but required approximately 40-fold higher concentrations than wild-type receptor, suggesting that the ligand-binding domain D163G variant has impaired ligand-binding activity. The responsiveness of PXR to activation by phthalate monoesters demonstrated here suggests that these ubiquitous environmental chemicals may, in part, exhibit their endocrine disruptor activities by altering PXR-regulated steroid hormone metabolism with potential adverse health effects in exposed individuals

  14. Messenger RNA Interferase RelE Controls relBE Transcription by Conditional Cooperativity

    DEFF Research Database (Denmark)

    Overgaard, Martin; Borch, Jonas; Jørgensen, Mikkel G

    2008-01-01

    Prokaryotic toxin-antitoxin (TA) loci consist of two genes in an operon that encodes a metabolically stable toxin and an unstable antitoxin. The antitoxin neutralises its cognate toxin by forming a tight complex with it. In all cases known, the antitoxin autoregulates TA operon transcription by b...... operator DNA. A mutational analysis of the operator-sites showed that RelE in excess counteracted cooperative binding of the RelB(2)*RelE complexes to the operator-sites. Thus, RelE controls relBE transcription by conditional cooperativity.......Prokaryotic toxin-antitoxin (TA) loci consist of two genes in an operon that encodes a metabolically stable toxin and an unstable antitoxin. The antitoxin neutralises its cognate toxin by forming a tight complex with it. In all cases known, the antitoxin autoregulates TA operon transcription...... by binding to one or more operators in the promoter region while the toxin functions as a co-repressor of transcription. Interestingly, the toxin can also stimulate TA operon transcription. Here we analyse mechanistic aspects of how RelE of Escherichia coli can function both as a co-repressor and derepressor...

  15. Mediator Undergoes a Compositional Change during Transcriptional Activation.

    Science.gov (United States)

    Petrenko, Natalia; Jin, Yi; Wong, Koon Ho; Struhl, Kevin

    2016-11-03

    Mediator is a transcriptional co-activator recruited to enhancers by DNA-binding activators, and it also interacts with RNA polymerase (Pol) II as part of the preinitiation complex (PIC). We demonstrate that a single Mediator complex associates with the enhancer and core promoter in vivo, indicating that it can physically bridge these transcriptional elements. However, the Mediator kinase module associates strongly with the enhancer, but not with the core promoter, and it dissociates from the enhancer upon depletion of the TFIIH kinase. Severing the kinase module from Mediator by removing the connecting subunit Med13 does not affect Mediator association at the core promoter but increases occupancy at enhancers. Thus, Mediator undergoes a compositional change in which the kinase module, recruited via Mediator to the enhancer, dissociates from Mediator to permit association with Pol II and the PIC. As such, Mediator acts as a dynamic bridge between the enhancer and core promoter. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. n-Butyrate inhibits Jun NH(2)-terminal kinase activation and cytokine transcription in mast cells

    International Nuclear Information System (INIS)

    Diakos, Christos; Prieschl, Eva E.; Saeemann, Marcus D.; Boehmig, Georg A.; Csonga, Robert; Sobanov, Yury; Baumruker, Thomas; Zlabinger, Gerhard J.

    2006-01-01

    Mast cells are well known to contribute to type I allergic conditions but only recently have been brought in association with chronic relapsing/remitting autoimmune diseases such as celiac disease and ulcerative colitis. Since the bacterial metabolite n-butyrate is considered to counteract intestinal inflammation we investigated the effects of this short chain fatty acid on mast cell activation. Using RNAse protection assays and reporter gene technology we show that n-butyrate downregulates TNF-α transcription. This correlates with an impaired activation of the Jun NH(2)-terminal kinase (JNK) but not other MAP kinases such as ERK and p38 that are largely unaffected by n-butyrate. As a consequence, we observed a decreased nuclear activity of AP-1 and NF-AT transcription factors. These results indicate that n-butyrate inhibits critical inflammatory mediators in mast cells by relatively selectively targeting the JNK signalling

  17. Transcription and splicing regulation in human umbilical vein endothelial cells under hypoxic stress conditions by exon array

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    Wu Yonghong

    2009-03-01

    Full Text Available Abstract Background The balance between endothelial cell survival and apoptosis during stress is an important cellular process for vessel integrity and vascular homeostasis, and it is also pivotal in angiogenesis during the development of many vascular diseases. However, the underlying molecular mechanisms remain largely unknown. Although both transcription and alternative splicing are important in regulating gene expression in endothelial cells under stress, the regulatory mechanisms underlying this state and their interactions have not yet been studied on a genome-wide basis. Results Human umbilical vein endothelial cells (HUVECs were treated with cobalt chloride (CoCl2 both to mimic hypoxia and to induce cell apoptosis and alternative splicing responses. Cell apoptosis rate analysis indicated that HUVECs exposed to 300 μM CoCl2 for 24 hrs were initially counterbalancing apoptosis with cell survival. We therefore used the Affymetrix exon array system to determine genome-wide transcript- and exon-level differential expression. Other than 1583 differentially expressed transcripts, 342 alternatively spliced exons were detected and classified by different splicing types. Sixteen alternatively spliced exons were validated by RT-PCR. Furthermore, direct evidence for the ongoing balance between HUVEC survival and apoptosis was provided by Gene Ontology (GO and protein function, as well as protein domain and pathway enrichment analyses of the differentially expressed transcripts. Importantly, a novel molecular module, in which the heat shock protein (HSP families play a significant role, was found to be activated under mimicked hypoxia conditions. In addition, 46% of the transcripts containing stress-modulated exons were differentially expressed, indicating the possibility of combinatorial regulation of transcription and splicing. Conclusion The exon array system effectively profiles gene expression and splicing on the genome-wide scale. Based on

  18. Active transcription and ultrastructural changes during Trypanosoma cruzi metacyclogenesis

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    Ludmila R.P. Ferreira

    2008-03-01

    Full Text Available The differentiation of proliferating epimastigote forms of Trypanosoma cruzi , the protozoan parasite that causes Chagas’ disease, into the infective and non-proliferating metacyclic forms can be reproduced in the laboratory by incubating the cells in a chemically-defined medium that mimics the urine of the insect vector. Epimastigotes have a spherical nucleus, a flagellum protruding from the middle of the protozoan cell, and a disk-shaped kinetoplast - an organelle that corresponds to the mitochondrial DNA. Metacyclic trypomastigotes have an elongated shape with the flagellum protruding from the posterior portion of the cell and associated with a spherical kinetoplast. Here we describe the morphological events of this transformation and characterize a novel intermediate stage by three-dimensional reconstruction of electron microscope serial sections. This new intermediate stage is characterized by a kinetoplast compressing an already elongated nucleus, indicating that metacyclogenesis involves active movements of the flagellar structure relative to the cell body. As transcription occurs more intensely in proliferating epimastigotes than in metacyclics, we also examined the presence of RNA polymerase II and measured transcriptional activity during the differentiation process. Both the presence of the enzyme and transcriptional activity remain unchanged during all steps of metacyclogenesis. RNA polymerase II levels and transcriptional activity only decrease after metacyclics are formed. We suggest that transcription is required during the epimastigote-to-metacyclic trypomastigote differentiation process, until the kinetoplast and flagellum reach the posterior position of the parasites in the infective form.A diferenciação de formas epimastigotas (proliferativas do Trypanosoma cruzi, parasita protozoário causador da doença de Chagas, em formas metacíclicas tripomastigotas (infectivas e não proliferativas, pode ser reproduzida em laborat

  19. 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...

  20. WRKY Transcription Factors Involved in Activation of SA Biosynthesis Genes

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    Bol John F

    2011-05-01

    Full Text Available Abstract Background Increased defense against a variety of pathogens in plants is achieved through activation of a mechanism known as systemic acquired resistance (SAR. The broad-spectrum resistance brought about by SAR is mediated through salicylic acid (SA. An important step in SA biosynthesis in Arabidopsis is the conversion of chorismate to isochorismate through the action of isochorismate synthase, encoded by the ICS1 gene. Also AVRPPHB SUSCEPTIBLE 3 (PBS3 plays an important role in SA metabolism, as pbs3 mutants accumulate drastically reduced levels of SA-glucoside, a putative storage form of SA. Bioinformatics analysis previously performed by us identified WRKY28 and WRKY46 as possible regulators of ICS1 and PBS3. Results Expression studies with ICS1 promoter::β-glucuronidase (GUS genes in Arabidopsis thaliana protoplasts cotransfected with 35S::WRKY28 showed that over expression of WRKY28 resulted in a strong increase in GUS expression. Moreover, qRT-PCR analyses indicated that the endogenous ICS1 and PBS3 genes were highly expressed in protoplasts overexpressing WRKY28 or WRKY46, respectively. Electrophoretic mobility shift assays indentified potential WRKY28 binding sites in the ICS1 promoter, positioned -445 and -460 base pairs upstream of the transcription start site. Mutation of these sites in protoplast transactivation assays showed that these binding sites are functionally important for activation of the ICS1 promoter. Chromatin immunoprecipitation assays with haemagglutinin-epitope-tagged WRKY28 showed that the region of the ICS1 promoter containing the binding sites at -445 and -460 was highly enriched in the immunoprecipitated DNA. Conclusions The results obtained here confirm results from our multiple microarray co-expression analyses indicating that WRKY28 and WRKY46 are transcriptional activators of ICS1 and PBS3, respectively, and support this in silico screening as a powerful tool for identifying new components of stress

  1. Hypoxia induces cyclophilin B through the activation of transcription factor 6 in gastric adenocarcinoma cells.

    Science.gov (United States)

    Jeong, Kwon; Kim, Kiyoon; Kim, Hunsung; Oh, Yoojung; Kim, Seong-Jin; Jo, Yunhee; Choe, Wonchae

    2015-06-01

    Hypoxia is an important form of physiological stress that induces cell death, due to the resulting endoplasmic reticulum (ER) stress, particularly in solid tumors. Although previous studies have indicated that cyclophilin B (CypB) plays a role in ER stress, there is currently no direct information supporting the mechanism of CypB involvement under hypoxic conditions. However, it has previously been demonstrated that ER stress positively regulates the expression of CypB. In the present study, it was demonstrated that CypB is transcriptionally regulated by hypoxia-mediated activation of transcription factor 6 (ATF6), an ER stress transcription factor. Subsequently, the effects of ATF6 on CypB promoter activity were investigated and an ATF6-responsive region in the promoter was identified. Hypoxia and ATF6 expression each increased CypB promoter activity. Collectively, these results demonstrate that ATF6 positively regulates the expression of CypB by binding to an ATF6-responsive region in the promoter, which may play an important role in the attenuation of apoptosis in the adaption to hypoxia. These results suggest that CypB may be a key molecule in the adaptation of cells to hypoxic conditions.

  2. Reference genes for accurate transcript normalization in citrus genotypes under different experimental conditions.

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    Valéria Mafra

    Full Text Available Real-time reverse transcription PCR (RT-qPCR has emerged as an accurate and widely used technique for expression profiling of selected genes. However, obtaining reliable measurements depends on the selection of appropriate reference genes for gene expression normalization. The aim of this work was to assess the expression stability of 15 candidate genes to determine which set of reference genes is best suited for transcript normalization in citrus in different tissues and organs and leaves challenged with five pathogens (Alternaria alternata, Phytophthora parasitica, Xylella fastidiosa and Candidatus Liberibacter asiaticus. We tested traditional genes used for transcript normalization in citrus and orthologs of Arabidopsis thaliana genes described as superior reference genes based on transcriptome data. geNorm and NormFinder algorithms were used to find the best reference genes to normalize all samples and conditions tested. Additionally, each biotic stress was individually analyzed by geNorm. In general, FBOX (encoding a member of the F-box family and GAPC2 (GAPDH was the most stable candidate gene set assessed under the different conditions and subsets tested, while CYP (cyclophilin, TUB (tubulin and CtP (cathepsin were the least stably expressed genes found. Validation of the best suitable reference genes for normalizing the expression level of the WRKY70 transcription factor in leaves infected with Candidatus Liberibacter asiaticus showed that arbitrary use of reference genes without previous testing could lead to misinterpretation of data. Our results revealed FBOX, SAND (a SAND family protein, GAPC2 and UPL7 (ubiquitin protein ligase 7 to be superior reference genes, and we recommend their use in studies of gene expression in citrus species and relatives. This work constitutes the first systematic analysis for the selection of superior reference genes for transcript normalization in different citrus organs and under biotic stress.

  3. 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.

  4. E-cadherin is transcriptionally activated via suppression of ZEB1 transcriptional repressor by small RNA-mediated gene silencing.

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    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.

  5. Activating transcription factor 3 regulates immune and metabolic homeostasis.

    Science.gov (United States)

    Rynes, Jan; Donohoe, Colin D; Frommolt, Peter; Brodesser, Susanne; Jindra, Marek; Uhlirova, Mirka

    2012-10-01

    Integration of metabolic and immune responses during animal development ensures energy balance, permitting both growth and defense. Disturbed homeostasis causes organ failure, growth retardation, and metabolic disorders. Here, we show that the Drosophila melanogaster activating transcription factor 3 (Atf3) safeguards metabolic and immune system homeostasis. Loss of Atf3 results in chronic inflammation and starvation responses mounted primarily by the larval gut epithelium, while the fat body suffers lipid overload, causing energy imbalance and death. Hyperactive proinflammatory and stress signaling through NF-κB/Relish, Jun N-terminal kinase, and FOXO in atf3 mutants deregulates genes important for immune defense, digestion, and lipid metabolism. Reducing the dose of either FOXO or Relish normalizes both lipid metabolism and gene expression in atf3 mutants. The function of Atf3 is conserved, as human ATF3 averts some of the Drosophila mutant phenotypes, improving their survival. The single Drosophila Atf3 may incorporate the diversified roles of two related mammalian proteins.

  6. Comprehensive Behavioral Analysis of Activating Transcription Factor 5-Deficient Mice

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    Mariko Umemura

    2017-07-01

    Full Text Available Activating transcription factor 5 (ATF5 is a member of the CREB/ATF family of basic leucine zipper transcription factors. We previously reported that ATF5-deficient (ATF5-/- mice demonstrated abnormal olfactory bulb development due to impaired interneuron supply. Furthermore, ATF5-/- mice were less aggressive than ATF5+/+ mice. Although ATF5 is widely expressed in the brain, and involved in the regulation of proliferation and development of neurons, the physiological role of ATF5 in the higher brain remains unknown. Our objective was to investigate the physiological role of ATF5 in the higher brain. We performed a comprehensive behavioral analysis using ATF5-/- mice and wild type littermates. ATF5-/- mice exhibited abnormal locomotor activity in the open field test. They also exhibited abnormal anxiety-like behavior in the light/dark transition test and open field test. Furthermore, ATF5-/- mice displayed reduced social interaction in the Crawley’s social interaction test and increased pain sensitivity in the hot plate test compared with wild type. Finally, behavioral flexibility was reduced in the T-maze test in ATF5-/- mice compared with wild type. In addition, we demonstrated that ATF5-/- mice display disturbances of monoamine neurotransmitter levels in several brain regions. These results indicate that ATF5 deficiency elicits abnormal behaviors and the disturbance of monoamine neurotransmitter levels in the brain. The behavioral abnormalities of ATF5-/- mice may be due to the disturbance of monoamine levels. Taken together, these findings suggest that ATF5-/- mice may be a unique animal model of some psychiatric disorders.

  7. 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

  8. 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...

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

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    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

  10. Mediator phosphorylation prevents stress response transcription during non-stress conditions.

    Science.gov (United States)

    Miller, Christian; Matic, Ivan; Maier, Kerstin C; Schwalb, Björn; Roether, Susanne; Strässer, Katja; Tresch, Achim; Mann, Matthias; Cramer, Patrick

    2012-12-28

    The multiprotein complex Mediator is a coactivator of RNA polymerase (Pol) II transcription that is required for the regulated expression of protein-coding genes. Mediator serves as an end point of signaling pathways and regulates Pol II transcription, but the mechanisms it uses are not well understood. Here, we used mass spectrometry and dynamic transcriptome analysis to investigate a functional role of Mediator phosphorylation in gene expression. Affinity purification and mass spectrometry revealed that Mediator from the yeast Saccharomyces cerevisiae is phosphorylated at multiple sites of 17 of its 25 subunits. Mediator phosphorylation levels change upon an external stimulus set by exposure of cells to high salt concentrations. Phosphorylated sites in the Mediator tail subunit Med15 are required for suppression of stress-induced changes in gene expression under non-stress conditions. Thus dynamic and differential Mediator phosphorylation contributes to gene regulation in eukaryotic cells.

  11. Fkh1 and Fkh2 associate with Sir2 to control CLB2 transcription under normal and oxidative stress conditions

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    Christian eLinke

    2013-07-01

    Full Text Available The Forkhead box family of transcription factors is evolutionary conserved from yeast to higher eukaryotes and its members are involved in many physiological processes including metabolism, DNA repair, cell cycle, stress resistance, apoptosis and aging. In budding yeast, four Forkhead transcription factors were identified, namely Fkh1, Fkh2, Fhl1, and Hcm1, which are implicated in chromatin silencing, cell cycle regulation and stress response. These factors impinge transcriptional regulation during cell cycle progression, and histone deacetylases play an essential role in this process, e.g. the nuclear localisation of Hcm1 depends on Sir2 activity, whereas Sin3/Rpd3 silence cell cycle specific gene transcription in G2/M phase. However, a direct involvement of Sir2 in Fkh1/Fkh2-dependent regulation of target genes is at present unknown. Here, we show that Fkh1 and Fkh2 associate with Sir2 in G1 and M phase, and that Fkh1/Fkh2-mediated activation of reporter genes is antagonized by Sir2. We further report that Sir2 overexpression strongly affects cell growth in an Fkh1/Fkh2-dependent manner. In addition, Sir2 regulates the expression of the mitotic cyclin Clb2 through Fkh1/Fkh2-mediated binding to the CLB2 promoter in G1 and M phase. We finally demonstrate that Sir2 is also enriched at the CLB2 promoter under stress conditions, and that the nuclear localization of Sir2 is dependent on Fkh1 and Fkh2. Taken together, our results show a functional interplay between Fkh1/Fkh2 and Sir2 suggesting a novel mechanism of cell cycle repression. Thus, in budding yeast, not only the regulation of G2/M gene expression but also the protective response against stress could be directly coordinated by Fkh1 and Fkh2.

  12. Bivariate Genomic Footprinting Detects Changes in Transcription Factor Activity

    Directory of Open Access Journals (Sweden)

    Songjoon Baek

    2017-05-01

    Full Text Available In response to activating signals, transcription factors (TFs bind DNA and regulate gene expression. TF binding can be measured by protection of the bound sequence from DNase digestion (i.e., footprint. Here, we report that 80% of TF binding motifs do not show a measurable footprint, partly because of a variable cleavage pattern within the motif sequence. To more faithfully portray the effect of TFs on chromatin, we developed an algorithm that captures two TF-dependent effects on chromatin accessibility: footprinting and motif-flanking accessibility. The algorithm, termed bivariate genomic footprinting (BaGFoot, efficiently detects TF activity. BaGFoot is robust to different accessibility assays (DNase-seq, ATAC-seq, all examined peak-calling programs, and a variety of cut bias correction approaches. BaGFoot reliably predicts TF binding and provides valuable information regarding the TFs affecting chromatin accessibility in various biological systems and following various biological events, including in cases where an absolute footprint cannot be determined.

  13. Global transcriptional regulatory network for Escherichia coli robustly connects gene expression to transcription factor activities

    DEFF Research Database (Denmark)

    Fang, Xin; Sastry, Anand; Mih, Nathan

    2017-01-01

    Transcriptional regulatory networks (TRNs) have been studied intensely for >25 y. Yet, even for the Escherichia coli TRN-probably the best characterized TRN-several questions remain. Here, we address three questions: (i) How complete is our knowledge of the E. coli TRN; (ii) how well can we predi...

  14. Activity-Based Anorexia Alters the Expression of BDNF Transcripts in the Mesocorticolimbic Reward Circuit.

    Science.gov (United States)

    Ho, Emily V; Klenotich, Stephanie J; McMurray, Matthew S; Dulawa, Stephanie C

    2016-01-01

    Anorexia nervosa (AN) is a complex eating disorder with severe dysregulation of appetitive behavior. The activity-based anorexia (ABA) paradigm is an animal model in which rodents exposed to both running wheels and scheduled feeding develop aspects of AN including paradoxical hypophagia, dramatic weight loss, and hyperactivity, while animals exposed to only one condition maintain normal body weight. Brain-derived neurotrophic factor (BDNF), an activity-dependent modulator of neuronal plasticity, is reduced in the serum of AN patients, and is a known regulator of feeding and weight maintenance. We assessed the effects of scheduled feeding, running wheel access, or both on the expression of BDNF transcripts within the mesocorticolimbic pathway. We also assessed the expression of neuronal cell adhesion molecule 1 (NCAM1) to explore the specificity of effects on BDNF within the mesocorticolimbic pathway. Scheduled feeding increased the levels of both transcripts in the hippocampus (HPC), increased NCAM1 mRNA expression in the ventral tegmental area (VTA), and decreased BDNF mRNA levels in the medial prefrontal cortex (mPFC). In addition, wheel running increased BDNF mRNA expression in the VTA. No changes in either transcript were observed in the nucleus accumbens (NAc). Furthermore, no changes in either transcript were induced by the combined scheduled feeding and wheel access condition. These data indicate that scheduled feeding or wheel running alter BDNF and NCAM1 expression levels in specific regions of the mesocorticolimbic pathway. These findings contribute to our current knowledge of the molecular alterations induced by ABA and may help elucidate possible mechanisms of AN pathology.

  15. 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...

  16. 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.

  17. Evaluation of new reference genes in papaya for accurate transcript normalization under different experimental conditions.

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    Xiaoyang Zhu

    Full Text Available Real-time reverse transcription PCR (RT-qPCR is a preferred method for rapid and accurate quantification of gene expression studies. Appropriate application of RT-qPCR requires accurate normalization though the use of reference genes. As no single reference gene is universally suitable for all experiments, thus reference gene(s validation under different experimental conditions is crucial for RT-qPCR analysis. To date, only a few studies on reference genes have been done in other plants but none in papaya. In the present work, we selected 21 candidate reference genes, and evaluated their expression stability in 246 papaya fruit samples using three algorithms, geNorm, NormFinder and RefFinder. The samples consisted of 13 sets collected under different experimental conditions, including various tissues, different storage temperatures, different cultivars, developmental stages, postharvest ripening, modified atmosphere packaging, 1-methylcyclopropene (1-MCP treatment, hot water treatment, biotic stress and hormone treatment. Our results demonstrated that expression stability varied greatly between reference genes and that different suitable reference gene(s or combination of reference genes for normalization should be validated according to the experimental conditions. In general, the internal reference genes EIF (Eukaryotic initiation factor 4A, TBP1 (TATA binding protein 1 and TBP2 (TATA binding protein 2 genes had a good performance under most experimental conditions, whereas the most widely present used reference genes, ACTIN (Actin 2, 18S rRNA (18S ribosomal RNA and GAPDH (Glyceraldehyde-3-phosphate dehydrogenase were not suitable in many experimental conditions. In addition, two commonly used programs, geNorm and Normfinder, were proved sufficient for the validation. This work provides the first systematic analysis for the selection of superior reference genes for accurate transcript normalization in papaya under different experimental

  18. Activating Transcription Factor 3 Regulates Immune and Metabolic Homeostasis

    Science.gov (United States)

    Rynes, Jan; Donohoe, Colin D.; Frommolt, Peter; Brodesser, Susanne; Jindra, Marek

    2012-01-01

    Integration of metabolic and immune responses during animal development ensures energy balance, permitting both growth and defense. Disturbed homeostasis causes organ failure, growth retardation, and metabolic disorders. Here, we show that the Drosophila melanogaster activating transcription factor 3 (Atf3) safeguards metabolic and immune system homeostasis. Loss of Atf3 results in chronic inflammation and starvation responses mounted primarily by the larval gut epithelium, while the fat body suffers lipid overload, causing energy imbalance and death. Hyperactive proinflammatory and stress signaling through NF-κB/Relish, Jun N-terminal kinase, and FOXO in atf3 mutants deregulates genes important for immune defense, digestion, and lipid metabolism. Reducing the dose of either FOXO or Relish normalizes both lipid metabolism and gene expression in atf3 mutants. The function of Atf3 is conserved, as human ATF3 averts some of the Drosophila mutant phenotypes, improving their survival. The single Drosophila Atf3 may incorporate the diversified roles of two related mammalian proteins. PMID:22851689

  19. Transcription factor PIF4 controls the thermosensory activation of flowering

    KAUST Repository

    Kumar, S. Vinod; Lucyshyn, Doris; Jaeger, Katja E.; Aló s, Enriqueta; Alvey, Elizabeth; Harberd, Nicholas P.; Wigge, Philip A.

    2012-01-01

    Plant growth and development are strongly affected by small differences in temperature. Current climate change has already altered global plant phenology and distribution, and projected increases in temperature pose a significant challenge to agriculture. Despite the important role of temperature on plant development, the underlying pathways are unknown. It has previously been shown that thermal acceleration of flowering is dependent on the florigen, FLOWERING LOCUS T (FT). How this occurs is, however, not understood, because the major pathway known to upregulate FT, the photoperiod pathway, is not required for thermal acceleration of flowering. Here we demonstrate a direct mechanism by which increasing temperature causes the bHLH transcription factor PHYTOCHROME INTERACTING FACTOR4 (PIF4) to activate FT. Our findings provide a new understanding of how plants control their timing of reproduction in response to temperature. Flowering time is an important trait in crops as well as affecting the life cycles of pollinator species. A molecular understanding of how temperature affects flowering will be important for mitigating the effects of climate change. © 2012 Macmillan Publishers Limited. All rights reserved.

  20. Transcriptional activation of Mina by Sp1/3 factors.

    Science.gov (United States)

    Lian, Shangli; Potula, Hari Hara S K; Pillai, Meenu R; Van Stry, Melanie; Koyanagi, Madoka; Chung, Linda; Watanabe, Makiko; Bix, Mark

    2013-01-01

    Mina is an epigenetic gene regulatory protein known to function in multiple physiological and pathological contexts, including pulmonary inflammation, cell proliferation, cancer and immunity. We showed previously that the level of Mina gene expression is subject to natural genetic variation linked to 21 SNPs occurring in the Mina 5' region. In order to explore the mechanisms regulating Mina gene expression, we set out to molecularly characterize the Mina promoter in the region encompassing these SNPs. We used three kinds of assays--reporter, gel shift and chromatin immunoprecipitation--to analyze a 2 kb genomic fragment spanning the upstream and intron 1 regions flanking exon 1. Here we discovered a pair of Mina promoters (P1 and P2) and a P1-specific enhancer element (E1). Pharmacologic inhibition and siRNA knockdown experiments suggested that Sp1/3 transcription factors trigger Mina expression through additive activity targeted to a cluster of four Sp1/3 binding sites forming the P1 promoter. These results set the stage for comprehensive analysis of Mina gene regulation from the context of tissue specificity, the impact of inherited genetic variation and the nature of upstream signaling pathways.

  1. Transcription factor PIF4 controls the thermosensory activation of flowering

    KAUST Repository

    Kumar, S. Vinod

    2012-03-21

    Plant growth and development are strongly affected by small differences in temperature. Current climate change has already altered global plant phenology and distribution, and projected increases in temperature pose a significant challenge to agriculture. Despite the important role of temperature on plant development, the underlying pathways are unknown. It has previously been shown that thermal acceleration of flowering is dependent on the florigen, FLOWERING LOCUS T (FT). How this occurs is, however, not understood, because the major pathway known to upregulate FT, the photoperiod pathway, is not required for thermal acceleration of flowering. Here we demonstrate a direct mechanism by which increasing temperature causes the bHLH transcription factor PHYTOCHROME INTERACTING FACTOR4 (PIF4) to activate FT. Our findings provide a new understanding of how plants control their timing of reproduction in response to temperature. Flowering time is an important trait in crops as well as affecting the life cycles of pollinator species. A molecular understanding of how temperature affects flowering will be important for mitigating the effects of climate change. © 2012 Macmillan Publishers Limited. All rights reserved.

  2. 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.

  3. 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.

  4. 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....

  5. Hypoxia-Inducible Factor 3 Is an Oxygen-Dependent Transcription Activator and Regulates a Distinct Transcriptional Response to Hypoxia

    Directory of Open Access Journals (Sweden)

    Peng Zhang

    2014-03-01

    Full Text Available Hypoxia-inducible factors (HIFs play key roles in the cellular response to hypoxia. It is widely accepted that whereas HIF-1 and HIF-2 function as transcriptional activators, HIF-3 inhibits HIF-1/2α action. Contrary to this idea, we show that zebrafish Hif-3α has strong transactivation activity. Hif-3α is degraded under normoxia. Mutation of P393, P493, and L503 inhibits this oxygen-dependent degradation. Transcriptomics and chromatin immunoprecipitation analyses identify genes that are regulated by Hif-3α, Hif-1α, or both. Under hypoxia or when overexpressed, Hif-3α binds to its target gene promoters and upregulates their expression. Dominant-negative inhibition and knockdown of Hif-3α abolish hypoxia-induced Hif-3α-promoter binding and gene expression. Hif-3α not only mediates hypoxia-induced growth and developmental retardation but also possesses hypoxia-independent activities. Importantly, transactivation activity is conserved and human HIF-3α upregulates similar genes in human cells. These findings suggest that Hif-3 is an oxygen-dependent transcription factor and activates a distinct transcriptional response to hypoxia.

  6. 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

  7. Inhibition of FoxO transcriptional activity prevents muscle fiber atrophy during cachexia and induces hypertrophy

    Science.gov (United States)

    Reed, Sarah A.; Sandesara, Pooja B.; Senf, Sarah M.; Judge, Andrew R.

    2012-01-01

    Cachexia is characterized by inexorable muscle wasting that significantly affects patient prognosis and increases mortality. Therefore, understanding the molecular basis of this muscle wasting is of significant importance. Recent work showed that components of the forkhead box O (FoxO) pathway are increased in skeletal muscle during cachexia. In the current study, we tested the physiological significance of FoxO activation in the progression of muscle atrophy associated with cachexia. FoxO-DNA binding dependent transcription was blocked in the muscles of mice through injection of a dominant negative (DN) FoxO expression plasmid prior to inoculation with Lewis lung carcinoma cells or the induction of sepsis. Expression of DN FoxO inhibited the increased mRNA levels of atrogin-1, MuRF1, cathepsin L, and/or Bnip3 and inhibited muscle fiber atrophy during cancer cachexia and sepsis. Interestingly, during control conditions, expression of DN FoxO decreased myostatin expression, increased MyoD expression and satellite cell proliferation, and induced fiber hypertrophy, which required de novo protein synthesis. Collectively, these data show that FoxO-DNA binding-dependent transcription is necessary for normal muscle fiber atrophy during cancer cachexia and sepsis, and further suggest that basal levels of FoxO play an important role during normal conditions to depress satellite cell activation and limit muscle growth.—Reed, S. A., Sandesara, P. B., Senf, S. F., Judge, A. R. Inhibition of FoxO transcriptional activity prevents muscle fiber atrophy during cachexia and induces hypertrophy. PMID:22102632

  8. 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.

  9. 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.

  10. The Antarctic krill Euphausia superba shows diurnal cycles of transcription under natural conditions.

    Directory of Open Access Journals (Sweden)

    Cristiano De Pittà

    Full Text Available BACKGROUND: Polar environments are characterized by extreme seasonal changes in day length, light intensity and spectrum, the extent of sea ice during the winter, and food availability. A key species of the Southern Ocean ecosystem, the Antarctic krill (Euphausia superba has evolved rhythmic physiological and behavioral mechanisms to adapt to daily and seasonal changes. The molecular organization of the clockwork underlying these biological rhythms is, nevertheless, still only partially understood. METHODOLOGY/PRINCIPAL FINDINGS: The genome sequence of the Antarctic krill is not yet available. A normalized cDNA library was produced and pyrosequenced in the attempt to identify large numbers of transcripts. All available E. superba sequences were then assembled to create the most complete existing oligonucleotide microarray platform with a total of 32,217 probes. Gene expression signatures of specimens collected in the Ross Sea at five different time points over a 24-hour cycle were defined, and 1,308 genes differentially expressed were identified. Of the corresponding transcripts, 609 showed a significant sinusoidal expression pattern; about 40% of these exibithed a 24-hour periodicity while the other 60% was characterized by a shorter (about 12-hour rhythm. We assigned the differentially expressed genes to functional categories and noticed that those concerning translation, proteolysis, energy and metabolic process, redox regulation, visual transduction and stress response, which are most likely related to daily environmental changes, were significantly enriched. Two transcripts of peroxiredoxin, thought to represent the ancestral timekeeping system that evolved about 2.5 billion years ago, were also identified as were two isoforms of the EsRh1 opsin and two novel arrestin1 sequences involved in the visual transduction cascade. CONCLUSIONS: Our work represents the first characterization of the krill diurnal transcriptome under natural conditions

  11. 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.

  12. 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.

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

    KAUST Repository

    Roy, S.; Schmeier, S.; Arner, E.; Alam, Tanvir; Parihar, S. P.; Ozturk, M.; Tamgue, O.; Kawaji, H.; de Hoon, M. J. L.; Itoh, M.; Lassmann, T.; Carninci, P.; Hayashizaki, Y.; Forrest, A. R. R.; Bajic, Vladimir B.; Guler, R.; Consortium, F.; Brombacher, F.; Suzuki, H.

    2015-01-01

    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

  14. 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.

  15. 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.

  16. Abscisic acid-dependent multisite phosphorylation regulates the activity of a transcription activator AREB1.

    Science.gov (United States)

    Furihata, Takashi; Maruyama, Kyonoshin; Fujita, Yasunari; Umezawa, Taishi; Yoshida, Riichiro; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

    2006-02-07

    bZIP-type transcription factors AREBs/ABFs bind an abscisic acid (ABA)-responsive cis-acting element named ABRE and transactivate downstream gene expression in Arabidopsis. Because AREB1 overexpression could not induce downstream gene expression, activation of AREB1 requires ABA-dependent posttranscriptional modification. We confirmed that ABA activated 42-kDa kinase activity, which, in turn, phosphorylated Ser/Thr residues of R-X-X-S/T sites in the conserved regions of AREB1. Amino acid substitutions of R-X-X-S/T sites to Ala suppressed transactivation activity, and multiple substitution of these sites resulted in almost complete suppression of transactivation activity in transient assays. In contrast, substitution of the Ser/Thr residues to Asp resulted in high transactivation activity without exogenous ABA application. A phosphorylated, transcriptionally active form was achieved by substitution of Ser/Thr in all conserved R-X-X-S/T sites to Asp. Transgenic plants overexpressing the phosphorylated active form of AREB1 expressed many ABA-inducible genes, such as RD29B, without ABA treatment. These results indicate that the ABA-dependent multisite phosphorylation of AREB1 regulates its own activation in plants.

  17. 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.

  18. Abscisic acid-dependent multisite phosphorylation regulates the activity of a transcription activator AREB1

    OpenAIRE

    Furihata, Takashi; Maruyama, Kyonoshin; Fujita, Yasunari; Umezawa, Taishi; Yoshida, Riichiro; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

    2006-01-01

    bZIP-type transcription factors AREBs/ABFs bind an abscisic acid (ABA)-responsive cis-acting element named ABRE and transactivate downstream gene expression in Arabidopsis. Because AREB1 overexpression could not induce downstream gene expression, activation of AREB1 requires ABA-dependent posttranscriptional modification. We confirmed that ABA activated 42-kDa kinase activity, which, in turn, phosphorylated Ser/Thr residues of R-X-X-S/T sites in the conserved regions of AREB1. Amino acid subs...

  19. 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

  20. 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.

  1. Culture conditions for equine bone marrow mesenchymal stem cells and expression of key transcription factors during their differentiation into osteoblasts

    Science.gov (United States)

    2013-01-01

    Background The use of equine bone marrow mesenchymal stem cells (BMSC) is a novel method to improve fracture healing in horses. However, additional research is needed to identify optimal culture conditions and to determine the mechanisms involved in regulating BMSC differentiation into osteoblasts. The objectives of the experiments were to determine: 1) if autologous or commercial serum is better for proliferation and differentiation of equine BMSC into osteoblasts, and 2) the expression of key transcription factors during the differentiation of equine BMSC into osteoblasts. Equine BMSC were isolated from the sterna of 3 horses, treated with purchased fetal bovine serum (FBS) or autologous horse serum (HS), and cell proliferation determined. To induce osteoblast differentiation, cells were incubated with L-ascorbic acid-2-phosphate and glycerol-2-phosphate in the presence or absence of human bone morphogenetic protein2 (BMP2), dexamethasone (DEX), or combination of the two. Alkaline phosphatase (ALP) activity, a marker of osteoblast differentiation, was determined by ELISA. Total RNA was isolated from differentiating BMSC between d 0 to 18 to determine expression of runt-related transcription factor2 (Runx2), osterix (Osx), and T-box3 (Tbx3). Data were analyzed by ANOVA. Results Relative to control, FBS and HS increased cell number (133 ± 5 and 116 ± 5%, respectively; P  0.8). Runt-related transcription factor2 expression increased 3-fold (P equine BMSC into osteoblasts. In addition, expression of Runx2 and osterix increased and expression of Tbx3 is reduced during differentiation. PMID:24169030

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

    Science.gov (United States)

    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.

  3. Direct interaction of natural and synthetic catechins with signal transducer activator of transcription 1 affects both its phosphorylation and activity

    KAUST Repository

    Menegazzi, Marta; Mariotto, Sofia; Dal Bosco, Martina; Darra, Elena; Vaiana, Nadia; Shoji, Kazuo; Safwat, Abdel Azeim; Marechal, Jean Didier; Perahia, David; Suzuki, Hisanori; Romeo, Sergio

    2013-01-01

    Our previous studies showed that (-)-epigallocatechin-3-gallate (EGCG) inhibits signal transducer activator of transcription 1 (STAT1) activation. Since EGCG may be a promising lead compound for new anti-STAT1 drug design, 15 synthetic catechins

  4. Synergistic cooperation of MDM2 and E2F1 contributes to TAp73 transcriptional activity

    International Nuclear Information System (INIS)

    Kasim, Vivi; Huang, Can; Zhang, Jing; Jia, Huizhen; Wang, Yunxia; Yang, Li; Miyagishi, Makoto; Wu, Shourong

    2014-01-01

    Highlights: • MDM2 is a novel positive regulator of TAp73 transcriptional activity. • MDM2 colocalizes together and physically interacts with E2F1. • Synergistic cooperation of MDM2 and E2F1 is crucial for TAp73 transcription. • MDM2 regulates TAp73 transcriptional activity in a p53-independent manner. - Abstract: TAp73, a structural homologue of p53, plays an important role in tumorigenesis. E2F1 had been reported as a transcriptional regulator of TAp73, however, the detailed mechanism remains to be elucidated. Here we reported that MDM2-silencing reduced the activities of the TAp73 promoters and the endogenous TAp73 expression level significantly; while MDM2 overexpression upregulated them. We further revealed that the regulation of TAp73 transcriptional activity occurs as a synergistic effect of MDM2 and E2F1, most probably through their physical interaction in the nuclei. Furthermore, we also suggested that MDM2 might be involved in DNA damage-induced TAp73 transcriptional activity. Finally, we elucidated that MDM2-silencing reduced the proliferation rate of colon carcinoma cells regardless of the p53 status. Our data show a synergistic effect of MDM2 and E2F1 on TAp73 transcriptional activity, suggesting a novel regulation pathway of TAp73

  5. Synergistic cooperation of MDM2 and E2F1 contributes to TAp73 transcriptional activity

    Energy Technology Data Exchange (ETDEWEB)

    Kasim, Vivi, E-mail: vivikasim78@gmail.com [The Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China); Huang, Can; Zhang, Jing; Jia, Huizhen; Wang, Yunxia [The Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China); Yang, Li [The Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China); The 111 Project Laboratory of Biomechanics and Tissue Repair, College of Bioengineering, Chongqing University, Chongqing 400044 (China); Miyagishi, Makoto [Molecular Composite Medicine Research Group, Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba 305-8566 (Japan); Wu, Shourong, E-mail: shourongwu@hotmail.com [The Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China); The 111 Project Laboratory of Biomechanics and Tissue Repair, College of Bioengineering, Chongqing University, Chongqing 400044 (China)

    2014-07-04

    Highlights: • MDM2 is a novel positive regulator of TAp73 transcriptional activity. • MDM2 colocalizes together and physically interacts with E2F1. • Synergistic cooperation of MDM2 and E2F1 is crucial for TAp73 transcription. • MDM2 regulates TAp73 transcriptional activity in a p53-independent manner. - Abstract: TAp73, a structural homologue of p53, plays an important role in tumorigenesis. E2F1 had been reported as a transcriptional regulator of TAp73, however, the detailed mechanism remains to be elucidated. Here we reported that MDM2-silencing reduced the activities of the TAp73 promoters and the endogenous TAp73 expression level significantly; while MDM2 overexpression upregulated them. We further revealed that the regulation of TAp73 transcriptional activity occurs as a synergistic effect of MDM2 and E2F1, most probably through their physical interaction in the nuclei. Furthermore, we also suggested that MDM2 might be involved in DNA damage-induced TAp73 transcriptional activity. Finally, we elucidated that MDM2-silencing reduced the proliferation rate of colon carcinoma cells regardless of the p53 status. Our data show a synergistic effect of MDM2 and E2F1 on TAp73 transcriptional activity, suggesting a novel regulation pathway of TAp73.

  6. Genome-wide transcriptional analysis of two soybean genotypes under dehydration and rehydration conditions

    Science.gov (United States)

    2013-01-01

    Background Soybean is an important crop that provides valuable proteins and oils for human use. Because soybean growth and development is extremely sensitive to water deficit, quality and crop yields are severely impacted by drought stress. In the face of limited water resources, drought-responsive genes are therefore of interest. Identification and analysis of dehydration- and rehydration-inducible differentially expressed genes (DEGs) would not only aid elucidation of molecular mechanisms of stress response, but also enable improvement of crop stress tolerance via gene transfer. Using Digital Gene Expression Tag profiling (DGE), a new technique based on Illumina sequencing, we analyzed expression profiles between two soybean genotypes to identify drought-responsive genes. Results Two soybean genotypes—drought-tolerant Jindou21 and drought-sensitive Zhongdou33—were subjected to dehydration and rehydration conditions. For analysis of DEGs under dehydration conditions, 20 cDNA libraries were generated from roots and leaves at two different time points under well-watered and dehydration conditions. We also generated eight libraries for analysis under rehydration conditions. Sequencing of the 28 libraries produced 25,000–33,000 unambiguous tags, which were mapped to reference sequences for annotation of expressed genes. Many genes exhibited significant expression differences among the libraries. DEGs in the drought-tolerant genotype were identified by comparison of DEGs among treatments and genotypes. In Jindou21, 518 and 614 genes were differentially expressed under dehydration in leaves and roots, respectively, with 24 identified both in leaves and roots. The main functional categories enriched in these DEGs were metabolic process, response to stresses, plant hormone signal transduction, protein processing, and plant-pathogen interaction pathway; the associated genes primarily encoded transcription factors, protein kinases, and other regulatory proteins. The

  7. DNA Topoisomerases Maintain Promoters in a State Competent for Transcriptional Activation in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Pedersen, Jakob Madsen; Fredsøe, Jacob Christian; Rødgaard, Morten Terpager

    2012-01-01

    To investigate the role of DNA topoisomerases in transcription, we have studied global gene expression in Saccharomyces cerevisiae cells deficient for topoisomerases I and II and performed single-gene analyses to support our findings. The genome-wide studies show a general transcriptional down......-regulation upon lack of the enzymes, which correlates with gene activity but not gene length. Furthermore, our data reveal a distinct subclass of genes with a strong requirement for topoisomerases. These genes are characterized by high transcriptional plasticity, chromatin regulation, TATA box presence......-depth analysis of the inducible PHO5 gene reveals that topoisomerases are essential for binding of the Pho4p transcription factor to the PHO5 promoter, which is required for promoter nucleosome removal during activation. In contrast, topoisomerases are dispensable for constitutive transcription initiation...

  8. Role of the σ54 Activator Interacting Domain in Bacterial Transcription Initiation

    Energy Technology Data Exchange (ETDEWEB)

    Siegel, Alexander R. [Univ. of California, Berkeley, CA (United States); Wemmer, David E. [Univ. of California, Berkeley, CA (United States)

    2016-10-11

    Bacterial sigma factors are subunits of RNA polymerase that direct the holoenzyme to specific sets of promoters in the genome and are a central element of regulating transcription. Most polymerase holoenzymes open the promoter and initiate transcription rapidly after binding. However, polymerase containing the members of the σ54 family must be acted on by a transcriptional activator before DNA opening and initiation occur. A key domain in these transcriptional activators forms a hexameric AAA + ATPase that acts through conformational changes brought on by ATP hydrolysis. Contacts between the transcriptional activator and σ54 are primarily made through an N-terminal σ54 activator interacting domain (AID). To better understand this mechanism of bacterial transcription initiation, we characterized the σ54 AID by NMR spectroscopy and other biophysical methods and show that it is an intrinsically disordered domain in σ54 alone. In this paper, we identified a minimal construct of the Aquifex aeolicus σ54 AID that consists of two predicted helices and retains native-like binding affinity for the transcriptional activator NtrC1. Using the NtrC1 ATPase domain, bound with the non-hydrolyzable ATP analog ADP-beryllium fluoride, we studied the NtrC1–σ54 AID complex using NMR spectroscopy. We show that the σ54 AID becomes structured after associating with the core loops of the transcriptional activators in their ATP state and that the primary site of the interaction is the first predicted helix. Finally, understanding this complex, formed as the first step toward initiation, will help unravel the mechanism of σ54 bacterial transcription initiation.

  9. 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.

  10. 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.

  11. Ubiquitin ligase activity of TFIIH and the transcriptional response to DNA damage.

    Science.gov (United States)

    Takagi, Yuichiro; Masuda, Claudio A; Chang, Wei-Hau; Komori, Hirofumi; Wang, Dong; Hunter, Tony; Joazeiro, Claudio A P; Kornberg, Roger D

    2005-04-15

    Core transcription factor (TF) IIH purified from yeast possesses an E3 ubiquitin (Ub) ligase activity, which resides, at least in part, in a RING finger (RNF) domain of the Ssl1 subunit. Yeast strains mutated in the Ssl1 RNF domain are sensitive to ultraviolet (UV) light and to methyl methanesulfonate (MMS). This increased sensitivity to DNA-damaging agents does not reflect a deficiency in nucleotide excision repair. Rather, it correlates with reduced transcriptional induction of genes involved in DNA repair, suggesting that the E3 Ub ligase activity of TFIIH mediates the transcriptional response to DNA damage.

  12. In vivo bioimaging with tissue-specific transcription factor activated luciferase reporters.

    OpenAIRE

    Buckley, SM; Delhove, JM; Perocheau, DP; Karda, R; Rahim, AA; Howe, SJ; Ward, NJ; Birrell, MA; Belvisi, MG; Arbuthnot, P; Johnson, MR; Waddington, SN; McKay, TR

    2015-01-01

    The application of transcription factor activated luciferase reporter cassettes in vitro is widespread but potential for in vivo application has not yet been realized. Bioluminescence imaging enables non-invasive tracking of gene expression in transfected tissues of living rodents. However the mature immune response limits luciferase expression when delivered in adulthood. We present a novel approach of tissue-targeted delivery of transcription factor activated luciferase reporter lentiviruse...

  13. 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.

  14. 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...

  15. Identification of cis-regulatory sequences that activate transcription in the suspensor of plant embryos.

    Science.gov (United States)

    Kawashima, Tomokazu; Wang, Xingjun; Henry, Kelli F; Bi, Yuping; Weterings, Koen; Goldberg, Robert B

    2009-03-03

    Little is known about the molecular mechanisms by which the embryo proper and suspensor of plant embryos activate specific gene sets shortly after fertilization. We analyzed the upstream region of the scarlet runner bean (Phaseolus coccineus) G564 gene to understand how genes are activated specifically within the suspensor during early embryo development. Previously, we showed that the G564 upstream region has a block of tandem repeats, which contain a conserved 10-bp motif (GAAAAG(C)/(T)GAA), and that deletion of these repeats results in a loss of suspensor transcription. Here, we use gain-of-function (GOF) experiments with transgenic globular-stage tobacco embryos to show that only 1 of the 5 tandem repeats is required to drive suspensor-specific transcription. Fine-scale deletion and scanning mutagenesis experiments with 1 tandem repeat uncovered a 54-bp region that contains all of the sequences required to activate transcription in the suspensor, including the 10-bp motif (GAAAAGCGAA) and a similar 10-bp-like motif (GAAAAACGAA). Site-directed mutagenesis and GOF experiments indicated that both the 10-bp and 10-bp-like motifs are necessary, but not sufficient to activate transcription in the suspensor, and that a sequence (TTGGT) between the 10-bp and the 10-bp-like motifs is also necessary for suspensor transcription. Together, these data identify sequences that are required to activate transcription in the suspensor of a plant embryo after fertilization.

  16. Active isotropic slabs: conditions for amplified reflection

    Science.gov (United States)

    Perez, Liliana I.; Matteo, Claudia L.; Etcheverry, Javier; Duplaá, María Celeste

    2012-12-01

    We analyse in detail the necessary conditions to obtain amplified reflection (AR) in isotropic interfaces when a plane wave propagates from a transparent medium towards an active one. First, we demonstrate analytically that AR is not possible if a single interface is involved. Then, we study the conditions for AR in a very simple configuration: normal incidence on an active slab immersed in transparent media. Finally, we develop an analysis in the complex plane in order to establish a geometrical method that not only describes the behaviour of active slabs but also helps to simplify the calculus.

  17. Active isotropic slabs: conditions for amplified reflection

    International Nuclear Information System (INIS)

    Perez, Liliana I; Duplaá, María Celeste; Matteo, Claudia L; Etcheverry, Javier

    2012-01-01

    We analyse in detail the necessary conditions to obtain amplified reflection (AR) in isotropic interfaces when a plane wave propagates from a transparent medium towards an active one. First, we demonstrate analytically that AR is not possible if a single interface is involved. Then, we study the conditions for AR in a very simple configuration: normal incidence on an active slab immersed in transparent media. Finally, we develop an analysis in the complex plane in order to establish a geometrical method that not only describes the behaviour of active slabs but also helps to simplify the calculus. (paper)

  18. 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.

  19. Modeling post-transcriptional regulation activity of small non-coding RNAs in Escherichia coli.

    Science.gov (United States)

    Wang, Rui-Sheng; Jin, Guangxu; Zhang, Xiang-Sun; Chen, Luonan

    2009-04-29

    Transcriptional regulation is a fundamental process in biological systems, where transcription factors (TFs) have been revealed to play crucial roles. In recent years, in addition to TFs, an increasing number of non-coding RNAs (ncRNAs) have been shown to mediate post-transcriptional processes and regulate many critical pathways in both prokaryotes and eukaryotes. On the other hand, with more and more high-throughput biological data becoming available, it is possible and imperative to quantitatively study gene regulation in a systematic and detailed manner. Most existing studies for inferring transcriptional regulatory interactions and the activity of TFs ignore the possible post-transcriptional effects of ncRNAs. In this work, we propose a novel framework to infer the activity of regulators including both TFs and ncRNAs by exploring the expression profiles of target genes and (post)transcriptional regulatory relationships. We model the integrated regulatory system by a set of biochemical reactions which lead to a log-bilinear problem. The inference process is achieved by an iterative algorithm, in which two linear programming models are efficiently solved. In contrast to available related studies, the effects of ncRNAs on transcription process are considered in this work, and thus more reasonable and accurate reconstruction can be expected. In addition, the approach is suitable for large-scale problems from the viewpoint of computation. Experiments on two synthesized data sets and a model system of Escherichia coli (E. coli) carbon source transition from glucose to acetate illustrate the effectiveness of our model and algorithm. Our results show that incorporating the post-transcriptional regulation of ncRNAs into system model can mine the hidden effects from the regulation activity of TFs in transcription processes and thus can uncover the biological mechanisms in gene regulation in a more accurate manner. The software for the algorithm in this paper is available

  20. Activated AMPK inhibits PPAR-{alpha} and PPAR-{gamma} transcriptional activity in hepatoma cells.

    Science.gov (United States)

    Sozio, Margaret S; Lu, Changyue; Zeng, Yan; Liangpunsakul, Suthat; Crabb, David W

    2011-10-01

    AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor-α (PPAR-α) are critical regulators of short-term and long-term fatty acid oxidation, respectively. We examined whether the activities of these molecules were coordinately regulated. H4IIEC3 cells were transfected with PPAR-α and PPAR-γ expression plasmids and a peroxisome-proliferator-response element (PPRE) luciferase reporter plasmid. The cells were treated with PPAR agonists (WY-14,643 and rosiglitazone), AMPK activators 5-aminoimidazole-4-carboxamide riboside (AICAR) and metformin, and the AMPK inhibitor compound C. Both AICAR and metformin decreased basal and WY-14,643-stimulated PPAR-α activity; compound C increased agonist-stimulated reporter activity and partially reversed the effect of the AMPK activators. Similar effects on PPAR-γ were seen, with both AICAR and metformin inhibiting PPRE reporter activity. Compound C increased basal PPAR-γ activity and rosiglitazone-stimulated activity. In contrast, retinoic acid receptor-α (RAR-α), another nuclear receptor that dimerizes with retinoid X receptor (RXR), was largely unaffected by the AMPK activators. Compound C modestly increased AM580 (an RAR agonist)-stimulated activity. The AMPK activators did not affect PPAR-α binding to DNA, and there was no consistent correlation between effects of the AMPK activators and inhibitor on PPAR and the nuclear localization of AMPK-α subunits. Expression of either a constitutively active or dominant negative AMPK-α inhibited basal and WY-14,643-stimulated PPAR-α activity and basal and rosiglitazone-stimulated PPAR-γ activity. We concluded that the AMPK activators AICAR and metformin inhibited transcriptional activities of PPAR-α and PPAR-γ, whereas inhibition of AMPK with compound C activated both PPARs. The effects of AMPK do not appear to be mediated through effects on RXR or on PPAR/RXR binding to DNA. These effects are independent of kinase activity and instead appear to

  1. Transcriptional activation by the thyroid hormone receptor through ligand-dependent receptor recruitment and chromatin remodelling.

    Science.gov (United States)

    Grøntved, Lars; Waterfall, Joshua J; Kim, Dong Wook; Baek, Songjoon; Sung, Myong-Hee; Zhao, Li; Park, Jeong Won; Nielsen, Ronni; Walker, Robert L; Zhu, Yuelin J; Meltzer, Paul S; Hager, Gordon L; Cheng, Sheue-yann

    2015-04-28

    A bimodal switch model is widely used to describe transcriptional regulation by the thyroid hormone receptor (TR). In this model, the unliganded TR forms stable, chromatin-bound complexes with transcriptional co-repressors to repress transcription. Binding of hormone dissociates co-repressors and facilitates recruitment of co-activators to activate transcription. Here we show that in addition to hormone-independent TR occupancy, ChIP-seq against endogenous TR in mouse liver tissue demonstrates considerable hormone-induced TR recruitment to chromatin associated with chromatin remodelling and activated gene transcription. Genome-wide footprinting analysis using DNase-seq provides little evidence for TR footprints both in the absence and presence of hormone, suggesting that unliganded TR engagement with repressive complexes on chromatin is, similar to activating receptor complexes, a highly dynamic process. This dynamic and ligand-dependent interaction with chromatin is likely shared by all steroid hormone receptors regardless of their capacity to repress transcription in the absence of ligand.

  2. Regulation of gene expression by manipulating transcriptional repressor activity using a novel CoSRI technology.

    Science.gov (United States)

    Xu, Yue; Li, Song Feng; Parish, Roger W

    2017-07-01

    Targeted gene manipulation is a central strategy for studying gene function and identifying related biological processes. However, a methodology for manipulating the regulatory motifs of transcription factors is lacking as these factors commonly possess multiple motifs (e.g. repression and activation motifs) which collaborate with each other to regulate multiple biological processes. We describe a novel approach designated conserved sequence-guided repressor inhibition (CoSRI) that can specifically reduce or abolish the repressive activities of transcription factors in vivo. The technology was evaluated using the chimeric MYB80-EAR transcription factor and subsequently the endogenous WUS transcription factor. The technology was employed to develop a reversible male sterility system applicable to hybrid seed production. In order to determine the capacity of the technology to regulate the activity of endogenous transcription factors, the WUS repressor was chosen. The WUS repression motif could be inhibited in vivo and the transformed plants exhibited the wus-1 phenotype. Consequently, the technology can be used to manipulate the activities of transcriptional repressor motifs regulating beneficial traits in crop plants and other eukaryotic organisms. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  3. Transcription control and neuronal differentiation by agents that activate the LXR nuclear receptor family.

    Science.gov (United States)

    Schmidt, A; Vogel, R; Holloway, M K; Rutledge, S J; Friedman, O; Yang, Z; Rodan, G A; Friedman, E

    1999-09-10

    LXR and PPAR receptors belong to the nuclear receptor superfamily of transcriptional activating factors. Using ligand-dependent transcription assays, we found that 5-tetradecyloxy-2-furancarboxylic acid (TOFA) transactivates chimeric receptors composed of the glucocorticoid receptor DNA binding domain and the ligand binding regions of PPARalpha, PPARbeta (NUC-1) and LXRbeta (NER) receptors. In the same assays, ligands for PPARs (oleic acid, WY-14643 and L-631,033) and LXRs (hydroxycholesterols) maintain their respective receptor selectivity. TOFA and hydroxycholesterols also stimulate transcription from a minimal fibrinogen promoter that is under the control of AP-1 or NF-kappaB transcription factor binding sites. In addition to their effects on transcription, these LXRbeta activators induce neuronal differentiation in rat pheochromocytoma cells. TOFA and the natural LXR agonist, 22 (R)-hydroxycholesterol, stimulate neurite outgrowth in 55 and 28% of cells, respectively. No neurite outgrowth was induced by the related 22(S)-hydroxycholesterol, which does not activate the LXR family. These results suggest that the hydroxycholesterol signaling pathway has a complex effect on transcription that mediates the activity of TOFA and hydroxycholesterol on neuronal differentiation in pheochromocytoma cells.

  4. Design, Engineering, and Characterization of Prokaryotic Ligand-Binding Transcriptional Activators as Biosensors in Yeast

    DEFF Research Database (Denmark)

    Ambri, Francesca; Snoek, Tim; Skjødt, Mette Louise

    2018-01-01

    process. In the yeast Saccharomyces cerevisiae, implementation of allosterically regulated transcription factors from prokaryotes as metabolite biosensors has proven a valuable strategy to alleviate this screening bottleneck. Here, we present a protocol to select and incorporate prokaryotic...... transcriptional activators as metabolite biosensors in S. cerevisiae. As an example, we outline the engineering and characterization of the LysR-type transcriptional regulator (LTTR) family member BenM from Acetinobacter sp. ADP1 for monitoring accumulation of cis,cis-muconic acid, a bioplast precursor, in yeast...

  5. Targeted HIV-1 Latency Reversal Using CRISPR/Cas9-Derived Transcriptional Activator Systems.

    Directory of Open Access Journals (Sweden)

    Julia K Bialek

    Full Text Available CRISPR/Cas9 technology is currently considered the most advanced tool for targeted genome engineering. Its sequence-dependent specificity has been explored for locus-directed transcriptional modulation. Such modulation, in particular transcriptional activation, has been proposed as key approach to overcome silencing of dormant HIV provirus in latently infected cellular reservoirs. Currently available agents for provirus activation, so-called latency reversing agents (LRAs, act indirectly through cellular pathways to induce viral transcription. However, their clinical performance remains suboptimal, possibly because reservoirs have diverse cellular identities and/or proviral DNA is intractable to the induced pathways. We have explored two CRISPR/Cas9-derived activator systems as targeted approaches to induce dormant HIV-1 proviral DNA. These systems recruit multiple transcriptional activation domains to the HIV 5' long terminal repeat (LTR, for which we have identified an optimal target region within the LTR U3 sequence. Using this target region, we demonstrate transcriptional activation of proviral genomes via the synergistic activation mediator complex in various in culture model systems for HIV latency. Observed levels of induction are comparable or indeed higher than treatment with established LRAs. Importantly, activation is complete, leading to production of infective viral particles. Our data demonstrate that CRISPR/Cas9-derived technologies can be applied to counteract HIV latency and may therefore represent promising novel approaches in the quest for HIV elimination.

  6. 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 ...

  7. DNA supercoiling: changes during cellular differentiation and activation of chromatin transcription

    International Nuclear Information System (INIS)

    Luchnik, A.N.; Bakayev, V.V.; Glaser, V.M.; Moscow State Univ., USSR)

    1983-01-01

    In this paper it is reported that elastic DNA torsional tension has been observed in a fraction of isolated SV40 minichromosomes, which are shown to be transcriptionally active, and that the number of DNA topological (titratable superhelical) turns in closed superhelical loops of nuclear DNA decreases during cellular differentiation, which, we propose, may be responsible for the coordinate switch in transcription of genes controlling cellular proliferation. 37 references, 6 figures, 2 tables

  8. The Effect of Pulsed Streamer-like Discharge in Liquid on Transcriptional Activation of Retrotransposon Genes of a Red Alga, Porphyra Yezoensis

    OpenAIRE

    Ohno, T.; Li, Z.; Lin, X.F.; Zhang, W.B.; Takano, H.; Takio, S.; Namihira, T.; Akiyama, H.; オオノ, ツヨシ; ナミヒラ, タカオ; アキヤマ, ヒデノリ; 大野, 剛史; 浪平, 隆男; 秋山, 秀典

    2007-01-01

    Retrotransposons are mobile genetic elements thataccomplished transposition via an RNA intermediate.These elements can be transcriptionally activated by stressfactors, such as UV light, ozone, pathogens, woundingand drought. A red alga, porphyra yezoensis has recentlybeen recognized as a model plant for fundamental andapplied study in marine biological science. In this paper,pulsed streamer-like discharge in liquid was used as a newstress condition, and the transcription level of a copia-like...

  9. 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

  10. The Transcription Factor STAT6 Mediates Direct Repression of Inflammatory Enhancers and Limits Activation of Alternatively Polarized Macrophages

    OpenAIRE

    Czimmerer, Zsolt; Daniel, Bence; Horvath, Attila; Rückerl, Dominik; Nagy, Gergely; Kiss, Mate; Peloquin, Matthew; Budai, Marietta M.; Cuaranta-Monroy, Ixchelt; Simandi, Zoltan; Steiner, Laszlo; Nagy, Bela; Poliska, Szilard; Banko, Csaba; Bacso, Zsolt

    2018-01-01

    Summary The molecular basis of signal-dependent transcriptional activation has been extensively studied in macrophage polarization, but our understanding remains limited regarding the molecular determinants of repression. Here we show that IL-4-activated STAT6 transcription factor is required for the direct transcriptional repression of a large number of genes during in vitro and in vivo alternative macrophage polarization. Repression results in decreased lineage-determining transcription fac...

  11. 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

  12. 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.

  13. Physiological and Pathological Transcriptional Activation of Endogenous Retroelements Assessed by RNA-Sequencing of B Lymphocytes

    Directory of Open Access Journals (Sweden)

    Jan Attig

    2017-12-01

    Full Text Available In addition to evolutionarily-accrued sequence mutation or deletion, endogenous retroelements (EREs in eukaryotic genomes are subject to epigenetic silencing, preventing or reducing their transcription, particularly in the germplasm. Nevertheless, transcriptional activation of EREs, including endogenous retroviruses (ERVs and long interspersed nuclear elements (LINEs, is observed in somatic cells, variably upon cellular differentiation and frequently upon cellular transformation. ERE transcription is modulated during physiological and pathological immune cell activation, as well as in immune cell cancers. However, our understanding of the potential consequences of such modulation remains incomplete, partly due to the relative scarcity of information regarding genome-wide ERE transcriptional patterns in immune cells. Here, we describe a methodology that allows probing RNA-sequencing (RNA-seq data for genome-wide expression of EREs in murine and human cells. Our analysis of B cells reveals that their transcriptional response during immune activation is dominated by induction of gene transcription, and that EREs respond to a much lesser extent. The transcriptional activity of the majority of EREs is either unaffected or reduced by B cell activation both in mice and humans, albeit LINEs appear considerably more responsive in the latter host. Nevertheless, a small number of highly distinct ERVs are strongly and consistently induced during B cell activation. Importantly, this pattern contrasts starkly with B cell transformation, which exhibits widespread induction of EREs, including ERVs that minimally overlap with those responsive to immune stimulation. The distinctive patterns of ERE induction suggest different underlying mechanisms and will help separate physiological from pathological expression.

  14. Mediator facilitates transcriptional activation and dynamic long-range contacts at the IgH locus during class switch recombination.

    Science.gov (United States)

    Thomas-Claudepierre, Anne-Sophie; Robert, Isabelle; Rocha, Pedro P; Raviram, Ramya; Schiavo, Ebe; Heyer, Vincent; Bonneau, Richard; Luo, Vincent M; Reddy, Janardan K; Borggrefe, Tilman; Skok, Jane A; Reina-San-Martin, Bernardo

    2016-03-07

    Immunoglobulin (Ig) class switch recombination (CSR) is initiated by the transcription-coupled recruitment of activation-induced cytidine deaminase (AID) to Ig switch regions (S regions). During CSR, the IgH locus undergoes dynamic three-dimensional structural changes in which promoters, enhancers, and S regions are brought to close proximity. Nevertheless, little is known about the underlying mechanisms. In this study, we show that Med1 and Med12, two subunits of the mediator complex implicated in transcription initiation and long-range enhancer/promoter loop formation, are dynamically recruited to the IgH locus enhancers and the acceptor regions during CSR and that their knockdown in CH12 cells results in impaired CSR. Furthermore, we show that conditional inactivation of Med1 in B cells results in defective CSR and reduced acceptor S region transcription. Finally, we show that in B cells undergoing CSR, the dynamic long-range contacts between the IgH enhancers and the acceptor regions correlate with Med1 and Med12 binding and that they happen at a reduced frequency in Med1-deficient B cells. Our results implicate the mediator complex in the mechanism of CSR and are consistent with a model in which mediator facilitates the long-range contacts between S regions and the IgH locus enhancers during CSR and their transcriptional activation. © 2016 Thomas-Claudepierre et al.

  15. 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.

  16. 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

  17. New Transcriptional Reporters to Quantify and Monitor PPARγ Activity

    Directory of Open Access Journals (Sweden)

    Séverine A. Degrelle

    2017-01-01

    Full Text Available The peroxisome-proliferator-activated-receptor-γ (PPARγ is a member of the nuclear receptor superfamily that plays a critical role in diverse biological processes, including adipogenesis, lipid metabolism, and placental development. To study the activity of PPARγ, we constructed two new reporter genes: a fluorescent GFP-tagged histone-2B (PPRE-H2B-eGFP and a secreted nanoluciferase (PPRE-pNL1.3[secNluc]. This study demonstrates their usage to monitor PPARγ activity in different cell types and screen for PPARγ’s potential ligands.

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

    Directory of Open Access Journals (Sweden)

    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.

  19. Cdk1 activity acts as a quantitative platform for coordinating cell cycle progression with periodic transcription

    Science.gov (United States)

    Banyai, Gabor; Baïdi, Feriel; Coudreuse, Damien; Szilagyi, Zsolt

    2016-01-01

    Cell proliferation is regulated by cyclin-dependent kinases (Cdks) and requires the periodic expression of particular gene clusters in different cell cycle phases. However, the interplay between the networks that generate these transcriptional oscillations and the core cell cycle machinery remains largely unexplored. In this work, we use a synthetic regulable Cdk1 module to demonstrate that periodic expression is governed by quantitative changes in Cdk1 activity, with different clusters directly responding to specific activity levels. We further establish that cell cycle events neither participate in nor interfere with the Cdk1-driven transcriptional program, provided that cells are exposed to the appropriate Cdk1 activities. These findings contrast with current models that propose self-sustained and Cdk1-independent transcriptional oscillations. Our work therefore supports a model in which Cdk1 activity serves as a quantitative platform for coordinating cell cycle transitions with the expression of critical genes to bring about proper cell cycle progression. PMID:27045731

  20. 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.

  1. 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.

  2. 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.

  3. 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

  4. 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...

  5. Rational Design of Mini-Cas9 for Transcriptional Activation.

    Science.gov (United States)

    Ma, Dacheng; Peng, Shuguang; Huang, Weiren; Cai, Zhiming; Xie, Zhen

    2018-04-20

    Nuclease dead Cas9 (dCas9) has been widely used for modulating gene expression by fusing with different activation or repression domains. However, delivery of the CRISPR/Cas system fused with various effector domains in a single adeno-associated virus (AAV) remains challenging due to the payload limit. Here, we engineered a set of downsized variants of Cas9 including Staphylococcus aureus Cas9 (SaCas9) that retained DNA binding activity by deleting conserved functional domains. We demonstrated that fusing FokI nuclease domain to the N-terminal of the minimal SaCas9 (mini-SaCas9) or to the middle of the split mini-SaCas9 can trigger efficient DNA cleavage. In addition, we constructed a set of compact transactivation domains based on the tripartite VPR activation domain and self-assembled arrays of split SpyTag:SpyCatch peptides, which are suitable for fusing to the mini-SaCas9. Lastly, we produced a single AAV containing the mini-SaCas9 fused with a downsized transactivation domain along with an optimized gRNA expression cassette, which showed efficient transactivation activity. Our results highlighted a practical approach to generate down-sized CRISPR/Cas9 and gene activation systems for in vivo applications.

  6. WRKY transcription factors involved in activation of SA biosynthesis genes

    NARCIS (Netherlands)

    van Verk, Marcel C; Bol, John F; Linthorst, Huub J M

    2011-01-01

    Increased defense against a variety of pathogens in plants is achieved through activation of a mechanism known as systemic acquired resistance (SAR). The broad-spectrum resistance brought about by SAR is mediated through salicylic acid (SA). An important step in SA biosynthesis in Arabidopsis is the

  7. Expression and Activation of STAT Transcription Factors in Breast Cancer

    Science.gov (United States)

    1998-05-08

    clinicians. J~, 273: 577-585, 1995. 183 Hundertmark 5, Buhler H, Rudolf M, Weitzel HK, Ragosch V: Inhibition of 11 beta-hydroxysteroid dehydrogenase...activated protein kinase through a Jakl-dependent pathway. Mol. Cell. Bioi., 17:3833-40, 1997. Stewart JF, Rubens RO, King RJ, Minton MJ, Steiner R

  8. MAML1 enhances the transcriptional activity of Runx2 and plays a role in bone development.

    Directory of Open Access Journals (Sweden)

    Takashi Watanabe

    Full Text Available Mastermind-like 1 (MAML1 is a transcriptional co-activator in the Notch signaling pathway. Recently, however, several reports revealed novel and unique roles for MAML1 that are independent of the Notch signaling pathway. We found that MAML1 enhances the transcriptional activity of runt-related transcription factor 2 (Runx2, a transcription factor essential for osteoblastic differentiation and chondrocyte proliferation and maturation. MAML1 significantly enhanced the Runx2-mediated transcription of the p6OSE2-Luc reporter, in which luciferase expression was controlled by six copies of the osteoblast specific element 2 (OSE2 from the Runx2-regulated osteocalcin gene promoter. Interestingly, a deletion mutant of MAML1 lacking the N-terminal Notch-binding domain also enhanced Runx2-mediated transcription. Moreover, inhibition of Notch signaling did not affect the action of MAML1 on Runx2, suggesting that the activation of Runx2 by MAML1 may be caused in a Notch-independent manner. Overexpression of MAML1 transiently enhanced the Runx2-mediated expression of alkaline phosphatase, an early marker of osteoblast differentiation, in the murine pluripotent mesenchymal cell line C3H10T1/2. MAML1(-/- embryos at embryonic day 16.5 (E16.5 had shorter bone lengths than wild-type embryos. The area of primary spongiosa of the femoral diaphysis was narrowed. At E14.5, extended zone of collagen type II alpha 1 (Col2a1 and Sox9 expression, markers of chondrocyte differentiation, and decreased zone of collagen type X alpha 1 (Col10a1 expression, a marker of hypertrophic chondrocyte, were observed. These observations suggest that chondrocyte maturation was impaired in MAML1(-/- mice. MAML1 enhances the transcriptional activity of Runx2 and plays a role in bone development.

  9. Palmitoylation regulates 17β-estradiol-induced estrogen receptor-α degradation and transcriptional activity.

    Science.gov (United States)

    La Rosa, Piergiorgio; Pesiri, Valeria; Leclercq, Guy; Marino, Maria; Acconcia, Filippo

    2012-05-01

    The estrogen receptor-α (ERα) is a transcription factor that regulates gene expression through the binding to its cognate hormone 17β-estradiol (E2). ERα transcriptional activity is regulated by E2-evoked 26S proteasome-mediated ERα degradation and ERα serine (S) residue 118 phosphorylation. Furthermore, ERα mediates fast cell responses to E2 through the activation of signaling cascades such as the MAPK/ERK and phosphoinositide-3-kinase/v-akt murine thymoma viral oncogene homolog 1 pathways. These E2 rapid effects require a population of the ERα located at the cell plasma membrane through palmitoylation, a dynamic enzymatic modification mediated by palmitoyl-acyl-transferases. However, whether membrane-initiated and transcriptional ERα activities integrate in a unique picture or represent parallel pathways still remains to be firmly clarified. Hence, we evaluated here the impact of ERα palmitoylation on E2-induced ERα degradation and S118 phosphorylation. The lack of palmitoylation renders ERα more susceptible to E2-dependent degradation, blocks ERα S118 phosphorylation and prevents E2-induced ERα estrogen-responsive element-containing promoter occupancy. Consequently, ERα transcriptional activity is prevented and the receptor addressed to the nuclear matrix subnuclear compartment. These data uncover a circuitry in which receptor palmitoylation links E2-dependent ERα degradation, S118 phosphorylation, and transcriptional activity in a unique molecular mechanism. We propose that rapid E2-dependent signaling could be considered as a prerequisite for ERα transcriptional activity and suggest an integrated model of ERα intracellular signaling where E2-dependent early extranuclear effects control late receptor-dependent nuclear actions.

  10. PEA3/ETV4-related transcription factors coupled with active ERK signalling are associated with poor prognosis in gastric adenocarcinoma

    LENUS (Irish Health Repository)

    Keld, R

    2011-06-28

    Background: Transcription factors often play important roles in tumourigenesis. Members of the PEA3 subfamily of ETS-domain transcription factors fulfil such a role and have been associated with tumour metastasis in several different cancers. Moreover, the activity of the PEA3 subfamily transcription factors is potentiated by Ras-ERK pathway signalling, which is itself often deregulated in tumour cells.\\r\

  11. 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.

  12. Facilitated recruitment of Pdc2p, a yeast transcriptional activator, in response to thiamin starvation.

    Science.gov (United States)

    Nosaka, Kazuto; Esaki, Hiroyoshi; Onozuka, Mari; Konno, Hiroyuki; Hattori, Yasunao; Akaji, Kenichi

    2012-05-01

    In Saccharomyces cerevisiae, genes involved in thiamin pyrophosphate (TPP) synthesis (THI genes) and the pyruvate decarboxylase structural gene PDC5 are transcriptionally induced in response to thiamin starvation. Three positive regulatory factors (Thi2p, Thi3p, and Pdc2p) are involved in the expression of THI genes, whereas only Pdc2p is required for the expression of PDC5. Thi2p and Pdc2p serve as transcriptional activators and each factor can interact with Thi3p. The target consensus DNA sequence of Thi2p has been deduced. When TPP is not bound to Thi3p, the interactions between the regulatory factors are increased and THI gene expression is upregulated. In this study, we demonstrated that Pdc2p interacts with the upstream region of THI genes and PDC5. The association of Pdc2p or Thi2p with THI gene promoters was enhanced by thiamin starvation, suggesting that Pdc2p and Thi2p assist each other in their recruitment to the THI promoters via interaction with Thi3p. It is highly likely that, under thiamin-deprived conditions, a ternary Thi2p/Thi3p/Pdc2p complex is formed and transactivates THI genes in yeast cells. On the other hand, the association of Pdc2p with PDC5 was unaffected by thiamin. We also identified a DNA element in the upstream region of PDC5, which can bind to Pdc2p and is required for the expression of PDC5. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  13. 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.

  14. Negative Correlation between the Diffusion Coefficient and Transcriptional Activity of the Glucocorticoid Receptor.

    Science.gov (United States)

    Mikuni, Shintaro; Yamamoto, Johtaro; Horio, Takashi; Kinjo, Masataka

    2017-08-25

    The glucocorticoid receptor (GR) is a transcription factor, which interacts with DNA and other cofactors to regulate gene transcription. Binding to other partners in the cell nucleus alters the diffusion properties of GR. Raster image correlation spectroscopy (RICS) was applied to quantitatively characterize the diffusion properties of EGFP labeled human GR (EGFP-hGR) and its mutants in the cell nucleus. RICS is an image correlation technique that evaluates the spatial distribution of the diffusion coefficient as a diffusion map. Interestingly, we observed that the averaged diffusion coefficient of EGFP-hGR strongly and negatively correlated with its transcriptional activities in comparison to that of EGFP-hGR wild type and mutants with various transcriptional activities. This result suggests that the decreasing of the diffusion coefficient of hGR was reflected in the high-affinity binding to DNA. Moreover, the hyper-phosphorylation of hGR can enhance the transcriptional activity by reduction of the interaction between the hGR and the nuclear corepressors.

  15. 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.

  16. 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.

  17. 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.

  18. PEA3activates CXCL12transcription in MCF-7breast cancer cells%PEA3 activates CXCL12 transcription in MCF-7 breast cancer cells

    Institute of Scientific and Technical Information of China (English)

    CHEN Li; CHEN Bo-bin; LI Jun-jie; JIN Wei; SHAO Zhi-min

    2011-01-01

    Objective To explore the activity of PEA3 ( polyomavirus enhancer activator 3 ) on CXCL12 (Chemokine CXC motif ligand 12) transcription and to reveal the role of PEA3 involved in CXCL12-mediated metastasis and angiogenesis in breast cancer. Methods Methods such as cell transfection, ChIP assay (chromatin immunoprecipitation ), and siRNA (small interfering RNA) were applied to demonstrate and confirm the interaction between PEA3 and CXCL12. Results Over-expression of PEA3 could increase the CXCL12 mRNA level and the CXCL12 promoter activity in human MCF-7 breast cancer cells. ChIP assay demonstrated that PEA3 could bind to the CXCL12 promoter in the cells transfected with PEA3 expression vector. PEA3 siRNA decreased CXCL12 promoter activity and the binding of PEA3 to the CXCL12 promoter in MCF-7 cells. Conclusions PEA3 could activate CXCL12 promoter transcription. It may be a potential mechanism of tumor angiogenesis and metastasis regarding of PEA3 and CXCL12.

  19. Requirement of Hsp105 in CoCl{sub 2}-induced HIF-1α accumulation and transcriptional activation

    Energy Technology Data Exchange (ETDEWEB)

    Mikami, Hiroki; Saito, Youhei, E-mail: ysaito@mb.kyoto-phu.ac.jp; Okamoto, Namiko; Kakihana, Ayana; Kuga, Takahisa; Nakayama, Yuji, E-mail: nakayama@mb.kyoto-phu.ac.jp

    2017-03-15

    The mammalian stress protein Hsp105α protects cells from stress conditions. Several studies have indicated that Hsp105α is overexpressed in many types of solid tumors, which contain hypoxic microenvironments. However, the role of Hsp105α in hypoxic tumors remains largely unknown. We herein demonstrated the involvement of Hsp105α in HIF-1 functions induced by the hypoxia-mimetic agent CoCl{sub 2}. While Hsp105α is mainly localized in the cytoplasm under normal conditions, a treatment with CoCl{sub 2} induces the nuclear localization of Hsp105α, which correlated with HIF-1α expression levels. The overexpression of degradation-resistant HIF-1α enhances the nuclear localization of Hsp105α without the CoCl{sub 2} treatment. The CoCl{sub 2}-dependent transcriptional activation of HIF-1, which is measured using a reporter gene containing a HIF-responsive element, is reduced by the knockdown of Hsp105α. Furthermore, the CoCl{sub 2}-induced accumulation of HIF-1α is enhanced by heat shock, which results in the nuclear localization of Hsp105, and is suppressed by the knockdown of Hsp105. Hsp105 associates with HIF-1α in CoCl{sub 2}-treated cells. These results suggest that Hsp105α plays an important role in the functions of HIF-1 under hypoxic conditions, in which Hsp105α enhances the accumulation and transcriptional activity of HIF-1 through the HIF-1α-mediated nuclear localization of Hsp105α. - Highlights: • Hsp105α is required for the CoCl{sub 2}-induced transcriptional activation and accumulation of HIF-1. • Hsp105α localizes to the nucleus and interacts with HIF-1α in CoCl{sub 2}-treated cells. • Hsp105 enhances the CoCl{sub 2}-induced accumulation of HIF-1α under heat shock conditions.

  20. Optimization of instrumental activation analysis conditions

    International Nuclear Information System (INIS)

    Guinn, V.; Gavrilas-Guinn, M.

    1993-01-01

    In instrumental neutron activation analysis (INAA) work, a good commonsense rule of thumb is that the opium conditions for the measurement of any induced activity, in a multi-element sample matrix, are an irradiation time, a decay time, and a counting time each approximately equal to the half-life of the radionuclide (if feasible). The INAA Advance Computer Program (APCP) was used to test this rule on ten reference materials. For the 280 radionuclide/material combinations traced through all 14 APCP condition sets, the rule predicted the best set for 67% of them, was off by one set for 31% of them, and was only off by two sets of 2% of them. (author) 6 refs.; 1 fig.; 3 tabs

  1. 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...

  2. 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.

  3. 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.

  4. 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.

  5. Evaluating Transcription Factor Activity Changes by Scoring Unexplained Target Genes in Expression Data.

    Directory of Open Access Journals (Sweden)

    Evi Berchtold

    Full Text Available Several methods predict activity changes of transcription factors (TFs from a given regulatory network and measured expression data. But available gene regulatory networks are incomplete and contain many condition-dependent regulations that are not relevant for the specific expression measurement. It is not known which combination of active TFs is needed to cause a change in the expression of a target gene. A method to systematically evaluate the inferred activity changes is missing. We present such an evaluation strategy that indicates for how many target genes the observed expression changes can be explained by a given set of active TFs. To overcome the problem that the exact combination of active TFs needed to activate a gene is typically not known, we assume a gene to be explained if there exists any combination for which the predicted active TFs can possibly explain the observed change of the gene. We introduce the i-score (inconsistency score, which quantifies how many genes could not be explained by the set of activity changes of TFs. We observe that, even for these minimal requirements, published methods yield many unexplained target genes, i.e. large i-scores. This holds for all methods and all expression datasets we evaluated. We provide new optimization methods to calculate the best possible (minimal i-score given the network and measured expression data. The evaluation of this optimized i-score on a large data compendium yields many unexplained target genes for almost every case. This indicates that currently available regulatory networks are still far from being complete. Both the presented Act-SAT and Act-A* methods produce optimal sets of TF activity changes, which can be used to investigate the difficult interplay of expression and network data. A web server and a command line tool to calculate our i-score and to find the active TFs associated with the minimal i-score is available from https://services.bio.ifi.lmu.de/i-score.

  6. 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.

  7. Stat1-Vitamin D Receptor Interactions Antagonize 1,25-Dihydroxyvitamin D Transcriptional Activity and Enhance Stat1-Mediated Transcription

    Science.gov (United States)

    Vidal, Marcos; Ramana, Chilakamarti V.; Dusso, Adriana S.

    2002-01-01

    The cytokine gamma interferon (IFN-γ) and the calcitropic steroid hormone 1,25-dihydroxyvitamin D (1,25D) are activators of macrophage immune function. In sarcoidosis, tuberculosis, and several granulomatoses, IFN-γ induces 1,25D synthesis by macrophages and inhibits 1,25D induction of 24-hydroxylase, a key enzyme in 1,25D inactivation, causing high levels of 1,25D in serum and hypercalcemia. This study delineates IFN-γ-1,25D cross talk in human monocytes-macrophages. Nuclear accumulation of Stat1 and vitamin D receptor (VDR) by IFN-γ and 1,25D promotes protein-protein interactions between Stat1 and the DNA binding domain of the VDR. This prevents VDR-retinoid X receptor (RXR) binding to the vitamin D-responsive element, thus diverting the VDR from its normal genomic target on the 24-hydroxylase promoter and antagonizing 1,25D-VDR transactivation of this gene. In contrast, 1,25D enhances IFN-γ action. Stat1-VDR interactions, by preventing Stat1 deactivation by tyrosine dephosphorylation, cooperate with IFN-γ/Stat1-induced transcription. This novel 1,25D-IFN-γ cross talk explains the pathogenesis of abnormal 1,25D homeostasis in granulomatous processes and provides new insights into 1,25D immunomodulatory properties. PMID:11909970

  8. 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

  9. Targeted deficiency of the transcriptional activator Hnf1alpha alters subnuclear positioning of its genomic targets.

    Directory of Open Access Journals (Sweden)

    Reini F Luco

    2008-05-01

    Full Text Available DNA binding transcriptional activators play a central role in gene-selective regulation. In part, this is mediated by targeting local covalent modifications of histone tails. Transcriptional regulation has also been associated with the positioning of genes within the nucleus. We have now examined the role of a transcriptional activator in regulating the positioning of target genes. This was carried out with primary beta-cells and hepatocytes freshly isolated from mice lacking Hnf1alpha, an activator encoded by the most frequently mutated gene in human monogenic diabetes (MODY3. We show that in Hnf1a-/- cells inactive endogenous Hnf1alpha-target genes exhibit increased trimethylated histone H3-Lys27 and reduced methylated H3-Lys4. Inactive Hnf1alpha-targets in Hnf1a-/- cells are also preferentially located in peripheral subnuclear domains enriched in trimethylated H3-Lys27, whereas active targets in wild-type cells are positioned in more central domains enriched in methylated H3-Lys4 and RNA polymerase II. We demonstrate that this differential positioning involves the decondensation of target chromatin, and show that it is spatially restricted rather than a reflection of non-specific changes in the nuclear organization of Hnf1a-deficient cells. This study, therefore, provides genetic evidence that a single transcriptional activator can influence the subnuclear location of its endogenous genomic targets in primary cells, and links activator-dependent changes in local chromatin structure to the spatial organization of the genome. We have also revealed a defect in subnuclear gene positioning in a model of a human transcription factor disease.

  10. 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

  11. 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

  12. 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.

  13. 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

  14. High resolution analysis of the human transcriptome: detection of extensive alternative splicing independent of transcriptional activity

    Directory of Open Access Journals (Sweden)

    Rouet Fabien

    2009-10-01

    transcriptional activity, indicating that the controls for transcript generation and transcription are distinct, and require novel tools in order to detect changes in specific transcript quantity. Our results demonstrate that the SpliceArray™ design will provide researchers with a robust platform to detect and quantify specific changes not only in overall gene expression, but also at the individual transcript level.

  15. 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...

  16. Commensal Streptococcus salivarius Modulates PPARγ Transcriptional Activity in Human Intestinal Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    Benoît Couvigny

    Full Text Available The impact of commensal bacteria in eukaryotic transcriptional regulation has increasingly been demonstrated over the last decades. A multitude of studies have shown direct effects of commensal bacteria from local transcriptional activity to systemic impact. The commensal bacterium Streptococcus salivarius is one of the early bacteria colonizing the oral and gut mucosal surfaces. It has been shown to down-regulate nuclear transcription factor (NF-кB in human intestinal cells, a central regulator of the host mucosal immune system response to the microbiota. In order to evaluate its impact on a further important transcription factor shown to link metabolism and inflammation in the intestine, namely PPARγ (peroxisome proliferator-activated receptor, we used human intestinal epithelial cell-lines engineered to monitor PPARγ transcriptional activity in response to a wide range of S. salivarius strains. We demonstrated that different strains from this bacterial group share the property to inhibit PPARγ activation independently of the ligand used. First attempts to identify the nature of the active compounds showed that it is a low-molecular-weight, DNase-, proteases- and heat-resistant metabolite secreted by S. salivarius strains. Among PPARγ-targeted metabolic genes, I-FABP and Angptl4 expression levels were dramatically reduced in intestinal epithelial cells exposed to S. salivarius supernatant. Both gene products modulate lipid accumulation in cells and down-regulating their expression might consequently affect host health. Our study shows that species belonging to the salivarius group of streptococci impact both host inflammatory and metabolic regulation suggesting a possible role in the host homeostasis and health.

  17. 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.

  18. An Increase of Abundance and Transcriptional Activity for Acinetobacter junii Post Wastewater Treatment

    Directory of Open Access Journals (Sweden)

    Muhammad Raihan Jumat

    2018-04-01

    Full Text Available A membrane bioreactor (MBR-based wastewater treatment plant (WWTP in Saudi Arabia is assessed over a five-month period in 2015 and once in 2017 for bacterial diversity and transcriptional activity using metagenomics, metatranscriptomics and real time quantitative polymerase chain reaction (RT-qPCR. Acinetobacter spp. are shown to be enriched in the chlorinated effluent. Members of the Acinetobacter genus are the most abundant in the effluent and chlorinated effluent. At the species level, Acinetobacter junii have higher relative abundances post MBR and chlorination. RNA-seq analysis show that, in A. junii, 288 genes and 378 genes are significantly upregulated in the effluent and chlorinated effluent, respectively, with 98 genes being upregulated in both. RT-qPCR of samples in 2015 and 2017 confirm the upregulation observed in RNA-seq. Analysis of the 98 genes show that majority of the upregulated genes are involved in cellular repair and metabolism followed by resistance, virulence, and signaling. Additionally, two different subpopulations of A. junii are observed in the effluent and chlorinated effluent. The upregulation of cellular repair and metabolism genes, and the formation of different subpopulations of A. junii in both effluents provide insights into the mechanisms employed by A. junii to persist in the conditions of a WWTP.

  19. An Increase of Abundance and Transcriptional Activity for Acinetobacter junii Post Wastewater Treatment

    KAUST Repository

    Jumat, Muhammad

    2018-04-11

    A membrane bioreactor (MBR)-based wastewater treatment plant (WWTP) in Saudi Arabia is assessed over a five-month period in 2015 and once in 2017 for bacterial diversity and transcriptional activity using metagenomics, metatranscriptomics and real time quantitative polymerase chain reaction (RT-qPCR). Acinetobacter spp. are shown to be enriched in the chlorinated effluent. Members of the Acinetobacter genus are the most abundant in the effluent and chlorinated effluent. At the species level, Acinetobacter junii have higher relative abundances post MBR and chlorination. RNA-seq analysis show that, in A. junii, 288 genes and 378 genes are significantly upregulated in the effluent and chlorinated effluent, respectively, with 98 genes being upregulated in both. RT-qPCR of samples in 2015 and 2017 confirm the upregulation observed in RNA-seq. Analysis of the 98 genes show that majority of the upregulated genes are involved in cellular repair and metabolism followed by resistance, virulence, and signaling. Additionally, two different subpopulations of A. junii are observed in the effluent and chlorinated effluent. The upregulation of cellular repair and metabolism genes, and the formation of different subpopulations of A. junii in both effluents provide insights into the mechanisms employed by A. junii to persist in the conditions of a WWTP.

  20. An Increase of Abundance and Transcriptional Activity for Acinetobacter junii Post Wastewater Treatment

    KAUST Repository

    Jumat, Muhammad; Haroon, Muhammad; Aljassim, Nada I.; Cheng, Hong; Hong, Pei-Ying

    2018-01-01

    A membrane bioreactor (MBR)-based wastewater treatment plant (WWTP) in Saudi Arabia is assessed over a five-month period in 2015 and once in 2017 for bacterial diversity and transcriptional activity using metagenomics, metatranscriptomics and real time quantitative polymerase chain reaction (RT-qPCR). Acinetobacter spp. are shown to be enriched in the chlorinated effluent. Members of the Acinetobacter genus are the most abundant in the effluent and chlorinated effluent. At the species level, Acinetobacter junii have higher relative abundances post MBR and chlorination. RNA-seq analysis show that, in A. junii, 288 genes and 378 genes are significantly upregulated in the effluent and chlorinated effluent, respectively, with 98 genes being upregulated in both. RT-qPCR of samples in 2015 and 2017 confirm the upregulation observed in RNA-seq. Analysis of the 98 genes show that majority of the upregulated genes are involved in cellular repair and metabolism followed by resistance, virulence, and signaling. Additionally, two different subpopulations of A. junii are observed in the effluent and chlorinated effluent. The upregulation of cellular repair and metabolism genes, and the formation of different subpopulations of A. junii in both effluents provide insights into the mechanisms employed by A. junii to persist in the conditions of a WWTP.

  1. 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.

  2. 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.

  3. Condensation of chromatin in transcriptional regions of an inactivated plant transgene: evidence for an active role of transcription in gene silencing.

    Science.gov (United States)

    van Blokland, R; ten Lohuis, M; Meyer, P

    1997-12-01

    The chromatin structures of two epigenetic alleles of a transgene were investigated by measuring the local accessibility of transgene chromatin to endonucleases. The two epialleles represented the active, hypomethylated state of a transgene in line 17-I of Petunia hybrida, and a transcriptionally inactive, hypermethylated derivative of the same transgene in line 17-IV. In nuclear preparations the inactive epiallele was significantly less sensitive to DNasel digestion and nuclease S7 digestion than the transcriptionally active epiallele, whereas no significant differences in accessibility were observed between naked DNA samples of the two epialleles. Our data suggest that a condensed chromatin structure is specifically imposed on transcribed regions of the construct in line 17-IV. In contrast, in both epialleles the plasmid region of the transgene, which is not transcriptionally active in plants, retains the same accessibility to endonucleases as the chromosomal integration site. These data suggest that transcriptional inactivation is linked to the process of transcription, and imply that control of transgene expression via the use of inducible or tissue-specific promoters might prevent transgene silencing and conserve the active state of transgenes during sexual propagation.

  4. 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.

  5. Tet1 Oxidase Regulates Neuronal Gene Transcription, Active DNA Hydroxy-methylation, Object Location Memory, and Threat Recognition Memory.

    Science.gov (United States)

    Kumar, Dinesh; Aggarwal, Milan; Kaas, Garrett A; Lewis, John; Wang, Jing; Ross, Daniel L; Zhong, Chun; Kennedy, Andrew; Song, Hongjun; Sweatt, J David

    2015-10-01

    A dynamic equilibrium between DNA methylation and demethylation of neuronal activity-regulated genes is crucial for memory processes. However, the mechanisms underlying this equilibrium remain elusive. Tet1 oxidase has been shown to play a key role in the active DNA demethylation in the CNS. In this study, we used Tet1 gene knockout (Tet1KO) mice to examine the involvement of Tet1 in memory consolidation and storage in the adult brain. We found that Tet1 ablation leads to: altered expression of numerous neuronal activity-regulated genes, compensatory upregulation of active demethylation pathway genes, and upregulation of various epigenetic modifiers. Moreover, Tet1KO mice showed an enhancement in the consolidation and storage of threat recognition (cued and contextual fear conditioning) and object location memories. We conclude that Tet1 plays a critical role in regulating neuronal transcription and in maintaining the epigenetic state of the brain associated with memory consolidation and storage.

  6. Tet1 oxidase regulates neuronal gene transcription, active DNA hydroxymethylation, object location memory, and threat recognition memory

    Directory of Open Access Journals (Sweden)

    Dinesh Kumar

    2015-10-01

    Full Text Available A dynamic equilibrium between DNA methylation and demethylation of neuronal activity-regulated genes is crucial for memory processes. However, the mechanisms underlying this equilibrium remain elusive. Tet1 oxidase has been shown to play a key role in the active DNA demethylation in the central nervous system. In this study, we used Tet1 gene knockout (Tet1KO mice to examine the involvement of Tet1 in memory consolidation and storage in the adult brain. We found that Tet1 ablation leads to altered expression of numerous neuronal activity-regulated genes, compensatory upregulation of active demethylation pathway genes, and upregulation of various epigenetic modifiers. Moreover, Tet1KO mice showed an enhancement in the consolidation and storage of threat recognition (cued and contextual fear conditioning and object location memories. We conclude that Tet1 plays a critical role in regulating neuronal transcription and in maintaining the epigenetic state of the brain associated with memory consolidation and storage.

  7. 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.

  8. 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.

  9. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex.

    Science.gov (United States)

    Konermann, Silvana; Brigham, Mark D; Trevino, Alexandro E; Joung, Julia; Abudayyeh, Omar O; Barcena, Clea; Hsu, Patrick D; Habib, Naomi; Gootenberg, Jonathan S; Nishimasu, Hiroshi; Nureki, Osamu; Zhang, Feng

    2015-01-29

    Systematic interrogation of gene function requires the ability to perturb gene expression in a robust and generalizable manner. Here we describe structure-guided engineering of a CRISPR-Cas9 complex to mediate efficient transcriptional activation at endogenous genomic loci. We used these engineered Cas9 activation complexes to investigate single-guide RNA (sgRNA) targeting rules for effective transcriptional activation, to demonstrate multiplexed activation of ten genes simultaneously, and to upregulate long intergenic non-coding RNA (lincRNA) transcripts. We also synthesized a library consisting of 70,290 guides targeting all human RefSeq coding isoforms to screen for genes that, upon activation, confer resistance to a BRAF inhibitor. The top hits included genes previously shown to be able to confer resistance, and novel candidates were validated using individual sgRNA and complementary DNA overexpression. A gene expression signature based on the top screening hits correlated with markers of BRAF inhibitor resistance in cell lines and patient-derived samples. These results collectively demonstrate the potential of Cas9-based activators as a powerful genetic perturbation technology.

  10. 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.

  11. 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

  12. Therapeutic doses of irradiation activate viral transcription and induce apoptosis in HIV-1 infected cells

    International Nuclear Information System (INIS)

    Iordanskiy, Sergey; Van Duyne, Rachel; Sampey, Gavin C; Woodson, Caitlin M; Fry, Kelsi; Saifuddin, Mohammed; Guo, Jia; Wu, Yuntao; Romerio, Fabio; Kashanchi, Fatah

    2015-01-01

    The highly active antiretroviral therapy reduces HIV-1 RNA in plasma to undetectable levels. However, the virus continues to persist in the long-lived resting CD4"+ T cells, macrophages and astrocytes which form a viral reservoir in infected individuals. Reactivation of viral transcription is critical since the host immune response in combination with antiretroviral therapy may eradicate the virus. Using the chronically HIV-1 infected T lymphoblastoid and monocytic cell lines, primary quiescent CD4"+ T cells and humanized mice infected with dual-tropic HIV-1 89.6, we examined the effect of various X-ray irradiation (IR) doses (used for HIV-related lymphoma treatment and lower doses) on HIV-1 transcription and viability of infected cells. Treatment of both T cells and monocytes with IR, a well-defined stress signal, led to increase of HIV-1 transcription, as evidenced by the presence of RNA polymerase II and reduction of HDAC1 and methyl transferase SUV39H1 on the HIV-1 promoter. This correlated with the increased GFP signal and elevated level of intracellular HIV-1 RNA in the IR-treated quiescent CD4"+ T cells infected with GFP-encoding HIV-1. Exposition of latently HIV-1infected monocytes treated with PKC agonist bryostatin 1 to IR enhanced transcription activation effect of this latency-reversing agent. Increased HIV-1 replication after IR correlated with higher cell death: the level of phosphorylated Ser46 in p53, responsible for apoptosis induction, was markedly higher in the HIV-1 infected cells following IR treatment. Exposure of HIV-1 infected humanized mice with undetectable viral RNA level to IR resulted in a significant increase of HIV-1 RNA in plasma, lung and brain tissues. Collectively, these data point to the use of low to moderate dose of IR alone or in combination with HIV-1 transcription activators as a potential application for the “Shock and Kill” strategy for latently HIV-1 infected cells. - Highlights: • X-ray irradiation (IR) increases

  13. Therapeutic doses of irradiation activate viral transcription and induce apoptosis in HIV-1 infected cells

    Energy Technology Data Exchange (ETDEWEB)

    Iordanskiy, Sergey [School of Systems Biology, Laboratory of Molecular Virology, George Mason University, Manassas, VA 20110 (United States); Van Duyne, Rachel [School of Systems Biology, Laboratory of Molecular Virology, George Mason University, Manassas, VA 20110 (United States); Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702 (United States); Sampey, Gavin C; Woodson, Caitlin M; Fry, Kelsi; Saifuddin, Mohammed; Guo, Jia; Wu, Yuntao [School of Systems Biology, Laboratory of Molecular Virology, George Mason University, Manassas, VA 20110 (United States); Romerio, Fabio [Department of Medicine, University of Maryland School of Medicine, Baltimore, MD 21201 (United States); Kashanchi, Fatah, E-mail: fkashanc@gmu.edu [School of Systems Biology, Laboratory of Molecular Virology, George Mason University, Manassas, VA 20110 (United States)

    2015-11-15

    The highly active antiretroviral therapy reduces HIV-1 RNA in plasma to undetectable levels. However, the virus continues to persist in the long-lived resting CD4{sup +} T cells, macrophages and astrocytes which form a viral reservoir in infected individuals. Reactivation of viral transcription is critical since the host immune response in combination with antiretroviral therapy may eradicate the virus. Using the chronically HIV-1 infected T lymphoblastoid and monocytic cell lines, primary quiescent CD4{sup +} T cells and humanized mice infected with dual-tropic HIV-1 89.6, we examined the effect of various X-ray irradiation (IR) doses (used for HIV-related lymphoma treatment and lower doses) on HIV-1 transcription and viability of infected cells. Treatment of both T cells and monocytes with IR, a well-defined stress signal, led to increase of HIV-1 transcription, as evidenced by the presence of RNA polymerase II and reduction of HDAC1 and methyl transferase SUV39H1 on the HIV-1 promoter. This correlated with the increased GFP signal and elevated level of intracellular HIV-1 RNA in the IR-treated quiescent CD4{sup +} T cells infected with GFP-encoding HIV-1. Exposition of latently HIV-1infected monocytes treated with PKC agonist bryostatin 1 to IR enhanced transcription activation effect of this latency-reversing agent. Increased HIV-1 replication after IR correlated with higher cell death: the level of phosphorylated Ser46 in p53, responsible for apoptosis induction, was markedly higher in the HIV-1 infected cells following IR treatment. Exposure of HIV-1 infected humanized mice with undetectable viral RNA level to IR resulted in a significant increase of HIV-1 RNA in plasma, lung and brain tissues. Collectively, these data point to the use of low to moderate dose of IR alone or in combination with HIV-1 transcription activators as a potential application for the “Shock and Kill” strategy for latently HIV-1 infected cells. - Highlights: • X-ray irradiation

  14. An interplay among FIS, H-NS and guanosine tetraphosphate modulates transcription of the Escherichia coli cspA gene under physiological growth conditions

    Directory of Open Access Journals (Sweden)

    Anna eBrandi

    2016-05-01

    Full Text Available CspA, the most characterized member of the csp gene family of Escherichia coli, is highly expressed not only in response to cold stress, but also during the early phase of growth at 37°C. Here, we investigate at molecular level the antagonistic role played by the nucleoid proteins FIS and H-NS in the regulation of cspA expression under non-stress conditions. By means of both probing experiments and immunological detection, we demonstrate in vitro the existence of binding sites for these proteins on the cspA regulatory region, in which FIS and H-NS bind simultaneously to form composite DNA-protein complexes. While the in vitro promoter activity of cspA is stimulated by FIS and repressed by H-NS, a compensatory effect is observed when both proteins are added in the transcription assay. Consistently with these findings, inactivation of fis and hns genes reversely affect the in vivo amount of cspA mRNA. In addition, by means of strains expressing a high level of the alarmone guanosine tetraphosphate ((pppGpp and in vitro transcription assays, we show that the cspA promoter is sensitive to (pppGpp inhibition. The (pppGpp-mediated expression of fis and hns genes is also analyzed, thus clarifying some aspects of the regulatory loop governing cspA transcription.

  15. Overexpression of the TaSHN1 transcription factor in bread wheat leads to leaf surface modifications, improved drought tolerance and no yield penalty under controlled growth conditions.

    Science.gov (United States)

    Bi, Huihui; Shi, Jianxin; Kovalchuk, Natalia; Luang, Sukanya; Bazanova, Natalia; Chirkova, Larissa; Zhang, Dabing; Shavrukov, Yuri; Stepanenko, Anton; Tricker, Penny; Langridge, Peter; Hrmova, Maria; Lopato, Sergiy; Borisjuk, Nikolai

    2018-05-14

    Transcription factors regulate multiple networks, mediating the responses of organisms to stresses, including drought. Here we investigated the role of the wheat transcription factor TaSHN1 in crop growth and drought tolerance. TaSHN1, isolated from bread wheat, was characterised for molecular interactions and functionality. The overexpression of TaSHN1 in wheat was followed by the evaluation of T 2 and T 3 transgenic lines for drought tolerance, growth and yield components. Leaf surface changes were analysed by light microscopy, SEM, TEM and GC-MS/GC-FID. TaSHN1 behaves as a transcriptional activator in a yeast transactivation assay and binds stress-related DNA cis-elements, determinants of which were revealed using 3D molecular modelling. The overexpression of TaSHN1 in transgenic wheat did not result in a yield penalty under the controlled plant growth conditions of a glasshouse. Transgenic lines had significantly lower stomatal density and leaf water loss, and exhibited improved recovery after severe drought, compared to control plants. The comparative analysis of cuticular waxes revealed an increased accumulation of alkanes in leaves of transgenic lines. Our data demonstrate that TaSHN1 may operate as a positive modulator of drought stress tolerance. Positive attributes could be mediated through an enhanced accumulation of alkanes and reduced stomatal density. This article is protected by copyright. All rights reserved.

  16. 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.

  17. Activating transcription factor 3 promotes loss of the acinar cell phenotype in response to cerulein-induced pancreatitis in mice.

    Science.gov (United States)

    Fazio, Elena N; Young, Claire C; Toma, Jelena; Levy, Michael; Berger, Kurt R; Johnson, Charis L; Mehmood, Rashid; Swan, Patrick; Chu, Alphonse; Cregan, Sean P; Dilworth, F Jeffrey; Howlett, Christopher J; Pin, Christopher L

    2017-09-01

    Pancreatitis is a debilitating disease of the exocrine pancreas that, under chronic conditions, is a major susceptibility factor for pancreatic ductal adenocarcinoma (PDAC). Although down-regulation of genes that promote the mature acinar cell fate is required to reduce injury associated with pancreatitis, the factors that promote this repression are unknown. Activating transcription factor 3 (ATF3) is a key mediator of the unfolded protein response, a pathway rapidly activated during pancreatic insult. Using chromatin immunoprecipitation followed by next-generation sequencing, we show that ATF3 is bound to the transcriptional regulatory regions of >30% of differentially expressed genes during the initiation of pancreatitis. Of importance, ATF3-dependent regulation of these genes was observed only upon induction of pancreatitis, with pathways involved in inflammation, acinar cell differentiation, and cell junctions being specifically targeted. Characterizing expression of transcription factors that affect acinar cell differentiation suggested that acinar cells lacking ATF3 maintain a mature cell phenotype during pancreatitis, a finding supported by maintenance of junctional proteins and polarity markers. As a result, Atf3 -/- pancreatic tissue displayed increased tissue damage and inflammatory cell infiltration at early time points during injury but, at later time points, showed reduced acinar-to-duct cell metaplasia. Thus our results reveal a critical role for ATF3 as a key regulator of the acinar cell transcriptional response during injury and may provide a link between chronic pancreatitis and PDAC. © 2017 Fazio et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  18. The Calmodulin-Binding Transcription Activator CAMTA1 Is Required for Long-Term Memory Formation in Mice

    Science.gov (United States)

    Bas-Orth, Carlos; Tan, Yan-Wei; Oliveira, Ana M. M.; Bengtson, C. Peter; Bading, Hilmar

    2016-01-01

    The formation of long-term memory requires signaling from the synapse to the nucleus to mediate neuronal activity-dependent gene transcription. Synapse-to-nucleus communication is initiated by influx of calcium ions through synaptic NMDA receptors and/or L-type voltage-gated calcium channels and involves the activation of transcription factors by…

  19. The role of factor inhibiting HIF (FIH-1 in inhibiting HIF-1 transcriptional activity in glioblastoma multiforme.

    Directory of Open Access Journals (Sweden)

    Enfeng Wang

    Full Text Available Glioblastoma multiforme (GBM accounts for about 38% of primary brain tumors in the United States. GBM is characterized by extensive angiogenesis induced by vascular growth factors and cytokines. The transcription of these growth factors and cytokines is regulated by the Hypoxia-Inducible-Factor-1(HIF-1, which is a key regulator mediating the cellular response to hypoxia. It is known that Factor Inhibiting HIF-1, or FIH-1, is also involved in the cellular response to hypoxia and has the capability to physically interact with HIF-1 and block its transcriptional activity under normoxic conditions. Delineation of the regulatory role of FIH-1 will help us to better understand the molecular mechanism responsible for tumor growth and progression and may lead to the design of new therapies targeting cellular pathways in response to hypoxia. Previous studies have shown that the chromosomal region of 10q24 containing the FIH-1 gene is often deleted in GBM, suggesting a role for the FIH-1 in GBM tumorigenesis and progression. In the current study, we found that FIH-1 is able to inhibit HIF-mediated transcription of GLUT1 and VEGF-A, even under hypoxic conditions in human glioblastoma cells. FIH-1 has been found to be more potent in inhibiting HIF function than PTEN. This observation points to the possibility that deletion of 10q23-24 and loss or decreased expression of FIH-1 gene may lead to a constitutive activation of HIF-1 activity, an alteration of HIF-1 targets such as GLUT-1 and VEGF-A, and may contribute to the survival of cancer cells in hypoxia and the development of hypervascularization observed in GBM. Therefore FIH-1 can be potential therapeutic target for the treatment of GBM patients with poor prognosis.

  20. Dimer formation and transcription activation in the sporulation response regulator Spo0A.

    Science.gov (United States)

    Lewis, Richard J; Scott, David J; Brannigan, James A; Ladds, Joanne C; Cervin, Marguerite A; Spiegelman, George B; Hoggett, James G; Barák, Imrich; Wilkinson, Anthony J

    2002-02-15

    The response regulator Spo0A is the master control element in the initiation of sporulation in Bacillus subtilis. Like many other multi-domain response regulators, the latent activity of the effector, C-terminal domain is stimulated by phosphorylation on a conserved aspartic acid residue in the regulatory, N-terminal domain. If a threshold concentration of phosphorylated Spo0A is achieved, the transcription of genes required for sporulation is activated, whereas the genes encoding stationary phase sentinels are repressed, and sporulation proceeds. Despite detailed genetic, biochemical and structural characterisation, it is not understood how the phosphorylation signal in the receiver domain is transduced into DNA binding and transcription activation in the distal effector domain. An obstacle to our understanding of Spo0A function is the uncertainty concerning changes in quaternary structure that accompany phosphorylation. Here we have revisited this question and shown unequivocally that Spo0A forms dimers upon phosphorylation and that the subunit interactions in the dimer are mediated principally by the receiver domain. Purified dimers of two mutants of Spo0A, in which the phosphorylatable aspartic acid residue has been substituted, activate transcription from the spoIIG promoter in vitro, whereas monomers do not. This suggests that dimers represent the activated form of Spo0A. Copyright 2002 Elsevier Science Ltd.

  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. 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.

  3. The reverse transcription inhibitor abacavir shows anticancer activity in prostate cancer cell lines.

    Directory of Open Access Journals (Sweden)

    Francesca Carlini

    Full Text Available BACKGROUND: Transposable Elements (TEs comprise nearly 45% of the entire genome and are part of sophisticated regulatory network systems that control developmental processes in normal and pathological conditions. The retroviral/retrotransposon gene machinery consists mainly of Long Interspersed Nuclear Elements (LINEs-1 and Human Endogenous Retroviruses (HERVs that code for their own endogenous reverse transcriptase (RT. Interestingly, RT is typically expressed at high levels in cancer cells. Recent studies report that RT inhibition by non-nucleoside reverse transcriptase inhibitors (NNRTIs induces growth arrest and cell differentiation in vitro and antagonizes growth of human tumors in animal model. In the present study we analyze the anticancer activity of Abacavir (ABC, a nucleoside reverse transcription inhibitor (NRTI, on PC3 and LNCaP prostate cancer cell lines. PRINCIPAL FINDINGS: ABC significantly reduces cell growth, migration and invasion processes, considerably slows S phase progression, induces senescence and cell death in prostate cancer cells. Consistent with these observations, microarray analysis on PC3 cells shows that ABC induces specific and dose-dependent changes in gene expression, involving multiple cellular pathways. Notably, by quantitative Real-Time PCR we found that LINE-1 ORF1 and ORF2 mRNA levels were significantly up-regulated by ABC treatment. CONCLUSIONS: Our results demonstrate the potential of ABC as anticancer agent able to induce antiproliferative activity and trigger senescence in prostate cancer cells. Noteworthy, we show that ABC elicits up-regulation of LINE-1 expression, suggesting the involvement of these elements in the observed cellular modifications.

  4. 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.

  5. The cell nuclei of skeletal muscle cells are transcriptionally active in hibernating edible dormice

    Directory of Open Access Journals (Sweden)

    Muller Sylviane

    2009-03-01

    Full Text Available Abstract Background Skeletal muscle is able to react in a rapid, dynamic way to metabolic and mechanical stimuli. In particular, exposure to either prolonged starvation or disuse results in muscle atrophy. At variance, in hibernating animals muscle atrophy may be scarce or absent after bouts of hibernation i.e., periods of prolonged (months inactivity and food deprivation, and muscle function is fully preserved at arousal. In this study, myocytes from the quadriceps muscle of euthermic and hibernating edible dormice were investigated by a combination of morphological, morphometrical and immunocytochemical analyses at the light and electron microscopy level. The focus was on cell nuclei and mitochondria, which are highly sensitive markers of changing metabolic rate. Results Findings presented herein demonstrate that: 1 the general histology of the muscle, inclusive of muscle fibre shape and size, and the ratio of fast and slow fibre types are not affected by hibernation; 2 the fine structure of cytoplasmic and nuclear constituents is similar in euthermia and hibernation but for lipid droplets, which accumulate during lethargy; 3 during hibernation, mitochondria are larger in size with longer cristae, and 4 myonuclei maintain the same amount and distribution of transcripts and transcription factors as in euthermia. Conclusion In this study we demonstrate that skeletal muscle cells of the hibernating edible dormouse maintain their structural and functional integrity in full, even after months in the nest. A twofold explanation for that is envisaged: 1 the maintenance, during hibernation, of low-rate nuclear and mitochondrial activity counterbalancing myofibre wasting, 2 the intensive muscle stimulation (shivering during periodic arousals in the nest, which would mimic physical exercise. These two factors would prevent muscle atrophy usually occurring in mammals after prolonged starvation and/or inactivity as a consequence of prevailing catabolism

  6. 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.

  7. Series active power filter in power conditioning

    Energy Technology Data Exchange (ETDEWEB)

    Turunen, J.

    2009-07-01

    Power quality has become an important issue nowadays for several reasons, e.g. modern society's growing dependence on electricity and the fact that poor power quality may generate significant economic losses in few moments. Probable power quality problems are, e.g. harmonics, flicker, voltage dips and supply interruptions. The power quality may be improved by using filters and compensators.The purpose of this thesis is to research the operation of the series active power filter (SAPF) in power conditioning. Therefore, this thesis presents a comparison of three series hybrid active power filters (SHAPFs) in current harmonics filtering. In addition to this, it is shown how the voltage dip compensation performance of the SAPF is improved in a unified power quality conditioner (UPQC) application.The three SHAPFs included in the comparison are series connected topology (SCT), filter connected topology (FCT) and electrically tuned LC shunt circuit (ETLC). The operating principle of these filters is to direct the harmonic currents produced by the load to flow in the LC shunt circuits instead of the supply. In the case of the SCT this phenomenon is boosted by applying so-called active resistance in the supply branch using the SAPF. In the case of the FCT a similar action is achieved by applying the compensation voltage in series with the LC shunt circuits using the SAPF. In the case of the ETLC the performance of the LC shunt circuit is enhanced by applying so-called active inductances in series with the LC shunt circuit using the SAPF. The SHAPFs are compared by searching for their best current filtering performance using various main circuit and control system configurations and loads. The operation of the SHAPFs is first analysed mathematically. After this, the current filtering performance of the SHAPFs is inspected using simulations and experimental tests. The experimental tests are carried out using SHAPF prototypes. As a result, it is shown that the current

  8. Overexpression of the transcription factor Sp1 activates the OAS-RNAse L-RIG-I pathway.

    Directory of Open Access Journals (Sweden)

    Valéryane Dupuis-Maurin

    Full Text Available Deregulated expression of oncogenes or transcription factors such as specificity protein 1 (Sp1 is observed in many human cancers and plays a role in tumor maintenance. Paradoxically in untransformed cells, Sp1 overexpression induces late apoptosis but the early intrinsic response is poorly characterized. In the present work, we studied increased Sp1 level consequences in untransformed cells and showed that it turns on an early innate immune transcriptome. Sp1 overexpression does not activate known cellular stress pathways such as DNA damage response or endoplasmic reticulum stress, but induces the activation of the OAS-RNase L pathway and the generation of small self-RNAs, leading to the upregulation of genes of the antiviral RIG-I pathway at the transcriptional and translational levels. Finally, Sp1-induced intrinsic innate immune response leads to the production of the chemokine CXCL4 and to the recruitment of inflammatory cells in vitro and in vivo. Altogether our results showed that increased Sp1 level in untransformed cells constitutes a novel danger signal sensed by the OAS-RNase L axis leading to the activation of the RIG-I pathway. These results suggested that the OAS-RNase L-RIG-I pathway may be activated in sterile condition in absence of pathogen.

  9. Benzimidazoles diminish ERE transcriptional activity and cell growth in breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Payton-Stewart, Florastina [Department of Chemistry, College of Arts and Sciences, Xavier University of Louisiana, New Orleans, LA (United States); Tilghman, Syreeta L. [Division of Basic Pharmaceutical Sciences, College of Pharmacy, Xavier University of Louisiana, New Orleans, LA (United States); Williams, LaKeisha G. [Division of Clinical and Administrative Sciences, College of Pharmacy Xavier University of Louisiana, New Orleans, LA (United States); Winfield, Leyte L., E-mail: lwinfield@spelman.edu [Department of Chemistry, Spelman College, Atlanta, GA (United States)

    2014-08-08

    Highlights: • The methyl-substituted benzimidazole was more effective at inhibiting growth in MDA-MB 231 cells. • The naphthyl-substituted benzimidazole was more effective at inhibiting growth in MCF-7 cells than ICI. • The benzimidazole molecules demonstrated a dose-dependent reduction in ERE transcriptional activity. • The benzimidazole molecules had binding mode in ERα and ERβ comparable to that of the co-crystallized ligand. - Abstract: Estrogen receptors (ERα and ERβ) are members of the nuclear receptor superfamily. They regulate the transcription of estrogen-responsive genes and mediate numerous estrogen related diseases (i.e., fertility, osteoporosis, cancer, etc.). As such, ERs are potentially useful targets for developing therapies and diagnostic tools for hormonally responsive human breast cancers. In this work, two benzimidazole-based sulfonamides originally designed to reduce proliferation in prostate cancer, have been evaluated for their ability to modulate growth in estrogen dependent and independent cell lines (MCF-7 and MDA-MB 231) using cell viability assays. The molecules reduced growth in MCF-7 cells, but differed in their impact on the growth of MDA-MB 231 cells. Although both molecules reduced estrogen response element (ERE) transcriptional activity in a dose dependent manner, the contrasting activity in the MDA-MB-231 cells seems to suggest that the molecules may act through alternate ER-mediated pathways. Further, the methyl analog showed modest selectivity for the ERβ receptor in an ER gene expression array panel, while the naphthyl analog did not significantly alter gene expression. The molecules were docked in the ligand binding domains of the ERα-antagonist and ERβ-agonist crystal structures to evaluate the potential of the molecules to interact with the receptors. The computational analysis complimented the results obtained in the assay of transcriptional activity and gene expression suggesting that the molecules

  10. Benzimidazoles diminish ERE transcriptional activity and cell growth in breast cancer cells

    International Nuclear Information System (INIS)

    Payton-Stewart, Florastina; Tilghman, Syreeta L.; Williams, LaKeisha G.; Winfield, Leyte L.

    2014-01-01

    Highlights: • The methyl-substituted benzimidazole was more effective at inhibiting growth in MDA-MB 231 cells. • The naphthyl-substituted benzimidazole was more effective at inhibiting growth in MCF-7 cells than ICI. • The benzimidazole molecules demonstrated a dose-dependent reduction in ERE transcriptional activity. • The benzimidazole molecules had binding mode in ERα and ERβ comparable to that of the co-crystallized ligand. - Abstract: Estrogen receptors (ERα and ERβ) are members of the nuclear receptor superfamily. They regulate the transcription of estrogen-responsive genes and mediate numerous estrogen related diseases (i.e., fertility, osteoporosis, cancer, etc.). As such, ERs are potentially useful targets for developing therapies and diagnostic tools for hormonally responsive human breast cancers. In this work, two benzimidazole-based sulfonamides originally designed to reduce proliferation in prostate cancer, have been evaluated for their ability to modulate growth in estrogen dependent and independent cell lines (MCF-7 and MDA-MB 231) using cell viability assays. The molecules reduced growth in MCF-7 cells, but differed in their impact on the growth of MDA-MB 231 cells. Although both molecules reduced estrogen response element (ERE) transcriptional activity in a dose dependent manner, the contrasting activity in the MDA-MB-231 cells seems to suggest that the molecules may act through alternate ER-mediated pathways. Further, the methyl analog showed modest selectivity for the ERβ receptor in an ER gene expression array panel, while the naphthyl analog did not significantly alter gene expression. The molecules were docked in the ligand binding domains of the ERα-antagonist and ERβ-agonist crystal structures to evaluate the potential of the molecules to interact with the receptors. The computational analysis complimented the results obtained in the assay of transcriptional activity and gene expression suggesting that the molecules

  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. 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.

  13. 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.

  14. SUMOylation regulates the transcriptional repression activity of FOG-2 and its association with GATA-4.

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    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.

  15. 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.

  16. Two transcriptional activators of N-acetylserotonin O-methyltransferase 2 and melatonin biosynthesis in cassava.

    Science.gov (United States)

    Wei, Yunxie; Liu, Guoyin; Bai, Yujing; Xia, Feiyu; He, Chaozu; Shi, Haitao; Foyer, Christine

    2017-10-13

    Similar to the situation in animals, melatonin biosynthesis is regulated by four sequential enzymatic steps in plants. Although the melatonin synthesis genes have been identified in various plants, the upstream transcription factors of them remain unknown. In this study on cassava (Manihot esculenta), we found that MeWRKY79 and heat-shock transcription factor 20 (MeHsf20) targeted the W-box and the heat-stress elements (HSEs) in the promoter of N-acetylserotonin O-methyltransferase 2 (MeASMT2), respectively. The interaction between MeWRKY79, MeHsf20, and the MeASMT2 promoter was evidenced by the activation of promoter activity and chromatin immunoprecipitation (ChIP) in cassava protoplasts, and by an in vitro electrophoretic mobility shift assay (EMSA). The transcripts of MeWRKY79, MeHsf20, and MeASMT2 were all regulated by a 22-amino acid flagellin peptide (flg22) and by Xanthomonas axonopodis pv manihotis (Xam). In common with the phenotype of MeASMT2, transient expression of MeWRKY79 and MeHsf20 in Nicotiana benthamiana leaves conferred improved disease resistance. Through virus-induced gene silencing (VIGS) in cassava, we found that MeWRKY79- and MeHsf20-silenced plants showed lower transcripts of MeASMT2 and less accumulation of melatonin, which resulted in disease sensitivity that could be reversed by exogenous melatonin. Taken together, these results indicate that MeASMT2 is a target of MeWRKY79 and MeHsf20 in plant disease resistance. This study identifies novel upstream transcription factors of melatonin synthesis genes in cassava, thus extending our knowledge of the complex modulation of melatonin synthesis in plant defense. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  17. A central regulatory system largely controls transcriptional activation and repression responses to phosphate starvation in Arabidopsis.

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    Regla Bustos

    2010-09-01

    Full Text Available Plants respond to different stresses by inducing or repressing transcription of partially overlapping sets of genes. In Arabidopsis, the PHR1 transcription factor (TF has an important role in the control of phosphate (Pi starvation stress responses. Using transcriptomic analysis of Pi starvation in phr1, and phr1 phr1-like (phl1 mutants and in wild type plants, we show that PHR1 in conjunction with PHL1 controls most transcriptional activation and repression responses to phosphate starvation, regardless of the Pi starvation specificity of these responses. Induced genes are enriched in PHR1 binding sequences (P1BS in their promoters, whereas repressed genes do not show such enrichment, suggesting that PHR1(-like control of transcriptional repression responses is indirect. In agreement with this, transcriptomic analysis of a transgenic plant expressing PHR1 fused to the hormone ligand domain of the glucocorticoid receptor showed that PHR1 direct targets (i.e., displaying altered expression after GR:PHR1 activation by dexamethasone in the presence of cycloheximide corresponded largely to Pi starvation-induced genes that are highly enriched in P1BS. A minimal promoter containing a multimerised P1BS recapitulates Pi starvation-specific responsiveness. Likewise, mutation of P1BS in the promoter of two Pi starvation-responsive genes impaired their responsiveness to Pi starvation, but not to other stress types. Phylogenetic footprinting confirmed the importance of P1BS and PHR1 in Pi starvation responsiveness and indicated that P1BS acts in concert with other cis motifs. All together, our data show that PHR1 and PHL1 are partially redundant TF acting as central integrators of Pi starvation responses, both specific and generic. In addition, they indicate that transcriptional repression responses are an integral part of adaptive responses to stress.

  18. Early growth response-1 negative feedback regulates skeletal muscle postprandial insulin sensitivity via activating Ptp1b transcription.

    Science.gov (United States)

    Wu, Jing; Tao, Wei-Wei; Chong, Dan-Yang; Lai, Shan-Shan; Wang, Chuang; Liu, Qi; Zhang, Tong-Yu; Xue, Bin; Li, Chao-Jun

    2018-03-15

    Postprandial insulin desensitization plays a critical role in maintaining whole-body glucose homeostasis by avoiding the excessive absorption of blood glucose; however, the detailed mechanisms that underlie how the major player, skeletal muscle, desensitizes insulin action remain to be elucidated. Herein, we report that early growth response gene-1 ( Egr-1) is activated by insulin in skeletal muscle and provides feedback inhibition that regulates insulin sensitivity after a meal. The inhibition of the transcriptional activity of Egr-1 enhanced the phosphorylation of the insulin receptor (InsR) and Akt, thus increasing glucose uptake in L6 myotubes after insulin stimulation, whereas overexpression of Egr-1 decreased insulin sensitivity. Furthermore, deletion of Egr-1 in the skeletal muscle improved systemic insulin sensitivity and glucose tolerance, which resulted in lower blood glucose levels after refeeding. Mechanistic analysis demonstrated that EGR-1 inhibited InsR phosphorylation and glucose uptake in skeletal muscle by binding to the proximal promoter region of protein tyrosine phosphatase-1B (PTP1B) and directly activating transcription. PTP1B knockdown largely restored insulin sensitivity and enhanced glucose uptake, even under conditions of EGR-1 overexpression. Our results indicate that EGR-1/PTP1B signaling negatively regulates postprandial insulin sensitivity and suggest a potential therapeutic target for the prevention and treatment of excessive glucose absorption.-Wu, J., Tao, W.-W., Chong, D.-Y., Lai, S.-S., Wang, C., Liu, Q., Zhang, T.-Y., Xue, B., Li, C.-J. Early growth response-1 negative feedback regulates skeletal muscle postprandial insulin sensitivity via activating Ptp1b transcription.

  19. Neuronal activity-regulated gene transcription: how are distant synaptic signals conveyed to the nucleus? [v1; ref status: indexed, http://f1000r.es/TYJStu

    Directory of Open Access Journals (Sweden)

    Miriam Matamales

    2012-12-01

    Full Text Available Synaptic activity can trigger gene expression programs that are required for the stable change of neuronal properties, a process that is essential for learning and memory. Currently, it is still unclear how the stimulation of dendritic synapses can be coupled to transcription in the nucleus in a timely way given that large distances can separate these two cellular compartments. Although several mechanisms have been proposed to explain long distance communication between synapses and the nucleus, the possible co-existence of these models and their relevance in physiological conditions remain elusive. One model suggests that synaptic activation triggers the translocation to the nucleus of certain transcription regulators localised at postsynaptic sites that function as synapto-nuclear messengers. Alternatively, it has been hypothesised that synaptic activity initiates propagating regenerative intracellular calcium waves that spread through dendrites into the nucleus where nuclear transcription machinery is thereby regulated. It has also been postulated that membrane depolarisation of voltage-gated calcium channels on the somatic membrane is sufficient to increase intracellular calcium concentration and activate transcription without the need for transported signals from distant synapses. Here I provide a critical overview of the suggested mechanisms for coupling synaptic stimulation to transcription, the underlying assumptions behind them and their plausible physiological significance.

  20. Generation of knockout rabbits using transcription activator-like effector nucleases

    Directory of Open Access Journals (Sweden)

    Yu Wang

    2014-01-01

    Full Text Available Zinc-finger nucleases and transcription activator-like effector nucleases are novel gene-editing platforms contributing to redefine the boundaries of modern biological research. They are composed of a non-specific cleavage domain and a tailor made DNA-binding module, which enables a broad range of genetic modifications by inducing efficient DNA double-strand breaks at desired loci. Among other remarkable uses, these nucleases have been employed to produce gene knockouts in mid-size and large animals, such as rabbits and pigs, respectively. This approach is cost effective, relatively quick, and can produce invaluable models for human disease studies, biotechnology or agricultural purposes. Here we describe a protocol for the efficient generation of knockout rabbits using transcription activator-like effector nucleases, and a perspective of the field.

  1. Generation of knockout rabbits using transcription activator-like effector nucleases.

    Science.gov (United States)

    Wang, Yu; Fan, Nana; Song, Jun; Zhong, Juan; Guo, Xiaogang; Tian, Weihua; Zhang, Quanjun; Cui, Fenggong; Li, Li; Newsome, Philip N; Frampton, Jon; Esteban, Miguel A; Lai, Liangxue

    2014-01-01

    Zinc-finger nucleases and transcription activator-like effector nucleases are novel gene-editing platforms contributing to redefine the boundaries of modern biological research. They are composed of a non-specific cleavage domain and a tailor made DNA-binding module, which enables a broad range of genetic modifications by inducing efficient DNA double-strand breaks at desired loci. Among other remarkable uses, these nucleases have been employed to produce gene knockouts in mid-size and large animals, such as rabbits and pigs, respectively. This approach is cost effective, relatively quick, and can produce invaluable models for human disease studies, biotechnology or agricultural purposes. Here we describe a protocol for the efficient generation of knockout rabbits using transcription activator-like effector nucleases, and a perspective of the field.

  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. Activation-induced deoxycytidine deaminase (AID) co-transcriptional scanning at single-molecule resolution

    Science.gov (United States)

    Senavirathne, Gayan; Bertram, Jeffrey G.; Jaszczur, Malgorzata; Chaurasiya, Kathy R.; Pham, Phuong; Mak, Chi H.; Goodman, Myron F.; Rueda, David

    2015-12-01

    Activation-induced deoxycytidine deaminase (AID) generates antibody diversity in B cells by initiating somatic hypermutation (SHM) and class-switch recombination (CSR) during transcription of immunoglobulin variable (IgV) and switch region (IgS) DNA. Using single-molecule FRET, we show that AID binds to transcribed dsDNA and translocates unidirectionally in concert with RNA polymerase (RNAP) on moving transcription bubbles, while increasing the fraction of stalled bubbles. AID scans randomly when constrained in an 8 nt model bubble. When unconstrained on single-stranded (ss) DNA, AID moves in random bidirectional short slides/hops over the entire molecule while remaining bound for ~5 min. Our analysis distinguishes dynamic scanning from static ssDNA creasing. That AID alone can track along with RNAP during transcription and scan within stalled transcription bubbles suggests a mechanism by which AID can initiate SHM and CSR when properly regulated, yet when unregulated can access non-Ig genes and cause cancer.

  4. Differential Transcriptional Activation of Genes Encoding Soluble Methane Monooxygenase in a Facultative Versus an Obligate Methanotroph.

    Science.gov (United States)

    Smirnova, Angela V; Dunfield, Peter F

    2018-03-06

    Methanotrophs are a specialized group of bacteria that can utilize methane (CH₄) as a sole energy source. A key enzyme responsible for methane oxidation is methane monooxygenase (MMO), of either a soluble, cytoplasmic type (sMMO), or a particulate, membrane-bound type (pMMO). Methylocella silvestris BL2 and Methyloferula stellata AR4 are closely related methanotroph species that oxidize methane via sMMO only. However, Methyloferula stellata is an obligate methanotroph, while Methylocella silvestris is a facultative methanotroph able to grow on several multicarbon substrates in addition to methane. We constructed transcriptional fusions of the mmo promoters of Methyloferula stellata and Methylocella silvestris to a promoterless gfp in order to compare their transcriptional regulation in response to different growth substrates, in the genetic background of both organisms. The following patterns were observed: (1) The mmo promoter of the facultative methanotroph Methylocella silvestris was either transcriptionally downregulated or repressed by any growth substrate other than methane in the genetic background of Methylocella silvetris ; (2) Growth on methane alone upregulated the mmo promoter of Methylocella silvetris in its native background but not in the obligate methanotroph Methyloferula stellata ; (3) The mmo promoter of Methyloferula stellata was constitutive in both organisms regardless of the growth substrate, but with much lower promoter activity than the mmo promoter of Methylocella silvetris . These results support a conclusion that a different mode of transcriptional regulation of sMMO contributes to the facultative lifestyle of Methylocella silvetris compared to the obligate methanotroph Methyloferula stellata .

  5. Differential Transcriptional Activation of Genes Encoding Soluble Methane Monooxygenase in a Facultative Versus an Obligate Methanotroph

    Directory of Open Access Journals (Sweden)

    Angela V. Smirnova

    2018-03-01

    Full Text Available Methanotrophs are a specialized group of bacteria that can utilize methane (CH4 as a sole energy source. A key enzyme responsible for methane oxidation is methane monooxygenase (MMO, of either a soluble, cytoplasmic type (sMMO, or a particulate, membrane-bound type (pMMO. Methylocella silvestris BL2 and Methyloferula stellata AR4 are closely related methanotroph species that oxidize methane via sMMO only. However, Methyloferula stellata is an obligate methanotroph, while Methylocella silvestris is a facultative methanotroph able to grow on several multicarbon substrates in addition to methane. We constructed transcriptional fusions of the mmo promoters of Methyloferula stellata and Methylocella silvestris to a promoterless gfp in order to compare their transcriptional regulation in response to different growth substrates, in the genetic background of both organisms. The following patterns were observed: (1 The mmo promoter of the facultative methanotroph Methylocella silvestris was either transcriptionally downregulated or repressed by any growth substrate other than methane in the genetic background of Methylocella silvetris; (2 Growth on methane alone upregulated the mmo promoter of Methylocella silvetris in its native background but not in the obligate methanotroph Methyloferula stellata; (3 The mmo promoter of Methyloferula stellata was constitutive in both organisms regardless of the growth substrate, but with much lower promoter activity than the mmo promoter of Methylocella silvetris. These results support a conclusion that a different mode of transcriptional regulation of sMMO contributes to the facultative lifestyle of Methylocella silvetris compared to the obligate methanotroph Methyloferula stellata.

  6. FATS is a transcriptional target of p53 and associated with antitumor activity

    Directory of Open Access Journals (Sweden)

    Zhang Xifeng

    2010-09-01

    Full Text Available Abstract Frequent mutations of p53 in human cancers exemplify its crucial role as a tumor suppressor transcription factor, and p21, a transcriptional target of p53, plays a central role in surveillance of cell-cycle checkpoints. Our previous study has shown that FATS stabilize p21 to preserve genome integrity. In this study we identified a novel transcript variant of FATS (GenBank: GQ499374 through screening a cDNA library from mouse testis, which uncovered the promoter region of mouse FATS. Mouse FATS was highly expressed in testis. The p53-responsive elements existed in proximal region of both mouse and human FATS promoters. Functional study indicated that the transcription of FATS gene was activated by p53, whereas such effect was abolished by site-directed mutagenesis in the p53-RE of FATS promoter. Furthermore, the expression of FATS increased upon DNA damage in a p53-dependent manner. FATS expression was silent or downregulated in human cancers, and overexpression of FATS suppressed tumorigenicity in vivo independently of p53. Our results reveal FATS as a p53-regulated gene to monitor genomic stability.

  7. A critique on nuclear factor-kappa B and signal transducer and activator of transcription 3: The key transcription factors in periodontal pathogenesis

    Directory of Open Access Journals (Sweden)

    Ranjith Ambili

    2017-01-01

    Full Text Available Periodontal disease is initiated by microorganisms in dental plaque, and host immunoinflammatory response to the microbial challenge helps in disease progression. Conventional periodontal therapy was mainly targeted on the elimination of microbial component. However, a better understanding of molecular aspects in host response will enable the clinicians to formulate effective host modulation therapy (HMT for the periodontal management. Inflammatory mediators were the main targets for HMT in the past. Transcription factors can regulate the production of multiple mediators simultaneously, and inhibition of these factors will be more beneficial than blocking individual molecule. Two important transcription factors implicated in chronic inflammatory diseases are nuclear factor kappa B (NF-κB and signal transducers and activators of transcription 3. The role of these factors in periodontal disease is a less explored area. This comprehensive review is aimed at unveiling the critical role of NF-κB and signal transducers and activators of transcription 3 in periodontal pathogenesis. An online search was performed using MEDLINE/PubMed database. All publications till 2016 related to NF-κB, signal transducer and activator of transcription 3 (STAT3, and inflammation were included in writing this review. A total of 27,390 references were published based on the search terms used. Out of these, 507 were related to the periodontal research published in English till 2016. Relevant papers were chosen after carefully reading the abstract. This review has attempted to comprehend the existing knowledge regarding the role of transcription factors NF-κB and STAT3 in periodontal disease. Moreover, it also provides a connecting molecular link for the periodontal medicine concept.

  8. Generation of knockout rabbits using transcription activator-like effector nucleases

    OpenAIRE

    Wang, Yu; Fan, Nana; Song, Jun; Zhong, Juan; Guo, Xiaogang; Tian, Weihua; Zhang, Quanjun; Cui, Fenggong; Li, Li; Newsome, Philip N; Frampton, Jon; Esteban, Miguel A; Lai, Liangxue

    2014-01-01

    Zinc-finger nucleases and transcription activator-like effector nucleases are novel gene-editing platforms contributing to redefine the boundaries of modern biological research. They are composed of a non-specific cleavage domain and a tailor made DNA-binding module, which enables a broad range of genetic modifications by inducing efficient DNA double-strand breaks at desired loci. Among other remarkable uses, these nucleases have been employed to produce gene knockouts in mid-size and large ...

  9. 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 ...

  10. 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

  11. Object-Location Training Elicits an Overlapping but Temporally Distinct Transcriptional Profile from Contextual Fear Conditioning

    OpenAIRE

    Poplawski, Shane G.; Schoch, Hannah; Wimmer, Mathieu; Hawk, Joshua D.; Walsh, Jennifer L.; Giese, Karl P.; Abel, Ted

    2014-01-01

    Hippocampus-dependent learning is known to induce changes in gene expression, but information on gene expression differences between different learning paradigms that require the hippocampus is limited. The bulk of studies investigating RNA expression after learning use the contextual fear conditioning task, which couples a novel environment with a footshock. Although contextual fear conditioning has been useful in discovering gene targets, gene expression after spatial memory tasks has recei...

  12. 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.

  13. Presence and transcriptional activity of anaerobic fungi in agricultural biogas plants.

    Science.gov (United States)

    Dollhofer, Veronika; Callaghan, Tony M; Griffith, Gareth W; Lebuhn, Michael; Bauer, Johann

    2017-07-01

    Bioaugmentation with anaerobic fungi (AF) is promising for improved biogas generation from lignocelluloses-rich substrates. However, before implementing AF into biogas processes it is necessary to investigate their natural occurrence, community structure and transcriptional activity in agricultural biogas plants. Thus, AF were detected with three specific PCR based methods: (i) Copies of their 18S genes were found in 7 of 10 biogas plants. (ii) Transcripts of a GH5 endoglucanase gene were present at low level in two digesters, indicating transcriptional cellulolytic activity of AF. (iii) Phylogeny of the AF-community was inferred with the 28S gene. A new Piromyces species was isolated from a PCR-positive digester. Evidence for AF was only found in biogas plants operated with high proportions of animal feces. Thus, AF were most likely transferred into digesters with animal derived substrates. Additionally, high process temperatures in combination with long retention times seemed to impede AF survival and activity. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. 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

  15. Low prevalence of transcriptionally active human papilloma virus in Indian patients with HNSCC and leukoplakia.

    Science.gov (United States)

    Bhosale, Priyanka G; Pandey, Manishkumar; Desai, Rajiv S; Patil, Asawari; Kane, Shubhada; Prabhash, Kumar; Mahimkar, Manoj B

    2016-11-01

    In the present study, we comprehensively analyzed the prevalence of transcriptionally active human papilloma virus (HPV) in tissue samples of Indian patients with leukoplakia, predominantly hyperplastic lesions and head and neck squamous cell carcinoma (HNSCC). In addition, saliva samples from patients with HNSCC were screened for HPV detection. P16 overexpression was analyzed by immunohistochemistry. Tissue samples of leukoplakia (n = 121) and HNSCC (n = 427) and saliva from patients with HNSCC (n = 215) were tested for HPV using nested polymerase chain reaction. Positive samples were sequenced for subtyping. The presence of HPV E6/E7 mRNA was confirmed by RNA in situ hybridization. P16 expression and HPV DNA were not detected in any of the leukoplakia specimens. Of the 427 HNSCC tumors, 9 showed p16 overexpression and 7/427 cases were positive for HPV16 DNA, in saliva or tissue. E6/E7 mRNA positivity was observed in 8 HNSCC samples, primarily from patients with no habit of tobacco consumption. The prevalence of high-risk HPV was restricted to oropharynx and larynx, with very little concordance between p16 overexpression and HPV positivity. All patients with HPV-positive saliva samples had transcriptionally active HPV present in their tumors. The presence of HPV DNA does not necessarily reflect transcriptionally active virus in tumors; hence, it is important to consider this fact while categorizing HPV-associated tumors. Copyright © 2016 Elsevier Inc. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  18. Recent behavioral history modifies coupling between cell activity and Arc gene transcription in hippocampal CA1 neurons.

    Science.gov (United States)

    Guzowski, John F; Miyashita, Teiko; Chawla, Monica K; Sanderson, Jennifer; Maes, Levi I; Houston, Frank P; Lipa, Peter; McNaughton, Bruce L; Worley, Paul F; Barnes, Carol A

    2006-01-24

    The ability of neurons to alter their transcriptional programs in response to synaptic input is of fundamental importance to the neuroplastic mechanisms underlying learning and memory. Because of technical limitations of conventional gene detection methods, the current view of activity-dependent neural transcription derives from experiments in which neurons are assumed quiescent until a signaling stimulus is given. The present study was designed to move beyond this static model by examining how earlier episodes of neural activity influence transcription of the immediate-early gene Arc. Using a sensitive FISH method that detects primary transcript at genomic alleles, the proportion of hippocampal CA1 neurons that activate transcription of Arc RNA was constant at approximately 40% in response to both a single novel exploration session and daily sessions repeated over 9 days. This proportion is similar to the percentage of active neurons defined electrophysiologically. However, this close correspondence was disrupted in rats exposed briefly, but repeatedly, to the same environment within a single day. Arc transcription in CA1 neurons declined dramatically after as few as four 5-min sessions, despite stable electrophysiological activity during all sessions. Additional experiments indicate that the decrement in Arc transcription occurred at the cellular, rather than synaptic level, and was not simply linked to habituation to novelty. Thus, the neural genomic response is governed by recent, but not remote, cell firing history in the behaving animal. This state-dependence of neuronal transcriptional coupling provides a mechanism of metaplasticity and may regulate capacity for synaptic modification in neural networks.

  19. BFV activates the NF-κB pathway through its transactivator (BTas) to enhance viral transcription

    International Nuclear Information System (INIS)

    Wang Jian; Tan Juan; Zhang Xihui; Guo Hongyan; Zhang Qicheng; Guo Tingting; Geng Yunqi; Qiao Wentao

    2010-01-01

    Multiple families of viruses have evolved sophisticated strategies to regulate nuclear factor-κB (NF-κB) signaling, which plays a pivotal role in diverse cellular events, including virus-host interactions. In this study, we report that bovine foamy virus (BFV) is able to activate the NF-κB pathway through the action of its transactivator, BTas. Both cellular IKKβ and IκBα also participate in this activation. In addition, we demonstrate that BTas induces the processing of p100, which implies that BTas can activate NF-κB through a noncanonical pathway as well. Co-immunoprecipitation analysis shows that BTas interacts with IKK catalytic subunits (IKKα and IKKβ), which may be responsible for regulation of IKK kinase activity and persistent NF-κB activation. Furthermore, our results indicate that the level of BTas-mediated LTR transcription correlates with the activity of cellular NF-κB. Together, this study suggests that BFV activates the NF-κB pathway through BTas to enhance viral transcription.

  20. 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

  1. CITED2 modulates estrogen receptor transcriptional activity in breast cancer cells

    International Nuclear Information System (INIS)

    Lau, Wen Min; Doucet, Michele; Huang, David; Weber, Kristy L.; Kominsky, Scott L.

    2013-01-01

    Highlights: •The effects of elevated CITED2 on ER function in breast cancer cells are examined. •CITED2 enhances cell growth in the absence of estrogen and presence of tamoxifen. •CITED2 functions as a transcriptional co-activator of ER in breast cancer cells. -- Abstract: Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 (CITED2) is a member of the CITED family of non-DNA binding transcriptional co-activators of the p300/CBP-mediated transcription complex. Previously, we identified CITED2 as being overexpressed in human breast tumors relative to normal mammary epithelium. Upon further investigation within the estrogen receptor (ER)-positive subset of these breast tumor samples, we found that CITED2 mRNA expression was elevated in those associated with poor survival. In light of this observation, we investigated the effect of elevated CITED2 levels on ER function. While ectopic overexpression of CITED2 in three ER-positive breast cancer cell lines (MCF-7, T47D, and CAMA-1) did not alter cell proliferation in complete media, growth was markedly enhanced in the absence of exogenous estrogen. Correspondingly, cells overexpressing CITED2 demonstrated reduced sensitivity to the growth inhibitory effects of the selective estrogen receptor modulator, 4-hydroxytamoxifen. Subsequent studies revealed that basal ER transcriptional activity was elevated in CITED2-overexpressing cells and was further increased upon the addition of estrogen. Similarly, basal and estrogen-induced expression of the ER-regulated genes trefoil factor 1 (TFF1) and progesterone receptor (PGR) was higher in cells overexpressing CITED2. Concordant with this observation, ChIP analysis revealed higher basal levels of CITED2 localized to the TFF-1 and PGR promoters in cells with ectopic overexpression of CITED2, and these levels were elevated further in response to estrogen stimulation. Taken together, these data indicate that CITED2 functions as a transcriptional co-activator

  2. Histone H4 Lys 20 methyltransferase SET8 promotes androgen receptor-mediated transcription activation in prostate cancer

    Energy Technology Data Exchange (ETDEWEB)

    Yao, Lushuai [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Li, Yanyan; Du, Fengxia [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China); Han, Xiao [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Li, Xiaohua [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China); Niu, Yuanjie [Chawnshang Chang Sex Hormone Research Center, Tianjin Institute of Urology, Tianjin Medical University, Tianjin 300070 (China); Ren, Shancheng, E-mail: renshancheng@gmail.com [Department of Urology, Shanghai Changhai Hospital, Second Military Medical University, Shanghai 200433 (China); Sun, Yingli, E-mail: sunyl@big.ac.cn [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China)

    2014-07-18

    Highlights: • Dihydrotestosterone stimulates H4K20me1 enrichment at the PSA promoter. • SET8 promotes AR-mediated transcription activation. • SET8 interacts with AR and promotes cell proliferation. - Abstract: Histone methylation status in different lysine residues has an important role in transcription regulation. The effect of H4K20 monomethylation (H4K20me1) on androgen receptor (AR)-mediated gene transcription remains unclear. Here we show that AR agonist stimulates the enrichment of H4K20me1 and SET8 at the promoter of AR target gene PSA in an AR dependent manner. Furthermore, SET8 is crucial for the transcription activation of PSA. Co-immunoprecipitation analyses demonstrate that SET8 interacts with AR. Therefore, we conclude that SET8 is involved in AR-mediated transcription activation, possibly through its interaction with AR and H4K20me1 modification.

  3. 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.

  4. 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.

  5. 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

  6. The role of RNA polymerase I transcription and embryonic genome activation in nucleolar development in bovine preimplantation embryos

    DEFF Research Database (Denmark)

    Østrup, Olga; Strejcek, F.; Petrovicova, I.

    2008-01-01

    The aim of the present study was to investigate the role of RNA polymerase I (RPI) transcription in nucleolar development during major transcriptional activation (MTA) in cattle. Late eight-cell embryos were cultured in the absence (control group) or presence of actinomycin D (AD) (RPI inhibition...

  7. 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.

  8. Cyclin D3 interacts with vitamin D receptor and regulates its transcription activity

    International Nuclear Information System (INIS)

    Jian Yongzhi; Yan Jun; Wang Hanzhou; Chen Chen; Sun Maoyun; Jiang Jianhai; Lu Jieqiong; Yang Yanzhong; Gu Jianxin

    2005-01-01

    D-type cyclins are essential for the progression through the G1 phase of the cell cycle. Besides serving as cell cycle regulators, D-type cyclins were recently reported to have transcription regulation functions. Here, we report that cyclin D3 is a new interacting partner of vitamin D receptor (VDR), a member of the superfamily of nuclear receptors for steroid hormones, thyroid hormone, and the fat-soluble vitamins A and D. The interaction was confirmed with methods of yeast two-hybrid system, in vitro binding analysis and in vivo co-immunoprecipitation. Cyclin D3 interacted with VDR in a ligand-independent manner, but treatment of the ligand, 1,25-dihydroxyvitamin D3, strengthened the interaction. Confocal microscopy analysis showed that ligand-activated VDR led to an accumulation of cyclin D3 in the nuclear region. Cyclin D3 up-regulated transcriptional activity of VDR and this effect was counteracted by overexpression of CDK4 and CDK6. These findings provide us a new clue to understand the transcription regulation functions of D-type cyclins

  9. 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.

  10. 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

  11. 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

  12. 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...

  13. 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

  14. Fucose-Mediated Transcriptional Activation of the fcs Operon by FcsR in Streptococcus pneumoniae.

    Science.gov (United States)

    Manzoor, Irfan; Shafeeq, Sulman; Afzal, Muhammad; Kuipers, Oscar P

    2015-01-01

    In this study, we explore the impact of fucose on the transcriptome of S. pneumoniae D39. The expression of various genes and operons, including the fucose uptake PTS and utilization operon (fcs operon) was altered in the presence of fucose. By means of quantitative RT-PCR and β-galactosidase analysis, we demonstrate the role of the transcriptional regulator FcsR, present upstream of the fcs operon, as a transcriptional activator of the fcs operon. We also predict a 19-bp putative FcsR regulatory site (5'-ATTTGAACATTATTCAAGT-3') in the promoter region of the fcs operon. The functionality of this predicted FcsR regulatory site was further confirmed by promoter-truncation experiments, where deletion of half of the FscR regulatory site or full deletion led to the abolition of expression of the fcs operon. © 2015 S. Karger AG, Basel.

  15. Molecular genetic analysis of activation-tagged transcription factors thought to be involved in photomorphogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Neff, Michael M.

    2011-06-23

    This is a final report for Department of Energy Grant No. DE-FG02-08ER15927 entitled “Molecular Genetic Analysis of Activation-Tagged Transcription Factors Thought to be Involved in Photomorphogenesis”. Based on our preliminary photobiological and genetic analysis of the sob1-D mutant, we hypothesized that OBP3 is a transcription factor involved in both phytochrome and cryptochrome-mediated signal transduction. In addition, we hypothesized that OBP3 is involved in auxin signaling and root development. Based on our preliminary photobiological and genetic analysis of the sob2-D mutant, we also hypothesized that a related gene, LEP, is involved in hormone signaling and seedling development.

  16. Androgen-androgen receptor system improves chronic inflammatory conditions by suppressing monocyte chemoattractant protein-1 gene expression in adipocytes via transcriptional regulation

    Energy Technology Data Exchange (ETDEWEB)

    Morooka, Nobukatsu, E-mail: amorooka@gunma-u.ac.jp [Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi, Gunma, 371-8512 (Japan); Ueguri, Kei [Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi, Gunma, 371-8512 (Japan); Yee, Karen Kar Lye [Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi, Gunma, 371-8512 (Japan); Human Resources Cultivation Center, Gunma University, 1-5-1 Tenjin-cho, Kiryushi, Gunma, 376-8515 (Japan); Yanase, Toshihiko [Department of Endocrinology and Diabetes Mellitus, School of Medicine, Fukuoka University, Jonan-ku, Fukuoka, 814-0180 (Japan); Sato, Takashi [Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi, Gunma, 371-8512 (Japan)

    2016-09-02

    Age-related decreases in sex hormones are closely related to chronic inflammation in obesity and metabolic diseases. Particularly, the molecular basis of androgen activity in regulating inflammation and controlling metabolism remains largely unknown. Obese adipocytes secrete monocyte chemoattractant protein-1 (MCP-1), a key chemokine that promotes the infiltration of monocytes/macrophages into adipose tissue, thereby leading to metabolic disorders. Here, we studied the role of androgen-androgen receptor (AR) action in regulating MCP-1 expression in adipose tissue. We observed the induction of Mcp-1 expression in 3T3-L1 adipocytes co-cultured with RAW264.7 macrophages. Additionally, Mcp-1 expression was upregulated by culturing in conditioned medium derived from inflammatory macrophages (M1-Mφ) containing tumor necrosis factor-alpha (TNF-α). We found that sex hormones downregulated TNF-α-induced Mcp-1 and interleukin (Il)-6 expression in 3T3-L1 adipocytes. Furthermore, luciferase-reporter analysis indicated that MCP-1 promoter activity was predominantly suppressed by dihydrotestosterone (DHT)-AR interactions through functional canonical nuclear factor-kappa B (NF-κB) sites, whereas non-canonical NF-κB site containing important flanking sequences exhibited minor contributions to DHT-AR transcriptional repression. These findings suggested that androgen-AR suppressed obesity-induced chronic inflammation in adipose tissue. - Highlights: • DHT, non-aromatizable androgen suppresses Mcp-1 expression in adipocytes. • Mcp-1 transcription was negatively regulated by DHT-AR action. • DHT-AR selectively regulates Mcp-1 transcription through distinct NF-κB sites.

  17. Androgen-androgen receptor system improves chronic inflammatory conditions by suppressing monocyte chemoattractant protein-1 gene expression in adipocytes via transcriptional regulation

    International Nuclear Information System (INIS)

    Morooka, Nobukatsu; Ueguri, Kei; Yee, Karen Kar Lye; Yanase, Toshihiko; Sato, Takashi

    2016-01-01

    Age-related decreases in sex hormones are closely related to chronic inflammation in obesity and metabolic diseases. Particularly, the molecular basis of androgen activity in regulating inflammation and controlling metabolism remains largely unknown. Obese adipocytes secrete monocyte chemoattractant protein-1 (MCP-1), a key chemokine that promotes the infiltration of monocytes/macrophages into adipose tissue, thereby leading to metabolic disorders. Here, we studied the role of androgen-androgen receptor (AR) action in regulating MCP-1 expression in adipose tissue. We observed the induction of Mcp-1 expression in 3T3-L1 adipocytes co-cultured with RAW264.7 macrophages. Additionally, Mcp-1 expression was upregulated by culturing in conditioned medium derived from inflammatory macrophages (M1-Mφ) containing tumor necrosis factor-alpha (TNF-α). We found that sex hormones downregulated TNF-α-induced Mcp-1 and interleukin (Il)-6 expression in 3T3-L1 adipocytes. Furthermore, luciferase-reporter analysis indicated that MCP-1 promoter activity was predominantly suppressed by dihydrotestosterone (DHT)-AR interactions through functional canonical nuclear factor-kappa B (NF-κB) sites, whereas non-canonical NF-κB site containing important flanking sequences exhibited minor contributions to DHT-AR transcriptional repression. These findings suggested that androgen-AR suppressed obesity-induced chronic inflammation in adipose tissue. - Highlights: • DHT, non-aromatizable androgen suppresses Mcp-1 expression in adipocytes. • Mcp-1 transcription was negatively regulated by DHT-AR action. • DHT-AR selectively regulates Mcp-1 transcription through distinct NF-κB sites.

  18. 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...

  19. The Transcriptional Signature of Active Tuberculosis Reflects Symptom Status in Extra-Pulmonary and Pulmonary Tuberculosis.

    Directory of Open Access Journals (Sweden)

    Simon Blankley

    Full Text Available Mycobacterium tuberculosis infection is a leading cause of infectious death worldwide. Gene-expression microarray studies profiling the blood transcriptional response of tuberculosis (TB patients have been undertaken in order to better understand the host immune response as well as to identify potential biomarkers of disease. To date most of these studies have focused on pulmonary TB patients with gene-expression profiles of extra-pulmonary TB patients yet to be compared to those of patients with pulmonary TB or sarcoidosis.A novel cohort of patients with extra-pulmonary TB and sarcoidosis was recruited and the transcriptional response of these patients compared to those with pulmonary TB using a variety of transcriptomic approaches including testing a previously defined 380 gene meta-signature of active TB.The 380 meta-signature broadly differentiated active TB from healthy controls in this new dataset consisting of pulmonary and extra-pulmonary TB. The top 15 genes from this meta-signature had a lower sensitivity for differentiating extra-pulmonary TB from healthy controls as compared to pulmonary TB. We found the blood transcriptional responses in pulmonary and extra-pulmonary TB to be heterogeneous and to reflect the extent of symptoms of disease.The transcriptional signature in extra-pulmonary TB demonstrated heterogeneity of gene expression reflective of symptom status, while the signature of pulmonary TB was distinct, based on a higher proportion of symptomatic individuals. These findings are of importance for the rational design and implementation of mRNA based TB diagnostics.

  20. 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.

  1. Object-Location Training Elicits an Overlapping but Temporally Distinct Transcriptional Profile from Contextual Fear Conditioning

    Science.gov (United States)

    Wimmer, Mathieu; Hawk, Joshua D.; Walsh, Jennifer L.; Giese, Karl P.; Abel, Ted

    2014-01-01

    Hippocampus-dependent learning is known to induce changes in gene expression, but information on gene expression differences between different learning paradigms that require the hippocampus is limited. The bulk of studies investigating RNA expression after learning use the contextual fear conditioning task, which couples a novel environment with a footshock. Although contextual fear conditioning has been useful in discovering gene targets, gene expression after spatial memory tasks has received less attention. In this study, we used the object-location memory task and studied gene expression at two time points after learning in a high-throughput manner using a microfluidic qPCR approach. We found that expression of the classic immediate-early genes changes after object-location training in a fashion similar to that observed after contextual fear conditioning. However, the temporal dynamics of gene expression are different between the two tasks, with object-location memory producing gene expression changes that last at least 2 hours. Our findings indicate that different training paradigms may give rise to distinct temporal dynamics of gene expression after learning. PMID:25242102

  2. Object-location training elicits an overlapping but temporally distinct transcriptional profile from contextual fear conditioning.

    Science.gov (United States)

    Poplawski, Shane G; Schoch, Hannah; Wimmer, Mathieu; Hawk, Joshua D; Walsh, Jennifer L; Giese, Karl P; Abel, Ted

    2014-12-01

    Hippocampus-dependent learning is known to induce changes in gene expression, but information on gene expression differences between different learning paradigms that require the hippocampus is limited. The bulk of studies investigating RNA expression after learning use the contextual fear conditioning task, which couples a novel environment with a footshock. Although contextual fear conditioning has been useful in discovering gene targets, gene expression after spatial memory tasks has received less attention. In this study, we used the object-location memory task and studied gene expression at two time points after learning in a high-throughput manner using a microfluidic qPCR approach. We found that expression of the classic immediate-early genes changes after object-location training in a fashion similar to that observed after contextual fear conditioning. However, the temporal dynamics of gene expression are different between the two tasks, with object-location memory producing gene expression changes that last at least 2 hours. Our findings indicate that different training paradigms may give rise to distinct temporal dynamics of gene expression after learning. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Regulation of hepatitis B virus ENI enhancer activity by hepatocyte-enriched transcription factor HNF3.

    Science.gov (United States)

    Chen, M; Hieng, S; Qian, X; Costa, R; Ou, J H

    1994-11-15

    Hepatitis B virus (HBV) ENI enhancer can activate the expression of HBV and non-HBV genes in a liver-specific manner. By performing the electrophoretic mobility-shift assays, we demonstrated that the three related, liver-enriched, transcription factors, HNF3 alpha, HNF3 beta, and HNF3 gamma could all bind to the 2c site of HBV ENI enhancer. Mutations introduced in the 2c site to abolish the binding by HNF3 reduced the enhancer activity approximately 15-fold. Moreover, expression of HNF3 antisense sequences to suppress the expression of HNF3 in Huh-7 hepatoma cells led to reduction of the ENI enhancer activity. These results indicate that HNF3 positively regulates the ENI enhancer activity and this regulation is most likely mediated through the 2c site. The requirement of HNF3 for the ENI enhancer activity could explain the liver specificity of this enhancer element.

  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. Mammalian transcriptional hotspots are enriched for tissue specific enhancers near cell type specific highly expressed genes and are predicted to act as transcriptional activator hubs.

    Science.gov (United States)

    Joshi, Anagha

    2014-12-30

    Transcriptional hotspots are defined as genomic regions bound by multiple factors. They have been identified recently as cell type specific enhancers regulating developmentally essential genes in many species such as worm, fly and humans. The in-depth analysis of hotspots across multiple cell types in same species still remains to be explored and can bring new biological insights. We therefore collected 108 transcription-related factor (TF) ChIP sequencing data sets in ten murine cell types and classified the peaks in each cell type in three groups according to binding occupancy as singletons (low-occupancy), combinatorials (mid-occupancy) and hotspots (high-occupancy). The peaks in the three groups clustered largely according to the occupancy, suggesting priming of genomic loci for mid occupancy irrespective of cell type. We then characterized hotspots for diverse structural functional properties. The genes neighbouring hotspots had a small overlap with hotspot genes in other cell types and were highly enriched for cell type specific function. Hotspots were enriched for sequence motifs of key TFs in that cell type and more than 90% of hotspots were occupied by pioneering factors. Though we did not find any sequence signature in the three groups, the H3K4me1 binding profile had bimodal peaks at hotspots, distinguishing hotspots from mono-modal H3K4me1 singletons. In ES cells, differentially expressed genes after perturbation of activators were enriched for hotspot genes suggesting hotspots primarily act as transcriptional activator hubs. Finally, we proposed that ES hotspots might be under control of SetDB1 and not DNMT for silencing. Transcriptional hotspots are enriched for tissue specific enhancers near cell type specific highly expressed genes. In ES cells, they are predicted to act as transcriptional activator hubs and might be under SetDB1 control for silencing.

  6. 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.

  7. A Taiwanese Propolis Derivative Induces Apoptosis through Inducing Endoplasmic Reticular Stress and Activating Transcription Factor-3 in Human Hepatoma Cells

    Directory of Open Access Journals (Sweden)

    Fat-Moon Suk

    2013-01-01

    Full Text Available Activating transcription factor-(ATF- 3, a stress-inducible transcription factor, is rapidly upregulated under various stress conditions and plays an important role in inducing cancer cell apoptosis. NBM-TP-007-GS-002 (GS-002 is a Taiwanese propolin G (PPG derivative. In this study, we examined the antitumor effects of GS-002 in human hepatoma Hep3B and HepG2 cells in vitro. First, we found that GS-002 significantly inhibited cell proliferation and induced cell apoptosis in dose-dependent manners. Several main apoptotic indicators were found in GS-002-treated cells, such as the cleaved forms of caspase-3, caspase-9, and poly(ADP-ribose polymerase (PARP. GS-002 also induced endoplasmic reticular (ER stress as evidenced by increases in ER stress-responsive proteins including glucose-regulated protein 78 (GRP78, growth arrest- and DNA damage-inducible gene 153 (GADD153, phosphorylated eukaryotic initiation factor 2α (eIF2α, phosphorylated protein endoplasmic-reticular-resident kinase (PERK, and ATF-3. The induction of ATF-3 expression was mediated by mitogen-activated protein kinase (MAPK signaling pathways in GS-002-treated cells. Furthermore, we found that GS-002 induced more cell apoptosis in ATF-3-overexpressing cells. These results suggest that the induction of apoptosis by the propolis derivative, GS-002, is partially mediated through ER stress and ATF-3-dependent pathways, and GS-002 has the potential for development as an antitumor drug.

  8. Downregulated long non-coding RNA MEG3 in breast cancer regulates proliferation, migration and invasion by depending on p53’s transcriptional activity

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Lin [West Biostatistics and Cost-effectiveness Research Center, Medical Insurance Office, West China Hospital of Sichuan University, 610041, Sichuan (China); Li, Yu [Department of Anesthesiology, West China Hospital, Sichuan University, 610041, Sichuan (China); Yang, Bangxiang, E-mail: b19933009@qq.coom [Department of Pain Management, West China Hospital of Sichuan University, 610041, Sichuan (China)

    2016-09-09

    Long non-coding RNAs (lncRNAs) was found to play critical roles in tumorigenesis, hence, screen of tumor-related lncRNAs, identification of their biological roles is important for understanding the processes of tumorigenesis. In this study, we identified the expressing difference of several tumor-related lncRNAs in breast cancer samples and found that, MEG3, which is downregulated in non-small cell lung cancer (NSCLC) tumor tissues, is also downregulated in breast cancer samples compared with adjacent tissues. For figuring out the effect of MEG3 in breast cancer cells MCF7 and MB231, we overexpressed MEG3 in these cells, and found that it resulted the inhibition of proliferation, colony formation, migration and invasion capacities by enhancing p53’s transcriptional activity on its target genes, including p21, Maspin and KAI1. MEG3 presented similar effects in MB157, which is a p53-null breast cancer cell line, when functional p53 but not p53R273H mutant, which lacks transcriptional activity, was introduced. Surprisingly, overexpression of MEG3 activates p53’s transcriptional activity by decreasing MDM2’s transcription level, and thus stabilizes and accumulates P53. Taken together, our findings indicate that MEG3 is downregulated in breast cancer tissues and affects breast cancer cells’ malignant behaviors, which indicate MEG3 a potential therapeutic target for breast cancer. - Highlights: • MEG3 RNA is widely downregulated in breast tumor tissue. • MEG3 regulates P53 indirectly through transcriptional regulation of MDM2. • Under unstressed condition, MEG3-related P53 accumulation transcriptionally activates p53’s target genes. • MEG3 expression level tightly regulates proliferation, colony formation, migration and invasion in breast tumor cells.

  9. 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.

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

    Directory of Open Access Journals (Sweden)

    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

  11. Transcriptional Response of Desulfovibrio vulgaris Hildenborough to Oxidative Stress Mimicking Environmental Conditions

    Energy Technology Data Exchange (ETDEWEB)

    Pereira, Patricia M.; He, Qiang; Xavier, Antonio V.; Zhou, Jizhong; Pereira, Ines A.C.; Louro, Ricardo O.

    2008-03-12

    Sulphate-reducing bacteria are anaerobes readily found in oxic-anoxic interfaces. Multiple defence pathways against oxidative conditions were identified in these organisms and proposed to be differentially expressed under different concentrations of oxygen, contributing to their ability to survive oxic conditions. In this study, Desulfovibrio vulgaris Hildenborough cells were exposed to the highest concentration of oxygen that sulphate-reducing bacteria are likely to encounter in natural habitats, and the global transcriptomic response was determined. 307 genes were responsive, with cellular roles in energy metabolism, protein fate, cell envelope and regulatory functions, including multiple genes encoding heat shock proteins, peptidases and proteins with heat shock promoters. Of the oxygen reducing mechanisms of D. vulgaris only the periplasmic hydrogen-dependent mechanism is up-regulated, involving the [NiFeSe]hydrogenase, formate dehydrogenase(s) and the Hmc membrane complex. The oxidative defence response concentrates on damage repair by metal-free enzymes. These data, together with the down regulation of the Fur operon, which restricts the availability of iron, and the lack of response of the PerR operon, suggest that a major effect of this oxygen stress is the inactivation and/or degradation of multiple metalloproteins present in D. vulgaris as a consequence of oxidative damage to their metal clusters.

  12. Epigenetic mediated transcriptional activation of WNT5A participates in arsenical-associated malignant transformation

    International Nuclear Information System (INIS)

    Jensen, Taylor J.; Wozniak, Ryan J.; Eblin, Kylee E.; Wnek, Sean M.; Gandolfi, A. Jay; Futscher, Bernard W.

    2009-01-01

    Arsenic is a human carcinogen with exposure associated with cancer of the lung, skin, and bladder. Many potential mechanisms have been implicated as playing a role in the process of arsenical-induced malignancy including the perturbation of signaling pathways and aberrant epigenetic regulation. We initiated studies to examine the role of a member of the non-canonical WNT signaling pathway, WNT5A, in UROtsa cells and arsenite [URO-ASSC] and monomethylarsonous acid [URO-MSC] malignantly transformed variants. We present data herein that suggest that WNT5A is transcriptionally activated during arsenical-induced malignant transformation. This WNT5A transcriptional activation is correlated with the enrichment of permissive histone modifications and the reduction of repressive modifications in the WNT5A promoter region. The epigenetic activation of WNT5A expression and acetylation of its promoter remain after the removal of the arsenical, consistent with the maintenance of an anchorage independent growth phenotype in these cells. Additionally, treatment with epigenetic modifying drugs supports a functional role for these epigenetic marks in controlling gene expression. Reduction of WNT5A using lentiviral shRNA greatly attenuated the ability of these cells to grow in an anchorage independent fashion. Extension of our model into human bladder cancer cell lines indicates that each of the cell lines examined also express WNT5A. Taken together, these data suggest that the epigenetic remodeling of the WNT5A promoter is correlated with its transcriptional activation and this upregulation likely participates in arsenical-induced malignant transformation

  13. 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.

  14. Localizing potentially active post-transcriptional regulations in the Ewing's sarcoma gene regulatory network

    Directory of Open Access Journals (Sweden)

    Delyon Bernard

    2010-11-01

    Full Text Available Abstract Background A wide range of techniques is now available for analyzing regulatory networks. Nonetheless, most of these techniques fail to interpret large-scale transcriptional data at the post-translational level. Results We address the question of using large-scale transcriptomic observation of a system perturbation to analyze a regulatory network which contained several types of interactions - transcriptional and post-translational. Our method consisted of post-processing the outputs of an open-source tool named BioQuali - an automatic constraint-based analysis mimicking biologist's local reasoning on a large scale. The post-processing relied on differences in the behavior of the transcriptional and post-translational levels in the network. As a case study, we analyzed a network representation of the genes and proteins controlled by an oncogene in the context of Ewing's sarcoma. The analysis allowed us to pinpoint active interactions specific to this cancer. We also identified the parts of the network which were incomplete and should be submitted for further investigation. Conclusions The proposed approach is effective for the qualitative analysis of cancer networks. It allows the integrative use of experimental data of various types in order to identify the specific information that should be considered a priority in the initial - and possibly very large - experimental dataset. Iteratively, new dataset can be introduced into the analysis to improve the network representation and make it more specific.

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

    Directory of Open Access Journals (Sweden)

    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.

  16. Transcriptional Activity of Nuclear Factor κB Family Genes in Patients with Systemic Sclerosis.

    Science.gov (United States)

    Lis-Święty, Anna; Gola, Joanna; Mazurek, Urszula; Brzezińska-Wcisło, Ligia

    2017-05-01

    Systemic sclerosis (SSc) is a connective tissue disease of unknown etiology and unclear pathogenesis. Evaluation of the activation of nuclear factor κB (NF-κB) family genes IκBα, p50, p52, p65, and c-Rel, potentially involved in the regulation of immunity, inflammation, angiogenesis, and tissue remodeling in SSc, was carried out. The study included 19 patients with limited SSc, 11 patients with early SSc, and 10 healthy persons constituting the control group. Real-time QRT-PCR was used to evaluate the mRNAs in peripheral blood samples. The patients with early SSc showed a decrease in transcriptional activity of IκBα inhibitor and c-Rel subunit. Transcriptional activity decrease in the other patients with limited SSc included genes encoding c-Rel and p50, subunits of NF-κB factor. Deregulation of intracellular signal transduction by NF-κB takes place at the beginning of SSc and in its fibrosis stage. Associations between clinical variables and NF-κB related gene expression as well as the activation of NF-κB family members in SSc patients should be addressed in future studies. © 2017 by the Association of Clinical Scientists, Inc.

  17. 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.

  18. Major differences observed in transcript profiles of blueberry during cold acclimation under field and cold room conditions.

    Science.gov (United States)

    Dhanaraj, Anik L; Alkharouf, Nadim W; Beard, Hunter S; Chouikha, Imed B; Matthews, Benjamin F; Wei, Hui; Arora, Rajeev; Rowland, Lisa J

    2007-02-01

    Our laboratory has been working toward increasing our understanding of the genetic control of cold hardiness in blueberry (Vaccinium section Cyanococcus) to ultimately use this information to develop more cold hardy cultivars for the industry. Here, we report using cDNA microarrays to monitor changes in gene expression at multiple times during cold acclimation under field and cold room conditions. Microarrays contained over 2,500 cDNA inserts, approximately half of which had been picked and single-pass sequenced from each of two cDNA libraries that were constructed from cold acclimated floral buds and non-acclimated floral buds of the fairly cold hardy cv. Bluecrop (Vaccinium corymbosum L.). Two biological samples were examined at each time point. Microarray data were analyzed statistically using t tests, ANOVA, clustering algorithms, and online analytical processing (OLAP). Interestingly, more transcripts were found to be upregulated under cold room conditions than under field conditions. Many of the genes induced only under cold room conditions could be divided into three major types: (1) genes associated with stress tolerance; (2) those that encode glycolytic and TCA cycle enzymes, and (3) those associated with protein synthesis machinery. A few of the genes induced only under field conditions appear to be related to light stress. Possible explanations for these differences are discussed in physiological context. Although many similarities exist in how plants respond during cold acclimation in the cold room and in the field environment, there are major differences suggesting caution should be taken in interpreting results based only on artificial, cold room conditions.

  19. Specificity and transcriptional activity of microbiota associated with low and high microbial abundance sponges from the Red Sea

    KAUST Repository

    Moitinho-Silva, Lucas; Bayer, Kristina; Cannistraci, Carlo; Giles, Emily; Ryu, Tae Woo; Seridi, Loqmane; Ravasi, Timothy; Hentschel, Ute T E

    2013-01-01

    Marine sponges are generally classified as high microbial abundance (HMA) and low microbial abundance (LMA) species. Here, 16S rRNA amplicon sequencing was applied to investigate the diversity, specificity and transcriptional activity of microbes

  20. Differential binding activity of the transcription factor LIL-Stat in immature and differentiated normal and leukemic myeloid cells

    NARCIS (Netherlands)

    Tuyt, LML; Bregman, K; Lummen, C; Dokter, WHA; Vellenga, E

    1998-01-01

    Cytokines and growth factors induce activation of the family of signal transducers and activators of transcription (Stats) that directly activate gene expression. Recently, constitutively activated Stat1, Stat3, and Stat5 were identified in nuclear extracts of acute myeloid leukemia (AML) patients,

  1. 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.

  2. 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.

  3. 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.

  4. Structural Features and Transcriptional Activity of Chicken PPARs (α, β, and γ

    Directory of Open Access Journals (Sweden)

    Ichiro Takada

    2013-01-01

    Full Text Available While an understanding of lipid metabolism in chickens is critical for a further improvement of food production, there are few studies concerning differences in lipid metabolism mechanisms between chickens and other species at a molecular level. Chickens have three PPAR gene subtypes (α, β, and γ that function differently from those present in humans and mice. The chicken PPAR-gamma (cPPARγ gene is shorter than that in humans and lacks a γ2 isoform. Moreover, in serum-free media, cPPARγ shows high transcriptional activity without exogenous ligands. Luciferase reporter assays were used to examine the effect of sera on cPPAR transcriptional activities and showed that adult bovine serum and chicken serum highly activate cPPARα and β functions. Moreover, we found that bezafibrate induces the transactivation function of cPPARβ, but not human PPARδ (human PPARβ ortholog. This ligand selectivity relies on one amino acid residue (chicken: Val419, human: Met444. These results show the possibilities for unique functions of cPPARs on chicken-specific lipid glucose metabolism. As such, a better understanding of the molecular mechanisms of lipid metabolism in chickens could result in higher productivity for the poultry industry.

  5. 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.

  6. 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

  7. Exercise induces transient transcriptional activation of the PGC-1a gene in human skeletal muscle

    DEFF Research Database (Denmark)

    Pilegaard, Henriette; Saltin, Bengt; Neufer, P. Darrell

    2003-01-01

    Endurance exercise training induces mitochondrial biogenesis in skeletal muscle. The peroxisome proliferator activated receptor co-activator 1a (PGC-1a) has recently been identified as a nuclear factor critical for coordinating the activation of genes required for mitochondrial biogenesis in cell...... culture and rodent skeletal muscle. To determine whether PGC-1a transcription is regulated by acute exercise and exercise training in human skeletal muscle, seven male subjects performed 4 weeks of one-legged knee extensor exercise training. At the end of training, subjects completed 3 h of two......-legged knee extensor exercise. Biopsies were obtained from the vastus lateralis muscle of both the untrained and trained legs before exercise and after 0, 2, 6 and 24 h of recovery. Time to exhaustion (2 min maximum resistance), as well as hexokinase II (HKII), citrate synthase and 3-hydroxyacyl...

  8. 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.

  9. 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.

  10. 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

  11. SUMOylation of the Forkhead transcription factor FOXL2 promotes its stabilization/activation through transient recruitment to PML bodies.

    Directory of Open Access Journals (Sweden)

    Adrien Georges

    Full Text Available BACKGROUND: FOXL2 is a transcription factor essential for ovarian development and maintenance. It is mutated in the genetic condition called Blepharophimosis Ptosis Epicantus inversus Syndrome (BPES and in cases of isolated premature ovarian failure. We and others have previously shown that FOXL2 undergoes several post-translational modifications. METHODS AND PRINCIPAL FINDINGS: Here, using cells in culture, we show that interference with FOXL2 SUMOylation leads to a robust inhibition of its transactivation ability, which correlates with a decreased stability. Interestingly, FOXL2 SUMOylation promotes its transient recruitment to subnuclear structures that we demonstrate to be PML (Promyelocytic Leukemia Nuclear Bodies. Since PML bodies are known to be sites where post-translational modifications of nuclear factors take place, we used tandem mass spectrometry to identify new post-translational modifications of FOXL2. Specifically, we detected four phosphorylated, one sulfated and three acetylated sites. CONCLUSIONS: By analogy with other transcription factors, we propose that PML Nuclear Bodies might transiently recruit FOXL2 to the vicinity of locally concentrated enzymes that could be involved in the post-translational maturation of FOXL2. FOXL2 acetylation, sulfation, phosphorylation as well as other modifications yet to be discovered might alter the transactivation capacity of FOXL2 and/or its stability, thus modulating its global intracellular activity.

  12. Identifying significant genetic regulatory networks in the prostate cancer from microarray data based on transcription factor analysis and conditional independency

    Directory of Open Access Journals (Sweden)

    Yeh Cheng-Yu

    2009-12-01

    Full Text Available Abstract Background Prostate cancer is a world wide leading cancer and it is characterized by its aggressive metastasis. According to the clinical heterogeneity, prostate cancer displays different stages and grades related to the aggressive metastasis disease. Although numerous studies used microarray analysis and traditional clustering method to identify the individual genes during the disease processes, the important gene regulations remain unclear. We present a computational method for inferring genetic regulatory networks from micorarray data automatically with transcription factor analysis and conditional independence testing to explore the potential significant gene regulatory networks that are correlated with cancer, tumor grade and stage in the prostate cancer. Results To deal with missing values in microarray data, we used a K-nearest-neighbors (KNN algorithm to determine the precise expression values. We applied web services technology to wrap the bioinformatics toolkits and databases to automatically extract the promoter regions of DNA sequences and predicted the transcription factors that regulate the gene expressions. We adopt the microarray datasets consists of 62 primary tumors, 41 normal prostate tissues from Stanford Microarray Database (SMD as a target dataset to evaluate our method. The predicted results showed that the possible biomarker genes related to cancer and denoted the androgen functions and processes may be in the development of the prostate cancer and promote the cell death in cell cycle. Our predicted results showed that sub-networks of genes SREBF1, STAT6 and PBX1 are strongly related to a high extent while ETS transcription factors ELK1, JUN and EGR2 are related to a low extent. Gene SLC22A3 may explain clinically the differentiation associated with the high grade cancer compared with low grade cancer. Enhancer of Zeste Homolg 2 (EZH2 regulated by RUNX1 and STAT3 is correlated to the pathological stage

  13. Identifying significant genetic regulatory networks in the prostate cancer from microarray data based on transcription factor analysis and conditional independency.

    Science.gov (United States)

    Yeh, Hsiang-Yuan; Cheng, Shih-Wu; Lin, Yu-Chun; Yeh, Cheng-Yu; Lin, Shih-Fang; Soo, Von-Wun

    2009-12-21

    Prostate cancer is a world wide leading cancer and it is characterized by its aggressive metastasis. According to the clinical heterogeneity, prostate cancer displays different stages and grades related to the aggressive metastasis disease. Although numerous studies used microarray analysis and traditional clustering method to identify the individual genes during the disease processes, the important gene regulations remain unclear. We present a computational method for inferring genetic regulatory networks from micorarray data automatically with transcription factor analysis and conditional independence testing to explore the potential significant gene regulatory networks that are correlated with cancer, tumor grade and stage in the prostate cancer. To deal with missing values in microarray data, we used a K-nearest-neighbors (KNN) algorithm to determine the precise expression values. We applied web services technology to wrap the bioinformatics toolkits and databases to automatically extract the promoter regions of DNA sequences and predicted the transcription factors that regulate the gene expressions. We adopt the microarray datasets consists of 62 primary tumors, 41 normal prostate tissues from Stanford Microarray Database (SMD) as a target dataset to evaluate our method. The predicted results showed that the possible biomarker genes related to cancer and denoted the androgen functions and processes may be in the development of the prostate cancer and promote the cell death in cell cycle. Our predicted results showed that sub-networks of genes SREBF1, STAT6 and PBX1 are strongly related to a high extent while ETS transcription factors ELK1, JUN and EGR2 are related to a low extent. Gene SLC22A3 may explain clinically the differentiation associated with the high grade cancer compared with low grade cancer. Enhancer of Zeste Homolg 2 (EZH2) regulated by RUNX1 and STAT3 is correlated to the pathological stage. We provide a computational framework to reconstruct

  14. 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.

  15. 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.

  16. Transcriptional and Cytokine Profiles Identify CXCL9 as a Biomarker of Disease Activity in Morphea.

    Science.gov (United States)

    O'Brien, Jack C; Rainwater, Yevgeniya Byekova; Malviya, Neeta; Cyrus, Nika; Auer-Hackenberg, Lorenz; Hynan, Linda S; Hosler, Gregory A; Jacobe, Heidi T

    2017-08-01

    IFN-related pathways have not been studied in morphea, and biomarkers are needed. We sought to characterize morphea serum cytokine imbalance and IFN-related gene expression in blood and skin to address this gap by performing a case-control study of 87 participants with morphea and 26 healthy control subjects. We used multiplexed immunoassays to determine serum cytokine concentrations, performed transcriptional profiling of whole blood and lesional morphea skin, and used double-staining immunohistochemistry to determine the cutaneous cellular source of CXCL9. We found that CXCL9 was present at increased concentrations in morphea serum (P morphea skin (fold change = 30.6, P = 0.006), and preliminary transcriptional profiling showed little evidence for IFN signature in whole blood. Double-staining immunohistochemistry showed CXCL9 co-localized with CD68 + dermal macrophages. In summary, inflammatory morphea is characterized by T helper type 1 cytokine imbalance in serum, particularly CXCL9, which is associated with disease activity. CXCL9 expression in lesional macrophages implicates the skin as the source of circulating cytokines. CXCL9 is a promising biomarker of disease activity in morphea. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  17. 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)

  18. Krüppel-like factor 4, a novel transcription factor regulates microglial activation and subsequent neuroinflammation

    Directory of Open Access Journals (Sweden)

    Das Sulagna

    2010-10-01

    Full Text Available Abstract Background Activation of microglia, the resident macrophages of the central nervous system (CNS, is the hallmark of neuroinflammation in neurodegenerative diseases and other pathological conditions associated with CNS infection. The activation of microglia is often associated with bystander neuronal death. Nuclear factor-κB (NF-κB is one of the important transcription factors known to be associated with microglial activation which upregulates the expression of inducible nitric oxide synthase (iNOS, cyclooxygenase-2 (Cox-2 and other pro-inflammatory cytokines. Recent studies have focused on the role of Krüppel-like factor 4 (Klf4, one of the zinc-finger transcription factors, in mediating inflammation. However, these studies were limited to peripheral system and its role in CNS is not understood. Our studies focused on the possible role of Klf4 in mediating CNS inflammation. Methods For in vitro studies, mouse microglial BV-2 cell lines were treated with 500 ng/ml Salmonella enterica lipopolysacchride (LPS. Brain tissues were isolated from BALB/c mice administered with 5 mg/kg body weight of LPS. Expressions of Klf4, Cox-2, iNOS and pNF-κB were evaluated using western blotting, quantitative real time PCR, and reverse transcriptase polymerase chain reactions (RT-PCRs. Klf4 knockdown was carried out using SiRNA specific for Klf4 mRNA and luciferase assays and electromobility shift assay (EMSA were performed to study the interaction of Klf4 to iNOS promoter elements in vitro. Co-immunoprecipitation of Klf4 and pNF-κB was done in order to study a possible interaction between the two transcription factors. Results LPS stimulation increased Klf4 expression in microglial cells in a time- and dose-dependent manner. Knockdown of Klf4 resulted in decreased levels of the pro-inflammatory cytokines TNF-α, MCP-1 and IL-6, along with a significant decrease in iNOS and Cox-2 expression. NO production also decreased as a result of Klf4 knockdown

  19. 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.

  20. Stimulation of ribosomal RNA gene promoter by transcription factor Sp1 involves active DNA demethylation by Gadd45-NER pathway.

    Science.gov (United States)

    Rajput, Pallavi; Pandey, Vijaya; Kumar, Vijay

    2016-08-01

    The well-studied Pol II transcription factor Sp1 has not been investigated for its regulatory role in rDNA transcription. Here, we show that Sp1 bound to specific sites on rDNA and localized into the nucleoli during the G1 phase of cell cycle to activate rDNA transcription. It facilitated the recruitment of Pol I pre-initiation complex and impeded the binding of nucleolar remodeling complex (NoRC) to rDNA resulting in the formation of euchromatin active state. More importantly, Sp1 also orchestrated the site-specific binding of Gadd45a-nucleotide excision repair (NER) complex resulting in active demethylation and transcriptional activation of rDNA. Interestingly, knockdown of Sp1 impaired rDNA transcription due to reduced engagement of the Gadd45a-NER complex and hypermethylation of rDNA. Thus, the present study unveils a novel role of Sp1 in rDNA transcription involving promoter demethylation. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. 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

  2. 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.

  3. Transcriptional Responses of Chilean Quinoa (Chenopodium quinoa Willd.) Under Water Deficit Conditions Uncovers ABA-Independent Expression Patterns.

    Science.gov (United States)

    Morales, Andrea; Zurita-Silva, Andres; Maldonado, Jonathan; Silva, Herman

    2017-01-01

    HIGHLIGHTS R49 genotype displayed best performance on selected physiological parameters and highest tolerance to drought.R49 drought over-represented transcripts has exhibited 19% of genes (306 contigs) that presented no homology to published databases.Expression pattern for canonical responses to drought such as ABA biosynthesis and other genes induced in response to drought were assessed by qPCR. Global freshwater shortage is one of the biggest challenges of our time, often associated to misuse, increased consumption demands and the effects of climate change, paralleled with the desertification of vast areas. Chenopodium quinoa (Willd.) represents a very promising species, due to both nutritional content and cultivation under water constraint. We characterized drought tolerance of three Chilean genotypes and selected Genotype R49 (Salares ecotype) based upon Relative Water Content (RWC), Electrolyte Leakage (EL) and maximum efficiency of photosystem II (F v /F m ) after drought treatment, when compared to another two genotypes. Exploratory RNA-Seq of R49 was generated by Illumina paired-ends method comparing drought and control irrigation conditions. We obtained 104.8 million reads, with 54 million reads for control condition and 51 million reads for drought condition. Reads were assembled in 150,952 contigs, were 31,523 contigs have a reading frame of at least 300 nucleotides (100 aminoacids). BLAST2GO annotation showed a 15% of genes without homology to NCBI proteins, but increased to 19% (306 contigs) when focused into drought-induced genes. Expression pattern for canonical drought responses such as ABA biosynthesis and other genes induced were assessed by qPCR, suggesting novelty of R49 drought responses.

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

    Directory of Open Access Journals (Sweden)

    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.

  5. Molecular cloning, transcriptional profiling, and subcellular localization of signal transducer and activator of transcription 2 (STAT2) ortholog from rock bream, Oplegnathus fasciatus.

    Science.gov (United States)

    Bathige, S D N K; Umasuthan, Navaneethaiyer; Priyathilaka, Thanthrige Thiunuwan; Thulasitha, William Shanthakumar; Jayasinghe, J D H E; Wan, Qiang; Nam, Bo-Hye; Lee, Jehee

    2017-08-30

    Signal transducer and activator of transcription 2 (STAT2) is a key element that transduces signals from the cell membrane to the nucleus via the type I interferon-signaling pathway. Although the structural and functional aspects of STAT proteins are well studied in mammals, information on teleostean STATs is very limited. In this study, a STAT paralog, which is highly homologous to the STAT2 members, was identified from a commercially important fish species called rock bream and designated as RbSTAT2. The RbSTAT2 gene was characterized at complementary DNA (cDNA) and genomic sequence levels, and was found to possess structural features common with its mammalian counterparts. The complete cDNA sequence was distributed into 24 exons in the genomic sequence. The promoter proximal region was analyzed and found to contain potential transcription factor binding sites to regulate the transcription of RbSTAT2. Phylogenetic studies and comparative genomic structure organization revealed the distinguishable evolution for fish and other vertebrate STAT2 orthologs. Transcriptional quantification was performed by SYBR Green quantitative real-time PCR (qPCR) and the ubiquitous expression of RbSTAT2 transcripts was observed in all tissues analyzed from healthy fish, with a remarkably high expression in blood cells. Significantly (Prock bream irido virus; RBIV), bacterial (Edwardsiella tarda and Streptococcus iniae), and immune stimulants (poly I:C and LPS). Antiviral potential was further confirmed by WST-1 assay, by measuring the viability of rock bream heart cells treated with RBIV. In addition, results of an in vitro challenge experiment signified the influence of rock bream interleukin-10 (RbIL-10) on transcription of RbSTAT2. Subcellular localization studies by transfection of pEGFP-N1/RbSTAT2 into rock bream heart cells revealed that the RbSTAT2 was usually located in the cytoplasm and translocated near to the nucleus upon poly I:C administration. Altogether, these

  6. A single, specific thymine mutation in the ComK-Binding site severely decreases binding and transcription activation by the competence transcription factor ComK of Bacillus subtilis

    NARCIS (Netherlands)

    Susanna, Kim A.; Mironczuk, Aleksandra M.; Smits, Wiep Klaas; Hamoen, Leendert W.; Kuipers, Oscar P.

    The competence transcription factor ComK plays a central role in competence development in Bacillus subtilis by activating the transcription of the K regulon. ComK-activated genes are characterized by the presence of a specific sequence to which ComK binds, a K-box, in their upstream DNA region.

  7. Transcriptional activation by the thyroid hormone receptor through ligand-dependent receptor recruitment and chromatin remodelling

    DEFF Research Database (Denmark)

    Grøntved, Lars; Waterfall, Joshua J; Kim, Dong Wook

    2015-01-01

    A bimodal switch model is widely used to describe transcriptional regulation by the thyroid hormone receptor (TR). In this model, the unliganded TR forms stable, chromatin-bound complexes with transcriptional co-repressors to repress transcription. Binding of hormone dissociates co...

  8. Kctd10 regulates heart morphogenesis by repressing the transcriptional activity of Tbx5a in zebrafish

    Science.gov (United States)

    Tong, Xiangjun; Zu, Yao; Li, Zengpeng; Li, Wenyuan; Ying, Lingxiao; Yang, Jing; Wang, Xin; He, Shuonan; Liu, Da; Zhu, Zuoyan; Chen, Jianming; Lin, Shuo; Zhang, Bo

    2014-01-01

    The T-box transcription factor Tbx5 (Tbx5a in zebrafish) plays a crucial role in the formation of cardiac chambers in a dose-dependent manner. Its deregulation leads to congenital heart disease. However, little is known regarding its regulation. Here we isolate a zebrafish mutant with heart malformations, called 34c. The affected gene is identified as kctd10, a member of the potassium channel tetramerization domain (KCTD)-containing family. In the mutant, the expressions of the atrioventricular canal marker genes, such as tbx2b, hyaluronan synthase 2 (has2), notch1b and bmp4, are changed. The knockdown of tbx5 rescues the ectopic expression of has2, and knockdown of either tbx5a or has2 alleviates the heart defects. We show that Kctd10 directly binds to Tbx5 to repress its transcriptional activity. Our results reveal a new essential factor for cardiac development and suggest that KCTD10 could be considered as a new causative gene of congenital heart disease.

  9. 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

  10. CacyBP/SIP binds ERK1/2 and affects transcriptional activity of Elk-1

    International Nuclear Information System (INIS)

    Kilanczyk, Ewa; Filipek, Slawomir; Jastrzebska, Beata; Filipek, Anna

    2009-01-01

    In this work we showed for the first time that mouse CacyBP/SIP interacts with extracellular signal regulated kinases 1 and 2 (ERK1/2). We also established that a calcium binding protein, S100A6, competes for this interaction. Moreover, the E217K mutant of CacyBP/SIP does not bind significantly to ERK1/2 although it retains the ability to interact with S100A6. Molecular modeling shows that the E217K mutation in the 189-219 CacyBP/SIP fragment markedly changes its electrostatic potential, suggesting that the binding with ERK1/2 might have an electrostatic character. We also demonstrate that CacyBP/SIP-ERK1/2 interaction inhibits phosphorylation of the Elk-1 transcription factor in vitro and in the nuclear fraction of NB2a cells. Altogether, our data suggest that the binding of CacyBP/SIP with ERK1/2 might regulate Elk-1 phosphorylation/transcriptional activity and that S100A6 might further modulate this effect via Ca 2+ -dependent interaction with CacyBP/SIP and competition with ERK1/2.

  11. 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.

  12. The purple cauliflower arises from activation of a MYB transcription factor.

    Science.gov (United States)

    Chiu, Li-Wei; Zhou, Xiangjun; Burke, Sarah; Wu, Xianli; Prior, Ronald L; Li, Li

    2010-11-01

    Anthocyanins are responsible for the color of many flowers, fruits, and vegetables. An interesting and unique Purple (Pr) gene mutation in cauliflower (Brassica oleracea var botrytis) confers an abnormal pattern of anthocyanin accumulation, giving the striking mutant phenotype of intense purple color in curds and a few other tissues. To unravel the nature of the Pr mutation in cauliflower, we isolated the Pr gene via a combination of candidate gene analysis and fine mapping. Pr encoded a R2R3 MYB transcription factor that exhibited tissue-specific expression, consistent with an abnormal anthocyanin accumulation pattern in the mutant. Transgenic Arabidopsis (Arabidopsis thaliana) and cauliflower plants expressing the Pr-D allele recapitulated the mutant phenotype, confirming the isolation of the Pr gene. Up-regulation of Pr specifically activated a basic helix-loop-helix transcription factor and a subset of anthocyanin structural genes encoding flavonoid 3'-hydroxylase, dihydroflavonol 4-reductase, and leucoanthocyanidin dioxygenase to confer ectopic accumulation of pigments in the purple cauliflower. Our results indicate that the genetic variation including a Harbinger DNA transposon insertion in the upstream regulatory region of the Pr-D allele is responsible for the up-regulation of the Pr gene in inducing phenotypic change in the plant. The successful isolation of Pr provides important information on the regulatory control of anthocyanin biosynthesis in Brassica vegetables, and offers a genetic resource for development of new varieties with enhanced health-promoting properties and visual appeal.

  13. The Purple Cauliflower Arises from Activation of a MYB Transcription Factor1[W][OA

    Science.gov (United States)

    Chiu, Li-Wei; Zhou, Xiangjun; Burke, Sarah; Wu, Xianli; Prior, Ronald L.; Li, Li

    2010-01-01

    Anthocyanins are responsible for the color of many flowers, fruits, and vegetables. An interesting and unique Purple (Pr) gene mutation in cauliflower (Brassica oleracea var botrytis) confers an abnormal pattern of anthocyanin accumulation, giving the striking mutant phenotype of intense purple color in curds and a few other tissues. To unravel the nature of the Pr mutation in cauliflower, we isolated the Pr gene via a combination of candidate gene analysis and fine mapping. Pr encoded a R2R3 MYB transcription factor that exhibited tissue-specific expression, consistent with an abnormal anthocyanin accumulation pattern in the mutant. Transgenic Arabidopsis (Arabidopsis thaliana) and cauliflower plants expressing the Pr-D allele recapitulated the mutant phenotype, confirming the isolation of the Pr gene. Up-regulation of Pr specifically activated a basic helix-loop-helix transcription factor and a subset of anthocyanin structural genes encoding flavonoid 3’-hydroxylase, dihydroflavonol 4-reductase, and leucoanthocyanidin dioxygenase to confer ectopic accumulation of pigments in the purple cauliflower. Our results indicate that the genetic variation including a Harbinger DNA transposon insertion in the upstream regulatory region of the Pr-D allele is responsible for the up-regulation of the Pr gene in inducing phenotypic change in the plant. The successful isolation of Pr provides important information on the regulatory control of anthocyanin biosynthesis in Brassica vegetables, and offers a genetic resource for development of new varieties with enhanced health-promoting properties and visual appeal. PMID:20855520

  14. 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.

  15. 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.

  16. Transcriptional signature of human adipose tissue-derived stem cells (hASCs) preconditioned for chondrogenesis in hypoxic conditions

    International Nuclear Information System (INIS)

    Pilgaard, L.; Lund, P.; Duroux, M.; Lockstone, H.; Taylor, J.; Emmersen, J.; Fink, T.; Ragoussis, J.; Zachar, V.

    2009-01-01

    Hypoxia is an important factor involved in the control of stem cells. To obtain a better insight into the phenotypical changes brought about by hypoxic preconditioning prior to chondrogenic differentiation; we have investigated growth, colony-forming and chondrogenic capacity, and global transcriptional responses of six adipose tissue-derived stem cell lines expanded at oxygen concentrations ranging from ambient to 1%. The assessment of cell proliferation and colony-forming potential revealed that the hypoxic conditions corresponding to 1% oxygen played a major role. The chondrogenic inducibility, examined by high-density pellet model, however, did not improve on hypoxic preconditioning. While the microarray analysis revealed a distinctive inter-donor variability, the exposure to 1% hypoxia superseded the biological variability and produced a specific expression profile with 2581 significantly regulated genes and substantial functional enrichment in the pathways of cell proliferation and apoptosis. Additionally, exposure to 1% oxygen resulted in upregulation of factors related to angiogenesis and cell growth. In particular, leptin (LEP), the key regulator of body weight and food intake was found to be highly upregulated. In conclusion, the results of this investigation demonstrate the significance of donor demographics and the importance of further studies into the use of regulated oxygen tension as a tool for preparation of ASCs in order to exploit their full potential.

  17. Simple ortho- and para-hydroquinones as compounds neuroprotective against oxidative stress in a manner associated with specific transcriptional activation

    International Nuclear Information System (INIS)

    Satoh, Takumi; Saitoh, Sachie; Hosaka, Manami; Kosaka, Kunio

    2009-01-01

    Electrophilic compounds protect neurons through the activation of the Keap1/Nrf2 pathway and the induction of phase-2 enzymes [T. Satoh, S.A. Lipton, Redox regulation of neuronal survival by electrophilic compounds, Trends Neurosci. 30 (2007) 38-45; T. Satoh, S. Okamoto, J. Cui, Y. Watanabe, K. Furuta, M. Suzuki, K. Tohyama, S.A. Lipton, Activation of the Keap1/Nrf2 pathway for neuroprotection by electrophilic phase II inducers. Proc. Natl. Acad. Sci. USA 103 (2006) 768-773]. Hydroquinone-type electrophilic compounds such as tert-butyl hydroquinone (TBHQ) and carnosic acid (CA) have attracted special attention, because the oxidative conversion of 'hydroquinone' to 'quinone' is essential for the transcriptional activation of the above-mentioned enzymes [T. Satoh, K. Kosaka, K. Itoh, A. Kobayashi, M. Yamamoto, Y. Shimojo, C. Kitajima, J. Cui, J. Kamins, S. Okamoto, T. Shirasawa, S.A. Lipton, Carnosic acid, a catechol-type electrophilic compound, protect neurons both in vitro and in vivo through activation of the Keap1/Nrf2 pathway via S-alkylation of specific cysteine, J. Neurochem. 104 (2008) 1161-1131; A.D. Kraft, D.A. Johnson, J.A. Johnson, Nuclear factor E2-related factor 2-dependent antioxidant response element activation by tert-butylhydroquinone and sulforaphane occurring preferentially in astrocytes conditions neurons against oxidative insult, J. Neurosci. 24 (2004) 1101-1112]. In the present study, we examined the relationship between electrophilicity and the protective effects afforded by electrophilic compounds. Electrophilicity was assessed in terms of the ability of a compound to bind to a cysteine on bovine serum albumin, by which we found that neuroprotective hydroquinones [TBHQ (para-) and CA (ortho-)] had distinctive patterns of cysteine binding compared with other electrophilic compounds. Further, we found that isomers of simple ortho- and para-hydroquinones such as 2-methylhydroquinone (para-) and 4-methyl-catechol (ortho-) [not in abstract] had

  18. Noncanonical ATM Activation and Signaling in Response to Transcription-Blocking DNA Damage.

    Science.gov (United States)

    Marteijn, Jurgen A; Vermeulen, Wim; Tresini, Maria

    2017-01-01

    Environmental genotoxins and metabolic byproducts generate DNA lesions that can cause genomic instability and disrupt tissue homeostasis. To ensure genomic integrity, cells employ mechanisms that convert signals generated by stochastic DNA damage into organized responses, including activation of repair systems, cell cycle checkpoints, and apoptotic mechanisms. DNA damage response (DDR) signaling pathways coordinate these responses and determine cellular fates in part, by transducing signals that modulate RNA metabolism. One of the master DDR coordinators, the Ataxia Telangiectasia Mutated (ATM) kinase, has a fundamental role in mediating DNA damage-induced changes in mRNA synthesis. ATM acts by modulating a variety of RNA metabolic pathways including nascent RNA splicing, a process catalyzed by the spliceosome. Interestingly, ATM and the spliceosome influence each other's activity in a reciprocal manner by a pathway that initiates when transcribing RNA polymerase II (RNAPII) encounters DNA lesions that prohibit forward translocation. In response to stalling of RNAPII assembly of late-stage spliceosomes is disrupted resulting in increased splicing factor mobility. Displacement of spliceosomes from lesion-arrested RNA polymerases facilitates formation of R-loops between the nascent RNA and DNA adjacent to the transcription bubble. R-loops signal for noncanonical ATM activation which in quiescent cells occurs in absence of detectable dsDNA breaks. In turn, activated ATM signals to regulate spliceosome dynamics and AS genome wide.This chapter describes the use of fluorescence microscopy methods that can be used to evaluate noncanonical ATM activation by transcription-blocking DNA damage. First, we present an immunofluorescence-detection method that can be used to evaluate ATM activation by autophosphorylation, in fixed cells. Second, we present a protocol for Fluorescence Recovery After Photobleaching (FRAP) of GFP-tagged splicing factors, a highly sensitive and

  19. Affected pathways and transcriptional regulators in gene expression response to an ultra-marathon trail: Global and independent activity approaches.

    Directory of Open Access Journals (Sweden)

    Maria Maqueda

    Full Text Available Gene expression (GE analyses on blood samples from marathon and half-marathon runners have reported significant impacts on the immune and inflammatory systems. An ultra-marathon trail (UMT represents a greater effort due to its more testing conditions. For the first time, we report the genome-wide GE profiling in a group of 16 runners participating in an 82 km UMT competition. We quantified their differential GE profile before and after the race using HuGene2.0st microarrays (Affymetrix Inc., California, US. The results obtained were decomposed by means of an independent component analysis (ICA targeting independent expression modes. We observed significant differences in the expression levels of 5,084 protein coding genes resulting in an overrepresentation of 14% of the human biological pathways from the Kyoto Encyclopedia of Genes and Genomes database. These were mainly clustered on terms related with protein synthesis repression, altered immune system and infectious diseases related mechanisms. In a second analysis, 27 out of the 196 transcriptional regulators (TRs included in the Open Regulatory Annotation database were overrepresented. Among these TRs, we identified transcription factors from the hypoxia-inducible factors (HIF family EPAS1 (p< 0.01 and HIF1A (p<0.001, and others jointly described in the gluconeogenesis program such as HNF4 (p< 0.001, EGR1 (p<0.001, CEBPA (p< 0.001 and a highly specific TR, YY1 (p<0.01. The five independent components, obtained from ICA, further revealed a down-regulation of 10 genes distributed in the complex I, III and V from the electron transport chain. This mitochondrial activity reduction is compatible with HIF-1 system activation. The vascular endothelial growth factor (VEGF pathway, known to be regulated by HIF, also emerged (p<0.05. Additionally, and related to the brain rewarding circuit, the endocannabinoid signalling pathway was overrepresented (p<0.05.

  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. Reactive oxygen species activate differentiation gene transcription of acute myeloid leukemia cells via the JNK/c-JUN signaling pathway.

    Science.gov (United States)

    Lam, Chung Fan; Yeung, Hoi Ting; Lam, Yuk Man; Ng, Ray Kit

    2018-05-01

    Reactive oxygen species (ROS) and altered cellular redox status are associated with many malignancies. Acute myeloid leukemia (AML) cells are maintained at immature state by differentiation blockade, which involves deregulation of transcription factors in myeloid differentiation. AML cells can be induced to differentiate by phorbol-12-myristate-13-acetate (PMA), which possesses pro-oxidative activity. However, the signaling events mediated by ROS in the activation of transcriptional program during AML differentiation has not been fully elucidated. Here, we investigated AML cell differentiation by treatment with PMA and ROS scavenger N-acetyl-l-cysteine (NAC). We observed elevation of intracellular ROS level in the PMA-treated AML cells, which correlated with differentiated cell morphology and increased CD11b + mature cell population. The effect of PMA can be abolished by NAC co-treatment, supporting the involvement of ROS in the process. Moreover, we demonstrated that short ROS elevation mediated cell cycle arrest, but failed to activate myeloid gene transcription; whereas prolonged ROS elevation activated JNK/c-JUN signaling pathway. Inhibition of JNK suppressed the expression of key myeloid transcriptional regulators c-JUN, SPI-1 and MAFB, and prevented AML cells from undergoing terminal differentiation. These findings provide new insights into the crucial role of JNK/c-Jun signaling pathway in the activation of transcriptional program during ROS-mediated AML differentiation. Copyright © 2018 Elsevier Ltd. All rights reserved.

  2. Downstream signaling mechanism of the C-terminal activation domain of transcriptional coactivator CoCoA

    OpenAIRE

    Kim, Jeong Hoon; Yang, Catherine K.; Stallcup, Michael R.

    2006-01-01

    The coiled-coil coactivator (CoCoA) is a transcriptional coactivator for nuclear receptors and enhances nuclear receptor function by the interaction with the bHLH-PAS domain (AD3) of p160 coactivators. The C-terminal activation domain (AD) of CoCoA possesses strong transactivation activity and is required for the coactivator function of CoCoA with nuclear receptors. To understand how CoCoA AD transmits its activating signal to the transcription machinery, we defined specific subregions, amino...

  3. Development of 1-aryl-3-furanyl/thienyl-imidazopyridine templates for inhibitors against hypoxia inducible factor (HIF)-1 transcriptional activity.

    Science.gov (United States)

    Fuse, Shinichiro; Ohuchi, Toshiaki; Asawa, Yasunobu; Sato, Shinichi; Nakamura, Hiroyuki

    2016-12-15

    1,3-Disubstituted-imidazopyridines were designed for developing inhibitors against HIF-1 transcriptional activity. Designed compounds were rapidly synthesized from a key aromatic scaffold via microwave-assisted Suzuki-Miyaura coupling/CH direct arylation sequence. Evaluation of ability to inhibit the hypoxia induced transcriptional activity of HIF-1 revealed that the compound 2i and 3a retained the same level of the inhibitory activity comparing with that of known inhibitor, YC-1 (1). Identified, readily accessible 1-aryl-3-furanyl/thienyl-imidazopyridine templates should be useful for future drug development. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. 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.

  5. Signal transducers and activators of transcription as regulators of growth, apoptosis and breast development

    International Nuclear Information System (INIS)

    Bromberg, Jacqueline

    2000-01-01

    STAT transcription factors were discovered 10 years ago as mediators of interferon-induced gene expression. They now form an important group, comprising seven members, that are activated by virtually every cytokine and growth factor. Their critical role in development and normal cell signaling has been largely determined through the analysis of transgenic mice lacking individual STAT genes. In addition, cell culture work has further delineated their importance in cellular transformation, apoptosis, differentiation and growth control. This review discusses the specific phenotypes of STAT-deficient animals with a focus on STAT5 and STAT3, as these two STAT molecules are required for normal breast development and involution, respectively, and may play an important role in breast carcinogenesis

  6. Comprehensive analysis of the specificity of transcription activator-like effector nucleases

    DEFF Research Database (Denmark)

    Juillerat, Alexandre; Dubois, Gwendoline; Valton, Julien

    2014-01-01

    A key issue when designing and using DNA-targeting nucleases is specificity. Ideally, an optimal DNA-targeting tool has only one recognition site within a genomic sequence. In practice, however, almost all designer nucleases available today can accommodate one to several mutations within...... their target site. The ability to predict the specificity of targeting is thus highly desirable. Here, we describe the first comprehensive experimental study focused on the specificity of the four commonly used repeat variable diresidues (RVDs; NI:A, HD:C, NN:G and NG:T) incorporated in transcription activator......-like effector nucleases (TALEN). The analysis of >15 500 unique TALEN/DNA cleavage profiles allowed us to monitor the specificity gradient of the RVDs along a TALEN/DNA binding array and to present a specificity scoring matrix for RVD/nucleotide association. Furthermore, we report that TALEN can only...

  7. 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...

  8. The ETS-5 transcription factor regulates activity states in Caenorhabditis elegans by controlling satiety

    DEFF Research Database (Denmark)

    Juozaityte, Vaida; Pladevall-Morera, David; Podolska, Agnieszka

    2017-01-01

    Animal behavior is shaped through interplay among genes, the environment, and previous experience. As in mammals, satiety signals induce quiescence in Caenorhabditis elegans Here we report that the C. elegans transcription factor ETS-5, an ortholog of mammalian FEV/Pet1, controls satiety......-induced quiescence. Nutritional status has a major influence on C. elegans behavior. When foraging, food availability controls behavioral state switching between active (roaming) and sedentary (dwelling) states; however, when provided with high-quality food, C. elegans become sated and enter quiescence. We show......-regulated behavioral state switching. Taken together, our results identify a neuronal mechanism for controlling intestinal fat stores and organismal behavioral states in C. elegans, and establish a paradigm for the elucidation of obesity-relevant mechanisms....

  9. Number of active transcription factor binding sites is essential for the Hes7 oscillator

    Directory of Open Access Journals (Sweden)

    de Angelis Martin

    2006-02-01

    Full Text Available Abstract Background It is commonly accepted that embryonic segmentation of vertebrates is regulated by a segmentation clock, which is induced by the cycling genes Hes1 and Hes7. Their products form dimers that bind to the regulatory regions and thereby repress the transcription of their own encoding genes. An increase of the half-life of Hes7 protein causes irregular somite formation. This was shown in recent experiments by Hirata et al. In the same work, numerical simulations from a delay differential equations model, originally invented by Lewis, gave additional support. For a longer half-life of the Hes7 protein, these simulations exhibited strongly damped oscillations with, after few periods, severely attenuated the amplitudes. In these simulations, the Hill coefficient, a crucial model parameter, was set to 2 indicating that Hes7 has only one binding site in its promoter. On the other hand, Bessho et al. established three regulatory elements in the promoter region. Results We show that – with the same half life – the delay system is highly sensitive to changes in the Hill coefficient. A small increase changes the qualitative behaviour of the solutions drastically. There is sustained oscillation and hence the model can no longer explain the disruption of the segmentation clock. On the other hand, the Hill coefficient is correlated with the number of active binding sites, and with the way in which dimers bind to them. In this paper, we adopt response functions in order to estimate Hill coefficients for a variable number of active binding sites. It turns out that three active transcription factor binding sites increase the Hill coefficient by at least 20% as compared to one single active site. Conclusion Our findings lead to the following crucial dichotomy: either Hirata's model is correct for the Hes7 oscillator, in which case at most two binding sites are active in its promoter region; or at least three binding sites are active, in which

  10. Selective transcription and cellular proliferation induced by PDGF require histone deacetylase activity

    International Nuclear Information System (INIS)

    Catania, Annunziata; Iavarone, Carlo; Carlomagno, Stella M.; Chiariello, Mario

    2006-01-01

    Histone deacetylases (HDACs) are key regulatory enzymes involved in the control of gene expression and their inhibition by specific drugs has been widely correlated to cell cycle arrest, terminal differentiation, and apoptosis. Here, we investigated whether HDAC activity was required for PDGF-dependent signal transduction and cellular proliferation. Exposure of PDGF-stimulated NIH3T3 fibroblasts to the HDAC inhibitor trichostatin A (TSA) potently repressed the expression of a group of genes correlated to PDGF-dependent cellular growth and pro-survival activity. Moreover, we show that TSA interfered with STAT3-dependent transcriptional activity induced by PDGF. Still, neither phosphorylation nor nuclear translocation and DNA-binding in vitro and in vivo of STAT3 were affected by using TSA to interfere with PDGF stimulation. Finally, TSA treatment resulted in the suppression of PDGF-dependent cellular proliferation without affecting cellular survival of NIH3T3 cells. Our data indicate that inhibition of HDAC activity antagonizes the mitogenic effect of PDGF, suggesting that these drugs may specifically act on the expression of STAT-dependent, PDGF-responsive genes

  11. Polymorphisms in signal transducer and activator of transcription 3 and lung function in asthma

    Directory of Open Access Journals (Sweden)

    Lazarus Ross

    2005-06-01

    Full Text Available Abstract Background Identifying genetic determinants for lung function is important in providing insight into the pathophysiology of asthma. Signal transducer and activator of transcription 3 is a transcription factor latent in the cytoplasm; the gene (STAT3 is activated by a wide range of cytokines, and may play a role in lung development and asthma pathogenesis. Methods We genotyped six single nucleotide polymorphisms (SNPs in the STAT3 gene in a cohort of 401 Caucasian adult asthmatics. The associations between each SNP and forced expiratory volume in 1 second (FEV1, as a percent of predicted, at the baseline exam were tested using multiple linear regression models. Longitudinal analyses involving repeated measures of FEV1 were conducted with mixed linear models. Haplotype analyses were conducted using imputed haplotypes. We completed a second association study by genotyping the same six polymorphisms in a cohort of 652 Caucasian children with asthma. Results We found that three polymorphisms were significantly associated with baseline FEV1: homozygotes for the minor alleles of each polymorphism had lower FEV1 than homozygotes for the major alleles. Moreover, these associations persisted when we performed an analysis on repeated measures of FEV1 over 8 weeks. A haplotypic analysis based on the six polymorphisms indicated that two haplotypes were associated with baseline FEV1. Among the childhood asthmatics, one polymorphism was associated with both baseline FEV1 and the repeated measures of FEV1 over 4 years. Conclusion Our results indicate that genetic variants in STAT3, independent of asthma treatment, are determinants of FEV1 in both adults and children with asthma, and suggest that STAT3 may participate in inflammatory pathways that have an impact on level of lung function.

  12. 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.

  13. 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

  14. Protection from cyanide-induced brain injury by the Nrf2 transcriptional activator carnosic acid.

    Science.gov (United States)

    Zhang, Dongxian; Lee, Brian; Nutter, Anthony; Song, Paul; Dolatabadi, Nima; Parker, James; Sanz-Blasco, Sara; Newmeyer, Traci; Ambasudhan, Rajesh; McKercher, Scott R; Masliah, Eliezer; Lipton, Stuart A

    2015-06-01

    Cyanide is a life-threatening, bioterrorist agent, preventing cellular respiration by inhibiting cytochrome c oxidase, resulting in cardiopulmonary failure, hypoxic brain injury, and death within minutes. However, even after treatment with various antidotes to protect cytochrome oxidase, cyanide intoxication in humans can induce a delayed-onset neurological syndrome that includes symptoms of Parkinsonism. Additional mechanisms are thought to underlie cyanide-induced neuronal damage, including generation of reactive oxygen species. This may account for the fact that antioxidants prevent some aspects of cyanide-induced neuronal damage. Here, as a potential preemptive countermeasure against a bioterrorist attack with cyanide, we tested the CNS protective effect of carnosic acid (CA), a pro-electrophilic compound found in the herb rosemary. CA crosses the blood-brain barrier to up-regulate endogenous antioxidant enzymes via activation of the Nrf2 transcriptional pathway. We demonstrate that CA exerts neuroprotective effects on cyanide-induced brain damage in cultured rodent and human-induced pluripotent stem cell-derived neurons in vitro, and in vivo in various brain areas of a non-Swiss albino mouse model of cyanide poisoning that simulates damage observed in the human brain. Cyanide, a potential bioterrorist agent, can produce a chronic delayed-onset neurological syndrome that includes symptoms of Parkinsonism. Here, cyanide poisoning treated with the proelectrophillic compound carnosic acid, results in reduced neuronal cell death in both in vitro and in vivo models through activation of the Nrf2/ARE transcriptional pathway. Carnosic acid is therefore a potential treatment for the toxic central nervous system (CNS) effects of cyanide poisoning. ARE, antioxidant responsive element; Nrf2 (NFE2L2, Nuclear factor (erythroid-derived 2)-like 2). © 2015 International Society for Neurochemistry.

  15. Expression of a splice variant of the platelet-activating factor receptor transcript 2 in various human cancer cell lines

    Directory of Open Access Journals (Sweden)

    Ibtissam Youlyouz

    2002-01-01

    Full Text Available Platelet-activating factor receptor (PAF-R transcripts were analysed by reverse transcriptase-polymerase chain reaction in five human cancer cell lines derived from the breast (BT20, SKBR3 and T47D cells, the pancreas (Miapaca cells and the bladder (5637 cells in order to confirm the existence of a splice variant of the PAF-R transcript 2. After cloning and sequencing, we confirmed its existence in all cell lines. It consisted of the PAF-R transcript 2 lengthening with 82 nucleotides from the 3' end of exon 1 of the PAF-R gene. The role of this elongated form of the tissue-type PAF-R transcript in cell physiology remains to be elucidated.

  16. The transcriptional activator NtrC controls the expression and activity of glutamine synthetase in Herbaspirillum seropedicae.

    Science.gov (United States)

    Persuhn, D C; Souza, E M; Steffens, M B; Pedrosa, F O; Yates, M G; Rigo, L U

    2000-11-15

    The role of the Ntr system in Herbaspirillum seropedicae was determined via ntrB and ntrC mutants. Three phenotypes were identified in these mutants: Nif(-), deficiency in growth using nitrate, and low glutamine synthetase (GS) activity. All phenotypes were restored by the plasmid pKRT1 containing the intact glnA, ntrB and ntrC genes of H. seropedicae. The promoter region of glnA was subcloned into a beta-galactosidase fusion vector and the results suggested that NtrC positively regulates the glnA promoter in response to low nitrogen. The H. seropedicae ntrC and ntrB mutant strains showed a deficiency of adenylylation/deadenylylation of GS, indicating that NtrC and NtrB are involved in both transcription and activity control of GS in this organism.

  17. 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...

  18. 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

  19. 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.

  20. 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.

  1. The Transcription Factor Nrf2 Protects Angiogenic Capacity of Endothelial Colony-Forming Cells in High-Oxygen Radical Stress Conditions

    Directory of Open Access Journals (Sweden)

    Hendrik Gremmels

    2017-01-01

    Full Text Available Background. Endothelial colony forming cells (ECFCs have shown a promise in tissue engineering of vascular constructs, where they act as endothelial progenitor cells. After implantation, ECFCs are likely to be subjected to elevated reactive oxygen species (ROS. The transcription factor Nrf2 regulates the expression of antioxidant enzymes in response to ROS. Methods. Stable knockdown of Nrf2 and Keap1 was achieved by transduction with lentiviral shRNAs; activation of Nrf2 was induced by incubation with sulforaphane (SFN. Expression of Nrf2 target genes was assessed by qPCR, oxidative stress was assessed using CM-DCFDA, and angiogenesis was quantified by scratch-wound and tubule-formation assays. Results. Nrf2 knockdown led to a reduction of antioxidant gene expression and increased ROS. Angiogenesis was disturbed after Nrf2 knockdown even in the absence of ROS. Conversely, angiogenesis was preserved in high ROS conditions after knockdown of Keap1. Preincubation of ECFCs with SFN reduced intracellular ROS in the presence of H2O2 and preserved scratch-wound closure and tubule-formation. Conclusion. The results of this study indicate that Nrf2 plays an important role in the angiogenic capacity of ECFCs, particularly under conditions of increased oxidative stress. Pretreatment of ECFCs with SFN prior to implantation may be a protective strategy for tissue-engineered constructs or cell therapies.

  2. 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.

  3. 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.

  4. 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

  5. Stat1-Vitamin D Receptor Interactions Antagonize 1,25-Dihydroxyvitamin D Transcriptional Activity and Enhance Stat1-Mediated Transcription

    OpenAIRE

    Vidal, Marcos; Ramana, Chilakamarti V.; Dusso, Adriana S.

    2002-01-01

    The cytokine gamma interferon (IFN-γ) and the calcitropic steroid hormone 1,25-dihydroxyvitamin D (1,25D) are activators of macrophage immune function. In sarcoidosis, tuberculosis, and several granulomatoses, IFN-γ induces 1,25D synthesis by macrophages and inhibits 1,25D induction of 24-hydroxylase, a key enzyme in 1,25D inactivation, causing high levels of 1,25D in serum and hypercalcemia. This study delineates IFN-γ-1,25D cross talk in human monocytes-macrophages. Nuclear accumulation of ...

  6. 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.

  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. Activation tagging in tomato identifies a transcriptional regulator of anthocyanin biosynthesis, modification, and transport.

    Science.gov (United States)

    Mathews, Helena; Clendennen, Stephanie K; Caldwell, Colby G; Liu, Xing Liang; Connors, Karin; Matheis, Nikolaus; Schuster, Debra K; Menasco, D J; Wagoner, Wendy; Lightner, Jonathan; Wagner, D Ry

    2003-08-01

    We have developed a high-throughput T-DNA insertional mutagenesis program in tomato using activation tagging to identify genes that regulate metabolic pathways. One of the activation-tagged insertion lines (ant1) showed intense purple pigmentation from the very early stage of shoot formation in culture, reflecting activation of the biosynthetic pathway leading to anthocyanin accumulation. The purple coloration resulted from the overexpression of a gene that encodes a MYB transcription factor. Vegetative tissues of ant1 plants displayed intense purple color, and the fruit showed purple spotting on the epidermis and pericarp. The gene-to-trait relationship of ant1 was confirmed by the overexpression of ANT1 in transgenic tomato and in tobacco under the control of a constitutive promoter. Suppression subtractive hybridization and RNA hybridization analysis of the purple tomato plants indicated that the overexpression of ANT1 caused the upregulation of genes that encode proteins in both the early and later steps of anthocyanidin biosynthesis as well as genes involved in the glycosylation and transport of anthocyanins into the vacuole.

  9. Tofacitinib Represses the Janus Kinase-Signal Transducer and Activators of Transcription Signalling Pathway in Keratinocytes.

    Science.gov (United States)

    Srivastava, Ankit; Ståhle, Mona; Pivarcsi, Andor; Sonkoly, Enikö

    2018-05-08

    Tofacitinib is a Janus kinase (JAK) inhibitor, which has shown efficacy in treating psoriasis. The mode of action of tofacitinib is not completely understood but it has been thought to be mediated by the inhibition of CD4+ T-cell activation. Here, we investigated whether the molecular targets of tofacitinib are expressed in keratinocytes, and whether tofacitinib can modulate the activity of the JAK/Signal Transducer and Activators of Transcription (STAT)-pathway in keratinocytes. Transcriptomic profiling of human keratinocytes treated with IL-22 in combination with tofacitinib revealed that tofacitinib could prevent the majority of IL-22-mediated gene expression changes. Pathway analysis of tofacitinib-regulated genes in keratinocytes revealed enrichment of genes involved in the JAK/STAT signalling pathway. Quantitative real-time-PCR confirmed the upregulation of S100A7 and downregulation of EGR1 expression by IL-22, which was prevented by tofacitinib pre-treatment. These results indicate a direct effect of tofacinitib on keratinocytes, which can have relevance for systemic as well as for topical treatment of psoriasis with tofacitinib.

  10. Unique Transcriptional Profile of Sustained Ligand-Activated Preconditioning in Pre- and Post-Ischemic Myocardium

    Science.gov (United States)

    Ashton, Kevin J.; Tupicoff, Amanda; Williams-Pritchard, Grant; Kiessling, Can J.; See Hoe, Louise E.; Headrick, John P.; Peart, Jason N.

    2013-01-01

    Background Opioidergic SLP (sustained ligand-activated preconditioning) induced by 3–5 days of opioid receptor (OR) agonism induces persistent protection against ischemia-reperfusion (I-R) injury in young and aged hearts, and is mechanistically distinct from conventional preconditioning responses. We thus applied unbiased gene-array interrogation to identify molecular effects of SLP in pre- and post-ischemic myocardium. Methodology/Principal Findings Male C57Bl/6 mice were implanted with 75 mg morphine or placebo pellets for 5 days. Resultant SLP did not modify cardiac function, and markedly reduced dysfunction and injury in perfused hearts subjected to 25 min ischemia/45 min reperfusion. Microarray analysis identified 14 up- and 86 down-regulated genes in normoxic hearts from SLP mice (≥1.3-fold change, FDR≤5%). Induced genes encoded sarcomeric/contractile proteins (Myh7, Mybpc3,Myom2,Des), natriuretic peptides (Nppa,Nppb) and stress-signaling elements (Csda,Ptgds). Highly repressed genes primarily encoded chemokines (Ccl2,Ccl4,Ccl7,Ccl9,Ccl13,Ccl3l3,Cxcl3), cytokines (Il1b,Il6,Tnf) and other proteins involved in inflammation/immunity (C3,Cd74,Cd83, Cd86,Hla-dbq1,Hla-drb1,Saa1,Selp,Serpina3), together with endoplasmic stress proteins (known: Dnajb1,Herpud1,Socs3; putative: Il6, Gadd45g,Rcan1) and transcriptional controllers (Egr2,Egr3, Fos,Hmox1,Nfkbid). Biological themes modified thus related to inflammation/immunity, together with cellular/cardiovascular movement and development. SLP also modified the transcriptional response to I-R (46 genes uniquely altered post-ischemia), which may influence later infarction/remodeling. This included up-regulated determinants of cellular resistance to oxidant (Mgst3,Gstm1,Gstm2) and other forms of stress (Xirp1,Ankrd1,Clu), and repression of stress-response genes (Hspa1a,Hspd1,Hsp90aa,Hsph1,Serpinh1) and Txnip. Conclusions Protection via SLP is associated with transcriptional repression of inflammation/immunity, up

  11. Unique transcriptional profile of sustained ligand-activated preconditioning in pre- and post-ischemic myocardium.

    Directory of Open Access Journals (Sweden)

    Kevin J Ashton

    Full Text Available BACKGROUND: Opioidergic SLP (sustained ligand-activated preconditioning induced by 3-5 days of opioid receptor (OR agonism induces persistent protection against ischemia-reperfusion (I-R injury in young and aged hearts, and is mechanistically distinct from conventional preconditioning responses. We thus applied unbiased gene-array interrogation to identify molecular effects of SLP in pre- and post-ischemic myocardium. METHODOLOGY/PRINCIPAL FINDINGS: Male C57Bl/6 mice were implanted with 75 mg morphine or placebo pellets for 5 days. Resultant SLP did not modify cardiac function, and markedly reduced dysfunction and injury in perfused hearts subjected to 25 min ischemia/45 min reperfusion. Microarray analysis identified 14 up- and 86 down-regulated genes in normoxic hearts from SLP mice (≥1.3-fold change, FDR≤5%. Induced genes encoded sarcomeric/contractile proteins (Myh7, Mybpc3,Myom2,Des, natriuretic peptides (Nppa,Nppb and stress-signaling elements (Csda,Ptgds. Highly repressed genes primarily encoded chemokines (Ccl2,Ccl4,Ccl7,Ccl9,Ccl13,Ccl3l3,Cxcl3, cytokines (Il1b,Il6,Tnf and other proteins involved in inflammation/immunity (C3,Cd74,Cd83, Cd86,Hla-dbq1,Hla-drb1,Saa1,Selp,Serpina3, together with endoplasmic stress proteins (known: Dnajb1,Herpud1,Socs3; putative: Il6, Gadd45g,Rcan1 and transcriptional controllers (Egr2,Egr3, Fos,Hmox1,Nfkbid. Biological themes modified thus related to inflammation/immunity, together with cellular/cardiovascular movement and development. SLP also modified the transcriptional response to I-R (46 genes uniquely altered post-ischemia, which may influence later infarction/remodeling. This included up-regulated determinants of cellular resistance to oxidant (Mgst3,Gstm1,Gstm2 and other forms of stress (Xirp1,Ankrd1,Clu, and repression of stress-response genes (Hspa1a,Hspd1,Hsp90aa,Hsph1,Serpinh1 and Txnip. CONCLUSIONS: Protection via SLP is associated with transcriptional repression of inflammation/immunity, up

  12. Involvement of the Transcriptional Coactivator ThMBF1 in the Biocontrol Activity of Trichoderma harzianum

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    M. Belén Rubio

    2017-11-01

    Full Text Available Trichoderma harzianum is a filamentous fungus well adapted to different ecological niches. Owing to its ability to antagonize a wide range of plant pathogens, it is used as a biological control agent in agriculture. Selected strains of T. harzianum are also able to increase the tolerance of plants to biotic and abiotic stresses. However, little is known about the regulatory elements of the T. harzianum transcriptional machinery and their role in the biocontrol by this species. We had previously reported the involvement of the transcription factor THCTF1 in the T. harzianum production of the secondary metabolite 6-pentyl-pyrone, an important volatile compound related to interspecies cross-talk. Here, we performed a subtractive hybridization to explore the genes regulated by THCTF1, allowing us to identify a multiprotein bridging factor 1 (mbf1 homolog. The gene from T. harzianum T34 was isolated and characterized, and the generated Thmbf1 overexpressing transformants were used to investigate the role of this gene in the biocontrol abilities of the fungus against two plant pathogens. The transformants showed a reduced antifungal activity against Fusarium oxysporum f. sp. lycopersici race 2 (FO and Botrytis cinerea (BC in confrontation assays on discontinuous medium, indicating that the Thmbf1 gene could affect T. harzianum production of volatile organic compounds (VOC with antifungal activity. Moreover, cellophane and dialysis membrane assays indicated that Thmbf1 overexpression affected the production of low molecular weight secreted compounds with antifungal activity against FO. Intriguingly, no correlation in the expression profiles, either in rich or minimal medium, was observed between Thmbf1 and the master regulator gene cross-pathway control (cpc1. Greenhouse assays allowed us to evaluate the biocontrol potential of T. harzianum strains against BC and FO on susceptible tomato plants. The wild type strain T34 significantly reduced the

  13. Involvement of the Transcriptional Coactivator ThMBF1 in the Biocontrol Activity of Trichoderma harzianum.

    Science.gov (United States)

    Rubio, M Belén; Pardal, Alonso J; Cardoza, Rosa E; Gutiérrez, Santiago; Monte, Enrique; Hermosa, Rosa

    2017-01-01

    Trichoderma harzianum is a filamentous fungus well adapted to different ecological niches. Owing to its ability to antagonize a wide range of plant pathogens, it is used as a biological control agent in agriculture. Selected strains of T. harzianum are also able to increase the tolerance of plants to biotic and abiotic stresses. However, little is known about the regulatory elements of the T. harzianum transcriptional machinery and their role in the biocontrol by this species. We had previously reported the involvement of the transcription factor THCTF1 in the T. harzianum production of the secondary metabolite 6-pentyl-pyrone, an important volatile compound related to interspecies cross-talk. Here, we performed a subtractive hybridization to explore the genes regulated by THCTF1, allowing us to identify a multiprotein bridging factor 1 ( mbf1 ) homolog. The gene from T. harzianum T34 was isolated and characterized, and the generated Thmbf1 overexpressing transformants were used to investigate the role of this gene in the biocontrol abilities of the fungus against two plant pathogens. The transformants showed a reduced antifungal activity against Fusarium oxysporum f. sp. lycopersici race 2 (FO) and Botrytis cinerea (BC) in confrontation assays on discontinuous medium, indicating that the Thmbf1 gene could affect T. harzianum production of volatile organic compounds (VOC) with antifungal activity. Moreover, cellophane and dialysis membrane assays indicated that Thmbf1 overexpression affected the production of low molecular weight secreted compounds with antifungal activity against FO. Intriguingly, no correlation in the expression profiles, either in rich or minimal medium, was observed between Thmbf1 and the master regulator gene cross-pathway control ( cpc1 ). Greenhouse assays allowed us to evaluate the biocontrol potential of T. harzianum strains against BC and FO on susceptible tomato plants. The wild type strain T34 significantly reduced the necrotic leaf

  14. Transcriptional Activation Domains of the Candida albicans Gcn4p and Gal4p Homologs▿ †

    OpenAIRE

    Martchenko, Mikhail; Levitin, Anastasia; Whiteway, Malcolm

    2006-01-01

    Many putative transcription factors in the pathogenic fungus Candida albicans contain sequence similarity to well-defined transcriptional regulators in the budding yeast Saccharomyces cerevisiae, but this sequence similarity is often limited to the DNA binding domains of the molecules. The Gcn4p and Gal4p proteins of Saccharomyces cerevisiae are highly studied and well-understood eukaryotic transcription factors of the basic leucine zipper (Gcn4p) and C6 zinc cluster (Gal4p) families; C. albi...

  15. RKIP Inhibits Local Breast Cancer Invasion by Antagonizing the Transcriptional Activation of MMP13.

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    Ila Datar

    Full Text Available Raf Kinase Inhibitory Protein or RKIP was initially identified as a Raf-1 binding protein using the yeast 2-hybrid screen. RKIP inhibits the activation phosphorylation of MEK by Raf-1 by competitively inhibiting the binding of MEK to Raf-1 and thus exerting an inhibitory effect on the Raf-MEK-Erk pathway. RKIP has been identified as a metastasis suppressor gene. Expression of RKIP is low in cancer metastases. Although primary tumor growth remains unaffected, re- expression of RKIP inhibits cancer metastasis. Mechanistically, RKIP constrains metastasis by inhibiting angiogenesis, local invasion, intravasation, and colonization. The molecular mechanism of how RKIP inhibits these individual steps remains undefined. In our present study, using an unbiased PCR based screening and by analyzing DNA microarray expression datasets we observe that the expression of multiple metalloproteases (MMPs including MMP1, MMP3, MMP10 and MMP13 are negatively correlated with RKIP expression in breast cancer cell lines and clinical samples. Since expression of MMPs by cancer cells is important for cancer metastasis, we hypothesize that RKIP may mediate suppression of breast cancer metastasis by inhibiting multiple MMPs. We show that the expression signature of RKIP and MMPs is better at predicting high metastatic risk than the individual gene. Using a combination of loss- and gain-of-function approaches, we find that MMP13 is the cause of RKIP-mediated inhibition of local cancer invasion. Interestingly expression of MMP13 alone is not sufficient to reverse the inhibition of breast cancer cell metastasis to the lung due to the expression of RKIP. We find that RKIP negatively regulates MMP13 through the Erk2 signaling pathway and the repression of MMP13 by RKIP is transcription factor AP-1 independent. Together, our findings indicate that RKIP inhibits cancer cell invasion, in part, via MMP13 inhibition. These data also implicate RKIP in the regulation of MMP

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

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    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.

  17. Signal transducer and activator of transcription 3 activation is associated with bladder cancer cell growth and survival

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    Hsieh Fu-Chuan

    2008-10-01

    Full Text Available Abstract Background Constitutive activation of signal transducer and activator of transcription 3 (Stat3 signaling pathway plays an important role in several human cancers. Activation of Stat3 is dependent on the phosphorylation at the tyrosine residue 705 by upstream kinases and subsequent nuclear translocation after dimerization. It remains unclear whether oncogenic Stat3 signaling pathway is involved in the oncogenesis of bladder cancer. Results We found that elevated Stat3 phosphorylation in 19 of 100 (19% bladder cancer tissues as well as bladder cancer cell lines, WH, UMUC-3 and 253J. To explore whether Stat3 activation is associated with cell growth and survival of bladder cancer, we targeted the Stat3 signaling pathway in bladder cancer cells using an adenovirus-mediated dominant-negative Stat3 (Y705F and a small molecule compound, STA-21. Both prohibited cell growth and induction of apoptosis in these bladder cancer cell lines but not in normal bladder smooth muscle cell (BdSMC. The survival inhibition might be mediated through apoptotic caspase 3, 8 and 9 pathways. Moreover, down-regulation of anti-apoptotic genes (Bcl-2, Bcl-xL and survivin and a cell cycle regulating gene (cyclin D1 was associated with the cell growth inhibition and apoptosis. Conclusion These results indicated that activation of Stat3 is crucial for bladder cancer cell growth and survival. Therefore, interference of Stat3 signaling pathway emerges as a potential therapeutic approach for bladder cancer.

  18. 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

  19. 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

  20. 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.

  1. 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

  2. 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.

  3. Resolving Early Signaling Events in T-Cell Activation Leading to IL-2 and FOXP3 Transcription

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    Jeffrey P. Perley

    2014-11-01

    Full Text Available Signal intensity and feedback regulation are known to be major factors in the signaling events stemming from the T-cell receptor (TCR and its various coreceptors, but the exact nature of these relationships remains in question. We present a mathematical model of the complex signaling network involved in T-cell activation with cross-talk between the Erk, calcium, PKC and mTOR signaling pathways. The model parameters are adjusted to fit new and published data on TCR trafficking, Zap70, calcium, Erk and Isignaling. The regulation of the early signaling events by phosphatases, CD45 and SHP1, and the TCR dynamics are critical to determining the behavior of the model. Additional model corroboration is provided through quantitative and qualitative agreement with experimental data collected under different stimulating and knockout conditions. The resulting model is analyzed to investigate how signal intensity and feedback regulation affect TCR- and coreceptor-mediated signal transduction and their downstream transcriptional profiles to predict the outcome for a variety of stimulatory and knockdown experiments. Analysis of the model shows that: (1 SHP1 negative feedback is necessary for preventing hyperactivity in TCR signaling; (2 CD45 is required for TCR signaling, but also partially suppresses it at high expression levels; and (3 elevated FOXP3 and reduced IL-2 signaling, an expression profile often associated with T regulatory cells (Tregs, is observed when the system is subjected to weak TCR and CD28 costimulation or a severe reduction in CD45 activity.

  4. VID22 is required for transcriptional activation of the PSD2 gene in the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Miyata, Non; Miyoshi, Takuya; Yamaguchi, Takanori; Nakazono, Toshimitsu; Tani, Motohiro; Kuge, Osamu

    2015-12-15

    Phosphatidylethanolamine (PE) in the yeast Saccharomyces cerevisiae is synthesized through decarboxylation of phosphatidylserine (PS), catalysed by PS decarboxylase 1 (Psd1p) and 2 (Psd2p) and the cytidine 5'-diphosphate (CDP)-ethanolamine (CDP-Etn) pathway. PSD1 null (psd1Δ) and PSD2 null (psd2Δ) mutants are viable in a synthetic minimal medium, but a psd1Δ psd2Δ double mutant exhibits Etn auxotrophy, which is incorporated into PE through the CDP-Etn pathway. We have previously shown that psd1Δ is synthetic lethal with deletion of VID22 (vid22Δ) [Kuroda et al. (2011) Mol. Microbiol. 80: , 248-265]. In the present study, we found that vid22Δ mutant exhibits Etn auxotrophy under PSD1-depressed conditions. Deletion of VID22 in wild-type and PSD1-depressed cells caused partial defects in PE formation through decarboxylation of PS. The enzyme activity of PS decarboxylase in an extract of vid22Δ cells was ∼70% of that in wild-type cells and similar to that in psd2Δ cells and the PS decarboxylase activity remaining in the PSD1-depressed cells became almost negligible with deletion of VID22. Thus, the vid22Δ mutation was suggested to cause a defect in the Psd2p activity. Furthermore, vid22Δ cells were shown to be defective in expression of the PSD2 gene tagged with 6×HA, the defect being ameliorated by replacement of the native promoter of the PSD2 gene with a CYC1 promoter. In addition, an α-galactosidase reporter assay revealed that the activity of the promoter of the PSD2 gene in vid22Δ cells was ∼5% of that in wild-type cells. These results showed that VID22 is required for transcriptional activation of the PSD2 gene. © 2015 Authors; published by Portland Press Limited.

  5. Sp1 transcriptional activity is up-regulated by phosphatase 2A in dividing T lymphocytes.

    Science.gov (United States)

    Lacroix, Isabelle; Lipcey, Carol; Imbert, Jean; Kahn-Perlès, Brigitte

    2002-03-15

    We have followed Sp1 expression in primary human T lymphocytes induced, via CD2 plus CD28 costimulation, to sustained proliferation and subsequent return to quiescence. Binding of Sp1 to wheat germ agglutinin lectin was not modified following activation, indicating that the overall glycosylation of the protein was unchanged. Sp1 underwent, instead, a major dephosphorylation that correlated with cyclin A expression and, thus, with cell cycle progression. A similar change was observed in T cells that re-entered cell cycle following secondary interleukin-2 stimulation, as well as in serum-induced proliferating NIH/3T3 fibroblasts. Phosphatase 2A (PP2A) appears involved because 1) treatment of dividing cells with okadaic acid or cantharidin inhibited Sp1 dephosphorylation and 2) PP2A dephosphorylated Sp1 in vitro and strongly interacted with Sp1 in vivo. Sp1 dephosphorylation is likely to increase its transcriptional activity because PP2A overexpression potentiated Sp1 site-driven chloramphenicol acetyltransferase expression in dividing Kit225 T cells and okadaic acid reversed this effect. This increase might be mediated by a stronger affinity of dephosphorylated Sp1 for DNA, as illustrated by the reduced DNA occupancy by hyperphosphorylated Sp factors from cantharidin- or nocodazole-treated cells. Finally, Sp1 dephosphorylation appears to occur throughout cell cycle except for mitosis, a likely common feature to all cycling cells.

  6. 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

  7. Targeted Editing of Myostatin Gene in Sheep by Transcription Activator-like Effector Nucleases

    Directory of Open Access Journals (Sweden)

    Xinxia Zhao

    2016-03-01

    Full Text Available Myostatin (MSTN is a secreted growth factor expressed in skeletal muscle and adipose tissue that negatively regulates skeletal muscle mass. Gene knockout of MSTN can result in increasing muscle mass in sheep. The objectives were to investigate whether myostatin gene can be edited in sheep by transcription activator-like effector nucleases (TALENs in tandem with single-stranded DNA oligonucleotides (ssODNs. We designed a pair of TALENs to target a highly conserved sequence in the coding region of the sheep MSTN gene. The activity of the TALENs was verified by using luciferase single-strand annealing reporter assay in HEK 293T cell line. Co-transfection of TALENs and ssODNs oligonucleotides induced precise gene editing of myostatin gene in sheep primary fibroblasts. MSTN gene-edited cells were successfully used as nuclear donors for generating cloned embryos. TALENs combined with ssDNA oligonucleotides provide a useful approach for precise gene modification in livestock animals.

  8. 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.

  9. Transcription Factors Synergistically Activated at the Crossing of the Restriction Point between G1 and S Cell Cycle Phases. Pathologic Gate Opening during Multi-Hit Malignant Transformation

    Directory of Open Access Journals (Sweden)

    Nicoletta Castagnino

    2016-12-01

    Full Text Available Transcription factors (TFs represent key regulators of gene-expression patterns controlling cell behavior. TFs are active at nuclear – chromatin levels. TFs do not act in isolation; small sets of TFs cooperate toward the transcription of sets of mRNAs and consequently the translation of new proteins (the molecular phenotypes of a cell. Most TFs are activated through a cascade of biochemical reactions mediated by receptors expressed on the target cell surface. Nuclear Receptors (NRs are transcription factors activated instead by small hydrophobic molecules capable of crossing the plasma membrane. The convergence of different pathways on TFs and their posttranslational modifications ensure that the external stimuli generate appropriate and integrated responses. The reconstruction of the molecular anatomy of these pathways through Molecular Interactions Maps (MIMs can depict these intricate interactions. A mathematical modeling approach simulates/mimics their mechanism of action in normal and pathological conditions. We can simulate the effect of virtual hits in neoplastic transformation as mutations/alterations in these pathways. We can also simulate the effect of targeted inhibitors on these deregulated pathways. This strategy can help to guide an appropriate combination of targeted drugs in the treatment of a cancer patient, a major innovative perspective of incoming years.

  10. Cross-talk between an activator of nuclear receptors-mediated transcription and the D1 dopamine receptor signaling pathway.

    Science.gov (United States)

    Schmidt, Azriel; Vogel, Robert; Rutledge, Su Jane; Opas, Evan E; Rodan, Gideon A; Friedman, Eitan

    2005-03-01

    Nuclear receptors are transcription factors that usually interact, in a ligand-dependent manner, with specific DNA sequences located within promoters of target genes. The nuclear receptors can also be controlled in a ligand-independent manner via the action of membrane receptors and cellular signaling pathways. 5-Tetradecyloxy-2-furancarboxylic acid (TOFA) was shown to stimulate transcription from the MMTV promoter via chimeric receptors that consist of the DNA binding domain of GR and the ligand binding regions of the PPARbeta or LXRbeta nuclear receptors (GR/PPARbeta and GR/LXRbeta). TOFA and hydroxycholesterols also modulate transcription from NF-kappaB- and AP-1-controlled reporter genes and induce neurite differentiation in PC12 cells. In CV-1 cells that express D(1) dopamine receptors, D(1) dopamine receptor stimulation was found to inhibit TOFA-stimulated transcription from the MMTV promoter that is under the control of chimeric GR/PPARbeta and GR/LXRbeta receptors. Treatment with the D(1) dopamine receptor antagonist, SCH23390, prevented dopamine-mediated suppression of transcription, and by itself increased transcription controlled by GR/LXRbeta. Furthermore, combined treatment of CV-1 cells with TOFA and SCH23390 increased transcription controlled by the GR/LXRbeta chimeric receptor synergistically. The significance of this in vitro synergy was demonstrated in vivo, by the observation that SCH23390 (but not haloperidol)-mediated catalepsy in rats was potentiated by TOFA, thus showing that an agent that mimics the in vitro activities of compounds that activate members of the LXR and PPAR receptor families can influence D1 dopamine receptor elicited responses.

  11. Light Controls Cytokinin Signaling via Transcriptional Regulation of Constitutively Active Sensor Histidine Kinase CKI1.

    Science.gov (United States)

    Dobisova, Tereza; Hrdinova, Vendula; Cuesta, Candela; Michlickova, Sarka; Urbankova, Ivana; Hejatkova, Romana; Zadnikova, Petra; Pernisova, Marketa; Benkova, Eva; Hejatko, Jan

    2017-05-01

    In plants, the multistep phosphorelay (MSP) pathway mediates a range of regulatory processes, including those activated by cytokinins. The cross talk between cytokinin response and light has been known for a long time. However, the molecular mechanism underlying the interaction between light and cytokinin signaling remains elusive. In the screen for upstream regulators we identified a LONG PALE HYPOCOTYL ( LPH ) gene whose activity is indispensable for spatiotemporally correct expression of CYTOKININ INDEPENDENT1 ( CKI1 ), encoding the constitutively active sensor His kinase that activates MSP signaling. lph is a new allele of HEME OXYGENASE1 ( HY1 ) that encodes the key protein in the biosynthesis of phytochromobilin, a cofactor of photoconvertible phytochromes. Our analysis confirmed the light-dependent regulation of the CKI1 expression pattern. We show that CKI1 expression is under the control of phytochrome A (phyA), functioning as a dual (both positive and negative) regulator of CKI1 expression, presumably via the phyA-regulated transcription factors (TF) PHYTOCHROME INTERACTING FACTOR3 and CIRCADIAN CLOCK ASSOCIATED1. Changes in CKI1 expression observed in lph / hy1 - 7 and phy mutants correlate with misregulation of MSP signaling, changed cytokinin sensitivity, and developmental aberrations that were previously shown to be associated with cytokinin and/or CKI1 action. Besides that, we demonstrate a novel role of phyA-dependent CKI1 expression in the hypocotyl elongation and hook development during skotomorphogenesis. Based on these results, we propose that the light-dependent regulation of CKI1 provides a plausible mechanistic link underlying the well-known interaction between light- and cytokinin-controlled plant development. © 2017 American Society of Plant Biologists. All Rights Reserved.

  12. Sertad1 encodes a novel transcriptional co-activator of SMAD1 in mouse embryonic hearts

    Energy Technology Data Exchange (ETDEWEB)

    Peng, Yin [Department of Genetics, The University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Zhao, Shaomin [Department of Genetics, The University of Alabama at Birmingham, Birmingham, AL 35294 (United States); School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069 (China); Song, Langying [Department of Genetics, The University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Wang, Manyuan [School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069 (China); Jiao, Kai, E-mail: kjiao@uab.edu [Department of Genetics, The University of Alabama at Birmingham, Birmingham, AL 35294 (United States)

    2013-11-29

    Highlights: •SERTAD1 interacts with SMAD1. •Sertad1 is expressed in mouse embryonic hearts. •SERTAD1 is localized in both cytoplasm and nucleus of cardiomyocytes. •SERTAD1 enhances expression of BMP target cardiogenic genes as a SMAD1 co-activator. -- Abstract: Despite considerable advances in surgical repairing procedures, congenital heart diseases (CHDs) remain the leading noninfectious cause of infant morbidity and mortality. Understanding the molecular/genetic mechanisms underlying normal cardiogenesis will provide essential information for the development of novel diagnostic and therapeutic strategies against CHDs. BMP signaling plays complex roles in multiple cardiogenic processes in mammals. SMAD1 is a canonical nuclear mediator of BMP signaling, the activity of which is critically regulated through its interaction partners. We screened a mouse embryonic heart yeast two-hybrid library using Smad1 as bait and identified SERTAD1 as a novel interaction partner of SMAD1. SERTAD1 contains multiple potential functional domains, including two partially overlapping transactivation domains at the C terminus. The SERTAD1-SMAD1 interaction in vitro and in mammalian cells was further confirmed through biochemical assays. The expression of Sertad1 in developing hearts was demonstrated using RT-PCR, western blotting and in situ hybridization analyses. We also showed that SERTAD1 was localized in both the cytoplasm and nucleus of immortalized cardiomyocytes and primary embryonic cardiomyocyte cultures. The overexpression of SERTAD1 in cardiomyocytes not only enhanced the activity of two BMP reporters in a dose-dependent manner but also increased the expression of several known BMP/SMAD regulatory targets. Therefore, these data suggest that SERTAD1 acts as a SMAD1 transcriptional co-activator to promote the expression of BMP target genes during mouse cardiogenesis.

  13. 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.

  14. Sequence and transcriptional analysis of the genes responsible for curdlan biosynthesis in Agrobacterium sp. ATCC 31749 under simulated dissolved oxygen gradients conditions.

    Science.gov (United States)

    Zhang, Hong-Tao; Zhan, Xiao-Bei; Zheng, Zhi-Yong; Wu, Jian-Rong; Yu, Xiao-Bin; Jiang, Yun; Lin, Chi-Chung

    2011-07-01

    Expression at the mRNA level of ten selected genes in Agrobacterium sp. ATCC 31749 under various dissolved oxygen (DO) levels during curdlan fermentation related to electron transfer chain (ETC), tricarboxylic acid (TCA) cycle, peptidoglycan/lipopolysaccharide biosynthesis, and uridine diphosphate (UDP)-glucose biosynthesis were determined by qRT-PCR. Experiments were performed at DO levels of 30%, 50%, and 75%, as well as under low-oxygen conditions. The effect of high cell density on transcriptional response of the above genes under low oxygen was also studied. Besides cytochrome d (cyd A), the transcription levels of all the other genes were increased at higher DO and reached maximum at 50% DO. Under 75% DO, the transcriptional levels of all the genes were repressed. In addition, transcription levels of icd, sdh, cyo A, and fix N genes did not exhibit significant fluctuation with high cell density culture under low oxygen. These results suggested a mechanism for DO regulation of curdlan synthesis through regulation of transcriptional levels of ETCs, TCA, and UDP-glucose synthesis genes during curdlan fermentation. To our knowledge, this is the first report that DO concentration apparently regulates curdlan biosynthesis in Agrobacterium sp. ATCC 31749 providing essential lead for the optimization of the fermentation at the industrial scale.

  15. The Mediator Complex MED15 Subunit Mediates Activation of Downstream Lipid-Related Genes by the WRINKLED1 Transcription Factor.

    Science.gov (United States)

    Kim, Mi Jung; Jang, In-Cheol; Chua, Nam-Hai

    2016-07-01

    The Mediator complex is known to be a master coordinator of transcription by RNA polymerase II, and this complex is recruited by transcription factors (TFs) to target promoters for gene activation or repression. The plant-specific TF WRINKLED1 (WRI1) activates glycolysis-related and fatty acid biosynthetic genes during embryogenesis. However, no Mediator subunit has yet been identified that mediates WRI1 transcriptional activity. Promoter-β-glucuronidase fusion experiments showed that MEDIATOR15 (MED15) is expressed in the same cells in the embryo as WRI1. We found that the Arabidopsis (Arabidopsis thaliana) MED15 subunit of the Mediator complex interacts directly with WRI1 in the nucleus. Overexpression of MED15 or WRI1 increased transcript levels of WRI1 target genes involved in glycolysis and fatty acid biosynthesis; these genes were down-regulated in wild-type or WRI1-overexpressing plants by silencing of MED15 However, overexpression of MED15 in the wri1 mutant also increased transcript levels of WRI1 target genes, suggesting that MED15 also may act with other TFs to activate downstream lipid-related genes. Chromatin immunoprecipitation assays confirmed the association of MED15 with six WRI1 target gene promoters. Additionally, silencing of MED15 resulted in reduced fatty acid content in seedlings and mature seeds, whereas MED15 overexpression increased fatty acid content in both developmental stages. Similar results were found in wri1 mutant and WRI1 overexpression lines. Together, our results indicate that the WRI1/MED15 complex transcriptionally regulates glycolysis-related and fatty acid biosynthetic genes during embryogenesis. © 2016 American Society of Plant Biologists. All Rights Reserved.

  16. 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

  17. Neuronal activity rapidly induces transcription of the CREB-regulated microRNA-132, in vivo

    DEFF Research Database (Denmark)

    Nudelman, Aaron Samuel; DiRocco, Derek P; Lambert, Talley J

    2010-01-01

    Activity-dependent changes in gene-expression are believed to underlie the molecular representation of memory. In this study, we report that in vivo activation of neurons rapidly induces the CREB-regulated microRNA miR-132. To determine if production of miR-132 is regulated by neuronal activity its......, olfactory bulb, and striatum by contextual fear conditioning, odor-exposure, and cocaine-injection, respectively, also increased pri-miR-132. Induction kinetics of pri-miR-132 were monitored and found to parallel those of immediate early genes, peaking at 45 min and returning to basal levels within 2 h...

  18. Activating transcription factor 3 is a target molecule linking hepatic steatosis to impaired glucose homeostasis.

    Science.gov (United States)

    Kim, Ji Yeon; Park, Keon Jae; Hwang, Joo-Yeon; Kim, Gyu Hee; Lee, DaeYeon; Lee, Yoo Jeong; Song, Eun Hyun; Yoo, Min-Gyu; Kim, Bong-Jo; Suh, Young Ho; Roh, Gu Seob; Gao, Bin; Kim, Won; Kim, Won-Ho

    2017-08-01

    Non-alcoholic fatty liver disease (NAFLD) contributes to impaired glucose tolerance, leading to type 2 diabetes (T2D); however, the precise mechanisms and target molecules that are involved remain unclear. Activating transcription factor 3 (ATF3) is associated with β-cell dysfunction that is induced by severe stress signals in T2D. We aimed to explore the exact functional role of ATF3 as a mechanistic link between hepatic steatosis and T2D development. Zucker diabetic fatty (ZDF) rats were utilized for animal experiments. An in vivo-jetPEI siRNA delivery system against ATF3 was used for loss-of-function experiments. We analyzed the baseline cross-sectional data derived from the biopsy-proven NAFLD registry (n=322). Human sera and liver tissues were obtained from 43 patients with biopsy-proven NAFLD and from seven healthy participants. ATF3 was highly expressed in the livers of ZDF rats and in human participants with NAFLD and/or T2D. Insulin resistance and hepatic steatosis were associated with increased ATF3 expression and decreased fatty acid oxidation via mitochondrial dysfunction and were attenuated by in vivo ATF3 silencing. Knockdown of ATF3 also ameliorated glucose intolerance, impaired insulin action, and inflammatory responses in ZDF rats. In patients with NAFLD and/or T2D, a significant positive correlation was observed between hepatic ATF3 expression and surrogate markers of T2D, mitochondrial dysfunction, and macrophage infiltration. Increased hepatic ATF3 expression is closely associated with hepatic steatosis and incident T2D; therefore, ATF3 may serve as a potential therapeutic target for NAFLD and hepatic steatosis-induced T2D. Hepatic activating transcription factor 3 (ATF3) may play an important role in oxidative stress-mediated hepatic steatosis and the development of type 2 diabetes (T2D) in a Zucker diabetic fatty (ZDF) rat model and in human patients with non-alcoholic fatty liver disease (NAFLD). Therefore, ATF3 may be a useful biomarker for

  19. 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.

  20. Management of nuclear PRs activity with optimal conditions

    International Nuclear Information System (INIS)

    Ohnishi, Teruaki

    1997-01-01

    A methodology is proposed to derive optimal conditions for the activity of nuclear public relations (PRs). With the use of data-bases available at present, expressions were derived which connect the budget allocated for the PRs activity with the intensity of stimulus for four types of activity of the advertisement in the press, the exclusive publicity, the pamphlet and the advertisement on television. Optimal conditions for the activity were determined by introducing a model describing a relation between the intensity of stimulus and the extent of the change of public's attitude to nuclear energy, namely the effect of PRs activity, and also by giving the optimal ratio of allocation of the budget among the four types of activity as a function of cost versus effectiveness of each type. Those optimal conditions, being for the ratio of allocation of the budget, the execution time and the intensity of each type of activity at that time, vary depending on the number of household in a target region, the target class of demography, the duration time of activity, and the amount of budget for the activity. It becomes clear from numerical calculation that the optimal conditions and the effect of activity show quite strong non-linearity with respect to the variation of those variables, and that the effect of PRs activity averaged over all public in the target region becomes to be maximum, in Japan, when the activity is executed with the optimal conditions determined for the target class of middle- and advanced-aged women. The management of nuclear PRs activity becomes possible by introducing such a method of fixation of optimal conditions for the activity as described here. (author)

  1. Direct interaction of natural and synthetic catechins with signal transducer activator of transcription 1 affects both its phosphorylation and activity

    KAUST Repository

    Menegazzi, Marta

    2013-12-10

    Our previous studies showed that (-)-epigallocatechin-3-gallate (EGCG) inhibits signal transducer activator of transcription 1 (STAT1) activation. Since EGCG may be a promising lead compound for new anti-STAT1 drug design, 15 synthetic catechins, characterized by the (-)-gallocatechin-3-gallate stereochemistry, were studied in the human mammary MDA-MB-231 cell line to identify the minimal structural features that preserve the anti-STAT1 activity. We demonstrate that the presence of three hydroxyl groups of B ring and one hydroxyl group in D ring is essential to preserve their inhibitory action. Moreover, a possible molecular target of these compounds in the STAT1 pathway was investigated. Our results demonstrate a direct interaction between STAT1 protein and catechins displaying anti-STAT1 activity. In particular, surface plasmon resonance (SPR) analysis and molecular modeling indicate the presence of two putative binding sites (a and b) with different affinity. Based on docking data, site-directed mutagenesis was performed, and interaction of the most active catechins with STAT1 was studied with SPR to test whether Gln518 on site a and His568 on site b could be important for the catechin-STAT1 interaction. Data indicate that site b has higher affinity for catechins than site a as the highest affinity constant disappears in the H568ASTAT1 mutant. Furthermore, Janus kinase 2 (JAK2) kinase assay data suggest that the contemporary presence in vitro of STAT1 and catechins inhibits JAK2-elicited STAT1 phosphorylation. The very tight catechin-STAT1 interaction prevents STAT1 phosphorylation and represents a novel, specific and efficient molecular mechanism for the inhibition of STAT1 activation. © Copyright 2014 Federation of European Biochemical Societies. All rights reserved.

  2. 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.

  3. 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

  4. CAR-mediated repression of Foxo1 transcriptional activity regulates the cell cycle inhibitor p21 in mouse livers

    International Nuclear Information System (INIS)

    Kazantseva, Yuliya A.; Yarushkin, Andrei A.; Pustylnyak, Vladimir O.

    2014-01-01

    Highlights: • CAR activation decreased the level of Foxo1 in mouse livers. • CAR activation decreased the level of p21 in mouse livers. • CAR activation inhibited Foxo1 transcriptional activity in mouse livers. - Abstract: 1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), an agonist of constitutive androstane receptor (CAR), is a well-known strong primary chemical mitogen for the mouse liver. Despite extensive investigation of the role of CAR in the regulation of cell proliferation, our knowledge of the intricate mediating mechanism is incomplete. In this study, we demonstrated that long-term CAR activation by TCPOBOP increased liver-to-body weight ratio and decreased tumour suppressor Foxo1 expression and transcriptional activity, which were correlated with reduced expression of genes regulated by Foxo1, including the cell-cycle inhibitor Cdkn1a(p21), and upregulation of the cell-cycle regulator Cyclin D1. Moreover, we demonstrated the negative regulatory effect of TCPOBOP-activated CAR on the association of Foxo1 with the target Foxo1 itself and Cdkn1a(p21) promoters. Thus, we identified CAR-mediated repression of cell cycle inhibitor p21, as mediated by repression of FOXO1 expression and transcriptional activity. CAR-FOXO1 cross-talk may provide new opportunities for understanding liver diseases and developing more effective therapeutic approaches to better drug treatments

  5. 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.

  6. 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.

  7. Transcriptional profiling of the dose response: a more powerful approach for characterizing drug activities.

    Directory of Open Access Journals (Sweden)

    Rui-Ru Ji

    2009-09-01

    Full Text Available The dose response curve is the gold standard for measuring the effect of a drug treatment, but is rarely used in genomic scale transcriptional profiling due to perceived obstacles of cost and analysis. One barrier to examining transcriptional dose responses is that existing methods for microarray data analysis can identify patterns, but provide no quantitative pharmacological information. We developed analytical methods that identify transcripts responsive to dose, calculate classical pharmacological parameters such as the EC50, and enable an in-depth analysis of coordinated dose-dependent treatment effects. The approach was applied to a transcriptional profiling study that evaluated four kinase inhibitors (imatinib, nilotinib, dasatinib and PD0325901 across a six-logarithm dose range, using 12 arrays per compound. The transcript responses proved a powerful means to characterize and compare the compounds: the distribution of EC50 values for the transcriptome was linked to specific targets, dose-dependent effects on cellular processes were identified using automated pathway analysis, and a connection was seen between EC50s in standard cellular assays and transcriptional EC50s. Our approach greatly enriches the information that can be obtained from standard transcriptional profiling technology. Moreover, these methods are automated, robust to non-optimized assays, and could be applied to other sources of quantitative data.

  8. Reverse transcriptase genes are highly abundant and transcriptionally active in marine plankton assemblages

    KAUST Repository

    Lescot, Magali

    2015-11-27

    Genes encoding reverse transcriptases (RTs) are found in most eukaryotes, often as a component of retrotransposons, as well as in retroviruses and in prokaryotic retroelements. We investigated the abundance, classification and transcriptional status of RTs based on Tara Oceans marine metagenomes and metatranscriptomes encompassing a wide organism size range. Our analyses revealed that RTs predominate large-size fraction metagenomes (>5 μm), where they reached a maximum of 13.5% of the total gene abundance. Metagenomic RTs were widely distributed across the phylogeny of known RTs, but many belonged to previously uncharacterized clades. Metatranscriptomic RTs showed distinct abundance patterns across samples compared with metagenomic RTs. The relative abundances of viral and bacterial RTs among identified RT sequences were higher in metatranscriptomes than in metagenomes and these sequences were detected in all metatranscriptome size fractions. Overall, these observations suggest an active proliferation of various RT-assisted elements, which could be involved in genome evolution or adaptive processes of plankton assemblage.

  9. The role of signal transducer and activator of transcription 3 in Rift Valley fever virus infection

    International Nuclear Information System (INIS)

    Pinkham, Chelsea; An, Soyeon; Lundberg, Lindsay; Bansal, Neha; Benedict, Ashwini; Narayanan, Aarthi; Kehn-Hall, Kylene

    2016-01-01

    Rift Valley fever (RVF) is a zoonotic disease that can cause severe illness in humans and livestock, triggering spontaneous abortion in almost 100% of pregnant ruminants. In this study, we demonstrate that signal transducer and activator of transcription 3 (STAT3) is phosphorylated on its conserved tyrosine residue (Y705) following RVFV infection. This phosphorylation was dependent on a major virulence factor, the viral nonstructural protein NSs. Loss of STAT3 had little effect on viral replication, but rather resulted in cells being more susceptible to RVFV-induced cell death. Phosphorylated STAT3 translocated to the nucleus, coinciding with inhibition of fos, jun, and nr4a2 gene expression, and the presence of STAT3 and NSs at the nr4a2 promoter. NSs was found predominantly in the cytoplasm of STAT3 null cells, indicating that STAT3 influences NSs nuclear localization. Collectively, these data demonstrate that STAT3 functions in a pro-survival capacity through modulation of NSs localization. - Highlights: • STAT3 is phosphorylated on tyrosine residue 705 following RVFV infection. • Phosphorylation of STAT3 was dependent on the viral protein NSs. • STAT3 -/- MEFs were more susceptible to RVFV-induced cell death. • Loss of STAT3 led to an increase in pro-apoptotic gene expression. • STAT3 functions in a pro-survival capacity by modulation of NSs localization.

  10. Constitutive cellulase production from glucose using the recombinant Trichoderma reesei strain overexpressing an artificial transcription activator.

    Science.gov (United States)

    Zhang, Xiaoyue; Li, Yonghao; Zhao, Xinqing; Bai, Fengwu

    2017-01-01

    The high cost of cellulase production presents biggest challenge in biomass deconstruction. Cellulase production by Trichoderma reesei using low cost carbon source is of great interest. In this study, an artificial transcription activator containing the Cre1 binding domain linked to the Xyr1 effector and binding domains was designed and constitutively overexpressed in T. reesei RUT C30. The recombinant strain T. reesei zxy-2 displayed constitutive cellulase production using glucose as a sole carbon source, and the production titer was 12.75-fold of that observed with T. reesei RUT C30 in shake flask culture. Moreover, FPase and xylanase titers of 2.63 and 108.72IU/mL, respectively, were achieved using glucose as sole carbon source within 48h in a 7-L fermenter by batch fermentation using T. reesei zxy-2. The crude enzyme obtained was used to hydrolyze alkali pretreated corn stover, and a high glucose yield of 99.18% was achieved. Copyright © 2016. Published by Elsevier Ltd.

  11. The role of signal transducer and activator of transcription 3 in Rift Valley fever virus infection

    Energy Technology Data Exchange (ETDEWEB)

    Pinkham, Chelsea; An, Soyeon; Lundberg, Lindsay; Bansal, Neha; Benedict, Ashwini; Narayanan, Aarthi; Kehn-Hall, Kylene, E-mail: kkehnhal@gmu.edu

    2016-09-15

    Rift Valley fever (RVF) is a zoonotic disease that can cause severe illness in humans and livestock, triggering spontaneous abortion in almost 100% of pregnant ruminants. In this study, we demonstrate that signal transducer and activator of transcription 3 (STAT3) is phosphorylated on its conserved tyrosine residue (Y705) following RVFV infection. This phosphorylation was dependent on a major virulence factor, the viral nonstructural protein NSs. Loss of STAT3 had little effect on viral replication, but rather resulted in cells being more susceptible to RVFV-induced cell death. Phosphorylated STAT3 translocated to the nucleus, coinciding with inhibition of fos, jun, and nr4a2 gene expression, and the presence of STAT3 and NSs at the nr4a2 promoter. NSs was found predominantly in the cytoplasm of STAT3 null cells, indicating that STAT3 influences NSs nuclear localization. Collectively, these data demonstrate that STAT3 functions in a pro-survival capacity through modulation of NSs localization. - Highlights: • STAT3 is phosphorylated on tyrosine residue 705 following RVFV infection. • Phosphorylation of STAT3 was dependent on the viral protein NSs. • STAT3 -/- MEFs were more susceptible to RVFV-induced cell death. • Loss of STAT3 led to an increase in pro-apoptotic gene expression. • STAT3 functions in a pro-survival capacity by modulation of NSs localization.

  12. 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.

  13. 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

  14. Reverse transcriptase genes are highly abundant and transcriptionally active in marine plankton assemblages

    KAUST Repository

    Lescot, Magali; Hingamp, Pascal; Kojima, Kenji K; Villar, Emilie; Romac, Sarah; Veluchamy, Alaguraj; Boccara, Martine; Jaillon, Olivier; Ludicone, Daniele; Bowler, Chris; Wincker, Patrick; Claverie, Jean-Michel; Ogata, Hiroyuki

    2015-01-01

    Genes encoding reverse transcriptases (RTs) are found in most eukaryotes, often as a component of retrotransposons, as well as in retroviruses and in prokaryotic retroelements. We investigated the abundance, classification and transcriptional status of RTs based on Tara Oceans marine metagenomes and metatranscriptomes encompassing a wide organism size range. Our analyses revealed that RTs predominate large-size fraction metagenomes (>5 μm), where they reached a maximum of 13.5% of the total gene abundance. Metagenomic RTs were widely distributed across the phylogeny of known RTs, but many belonged to previously uncharacterized clades. Metatranscriptomic RTs showed distinct abundance patterns across samples compared with metagenomic RTs. The relative abundances of viral and bacterial RTs among identified RT sequences were higher in metatranscriptomes than in metagenomes and these sequences were detected in all metatranscriptome size fractions. Overall, these observations suggest an active proliferation of various RT-assisted elements, which could be involved in genome evolution or adaptive processes of plankton assemblage.

  15. FATS is a transcriptional target of p53 and associated with antitumor activity

    OpenAIRE

    Zhang Xifeng; Zhang Qian; Zhang Jun; Qiu Li; Yan Shuang-shuang; Feng Juling; Sun Yan; Huang Xingxu; Lu Karen H; Li Zheng

    2010-01-01

    Abstract Frequent mutations of p53 in human cancers exemplify its crucial role as a tumor suppressor transcription factor, and p21, a transcriptional target of p53, plays a central role in surveillance of cell-cycle checkpoints. Our previous study has shown that FATS stabilize p21 to preserve genome integrity. In this study we identified a novel transcript variant of FATS (GenBank: GQ499374) through screening a cDNA library from mouse testis, which uncovered the promoter region of mouse FATS....

  16. 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.

  17. Hypoxia-induced oxidative base modifications in the VEGF hypoxia-response element are associated with transcriptionally active nucleosomes.

    Science.gov (United States)

    Ruchko, Mykhaylo V; Gorodnya, Olena M; Pastukh, Viktor M; Swiger, Brad M; Middleton, Natavia S; Wilson, Glenn L; Gillespie, Mark N

    2009-02-01

    Reactive oxygen species (ROS) generated in hypoxic pulmonary artery endothelial cells cause transient oxidative base modifications in the hypoxia-response element (HRE) of the VEGF gene that bear a conspicuous relationship to induction of VEGF mRNA expression (K.A. Ziel et al., FASEB J. 19, 387-394, 2005). If such base modifications are indeed linked to transcriptional regulation, then they should be detected in HRE sequences associated with transcriptionally active nucleosomes. Southern blot analysis of the VEGF HRE associated with nucleosome fractions prepared by micrococcal nuclease digestion indicated that hypoxia redistributed some HRE sequences from multinucleosomes to transcriptionally active mono- and dinucleosome fractions. A simple PCR method revealed that VEGF HRE sequences harboring oxidative base modifications were found exclusively in mononucleosomes. Inhibition of hypoxia-induced ROS generation with myxathiozol prevented formation of oxidative base modifications but not the redistribution of HRE sequences into mono- and dinucleosome fractions. The histone deacetylase inhibitor trichostatin A caused retention of HRE sequences in compacted nucleosome fractions and prevented formation of oxidative base modifications. These findings suggest that the hypoxia-induced oxidant stress directed at the VEGF HRE requires the sequence to be repositioned into mononucleosomes and support the prospect that oxidative modifications in this sequence are an important step in transcriptional activation.

  18. Scleraxis is a transcriptional activator that regulates the expression of Tenomodulin, a marker of mature tenocytes and ligamentocytes.

    Science.gov (United States)

    Shukunami, Chisa; Takimoto, Aki; Nishizaki, Yuriko; Yoshimoto, Yuki; Tanaka, Seima; Miura, Shigenori; Watanabe, Hitomi; Sakuma, Tetsushi; Yamamoto, Takashi; Kondoh, Gen; Hiraki, Yuji

    2018-02-16

    Tenomodulin (Tnmd) is a type II transmembrane glycoprotein predominantly expressed in tendons and ligaments. We found that scleraxis (Scx), a member of the Twist-family of basic helix-loop-helix transcription factors, is a transcriptional activator of Tnmd expression in tenocytes. During embryonic development, Scx expression preceded that of Tnmd. Tnmd expression was nearly absent in tendons and ligaments of Scx-deficient mice generated by transcription activator-like effector nucleases-mediated gene disruption. Tnmd mRNA levels were dramatically decreased during serial passages of rat tenocytes. Scx silencing by small interfering RNA significantly suppressed endogenous Tnmd mRNA levels in tenocytes. Mouse Tnmd contains five E-box sites in the ~1-kb 5'-flanking region. A 174-base pair genomic fragment containing a TATA box drives transcription in tenocytes. Enhancer activity was increased in the upstream region (-1030 to -295) of Tnmd in tenocytes, but not in NIH3T3 and C3H10T1/2 cells. Preferential binding of both Scx and Twist1 as a heterodimer with E12 or E47 to CAGATG or CATCTG and transactivation of the 5'-flanking region were confirmed by electrophoresis mobility shift and dual luciferase assays, respectively. Scx directly transactivates Tnmd via these E-boxes to positively regulate tenocyte differentiation and maturation.

  19. C/EBPβ contributes to transcriptional activation of long non-coding RNA NEAT1 during APL cell differentiation.

    Science.gov (United States)

    Wang, Yewei; Fu, Lei; Sun, Ailian; Tang, Doudou; Xu, Yunxiao; Li, Zheyuan; Chen, Mingjie; Zhang, Guangsen

    2018-05-05

    Emerging evidences have shown that long non-coding RNAs (lncRNAs) play critical roles in cancer development and cancer therapy. LncRNA Nuclear Enriched Abundant Transcript 1 (NEAT1) is indispensable during acute promyelocytic leukemia (APL) cell differentiation induced by all-trans retinoic acid (ATRA). However, the precise mechanism of NEAT1 upregulation has not been fully understood. In this study, we performed chromatin immunoprecipitation and luciferase reporter assays to demonstrate that C/EBP family transcription factor C/EBPβ bind to and transactivate the promoter of lncRNA NEAT1 through the C/EBPβ binding sites both around -54 bp and -1453 bp upstream of the transcription start site. Moreover, the expression of C/EBPβ was increased after ATRA treatment, and the binding of C/EBPβ in the NEAT1 promoter was also dramatically increased. Finally, knockdown of C/EBPβ significantly reduced the ATRA-induced upregulation of NEAT1. In conclusion, C/EBPβ directly activates the expression of NEAT1 through binding to the promoter of NEAT1. Knockdown of C/EBPβ impairs ATRA-induced transcriptional activation of NEAT1. Our data indicate that C/EBPβ contributes to ATRA-induced activation of NEAT1 during APL cell differentiation. Our results enrich our knowledge on the regulation of lncRNAs and the regulatory role of C/EBPβ in APL cell differentiation. Copyright © 2017. Published by Elsevier Inc.

  20. UNTAGGED MUTATION IN RICE GAL4/VP16 TRANSCRIPTIONAL ACTIVATOR FACILITATED-ENHANCER TRAP LINES

    Directory of Open Access Journals (Sweden)

    Sri Koerniati

    2013-04-01

    Full Text Available An enhancer trap system is an insertional mutagenesis based upon gene expression, instead of gene knock-out, so its insertion in genome is  expected not linked to any dramatic changes in plant phenotypes. Gene  knock-out, leading to lossof- function (LoF mutation, is a dominant  approach for rice functional genomic studies. The objective of this study was to find out whether Transcriptional Activator-Facilitated Enhancer Trap (TAFET T-DNA insertion inducing mutant phenotypes in rice TAFET population. Materials used in this experiment were T1 generation of 270 rice TAFET lines. Eight plants of each were grown in the greenhouse and observed for any mutant phenotypes. Phenotypic, histochemical, Southernblot analyses were carried out to define a mutant of pSKC66.1- 8e. Result showed that about 10% of the 270 lines produced chlorophyll-deficient  leaves, ranged from yellowish green (viridis, white stripe green zebra-like stripe to completely white (albino. Albino plants died after two weeks,  whilst white stripe or viridis mutants became normal in the next generation(T2. Another mutant was pSKC66.1-8e line which had floral dramatic phenotype change with various spikelet shapes and number of organs, and had a single twisted culm. The flower of mutant also had gus gene expression. Plants with wild type did not express gus gene and had six or more straight culms. Molecular, histochemical and phenotypic analyses of this particular line for three generations indicated that mutant phenotype was not due to the T-DNA insertion. Since there was approved that Tos17 is activated during tissue culture and induced mutant phenotype, this line might relate to Tos17 insertion, but it needs further investigation to gain such conclusion.

  1. Improved somatic mutagenesis in zebrafish using transcription activator-like effector nucleases (TALENs.

    Directory of Open Access Journals (Sweden)

    Finola E Moore

    Full Text Available Zinc Finger Nucleases (ZFNs made by Context-Dependent Assembly (CoDA and Transcription Activator-Like Effector Nucleases (TALENs provide robust and user-friendly technologies for efficiently inactivating genes in zebrafish. These designer nucleases bind to and cleave DNA at particular target sites, inducing error-prone repair that can result in insertion or deletion mutations. Here, we assess the relative efficiencies of these technologies for inducing somatic DNA mutations in mosaic zebrafish. We find that TALENs exhibited a higher success rate for obtaining active nucleases capable of inducing mutations than compared with CoDA ZFNs. For example, all six TALENs tested induced DNA mutations at genomic target sites while only a subset of CoDA ZFNs exhibited detectable rates of mutagenesis. TALENs also exhibited higher mutation rates than CoDA ZFNs that had not been pre-screened using a bacterial two-hybrid assay, with DNA mutation rates ranging from 20%-76.8% compared to 1.1%-3.3%. Furthermore, the broader targeting range of TALENs enabled us to induce mutations at the methionine translation start site, sequences that were not targetable using the CoDA ZFN platform. TALENs exhibited similar toxicity to CoDA ZFNs, with >50% of injected animals surviving to 3 days of life. Taken together, our results suggest that TALEN technology provides a robust alternative to CoDA ZFNs for inducing targeted gene-inactivation in zebrafish, making it a preferred technology for creating targeted knockout mutants in zebrafish.

  2. The loss of activating transcription factor 4 (ATF4) reduces bone toughness and fracture toughness.

    Science.gov (United States)

    Makowski, Alexander J; Uppuganti, Sasidhar; Wadeer, Sandra A; Whitehead, Jack M; Rowland, Barbara J; Granke, Mathilde; Mahadevan-Jansen, Anita; Yang, Xiangli; Nyman, Jeffry S

    2014-05-01

    Even though age-related changes to bone tissue affecting fracture risk are well characterized, only a few matrix-related factors have been identified as important to maintaining fracture resistance. As a gene critical to osteoblast differentiation, activating transcription factor 4 (ATF4) is possibly one of these important factors. To test the hypothesis that the loss of ATF4 affects the fracture resistance of bone beyond bone mass and structure, we harvested bones from Atf4+/+ and Atf4-/- littermates at 8 and 20 weeks of age (n≥9 per group) for bone assessment across several length scales. From whole bone mechanical tests in bending, femurs from Atf4-/- mice were found to be brittle with reduced toughness and fracture toughness compared to femurs from Atf4+/+ mice. However, there were no differences in material strength and in tissue hardness, as determined by nanoindentation, between the genotypes, irrespective of age. Tissue mineral density of the cortex at the point of loading as determined by micro-computed tomography was also not significantly different. However, by analyzing local composition by Raman Spectroscopy (RS), bone tissue of Atf4-/- mice was found to have higher mineral to collagen ratio compared to wild-type tissue, primarily at 20 weeks of age. From RS analysis of intact femurs at 2 orthogonal orientations relative to the polarization axis of the laser, we also found that the organizational-sensitive peak ratio, ν1Phosphate per Amide I, changed to a greater extent upon bone rotation for Atf4-deficient tissue, implying bone matrix organization may contribute to the brittleness phenotype. Target genes of ATF4 activity are not only important to osteoblast differentiation but also in maintaining bone toughness and fracture toughness. Published by Elsevier Inc.

  3. The Loss of Activating Transcription Factor 4 (ATF4) Reduces Bone Toughness and Fracture Toughness

    Science.gov (United States)

    Makowski, Alexander J.; Uppuganti, Sasidhar; Waader, Sandra A.; Whitehead, Jack M.; Rowland, Barbara J.; Granke, Mathilde; Mahadevan-Jansen, Anita; Yang, Xiangli; Nyman, Jeffry S.

    2014-01-01

    Even though age-related changes to bone tissue affecting fracture risk are well characterized, only a few matrix-related factors have been identified as important to maintaining fracture resistance. As a gene critical to osteoblast differentiation, activating transcription factor 4 (ATF4) is possibly one of the seimportant factors. To test the hypothesis that the loss of ATF4 affects the fracture resistance of bone beyond bone mass and structure, we harvested bones from Atf4+/+ and Atf4−/− littermates at 8 and 20 weeks of age (n≥9 per group) for bone assessment across several length scales. From whole bone mechanical tests in bending, femurs from Atf4−/− mice were found to be brittle with reduced toughness and fracture toughness compared to femurs from Atf4+/+ mice. However, there were no differences in material strength and in tissue hardness, as determined by nanoindentation, between the genotypes, irrespective age. Tissue mineral density of the cortex at the point of loading as determined by micro-computed tomography was also not significantly different. However, by analyzing local composition by Raman Spectroscopy (RS), bone tissue of Atf4−/− mice was found to have higher mineral to collagen ratio compared to wild-type tissue, primarily at 20 weeks of age. From RS analysis of intact femurs at 2 orthogonal orientations relative to the polarization axis of the laser, we also found that the organizational-sensitive peak ratio, ν1 Phosphate per Amide I, changed to a greater extent upon bone rotation for Atf4-deficient tissue, implying bone matrix organization may contribute to the brittleness phenotype. Target genes of ATF4 activity are not only important to osteoblast differentiation but also maintaining bone toughness and fracture toughness. PMID:24509412

  4. Mechanisms of transcriptional regulation and prognostic significance of activated leukocyte cell adhesion molecule in cancer

    Directory of Open Access Journals (Sweden)

    Chen Hairu

    2010-10-01

    Full Text Available Abstract Background Activated leukocyte cell adhesion molecule (ALCAM is implicated in the prognosis of multiple cancers with low level expression associated with metastasis and early death in breast cancer. Despite this significance, mechanisms that regulate ALCAM gene expression and ALCAM's role in adhesion of pre-metastatic circulating tumor cells have not been defined. We studied ALCAM expression in 20 tumor cell lines by real-time PCR, western blot and immunochemistry. Epigenetic alterations of the ALCAM promoter were assessed using methylation-specific PCR and bisulfite sequencing. ALCAM's role in adhesion of tumor cells to the vascular wall was studied in isolated perfused lungs. Results A common site for transcription initiation of the ALCAM gene was identified and the ALCAM promoter sequenced. The promoter contains multiple cis-active elements including a functional p65 NF-κB motif, and it harbors an extensive array of CpG residues highly methylated exclusively in ALCAM-negative tumor cells. These CpG residues were modestly demethylated after 5-aza-2-deoxycytidine treatment. Restoration of high-level ALCAM expression using an ALCAM cDNA increased clustering of MDA-MB-435 tumor cells perfused through the pulmonary vasculature of ventilated rat lungs. Anti-ALCAM antibodies reduced the number of intravascular tumor cell clusters. Conclusion Our data suggests that loss of ALCAM expression, due in part to DNA methylation of extensive segments of the promoter, significantly impairs the ability of circulating tumor cells to adhere to each other, and may therefore promote metastasis. These findings offer insight into the mechanisms for down-regulation of ALCAM gene expression in tumor cells, and for the positive prognostic value of high-level ALCAM in breast cancer.

  5. 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.

  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. 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.

  8. 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.

  9. 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.

  10. 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

  11. c-Abl phosphorylation of Yin Yang 1's conserved tyrosine 254 in the spacer region modulates its transcriptional activity.

    Science.gov (United States)

    Daraiseh, Susan I; Kassardjian, Ari; Alexander, Karen E; Rizkallah, Raed; Hurt, Myra M

    2018-05-25

    Yin Yang 1 (YY1) is a multifunctional transcription factor that can activate or repress transcription depending on the promotor and/or the co-factors recruited. YY1 is phosphorylated in various signaling pathways and is critical for different biological functions including embryogenesis, apoptosis, proliferation, cell-cycle regulation and tumorigenesis. Here we report that YY1 is a substrate for c-Abl kinase phosphorylation at conserved residue Y254 in the spacer region. Pharmacological inhibition of c-Abl kinase by imatinib, nilotinib and GZD824, knock-down of c-Abl using siRNA, and the use of c-Abl kinase-dead drastically reduces tyrosine phosphorylation of YY1. Both radioactive and non-radioactive in vitro kinase assays, as well as co-immunoprecipitation in different cell lines, show that the target of c-Abl phosphorylation is tyrosine residue 254. c-Abl phosphorylation has little effect on YY1 DNA binding ability or cellular localization in asynchronous cells. However, functional studies reveal that c-Abl mediated phosphorylation of YY1 regulates YY1's transcriptional ability in vivo. In conclusion, we demonstrate the novel role of c-Abl kinase in regulation of YY1's transcriptional activity, linking YY1 regulation with c-Abl tyrosine kinase signaling pathways. Copyright © 2018. Published by Elsevier B.V.

  12. Functional Genomic investigation of Peroxisome Proliferator-Activated Receptor Gamma (PPARG mediated transcription response in gastric cancer

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    Karthikeyan Selvarasu

    2017-10-01

    Full Text Available Cancer is a complex and progressive multi-step disorder that results from the transformation of normal cells to malignant derivatives. Several oncogenic signaling pathways are involved in this transformation. PPARG (Peroxisome proliferator-activated receptor gamma mediated transcription and signaling is involved in few cancers. We have investigated the PPARG in gastric tumors. The objective of the present study was to investigate the PPARG mediated transcriptional response in gastric tumors. Gene-set based and pathway focused gene-set enrichment analysis of available PPARG signatures in gastric tumor mRNA profiles shows that PPARG mediated transcription is highly activated in intestinal sub-type of gastric tumors. Further, we have derived the PPARG associated genes in gastric cancer and their expression was identified for the association with the better survival of the patients. Analysis of the PPARG associated genes reveals their involvement in mitotic cell cycle process, chromosome organization and nuclear division. Towards identifying the association with other oncogenic signaling process, E2F regulated genes were found associated with PPARG mediated transcription. The current results reveal the possible stratification of gastric tumors based on the PPARG gene expression and the possible development of PPARG targeted gastric cancer therapeutics. The identified PPARG regulated genes were identified to be targetable by pioglitazone and rosiglitazone. The identification of PPARG genes also in the normal stomach tissues reveal the possible involvement of these genes in the normal physiology of stomach and needs to be investigated.

  13. 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.

  14. 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.

  15. Kurarinol induces hepatocellular carcinoma cell apoptosis through suppressing cellular signal transducer and activator of transcription 3 signaling

    International Nuclear Information System (INIS)

    Shu, Guangwen; Yang, Jing; Zhao, Wenhao; Xu, Chan; Hong, Zongguo; Mei, Zhinan; Yang, Xinzhou

    2014-01-01

    Kurarinol is a flavonoid isolated from roots of the medical plant Sophora flavescens. However, its cytotoxic activity against hepatocellular carcinoma (HCC) cells and toxic effects on mammalians remain largely unexplored. Here, the pro-apoptotic activities of kurarinol on HCC cells and its toxic impacts on tumor-bearing mice were evaluated. The molecular mechanisms underlying kurarinol-induced HCC cell apoptosis were also investigated. We found that kurarinol dose-dependently provoked HepG2, Huh-7 and H22 HCC cell apoptosis. In addition, kurarinol gave rise to a considerable decrease in the transcriptional activity of signal transducer and activator of transcription 3 (STAT3) in HCC cells. Suppression of STAT3 signaling is involved in kurarinol-induced HCC cell apoptosis. In vivo studies showed that kurarinol injection substantially induced transplanted H22 cell apoptosis with low toxic impacts on tumor-bearing mice. Similarly, the transcriptional activity of STAT3 in transplanted tumor tissues was significantly suppressed after kurarinol treatment. Collectively, our current research demonstrated that kurarinol has the capacity of inducing HCC cell apoptosis both in vitro and in vivo with undetectable toxic impacts on the host. Suppressing STAT3 signaling is implicated in kurarinol-mediated HCC cell apoptosis. - Highlights: • Kurarinol induces hepatocellular carcinoma (HCC) cell apoptosis. • Kurarinol induces HCC cell apoptosis via inhibiting STAT3. • Kurarinol exhibits low toxic effects on tumor-bearing animals

  16. DAX-1 Inhibits Hepatocellular Carcinoma Proliferation by Inhibiting β-Catenin Transcriptional Activity

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    Hong-Lei Jiang

    2014-08-01

    Full Text Available Background/Aims: Hepatocellular carcinoma (HCC represents the most common type of liver cancer. DAX1 (dosage-sensitive sex reversal adrenal hypoplasia congenital critical region on X chromosome, gene 1, an atypical member of the nuclear receptor family due to lack of classical DNA-binding domains, has been known for its fundamental roles in the development, especially in the sex determination and steroidogenesis. Previous studies also showed that DAX-1 played a critical role in endocrine and sex steroid-dependent neoplasms such as adrenocortical, pituitary, endometrial, and ovarian tumors. However, its biological roles in the development of HCC remain largely unexplored. Methods: Real-time PCR and Western blot were used to detect the expression of DAX-1 in HCC tissues and cell lines. Immunoprecipitation (IP assay was used to show the interaction between DAX-1 and β-Catenin. Small interfering RNA (siRNA was used to silence the expression of DAX-1. BrdU incorporation and Cell-cycle assays were used to detect the role of DAX-1 in HCC cells proliferation. Migration and invasion assays were carried out to test the metastasis ability of DAX-1 in HCC cells. Results: In the present study, we found that mRNA and protein levels of DAX-1 were down-regulated in HCC tissues and cell lines. Furthermore, overexpression of DAX-1 could inhibit while its knockdown using small interfering RNA promoted cell proliferation in several HCC cell lines. At the molecular level, we demonstrated that DAX-1 could interact with β-Catenin and attenuate its transcriptional activity. Conclusion: Therefore, our results suggest a previously unknown DAX-1/β-Catenin molecular network controlling HCC development.

  17. The transcriptional activator LdtR from 'Candidatus Liberibacter asiaticus' mediates osmotic stress tolerance.

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    Fernando A Pagliai

    2014-04-01

    Full Text Available The causal agent of Huanglongbing disease, 'Candidatus Liberibacter asiaticus', is a non-culturable, gram negative, phloem-limited α-proteobacterium. Current methods to control the spread of this disease are still limited to the removal and destruction of infected trees. In this study, we identified and characterized a regulon from 'Ca. L. asiaticus' involved in cell wall remodeling, that contains a member of the MarR family of transcriptional regulators (ldtR, and a predicted L,D-transpeptidase (ldtP. In Sinorhizobium meliloti, mutation of ldtR resulted in morphological changes (shortened rod-type phenotype and reduced tolerance to osmotic stress. A biochemical approach was taken to identify small molecules that modulate LdtR activity. The LdtR ligands identified by thermal shift assays were validated using DNA binding methods. The biological impact of LdtR inactivation by the small molecules was then examined in Sinorhizobium meliloti and Liberibacter crescens, where a shortened-rod phenotype was induced by growth in presence of the ligands. A new method was also developed to examine the effects of small molecules on the viability of 'Ca. Liberibacter asiaticus', using shoots from HLB-infected orange trees. Decreased expression of ldtRLas and ldtPLas was observed in samples taken from HLB-infected shoots after 6 h of incubation with the LdtR ligands. These results provide strong proof of concept for the use of small molecules that target LdtR, as a potential treatment option for Huanglongbing disease.

  18. Transcriptional activity of the giant barrel sponge, Xestospongia muta Holobiont: Molecular Evidence for Metabolic Interchange

    Directory of Open Access Journals (Sweden)

    Cara L Fiore

    2015-04-01

    Full Text Available Compared to our understanding of the taxonomic composition of the symbiotic microbes in marine sponges, the functional diversity of these symbionts is largely unknown. Furthermore, the application of genomic, transcriptomic, and proteomic techniques to functional questions on sponge host-symbiont interactions is in its infancy. In this study, we generated a transcriptome for the host and a metatranscriptome of its microbial symbionts for the giant barrel sponge, Xestospongia muta, from the Caribbean. In combination with a gene-specific approach, our goals were to 1 characterize genetic evidence for nitrogen cycling in X. muta, an important limiting nutrient on coral reefs 2 identify which prokaryotic symbiont lineages are metabolically active and, 3 characterize the metabolic potential of the prokaryotic community. Xestospongia muta expresses genes from multiple nitrogen transformation pathways that when combined with the abundance of this sponge, and previous data on dissolved inorganic nitrogen fluxes, shows that this sponge is an important contributor to nitrogen cycling on coral reefs. Additionally, we observed significant differences in gene expression of the archaeal amoA gene, which is involved in ammonia oxidation, between coral reef locations consistent with differences in the fluxes of dissolved inorganic nitrogen previously reported. In regards to symbiont metabolic potential, the genes in the biosynthetic pathways of several amino acids were present in the prokaryotic metatranscriptome dataset but in the host-derived transcripts only the catabolic reactions for these amino acids were present. A similar pattern was observed for the B vitamins (riboflavin, biotin, thiamin, cobalamin. These results expand our understanding of biogeochemical cycling in sponges, and the metabolic interchange highlighted here advances the field of symbiont physiology by elucidating specific metabolic pathways where there is high potential for host

  19. Thyroid hormone receptor inhibits hepatoma cell migration through transcriptional activation of Dickkopf 4

    Energy Technology Data Exchange (ETDEWEB)

    Chi, Hsiang-Cheng; Liao, Chen-Hsin [Department of Biochemistry, School of Medicine, Chang-Gung University, Taoyuan 333, Taiwan, ROC (China); Huang, Ya-Hui [Medical Research Central, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan, ROC (China); Wu, Sheng-Ming; Tsai, Chung-Ying; Liao, Chia-Jung; Tseng, Yi-Hsin; Lin, Yang-Hsiang; Chen, Cheng-Yi; Chung, I-Hsiao; Wu, Tzu-I [Department of Biochemistry, School of Medicine, Chang-Gung University, Taoyuan 333, Taiwan, ROC (China); Chen, Wei-Jan [First Cardiovascular Division, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan, ROC (China); Lin, Kwang-Huei, E-mail: khlin@mail.cgu.edu.tw [Department of Biochemistry, School of Medicine, Chang-Gung University, Taoyuan 333, Taiwan, ROC (China)

    2013-09-13

    Highlights: •T{sub 3} affects DKK4 mRNA and protein expression in HepG2-TR cells. •Regulation of DKK4 by T{sub 3} is at transcriptional level. •DKK4 overexpression suppresses hepatoma cell metastasis. -- Abstract: Triiodothyronine (T{sub 3}) is a potent form of thyroid hormone mediates several physiological processes including cellular growth, development, and differentiation via binding to the nuclear thyroid hormone receptor (TR). Recent studies have demonstrated critical roles of T{sub 3}/TR in tumor progression. Moreover, long-term hypothyroidism appears to be associated with the incidence of human hepatocellular carcinoma (HCC), independent of other major HCC risk factors. Dickkopf (DKK) 4, a secreted protein that antagonizes the canonical Wnt signaling pathway, is induced by T{sub 3} at both mRNA and protein levels in HCC cell lines. However, the mechanism underlying T{sub 3}-mediated regulation of DKK4 remains unknown. In the present study, the 5′ promoter region of DKK4 was serially deleted, and the reporter assay performed to localize the T{sub 3} response element (TRE). Consequently, we identified an atypical direct repeat TRE between nucleotides −1645 and −1629 conferring T{sub 3} responsiveness to the DKK4 gene. This region was further validated using chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA). Stable DKK4 overexpression in SK-Hep-1 cells suppressed cell invasion and metastatic potential, both in vivo andin vitro, via reduction of matrix metalloproteinase-2 (MMP-2) expression. Our findings collectively suggest that DKK4 upregulated by T{sub 3}/TR antagonizes the Wnt signal pathway to suppress tumor cell progression, thus providing new insights into the molecular mechanism underlying thyroid hormone activity in HCC.

  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

  1. Immunohistological Analysis of the Jun Family and the Signal Transducers and Activators of Transcription in Thymus

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    Alexandra Papoudou-Bai

    2015-01-01

    Full Text Available The Jun family and the signal transducers and activators of transcription (STAT are involved in proliferation and apoptosis. Moreover, c-Jun and STAT3 cooperate to regulate apoptosis. Therefore, we used double immunostaining to investigate the immunotopographical distribution of phospho-c-Jun (p-c-Jun, JunB, JunD, p-STAT3, p-STAT5, and p-STAT6 in human thymus. JunD was frequently expressed by thymocytes with higher expression in medullary compared to cortical thymocytes. p-c-Jun was frequently expressed by cortical and medullary thymic epithelial cells (TEC and Hassall bodies (HB. p-STAT3 was frequently expressed by TEC with higher expression in cortical compared to medullary TEC and HB. p-c-Jun, JunB, p-STAT3, p-STAT5, and p-STAT6 were rarely expressed by thymocytes. JunB and JunD were expressed by rare cortical TEC with higher expression in medullary TEC. p-STAT5 and p-STAT6 were expressed by rare cortical and medullary TEC. Double immunostaining revealed p-c-Jun and JunD expression in rare CD11c positive dendritic cells. Our findings suggest a notable implication of JunD in the physiology of thymocytes and p-c-Jun and p-STAT3 in the physiology of TEC. The diversity of the immunotopographical distribution and the expression levels of p-c-Jun, JunB, JunD, p-STAT3, p-STAT5, and p-STAT6 indicates that they are differentially involved in the differentiation of TEC, thymocytes, and dendritic cells.

  2. Peripheral CLOCK regulates target-tissue glucocorticoid receptor transcriptional activity in a circadian fashion in man.

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    Evangelia Charmandari

    Full Text Available Circulating cortisol fluctuates diurnally under the control of the "master" circadian CLOCK, while the peripheral "slave" counterpart of the latter regulates the transcriptional activity of the glucocorticoid receptor (GR at local glucocorticoid target tissues through acetylation. In this manuscript, we studied the effect of CLOCK-mediated GR acetylation on the sensitivity of peripheral tissues to glucocorticoids in humans.We examined GR acetylation and mRNA expression of GR, CLOCK-related and glucocorticoid-responsive genes in peripheral blood mononuclear cells (PBMCs obtained at 8 am and 8 pm from 10 healthy subjects, as well as in PBMCs obtained in the morning and cultured for 24 hours with exposure to 3-hour hydrocortisone pulses every 6 hours. We used EBV-transformed lymphocytes (EBVLs as non-synchronized controls.GR acetylation was higher in the morning than in the evening in PBMCs, mirroring the fluctuations of circulating cortisol in reverse phase. All known glucocorticoid-responsive genes tested responded as expected to hydrocortisone in non-synchronized EBVLs, however, some of these genes did not show the expected diurnal mRNA fluctuations in PBMCs in vivo. Instead, their mRNA oscillated in a Clock- and a GR acetylation-dependent fashion in naturally synchronized PBMCs cultured ex vivo in the absence of the endogenous glucocorticoid, suggesting that circulating cortisol might prevent circadian GR acetylation-dependent effects in some glucocorticoid-responsive genes in vivo.Peripheral CLOCK-mediated circadian acetylation of the human GR may function as a target-tissue, gene-specific counter regulatory mechanism to the actions of diurnally fluctuating cortisol, effectively decreasing tissue sensitivity to glucocorticoids in the morning and increasing it at night.

  3. Peripheral CLOCK Regulates Target-Tissue Glucocorticoid Receptor Transcriptional Activity in a Circadian Fashion in Man

    Science.gov (United States)

    Charmandari, Evangelia; Chrousos, George P.; Lambrou, George I.; Pavlaki, Aikaterini; Koide, Hisashi; Ng, Sinnie Sin Man; Kino, Tomoshige

    2011-01-01

    Context and Objective Circulating cortisol fluctuates diurnally under the control of the “master” circadian CLOCK, while the peripheral “slave” counterpart of the latter regulates the transcriptional activity of the glucocorticoid receptor (GR) at local glucocorticoid target tissues through acetylation. In this manuscript, we studied the effect of CLOCK-mediated GR acetylation on the sensitivity of peripheral tissues to glucocorticoids in humans. Design and Participants We examined GR acetylation and mRNA expression of GR, CLOCK-related and glucocorticoid-responsive genes in peripheral blood mononuclear cells (PBMCs) obtained at 8 am and 8 pm from 10 healthy subjects, as well as in PBMCs obtained in the morning and cultured for 24 hours with exposure to 3-hour hydrocortisone pulses every 6 hours. We used EBV-transformed lymphocytes (EBVLs) as non-synchronized controls. Results GR acetylation was higher in the morning than in the evening in PBMCs, mirroring the fluctuations of circulating cortisol in reverse phase. All known glucocorticoid-responsive genes tested responded as expected to hydrocortisone in non-synchronized EBVLs, however, some of these genes did not show the expected diurnal mRNA fluctuations in PBMCs in vivo. Instead, their mRNA oscillated in a Clock- and a GR acetylation-dependent fashion in naturally synchronized PBMCs cultured ex vivo in the absence of the endogenous glucocorticoid, suggesting that circulating cortisol might prevent circadian GR acetylation-dependent effects in some glucocorticoid-responsive genes in vivo. Conclusions Peripheral CLOCK-mediated circadian acetylation of the human GR may function as a target-tissue, gene-specific counter regulatory mechanism to the actions of diurnally fluctuating cortisol, effectively decreasing tissue sensitivity to glucocorticoids in the morning and increasing it at night. PMID:21980503

  4. Signal transducer and activator of transcription and the risk of rheumatoid arthritis and thyroid autoimmune disorders.

    Science.gov (United States)

    Ben Hamad, M; Cornelis, F; Mbarek, H; Chabchoub, G; Marzouk, S; Bahloul, Z; Rebai, A; Fakhfakh, F; Ayadi, H; Petit-Teixeira, E; Maalej, A

    2011-01-01

    The signal transducer and activator of transcription 4 (STAT4) gene localised on chromosome 2q32.2-q32.3 is known to be essential for mediating responses to interleukin 12 in lymphocytes and regulating the differentiation of T helper cells. The aim of this study was to investigate the role of the STAT4 gene in susceptibility to rheumatoid arthritis (RA) and autoimmune thyroid diseases (AITDs) in Tunisian case control studies. Genotyping of STAT4 rs7574865 single nucleotide polymorphism (SNP) was performed in 140 patients affected with RA, 159 patients affected with AITDs and 200 healthy controls using TaqMan® allelic discrimination assay. Data were analysed by χ2-test, genotype relative risk (GRR) and odds ratio (OR). Our results revealed that frequencies of the T allele and the T/T genotype were significantly higher among RA patients compared to controls (p=0.008; p=0.003, respectively). However, no significant associations with the risk of autoimmune thyroid diseases were detected. Moreover, the stratification of RA patients subgroups revealed a significant association of both T allele and T/T genotype in patients presented erosion (p=0.003; p=0.004, respectively) as well as anti-cyclic peptides-negative RA (ACPA-) (p=0.002; p=0.0003, respectively). Furthermore, genotypic association was found according to the absence of rheumatoid factor antibody (RF) (p=0.0014). But, no significant differences in allele and genotype frequencies of STAT4 rs7574865 polymorphism were detected according to the presence of another autoimmune disease, nodules and in HLA-DRB1*04 and HLA-DRB1*0404 positive subgroups. Our results support involvement of the STAT4 gene in the genetic susceptibility to RA but not to AITDs in the Tunisian population.

  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. Susceptibility to bystander DNA damage is influenced by replication and transcriptional activity

    Science.gov (United States)

    Dickey, Jennifer S.; Baird, Brandon J.; Redon, Christophe E.; Avdoshina, Valeriya; Palchik, Guillermo; Wu, Junfang; Kondratyev, Alexei; Bonner, William M.; Martin, Olga A.

    2012-01-01

    Direct cellular DNA damage may lead to genome destabilization in unexposed, bystander, cells sharing the same milieu with directly damaged cells by means of the bystander effect. One proposed mechanism involves double strand break (DSB) formation in S phase cells at sites of single strand lesions in the DNA of replication complexes, which has a more open structure compared with neighboring DNA. The DNA in transcription complexes also has a more open structure, and hence may be susceptible to bystander DSB formation from single strand lesions. To examine whether transcription predisposes non-replicating cells to bystander effect-induced DNA DSBs, we examined two types of primary cells that exhibit high levels of transcription in the absence of replication, rat neurons and human lymphocytes. We found that non-replicating bystander cells with high transcription rates exhibited substantial levels of DNA DSBs, as monitored by γ-H2AX foci formation. Additionally, as reported in proliferating cells, TGF-β and NO were found to mimic bystander effects in cell populations lacking DNA synthesis. These results indicate that cell vulnerability to bystander DSB damage may result from transcription as well as replication. The findings offer insights into which tissues may be vulnerable to bystander genomic destabilization in vivo. PMID:22941641

  7. Mechanism of transcription activation at the comG promoter by the competence transcription factor ComK of Bacillus subtilis

    NARCIS (Netherlands)

    Susanna, KA; van der Werff, AF; den Hengst, CD; Calles, B; Salas, M; Venema, G; Hamoen, LW; Kuipers, OP

    The development of genetic competence in Bacillus subtilis is regulated by a complex signal transduction cascade, which results in the synthesis of the competence transcription factor, encoded by comK. ComK is required for the transcription of the late competence genes that encode the DNA binding

  8. A transcriptionally active estrogen receptor mutant is a novel type of dominant negative inhibitor of estrogen action.

    Science.gov (United States)

    McInerney, E M; Ince, B A; Shapiro, D J; Katzenellenbogen, B S

    1996-12-01

    We have characterized a human estrogen receptor (ER) mutant, V364E, which has a single amino acid substitution in its hormone-binding domain. This ER mutant is fully active or even superactive at saturating levels of estradiol (10(-8) M E2) yet has the capacity to act as a strong dominant negative inhibitor of the wild type ER. In transient transfection assays using ER-negative Chinese hamster ovary (CHO) cells and two different estrogen response element (ERE)-containing promoter reporter genes, V364E treated with 10(-8) M E2 exhibited approximately 250% and 100% of the activity of the wild type ER with these two promoter contexts, respectively. Despite the high activity of V364E when present alone in cells, coexpression of both V364E and wild type ER causes a significant decrease in overall ER-mediated transcriptional activity. On the TATA promoter, where V364E was more inhibitory, estrogen-stimulated activity was reduced by approximately 50% at a 1:1 ratio of mutant to wild type ER expression vector, and at a 10:1 ratio, 75% of ER activity was inhibited. V364E was expressed at lower levels than wild type ER and has a approximately 40-fold lower affinity for E2 compared with wild type ER. In promoter interference assays, V364E exhibited a strict dependence upon E2 for binding to an ERE. Surprisingly, even when V364E was unable to bind to ERE DNA (i.e. either at low E2 concentration or by mutation of its DNA-binding domain), this mutant retained full dominant negative activity. This highly active ER mutant is, thus, able to repress ER-mediated transcription when the mutant and wild type ER are present together in cells, even without DNA binding. Since competition for ERE binding and the formation of inactive heterodimers cannot fully account for the dominant negative activity of V364E, it is probable that altered interactions with proteins important in ER-mediated transcription play a key role in the repression of transcription by V364E. The properties and probable

  9. 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.

  10. 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.

  11. MANAGEMENT OF INVESTMENT ACTIVITY OF THE ENTERPRISE UNDER CRISIS CONDITIONS

    Directory of Open Access Journals (Sweden)

    O. Naumova

    2016-04-01

    Full Text Available The method of comparison, analysis and synthesis, induction and deduction in devoted paper for the purpose of summarized modern theoretical approaches to the investment activity of the enterprise are used. The research is based on the analysis of scientific papers of leading scientists about investment activity of the enterprise under crisis economic conditions. The relationship between corporate management, technological modification of the production process, the government protection of the property rights of investors under crisis conditions and investment activity of Ukrainian enterprises are analyzed. The paper is shown that lack of the investment activity in Ukraine is a negative consequence of the presence a high degree of risk as a result of banking crisis and serious legal violations in the activity of enterprises.

  12. Alteration of the exopolysaccharide production and the transcriptional profile of free-living Frankia strain CcI3 under nitrogen-fixing conditions.

    Science.gov (United States)

    Lee, Hae-In; Donati, Andrew J; Hahn, Dittmar; Tisa, Louis S; Chang, Woo-Suk

    2013-12-01

    We investigated the effect of different nitrogen (N) sources on exopolysaccharide (EPS) production and composition by Frankia strain CcI3, a N2-fixing actinomycete that forms root nodules with Casuarina species. Frankia cells grown in the absence of NH4Cl (i.e., under N2-fixing conditions) produced 1.7-fold more EPS, with lower galactose (45.1 vs. 54.7 mol%) and higher mannose (17.3 vs. 9.7 mol%) contents than those grown in the presence of NH4Cl as a combined N-source. In the absence of the combined N-source, terminally linked and branched residue contents were nearly twice as high with 32.8 vs. 15.1 mol% and 15.1 vs. 8.7 mol%, respectively, than in its presence, while the content of linearly linked residues was lower with 52.1 mol% compared to 76.2 mol%. To find out clues for the altered EPS production at the transcriptional level, we performed whole-gene expression profiling using quantitative reverse transcription PCR and microarray technology. The transcription profiles of Frankia strain CcI3 grown in the absence of NH4Cl revealed up to 2 orders of magnitude higher transcription of nitrogen fixation-related genes compared to those of CcI3 cells grown in the presence of NH4Cl. Unexpectedly, microarray data did not provide evidence for transcriptional regulation as a mechanism for differences in EPS production. These findings indicate effects of nitrogen fixation on the production and composition of EPS in Frankia strain CcI3 and suggest posttranscriptional regulation of enhanced EPS production in the absence of the combined N-source.

  13. Transcriptional regulator-mediated activation of adaptation genes triggers CRISPR de novo spacer acquisition

    DEFF Research Database (Denmark)

    Liu, Tao; Li, Yingjun; Wang, Xiaodi

    2015-01-01

    Acquisition of de novo spacer sequences confers CRISPR-Cas with a memory to defend against invading genetic elements. However, the mechanism of regulation of CRISPR spacer acquisition remains unknown. Here we examine the transcriptional regulation of the conserved spacer acquisition genes in Type I......, it was demonstrated that the transcription level of csa1, cas1, cas2 and cas4 was significantly enhanced in a csa3a-overexpression strain and, moreover, the Csa1 and Cas1 protein levels were increased in this strain. Furthermore, we demonstrated the hyperactive uptake of unique spacers within both CRISPR loci...... in the presence of the csa3a overexpression vector. The spacer acquisition process is dependent on the CCN PAM sequence and protospacer selection is random and non-directional. These results suggested a regulation mechanism of CRISPR spacer acquisition where a single transcriptional regulator senses the presence...

  14. Active Center Control of Termination by RNA Polymerase III and tRNA Gene Transcription Levels In Vivo.

    Directory of Open Access Journals (Sweden)

    Keshab Rijal

    2016-08-01

    Full Text Available The ability of RNA polymerase (RNAP III to efficiently recycle from termination to reinitiation is critical for abundant tRNA production during cellular proliferation, development and cancer. Yet understanding of the unique termination mechanisms used by RNAP III is incomplete, as is its link to high transcription output. We used two tRNA-mediated suppression systems to screen for Rpc1 mutants with gain- and loss- of termination phenotypes in S. pombe. 122 point mutation mutants were mapped to a recently solved 3.9 Å structure of yeast RNAP III elongation complex (EC; they cluster in the active center bridge helix and trigger loop, as well as the pore and funnel, the latter of which indicate involvement of the RNA cleavage domain of the C11 subunit in termination. Purified RNAP III from a readthrough (RT mutant exhibits increased elongation rate. The data strongly support a kinetic coupling model in which elongation rate is inversely related to termination efficiency. The mutants exhibit good correlations of terminator RT in vitro and in vivo, and surprisingly, amounts of transcription in vivo. Because assessing in vivo transcription can be confounded by various parameters, we used a tRNA reporter with a processing defect and a strong terminator. By ruling out differences in RNA decay rates, the data indicate that mutants with the RT phenotype synthesize more RNA than wild type cells, and than can be accounted for by their increased elongation rate. Finally, increased activity by the mutants appears unrelated to the RNAP III repressor, Maf1. The results show that the mobile elements of the RNAP III active center, including C11, are key determinants of termination, and that some of the mutations activate RNAP III for overall transcription. Similar mutations in spontaneous cancer suggest this as an unforeseen mechanism of RNAP III activation in disease.

  15. Arabidopsis basic leucine zipper transcription factors involved in an abscisic acid-dependent signal transduction pathway under drought and high-salinity conditions.

    Science.gov (United States)

    Uno, Y; Furihata, T; Abe, H; Yoshida, R; Shinozaki, K; Yamaguchi-Shinozaki, K

    2000-10-10

    The induction of the dehydration-responsive Arabidopsis gene, rd29B, is mediated mainly by abscisic acid (ABA). Promoter analysis of rd29B indicated that two ABA-responsive elements (ABREs) are required for the dehydration-responsive expression of rd29B as cis-acting elements. Three cDNAs encoding basic leucine zipper (bZIP)-type ABRE-binding proteins were isolated by using the yeast one-hybrid system and were designated AREB1, AREB2, and AREB3 (ABA-responsive element binding protein). Transcription of the AREB1 and AREB2 genes is up-regulated by drought, NaCl, and ABA treatment in vegetative tissues. In a transient transactivation experiment using Arabidopsis leaf protoplasts, both the AREB1 and AREB2 proteins activated transcription of a reporter gene driven by ABRE. AREB1 and AREB2 required ABA for their activation, because their transactivation activities were repressed in aba2 and abi1 mutants and enhanced in an era1 mutant. Activation of AREBs by ABA was suppressed by protein kinase inhibitors. These results suggest that both AREB1 and AREB2 function as transcriptional activators in the ABA-inducible expression of rd29B, and further that ABA-dependent posttranscriptional activation of AREB1 and AREB2, probably by phosphorylation, is necessary for their maximum activation by ABA. Using cultured Arabidopsis cells, we demonstrated that a specific ABA-activated protein kinase of 42-kDa phosphorylated conserved N-terminal regions in the AREB proteins.

  16. Can AtTZF1 act as a transcriptional activator or repressor in plants?

    OpenAIRE

    Pomeranz, Marcelo; Zhang, Li; Finer, John; Jang, Jyan-Chyun

    2011-01-01

    In animals, Tandem CCCH Zinc Finger (TZF) proteins can affect gene expression at both transcriptional and post-transcriptional levels. In Arabidopsis thaliana, AtTZF1 is a member of the TZF family characterized by a plant-unique tandem zinc finger motif. AtTZF1 can bind both DNA and RNA in vitro, and it can traffic between the nucleus and cytoplasmic foci. However, no in vivo DNA/RNA targets have been identified so far, and little is known about the molecular mechanisms underlying AtTZF1's pr...

  17. The forkhead transcription factor Foxl2 is sumoylated in both human and mouse: sumoylation affects its stability, localization, and activity.

    Directory of Open Access Journals (Sweden)

    Mara Marongiu

    2010-03-01

    Full Text Available The FOXL2 forkhead transcription factor is expressed in ovarian granulosa cells, and mutated FOXL2 causes the blepharophimosis, ptosis and epicanthus inversus syndrome (BPES and predisposes to premature ovarian failure. Inactivation of Foxl2 in mice demonstrated its indispensability for female gonadal sex determination and ovary development and revealed its antagonism of Sox9, the effector of male testis development. To help to define the regulatory activities of FOXL2, we looked for interacting proteins. Based on yeast two-hybrid screening, we found that FOXL2 interacts with PIAS1 and UBC9, both parts of the sumoylation machinery. We showed that human FOXL2 is sumoylated in transfected cell lines, and that endogenous mouse Foxl2 is comparably sumoylated. This modification changes its cellular localization, stability and transcriptional activity. It is intriguing that similar sumoylation and regulatory consequences have also been reported for SOX9, the male counterpart of FOXL2 in somatic gonadal tissues.

  18. In vivo evidence suggesting reciprocal renal hypoxia-inducible factor-1 upregulation and signal transducer and activator of transcription 3 activation in response to hypoxic and non-hypoxic stimuli.

    Science.gov (United States)

    Nechemia-Arbely, Yael; Khamaisi, Mogher; Rosenberger, Christian; Koesters, Robert; Shina, Ahuva; Geva, Carmit; Shriki, Anat; Klaus, Stephen; Rosen, Seymour; Rose-John, Stefan; Galun, Eithan; Axelrod, Jonathan H; Heyman, Samuel N

    2013-04-01

    In vitro studies suggest that combined activation of hypoxia-inducible factor (HIF) and signal transducer and activator of transcription 3 (STAT3) promotes the hypoxia response. However, their interrelationship in vivo remains poorly defined. The present study investigated the possible relationship between HIF-1 upregulation and STAT3 activation in the rodent kidney in vivo. Activation of HIF-1 and STAT3 was analysed by immunohistochemical staining and western blot analysis in: (i) models of hypoxia-associated kidney injury induced by radiocontrast media or rhabdomyolysis; (ii) following activation of STAT3 by the interleukin (IL)-6-soluble IL-6 receptor complex; or (iii) following HIF-1α stabilization using hypoxic and non-hypoxic stimuli (mimosine, FG-4497, CO, CoCl(2)) and in targeted von Hippel-Lindau-knockout mice. Western blot analysis and immunostaining revealed marked induction of both transcription factors under all conditions tested, suggesting that in vivo STAT3 can trigger HIF and vice versa. Colocalization of HIF-1α and phosphorylated STAT3 was detected in some, but not all, renal cell types, suggesting that in some cells a paracrine mechanism may be responsible for the reciprocal activation of the two transcription factors. Nevertheless, in several cell types spatial concordance was observed under the majority of conditions tested, suggesting that HIF-1 and STAT3 may act as cotranscription factors. These in vivo studies suggest that, in response to renal hypoxic-stress, upregulation of HIF-1 and activation of STAT3 may be both reciprocal and cell type dependent. © 2013 The Authors Clinical and Experimental Pharmacology and Physiology © 2013 Wiley Publishing Asia Pty Ltd.

  19. Signal transduction and HIV transcriptional activation after exposure to ultraviolet light and other DNA-damaging agents

    International Nuclear Information System (INIS)

    Valerie, K.; Laster, W.S.; Luhua Cheng; Kirkham, J.C.; Reavey, Peter; Kuemmerle, N.B.

    1996-01-01

    Short wavelength (254 nm) ultraviolet light (UVC) radiation was much more potent in activating transcription of human immunodeficiency virus 1 (HIV) reporter genes stably integrated into the genomes of human and monkey cells than ionizing radiation (IR) from a 137 Cs source at similarly cytotoxic doses. A similar differential was also observed when c-jun transcription levels were examined. However, these transcription levels do not correlate with activation of nuclear factor (NF)-kB and AP-1 measured by band-shift assays, i.e. both types of radiation produce similar increases in NF-kB and AP-1 activity, suggesting existence of additional levels of regulation during these responses. Because of the well-established involvement of cytoplasmic signaling pathways in the cellular response to tumor necrosis factor-α (TNF-α), UVC, and IR using other types of assays, the role of TNF-α in the UVC response of HIV and c-jun was investigated in our cell system. We demonstrate that UVC and TNF-α activate HIV gene expression in a synergistic fashion, suggesting that it is unlikely that TNF-α is involved in UVC activation of HIV transcription in stably transfected HeLa cells. Moreover, maximum TNF-α stimulation resulted in one order of magnitude lower levels of HIV expression than that observed after UVC exposure. We also observed an additive effect of UVC and TNF-α on c-jun steady-state mRNA levels, suggestive of a partial overlap in activation mechanism of c-jun by UVC and TNF-α; yet these responses are distinct to some extent. Our results indicate that the HIV, and to some extent also the c-jun, transcriptional responses to UVC are not the result of TNF-α stimulation and subsequent downstream cytoplasmic signaling events in HeLa cells. In addition to the new data, this report also summarizes our current views regarding UVC-induced activations of HIV gene expression in stably transfected cells. (Author)

  20. BRD4 Regulates Transcription via Intrinsic HAT Activity | Center for Cancer Research

    Science.gov (United States)

    In order to express a gene, its DNA must be accessible to the transcription machinery. This requires chromatin de-compaction, which depends on the addition of acetyl groups to lysine residues on histones, thereby weakening interactions between histones and DNA and between adjacent nucleosomes.

  1. 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

  2. Condition of esthetic component of motive activity in aerobic gymnastics

    Directory of Open Access Journals (Sweden)

    Valentina Todorova

    2017-02-01

    Full Text Available Purpose: to prove condition of esthetic component of motive activity of sportsmen in aerobic gymnastics at various stages of sports preparation. Material & Methods: analysis of video records, method of expert assessment, methods of mathematical statistics. Results: the level of criteria of esthetic component ("musicality", "dancing", "illustrative expressiveness", and "emotional expressiveness" with use of the offered rating scale is determined. Conclusions: indicators of motive activities of sportsmen for aerobic gymnastics are defined at various stages of sports preparation.

  3. Role of the N-terminal activation domain of coactivator CoCoA in mediating transcriptional activation by β-catenin*

    OpenAIRE

    Yang, Catherine K.; Kim, Jeong Hoon; Stallcup, Michael R.

    2006-01-01

    The coiled-coil coactivator (CoCoA) is involved in transcriptional activation of target genes by nuclear receptors and the xenobiotic aryl hydrocarbon receptor, as well as target genes of the Wnt signaling pathway, which is mediated by the lymphocyte enhancer factor (LEF)/T cell factor transcription factors and the coactivator β-catenin. The recruitment of CoCoA by nuclear receptors is accomplished by the interaction of the central coiled-coiled domain of CoCoA with p160 coactivators; the C-t...

  4. Structures of BmrR-Drug Complexes Reveal a Rigid Multidrug Binding Pocket And Transcription Activation Through Tyrosine Expulsion

    Energy Technology Data Exchange (ETDEWEB)

    Newberry, K.J.; Huffman, J.L.; Miller, M.C.; Vazquez-Laslop, N.; Neyfakh, A.A.; Brennan, R.G.

    2009-05-22

    BmrR is a member of the MerR family and a multidrug binding transcription factor that up-regulates the expression of the bmr multidrug efflux transporter gene in response to myriad lipophilic cationic compounds. The structural mechanism by which BmrR binds these chemically and structurally different drugs and subsequently activates transcription is poorly understood. Here, we describe the crystal structures of BmrR bound to rhodamine 6G (R6G) or berberine (Ber) and cognate DNA. These structures reveal each drug stacks against multiple aromatic residues with their positive charges most proximal to the carboxylate group of Glu-253 and that, unlike other multidrug binding pockets, that of BmrR is rigid. Substitution of Glu-253 with either alanine (E253A) or glutamine (E253Q) results in unpredictable binding affinities for R6G, Ber, and tetraphenylphosphonium. Moreover, these drug binding studies reveal that the negative charge of Glu-253 is not important for high affinity binding to Ber and tetraphenylphosphonium but plays a more significant, but unpredictable, role in R6G binding. In vitro transcription data show that E253A and E253Q are constitutively active, and structures of the drug-free E253A-DNA and E253Q-DNA complexes support a transcription activation mechanism requiring the expulsion of Tyr-152 from the multidrug binding pocket. In sum, these data delineate the mechanism by which BmrR binds lipophilic, monovalent cationic compounds and suggest the importance of the redundant negative electrostatic nature of this rigid drug binding pocket that can be used to discriminate against molecules that are not substrates of the Bmr multidrug efflux pump.

  5. 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 ad