The C'-terminal interaction domain of the thyroid hormone receptor confers the ability of the DNA site to dictate positive or negative transcriptional activity

International Nuclear Information System (INIS)

Holloway, J.M.; Glass, C.K.; Adler, S.; Nelson, C.A.; Rosenfeld, M.G.

1990-01-01

To investigate mechanisms responsible for positive and negative transcriptional control, the authors have utilized two types of promoters that are diffferentially regulated by thyroid hormone (T 3 ) receptors. Promoters containing the palindromic T 3 response element TCAGGTCA TGACCTGA are positively regulated by the T 3 receptor after the administration of T 3 , whereas otherwise identical promoters containing the estrogen response element TCAGGTCA CTG TGACCTGA can be regulated negatively; converse effects are observed with the estrogen receptor. They describe evidence that the transcriptional inhibitory effects of the T 3 or estrogen receptors on the estrogen or T 3 response elements, respectively, are imposed by amino acid sequences in the C'-terminal region that colocalize with dimerization and hormone-binding domains and that these sequences can transfer inhibitory functions to other classes of transcription factors. Removal of the C'-terminal dimerization and hormone-binding domains of either the αT 3 or estrogen receptors permits each receptor to act constitutively to enhance transcription on both T 3 and estrogen response elements. It is, therefore, suggested that protein-protein interactions between receptor C' termini limit the subset of DNA binding sites on which transcriptional activation occurs

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

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Kristin E Murphy

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

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

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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.

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

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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.

Functional Interaction Map of Lyssavirus Phosphoprotein: Identification of the Minimal Transcription Domains

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Jacob, Yves; Real, Eléonore; Tordo, Noël

2001-01-01

Lyssaviruses, the causative agents of rabies encephalitis, are distributed in seven genotypes. The phylogenetically distant rabies virus (PV strain, genotype 1) and Mokola virus (genotype 3) were used to develop a strategy to identify functional homologous interactive domains from two proteins (P and N) which participate in the viral ribonucleoprotein (RNP) transcription-replication complex. This strategy combined two-hybrid and green fluorescent protein–reverse two-hybrid assays in Saccharomyces cerevisiae to analyze protein-protein interactions and a reverse genetic assay in mammalian cells to study the transcriptional activity of the reconstituted RNP complex. Lyssavirus P proteins contain two N-binding domains (N-BDs), a strong one encompassing amino acid (aa) 176 to the C terminus and a weak one in the 189 N-terminal aa. The N-terminal portion of P (aa 52 to 189) also contains a homomultimerization site. Here we demonstrate that N-P interactions, although weaker, are maintained between proteins of the different genotypes. A minimal transcriptional module of the P protein was obtained by fusing the first 60 N-terminal aa containing the L protein binding site to the C-terminal strong N-BD. Random mutation of the strong N-BD on P protein identified three highly conserved K residues crucial for N-P interaction. Their mutagenesis in full-length P induced a transcriptionally defective RNP. The analysis of homologous interactive domains presented here and previously reported dissections of the P protein allowed us to propose a model of the functional interaction network of the lyssavirus P protein. This model underscores the central role of P at the interface between L protein and N-RNA template. PMID:11559793

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

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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.

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....

Crystallization and preliminary X-ray analysis of the ATPase domain of the σ(54)-dependent transcription activator NtrC1 from Aquifex aeolicus bound to the ATP analog ADP-BeFx.

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Sysoeva, Tatyana A; Yennawar, Neela; Allaire, Marc; Nixon, B Tracy

2013-12-01

One way that bacteria regulate the transcription of specific genes to adapt to environmental challenges is to use different σ factors that direct the RNA polymerase holoenzyme to distinct promoters. Unlike σ(70) RNA polymerase (RNAP), σ(54) RNAP is unable to initiate transcription without an activator: enhancer-binding protein (EBP). All EBPs contain one ATPase domain that belongs to the family of ATPases associated with various cellular activities (AAA+ ATPases). AAA+ ATPases use the energy of ATP hydrolysis to remodel different target macromolecules to perform distinct functions. These mechanochemical enzymes are known to form ring-shaped oligomers whose conformations strongly depend upon nucleotide status. Here, the crystallization of the AAA+ ATPase domain of an EBP from Aquifex aeolicus, NtrC1, in the presence of the non-hydrolyzable ATP analog ADP-BeFx is reported. X-ray diffraction data were collected from two crystals from two different protein fractions of the NtrC1 ATPase domain. Previously, this domain was co-crystallized with ADP and ATP, but the latter crystals were grown from the Walker B substitution variant E239A. Therefore, the new data sets are the first for a wild-type EBP ATPase domain co-crystallized with an ATP analog and they reveal a new crystal form. The resulting structure(s) will shed light on the mechanism of EBP-type transcription activators.

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

An activation domain within the walleye dermal sarcoma virus retroviral cyclin protein is essential for inhibition of the viral promoter

International Nuclear Information System (INIS)

Rovnak, Joel; Hronek, Brett W.; Ryan, Sean O.; Cai, Sumin; Quackenbush, Sandra L.

2005-01-01

Walleye dermal sarcoma virus (WDSV) is a complex retrovirus associated with seasonal dermal sarcomas. Developing tumors have low levels of accessory gene transcripts, A1 and B, and regressing tumors have high levels of full-length and spliced transcripts. Transcript A1 encodes a retroviral cyclin (rv-cyclin) with limited homology to host cyclins. The rv-cyclin is physically linked to components of the transcriptional co-activator complex, Mediator, and regulates transcription. In walleye fibroblasts, it inhibits the WDSV promoter independently of cis-acting DNA sequences. The rv-cyclin activates transcription from GAL4 promoters when fused to the GAL4 DNA binding domain. A 30 a.a. activation domain in the carboxy region can be inactivated by single point mutations, and these mutations diminish the ability of the rv-cyclin to inhibit the WDSV promoter. When fused to glutathione S-transferase, the rv-cyclin, its carboxy region, and the activation domain pull down components of transcription complexes from nuclear extracts, and pulldown is lost by mutation of the activation domain

Changes in signal transducer and activator of transcription 3 (STAT3) dynamics induced by complexation with pharmacological inhibitors of Src homology 2 (SH2) domain dimerization.

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Resetca, Diana; Haftchenary, Sina; Gunning, Patrick T; Wilson, Derek J

2014-11-21

The activity of the transcription factor signal transducer and activator of transcription 3 (STAT3) is dysregulated in a number of hematological and solid malignancies. Development of pharmacological STAT3 Src homology 2 (SH2) domain interaction inhibitors holds great promise for cancer therapy, and a novel class of salicylic acid-based STAT3 dimerization inhibitors that includes orally bioavailable drug candidates has been recently developed. The compounds SF-1-066 and BP-1-102 are predicted to bind to the STAT3 SH2 domain. However, given the highly unstructured and dynamic nature of the SH2 domain, experimental confirmation of this prediction was elusive. We have interrogated the protein-ligand interaction of STAT3 with these small molecule inhibitors by means of time-resolved electrospray ionization hydrogen-deuterium exchange mass spectrometry. Analysis of site-specific evolution of deuterium uptake induced by the complexation of STAT3 with SF-1-066 or BP-1-102 under physiological conditions enabled the mapping of the in silico predicted inhibitor binding site to the STAT3 SH2 domain. The binding of both inhibitors to the SH2 domain resulted in significant local decreases in dynamics, consistent with solvent exclusion at the inhibitor binding site and increased rigidity of the inhibitor-complexed SH2 domain. Interestingly, inhibitor binding induced hot spots of allosteric perturbations outside of the SH2 domain, manifesting mainly as increased deuterium uptake, in regions of STAT3 important for DNA binding and nuclear localization. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

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

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

2009-02-01

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

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

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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.

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

RNA-guided transcriptional regulation in planta via synthetic dCas9-based transcription factors

KAUST Repository

Piatek, Agnieszka Anna

2014-11-14

Targeted genomic regulation is a powerful approach to accelerate trait discovery and development in agricultural biotechnology. Bacteria and archaea use clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) regulatory systems for adaptive molecular immunity against foreign nucleic acids introduced by invading phages and conjugative plasmids. The type II CRISPR/Cas system has been adapted for genome editing in many cell types and organisms. A recent study used the catalytically inactive Cas9 (dCas9) protein combined with guide-RNAs (gRNAs) as a DNA-targeting platform to modulate gene expression in bacterial, yeast, and human cells. Here, we modified this DNA-targeting platform for targeted transcriptional regulation in planta by developing chimeric dCas9-based transcriptional activators and repressors. To generate transcriptional activators, we fused the dCas9 C-terminus with the activation domains of EDLL and TAL effectors. To generate a transcriptional repressor, we fused the dCas9 C-terminus with the SRDX repression domain. Our data demonstrate that dCas9 fusion with the EDLL activation domain (dCas9:EDLL) and the TAL activation domain (dCas9:TAD), guided by gRNAs complementary to selected promoter elements, induce strong transcriptional activation on Bs3

RNA-guided transcriptional regulation in planta via synthetic dCas9-based transcription factors

KAUST Repository

Piatek, Agnieszka Anna; Ali, Zahir; Baazim, Hatoon; Li, Lixin; Abulfaraj, Aala A.; Alshareef, Sahar; Aouida, Mustapha; Mahfouz, Magdy M.

2014-01-01

Targeted genomic regulation is a powerful approach to accelerate trait discovery and development in agricultural biotechnology. Bacteria and archaea use clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) regulatory systems for adaptive molecular immunity against foreign nucleic acids introduced by invading phages and conjugative plasmids. The type II CRISPR/Cas system has been adapted for genome editing in many cell types and organisms. A recent study used the catalytically inactive Cas9 (dCas9) protein combined with guide-RNAs (gRNAs) as a DNA-targeting platform to modulate gene expression in bacterial, yeast, and human cells. Here, we modified this DNA-targeting platform for targeted transcriptional regulation in planta by developing chimeric dCas9-based transcriptional activators and repressors. To generate transcriptional activators, we fused the dCas9 C-terminus with the activation domains of EDLL and TAL effectors. To generate a transcriptional repressor, we fused the dCas9 C-terminus with the SRDX repression domain. Our data demonstrate that dCas9 fusion with the EDLL activation domain (dCas9:EDLL) and the TAL activation domain (dCas9:TAD), guided by gRNAs complementary to selected promoter elements, induce strong transcriptional activation on Bs3

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

Structural and functional aspects of winged-helix domains at the core of transcription initiation complexes.

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Teichmann, Martin; Dumay-Odelot, Hélène; Fribourg, Sébastien

2012-01-01

The winged helix (WH) domain is found in core components of transcription systems in eukaryotes and prokaryotes. It represents a sub-class of the helix-turn-helix motif. The WH domain participates in establishing protein-DNA and protein-protein-interactions. Here, we discuss possible explanations for the enrichment of this motif in transcription systems.

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

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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.

Functional interaction of the DNA-binding transcription factor Sp1 through its DNA-binding domain with the histone chaperone TAF-I.

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Suzuki, Toru; Muto, Shinsuke; Miyamoto, Saku; Aizawa, Kenichi; Horikoshi, Masami; Nagai, Ryozo

2003-08-01

Transcription involves molecular interactions between general and regulatory transcription factors with further regulation by protein-protein interactions (e.g. transcriptional cofactors). Here we describe functional interaction between DNA-binding transcription factor and histone chaperone. Affinity purification of factors interacting with the DNA-binding domain of the transcription factor Sp1 showed Sp1 to interact with the histone chaperone TAF-I, both alpha and beta isoforms. This interaction was specific as Sp1 did not interact with another histone chaperone CIA nor did other tested DNA-binding regulatory factors (MyoD, NFkappaB, p53) interact with TAF-I. Interaction of Sp1 and TAF-I occurs both in vitro and in vivo. Interaction with TAF-I results in inhibition of DNA-binding, and also likely as a result of such, inhibition of promoter activation by Sp1. Collectively, we describe interaction between DNA-binding transcription factor and histone chaperone which results in negative regulation of the former. This novel regulatory interaction advances our understanding of the mechanisms of eukaryotic transcription through DNA-binding regulatory transcription factors by protein-protein interactions, and also shows the DNA-binding domain to mediate important regulatory interactions.

RNA-guided transcriptional activation via CRISPR/dCas9 mimics overexpression phenotypes in Arabidopsis.

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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.

GCR1, a transcriptional activator in Saccharomyces cerevisiae, complexes with RAP1 and can function without its DNA binding domain.

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Tornow, J; Zeng, X; Gao, W; Santangelo, G M

1993-01-01

In Saccharomyces cerevisiae, efficient expression of glycolytic and translational component genes requires two DNA binding proteins, RAP1 (which binds to UASRPG) and GCR1 (which binds to the CT box). We generated deletions in GCR1 to test the validity of several different models for GCR1 function. We report here that the C-terminal half of GCR1, which includes the domain required for DNA binding to the CT box in vitro, can be removed without affecting GCR1-dependent transcription of either the glycolytic gene ADH1 or the translational component genes TEF1 and TEF2. We have also identified an activation domain within a segment of the GCR1 protein (the N-terminal third) that is essential for in vivo function. RAP1 and GCR1 can be co-immunoprecipitated from whole cell extracts, suggesting that they form a complex in vivo. The data are most consistent with a model in which GCR1 is attracted to DNA through contact with RAP1. Images PMID:8508768

DNA residence time is a regulatory factor of transcription repression

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Clauß, Karen; Popp, Achim P.; Schulze, Lena; Hettich, Johannes; Reisser, Matthias; Escoter Torres, Laura; Uhlenhaut, N. Henriette

2017-01-01

Abstract Transcription comprises a highly regulated sequence of intrinsically stochastic processes, resulting in bursts of transcription intermitted by quiescence. In transcription activation or repression, a transcription factor binds dynamically to DNA, with a residence time unique to each factor. Whether the DNA residence time is important in the transcription process is unclear. Here, we designed a series of transcription repressors differing in their DNA residence time by utilizing the modular DNA binding domain of transcription activator-like effectors (TALEs) and varying the number of nucleotide-recognizing repeat domains. We characterized the DNA residence times of our repressors in living cells using single molecule tracking. The residence times depended non-linearly on the number of repeat domains and differed by more than a factor of six. The factors provoked a residence time-dependent decrease in transcript level of the glucocorticoid receptor-activated gene SGK1. Down regulation of transcription was due to a lower burst frequency in the presence of long binding repressors and is in accordance with a model of competitive inhibition of endogenous activator binding. Our single molecule experiments reveal transcription factor DNA residence time as a regulatory factor controlling transcription repression and establish TALE-DNA binding domains as tools for the temporal dissection of transcription regulation. PMID:28977492

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

MADS interactomics : towards understanding the molecular mechanisms of plant MADS-domain transcription factor function

NARCIS (Netherlands)

Smaczniak, C.D.

2013-01-01

Protein-protein and protein-DNA interactions are essential for the molecular action of transcription factors. By combinatorial binding to target gene promoters, transcription factors are able to up- or down-regulate the expression of these genes. MADS-domain proteins comprise a large family of

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

The DNA binding and activation domains of Gal4p are sufficient for conveying its regulatory signals.

OpenAIRE

Ding, W V; Johnston, S A

1997-01-01

The transcriptional activation function of the Saccharomyces cerevisiae activator Gal4p is known to rely on a DNA binding activity at its amino terminus and an activation domain at its carboxy terminus. Although both domains are required for activation, truncated forms of Gal4p containing only these domains activate poorly in vivo. Also, mutations in an internal conserved region of Gal4p inactivate the protein, suggesting that this internal region has some function critical to the activity of...

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.

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

Transcriptional machinery of TNF-α-inducible YTH domain containing 2 (YTHDC2) gene.

Science.gov (United States)

Tanabe, Atsushi; Konno, Junpei; Tanikawa, Kenya; Sahara, Hiroeki

2014-02-01

We previously demonstrated that a cellular factor, cyclosporin A (CsA) associated helicase-like protein (CAHL) that is identical to YTH domain containing 2 (YTHDC2), forms trimer complex with cyclophilin B and NS5B of hepatitis C virus (HCV) and facilitates HCV genome replication. Gene expression of YTHDC2 was shown in tumor cell lines and tumor necrosis factor (TNF)-α-treated hepatocytes, but not in untreated. However, the function of YTHDC2 in the tumor cells and the mechanism by which the YTHDC2 gene is transcribed in these cells is largely unknown. We first evaluated that the role of YTHDC2 in the proliferation of hepatocellular carcinoma (HCC) cell line Huh7 using RNA interference and found that YTHDC2-downregulated Huh7 were significantly decreased cell growth as compared to control. We next demonstrated that the cAMP response element (CRE) site in the promoter region of the YTHDC2 gene is critical for YTHDC2 transcription. To further investigate the transcription factors bound to the CRE site, we performed chromatin immunoprecipitation assays. Our findings demonstrate that c-Jun and ATF-2 bind to the CRE site in Huh7, and that TNF-α induces the biological activity of these transcription factors in hepatocytes as well as Huh7. Moreover, treatment with the HDAC inhibitor, trichostatin A (TSA), reduces YTHDC2 expression in Huh7 and in TNF-α-stimulated hepatocytes. Collectively, these data show that YTHDC2 plays an important role in tumor cells growth and activation/recruitment of c-Jun and ATF-2 to the YTHDC2 promoter is necessary for the transcription of YTHDC2, and that HDAC activity is required for the efficient expression of YTHDC2 in both of hepatocyte and HCC cells. Copyright © 2013 Elsevier B.V. All rights reserved.

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.

Human I-mfa domain proteins specifically interact with KSHV LANA and affect its regulation of Wnt signaling-dependent transcription

Energy Technology Data Exchange (ETDEWEB)

Kusano, Shuichi, E-mail: skusano@m2.kufm.kagoshima-u.ac.jp [Division of Persistent and Oncogenic Viruses, Center for Chronic Viral Diseases, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Eizuru, Yoshito [Division of Persistent and Oncogenic Viruses, Center for Chronic Viral Diseases, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan)

2010-06-04

Kaposi's sarcoma-associated herpes virus (KSHV)-encoded latency-associated nuclear antigen (LANA) protein has been reported to interact with glycogen synthase kinase 3{beta} (GSK-3{beta}) and to negatively regulate its activity, leading to stimulation of GSK-3{beta}-dependent {beta}-catenin degradation. We show here that the I-mfa domain proteins, HIC (human I-mfa domain-containing protein) and I-mfa (inhibitor of MyoD family a), interacted in vivo with LANA through their C-terminal I-mfa domains. This interaction affected the intracellular localization of HIC, inhibited the LANA-dependent transactivation of a {beta}-catenin-regulated reporter construct, and decreased the level of the LANA.GSK-3{beta} complex. These data reveal for the first time that I-mfa domain proteins interact with LANA and negatively regulate LANA-mediated activation of Wnt signaling-dependent transcription by inhibiting the formation of the LANA.GSK-3{beta} complex.

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.

Separation of replication and transcription domains in nucleoli.

Science.gov (United States)

Smirnov, E; Borkovec, J; Kováčik, L; Svidenská, S; Schröfel, A; Skalníková, M; Švindrych, Z; Křížek, P; Ovesný, M; Hagen, G M; Juda, P; Michalová, K; Cardoso, M C; Cmarko, D; Raška, I

2014-12-01

In mammalian cells, active ribosomal genes produce the 18S, 5.8S and 28S RNAs of ribosomal particles. Transcription levels of these genes are very high throughout interphase, and the cell needs a special strategy to avoid collision of the DNA polymerase and RNA polymerase machineries. To investigate this problem, we measured the correlation of various replication and transcription signals in the nucleoli of HeLa, HT-1080 and NIH 3T3 cells using a specially devised software for analysis of confocal images. Additionally, to follow the relationship between nucleolar replication and transcription in living cells, we produced a stable cell line expressing GFP-RPA43 (subunit of RNA polymerase I, pol I) and RFP-PCNA (the sliding clamp protein) based on human fibrosarcoma HT-1080 cells. We found that replication and transcription signals are more efficiently separated in nucleoli than in the nucleoplasm. In the course of S phase, separation of PCNA and pol I signals gradually increased. During the same period, separation of pol I and incorporated Cy5-dUTP signals decreased. Analysis of single molecule localization microscopy (SMLM) images indicated that transcriptionally active FC/DFC units (i.e. fibrillar centers with adjacent dense fibrillar components) did not incorporate DNA nucleotides. Taken together, our data show that replication of the ribosomal genes is spatially separated from their transcription, and FC/DFC units may provide a structural basis for that separation. Copyright © 2014 Elsevier Inc. All rights reserved.

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

Crystal structure of the C-terminal domain of the RAP74 subunit of human transcription factor IIF

Energy Technology Data Exchange (ETDEWEB)

Kamada, Katsuhiko; De Angelis, Jacqueline; Roeder, Robert G.; Burley, Stephen K. (Rockefeller)

2012-12-13

The x-ray structure of a C-terminal fragment of the RAP74 subunit of human transcription factor (TF) IIF has been determined at 1.02-{angstrom} resolution. The {alpha}/{beta} structure is strikingly similar to the globular domain of linker histone H5 and the DNA-binding domain of hepatocyte nuclear factor 3{gamma} (HNF-3{gamma}), making it a winged-helix protein. The surface electrostatic properties of this compact domain differ significantly from those of bona fide winged-helix transcription factors (HNF-3{gamma} and RFX1) and from the winged-helix domains found within the RAP30 subunit of TFIIF and the {beta} subunit of TFIIE. RAP74 has been shown to interact with the TFIIF-associated C-terminal domain phosphatase FCP1, and a putative phosphatase binding site has been identified within the RAP74 winged-helix domain.

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.

TALE-mediated modulation of transcriptional enhancers in vivo.

Science.gov (United States)

Crocker, Justin; Stern, David L

2013-08-01

We tested whether transcription activator-like effectors (TALEs) could mediate repression and activation of endogenous enhancers in the Drosophila genome. TALE repressors (TALERs) targeting each of the five even-skipped (eve) stripe enhancers generated repression specifically of the focal stripes. TALE activators (TALEAs) targeting the eve promoter or enhancers caused increased expression primarily in cells normally activated by the promoter or targeted enhancer, respectively. This effect supports the view that repression acts in a dominant fashion on transcriptional activators and that the activity state of an enhancer influences TALE binding or the ability of the VP16 domain to enhance transcription. In these assays, the Hairy repression domain did not exhibit previously described long-range transcriptional repression activity. The phenotypic effects of TALER and TALEA expression in larvae and adults are consistent with the observed modulations of eve expression. TALEs thus provide a novel tool for detection and functional modulation of transcriptional enhancers in their native genomic context.

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.

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.

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.

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 ...

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

Science.gov (United States)

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

2014-10-16

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

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.

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.

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...

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\

Changes in pH and NADPH regulate the DNA binding activity of neuronal PAS domain protein 2, a mammalian circadian transcription factor.

Science.gov (United States)

Yoshii, Katsuhiro; Tajima, Fumihisa; Ishijima, Sumio; Sagami, Ikuko

2015-01-20

Neuronal PAS domain protein 2 (NPAS2) is a core clock transcription factor that forms a heterodimer with BMAL1 to bind the E-box in the promoter of clock genes and is regulated by various environmental stimuli such as heme, carbon monoxide, and NAD(P)H. In this study, we investigated the effects of pH and NADPH on the DNA binding activity of NPAS2. In an electrophoretic mobility shift (EMS) assay, the pH of the reaction mixture affected the DNA binding activity of the NPAS2/BMAL1 heterodimer but not that of the BMAL1/BMAL1 homodimer. A change in pH from 7.0 to 7.5 resulted in a 1.7-fold increase in activity in the absence of NADPH, and NADPH additively enhanced the activity up to 2.7-fold at pH 7.5. The experiments using truncated mutants revealed that N-terminal amino acids 1-61 of NPAS2 were sufficient to sense the change in both pH and NADPH. We further analyzed the kinetics of formation and DNA binding of the NPAS2/BMAL1 heterodimer at various pH values. In the absence of NADPH, a change in pH from 6.5 to 8.0 decreased the KD(app) value of the E-box from 125 to 22 nM, with an 8-fold increase in the maximal level of DNA binding for the NPAS2/BMAL1 heterodimer. The addition of NADPH resulted in a further decrease in KD(app) to 9 nM at pH 8.0. Furthermore, NPAS2-dependent transcriptional activity in a luciferase assay using NIH3T3 cells also increased with the pH of the culture medium. These results suggest that NPAS2 has a role as a pH and metabolite sensor in regulating circadian rhythms.

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

International Nuclear Information System (INIS)

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

1989-01-01

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

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.

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.

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

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

Directory of Open Access Journals (Sweden)

Gary T ZeRuth

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

MrkH, a novel c-di-GMP-dependent transcriptional activator, controls Klebsiella pneumoniae biofilm formation by regulating type 3 fimbriae expression.

Directory of Open Access Journals (Sweden)

Jonathan J Wilksch

2011-08-01

Full Text Available Klebsiella pneumoniae causes significant morbidity and mortality worldwide, particularly amongst hospitalized individuals. The principle mechanism for pathogenesis in hospital environments involves the formation of biofilms, primarily on implanted medical devices. In this study, we constructed a transposon mutant library in a clinical isolate, K. pneumoniae AJ218, to identify the genes and pathways implicated in biofilm formation. Three mutants severely defective in biofilm formation contained insertions within the mrkABCDF genes encoding the main structural subunit and assembly machinery for type 3 fimbriae. Two other mutants carried insertions within the yfiN and mrkJ genes, which encode GGDEF domain- and EAL domain-containing c-di-GMP turnover enzymes, respectively. The remaining two isolates contained insertions that inactivated the mrkH and mrkI genes, which encode for novel proteins with a c-di-GMP-binding PilZ domain and a LuxR-type transcriptional regulator, respectively. Biochemical and functional assays indicated that the effects of these factors on biofilm formation accompany concomitant changes in type 3 fimbriae expression. We mapped the transcriptional start site of mrkA, demonstrated that MrkH directly activates transcription of the mrkA promoter and showed that MrkH binds strongly to the mrkA regulatory region only in the presence of c-di-GMP. Furthermore, a point mutation in the putative c-di-GMP-binding domain of MrkH completely abolished its function as a transcriptional activator. In vivo analysis of the yfiN and mrkJ genes strongly indicated their c-di-GMP-specific function as diguanylate cyclase and phosphodiesterase, respectively. In addition, in vitro assays showed that purified MrkJ protein has strong c-di-GMP phosphodiesterase activity. These results demonstrate for the first time that c-di-GMP can function as an effector to stimulate the activity of a transcriptional activator, and explain how type 3 fimbriae

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.

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.

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

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

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

Tat-dependent repression of human immunodeficiency virus type 1 long terminal repeat promoter activity by fusion of cellular transcription factors

International Nuclear Information System (INIS)

Zhao Cunyou; Chen Yali; Park, Jiyoung; Kim, Jae Bum; Tang Hong

2004-01-01

Transcription initiation from HIV-1 long terminal repeat (LTR) promoter requires the virally encoded transactivator, Tat, and several cellular co-factors to accomplish the Tat-dependent processive transcription elongation. Individual cellular transcription activators, LBP-1b and Oct-1, on the other hand, have been shown to inhibit LTR promoter activities probably via competitive binding against TFIID to the TATA-box in LTR promoter. To explore the genetic interference strategies against the viral replication, we took advantage of the existence of the bipartite DNA binding domains and the repression domains of LBP-1b and Oct-1 factors to generate a chimeric transcription repressor. Our results indicated that the fusion protein of LBP-1b and Oct-1 exhibited higher DNA binding affinity to the viral promoter than the individual factors, and little interference with the host cell gene expression due to its anticipated rare cognate DNA sites in the host cell genome. Moreover, the chimera exerted increased Tat-dependent repression of transcription initiation at the LTR promoter both in vitro and in vivo compared to LBP-1b, Oct-1 or combination of LBP-1b and Oct-1. These results might provide the lead in generating a therapeutic reagent useful to suppress HIV-1 replication

Structural studies of the activation of the two component receiver domain NTRC by multidimensional heteronuclear NMR

Energy Technology Data Exchange (ETDEWEB)

Nohaile, Michael James [Univ. of California, Berkeley, CA (United States). Dept. of Chemistry

1996-05-01

Multidimensional heteronuclear NMR spectroscopy was used to investigate the N-terminal domain of the transcriptional enhancer NTRC (NiTrogen Regulatory protein C). This domain belongs to the family of receiver domains of two-component regulatory systems involved in signal transduction. Phosphorylation of NTRC at D54 leads to an activated form of the molecule which stimulates transcription of genes involved in nitrogen regulation. Three and four dimensional NMR techniques were used to determine an intermediate resolution structure of the unphosphorylated, inactive form of the N-terminal domain of NTRC. The structure is comprised of five α-helices and a five-stranded β-sheet in a (β/α)₅ topology. Analysis of the backbone dynamics of NTRC indicate that helix 4 and strand 5 are significantly more flexible than the rest of the secondary structure of the protein and that the loops making up the active site are flexible. The short lifetime of phospho-NTRC hampers the study of this form. However, conditions for determining the resonance assignments and, possibly, the three dimensional structure of phosphorylated NTRC have been obtained. Tentative assignments of the phosphorylated form indicate that the majority of the changes that NTRC experiences upon phosphorylation occur in helix 3, strand 4, helix 4, strand 5, and the loop between strand 5 and helix 5 (the 3445 face of NTRC) as well as near the site of phosphorylation. In order to examine a stable, activated form of the protein, constitutively active mutants of NTRC were investigated.

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.

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.

Targeted transcriptional repression using a chimeric TALE-SRDX repressor protein

KAUST Repository

Mahfouz, Magdy M.

2011-12-14

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

Targeted transcriptional repression using a chimeric TALE-SRDX repressor protein

KAUST Repository

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

2011-01-01

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

Proto-oncogene FBI-1 (Pokemon) and SREBP-1 Synergistically Activate Transcription of Fatty-acid Synthase Gene (FASN)*S⃞

Science.gov (United States)

Choi, Won-Il; Jeon, Bu-Nam; Park, Hyejin; Yoo, Jung-Yoon; Kim, Yeon-Sook; Koh, Dong-In; Kim, Myung-Hwa; Kim, Yu-Ri; Lee, Choong-Eun; Kim, Kyung-Sup; Osborne, Timothy F.; Hur, Man-Wook

2008-01-01

FBI-1 (Pokemon/ZBTB7A) is a proto-oncogenic transcription factor of the BTB/POZ (bric-à-brac, tramtrack, and broad complex and pox virus zinc finger) domain family. Recent evidence suggested that FBI-1 might be involved in adipogenic gene expression. Coincidentally, expression of FBI-1 and fatty-acid synthase (FASN) genes are often increased in cancer and immortalized cells. Both FBI-1 and FASN are important in cancer cell proliferation. SREBP-1 is a major regulator of many adipogenic genes, and FBI-1 and SREBP-1 (sterol-responsive element (SRE)-binding protein 1) interact with each other directly via their DNA binding domains. FBI-1 enhanced the transcriptional activation of SREBP-1 on responsive promoters, pGL2-6x(SRE)-Luc and FASN gene. FBI-1 and SREBP-1 synergistically activate transcription of the FASN gene by acting on the proximal GC-box and SRE/E-box. FBI-1, Sp1, and SREBP-1 can bind to all three SRE, GC-box, and SRE/E-box. Binding competition among the three transcription factors on the GC-box and SRE/E-box appears important in the transcription regulation. FBI-1 is apparently changing the binding pattern of Sp1 and SREBP-1 on the two elements in the presence of induced SREBP-1 and drives more Sp1 binding to the proximal promoter with less of an effect on SREBP-1 binding. The changes induced by FBI-1 appear critical in the synergistic transcription activation. The molecular mechanism revealed provides insight into how proto-oncogene FBI-1 may attack the cellular regulatory mechanism of FASN gene expression to provide more phospholipid membrane components needed for rapid cancer cell proliferation. PMID:18682402

Multiple Taf subunits of TFIID interact with Ino2 activation domains and contribute to expression of genes required for yeast phospholipid biosynthesis.

Science.gov (United States)

Hintze, Stefan; Engelhardt, Maike; van Diepen, Laura; Witt, Eric; Schüller, Hans-Joachim

2017-12-01

Expression of phospholipid biosynthetic genes in yeast requires activator protein Ino2 which can bind to the UAS element inositol/choline-responsive element (ICRE) and trigger activation of target genes, using two separate transcriptional activation domains, TAD1 and TAD2. However, it is still unknown which cofactors mediate activation by TADs of Ino2. Here, we show that multiple subunits of basal transcription factor TFIID (TBP-associated factors Taf1, Taf4, Taf6, Taf10 and Taf12) are able to interact in vitro with activation domains of Ino2. Interaction was no longer observed with activation-defective variants of TAD1. We were able to identify two nonoverlapping regions in the N-terminus of Taf1 (aa 1-100 and aa 182-250) each of which could interact with TAD1 of Ino2 as well as with TAD4 of activator Adr1. Specific missense mutations within Taf1 domain aa 182-250 affecting basic and hydrophobic residues prevented interaction with wild-type TAD1 and caused reduced expression of INO1. Using chromatin immunoprecipitation we demonstrated Ino2-dependent recruitment of Taf1 and Taf6 to ICRE-containing promoters INO1 and CHO2. Transcriptional derepression of INO1 was no longer possible with temperature-sensitive taf1 and taf6 mutants cultivated under nonpermissive conditions. This result supports the hypothesis of Taf-dependent expression of structural genes activated by Ino2. © 2017 John Wiley & Sons Ltd.

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

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

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

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

International Nuclear Information System (INIS)

Lainšček, Duško; Lebar, Tina; Jerala, Roman

2017-01-01

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

Conformational control of the binding of the transactivation domain of the MLL protein and c-Myb to the KIX domain of CREB.

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Elif Nihal Korkmaz

Full Text Available The KIX domain of CBP is a transcriptional coactivator. Concomitant binding to the activation domain of proto-oncogene protein c-Myb and the transactivation domain of the trithorax group protein mixed lineage leukemia (MLL transcription factor lead to the biologically active ternary MLL∶KIX∶c-Myb complex which plays a role in Pol II-mediated transcription. The binding of the activation domain of MLL to KIX enhances c-Myb binding. Here we carried out molecular dynamics (MD simulations for the MLL∶KIX∶c-Myb ternary complex, its binary components and KIX with the goal of providing a mechanistic explanation for the experimental observations. The dynamic behavior revealed that the MLL binding site is allosterically coupled to the c-Myb binding site. MLL binding redistributes the conformational ensemble of KIX, leading to higher populations of states which favor c-Myb binding. The key element in the allosteric communication pathways is the KIX loop, which acts as a control mechanism to enhance subsequent binding events. We tested this conclusion by in silico mutations of loop residues in the KIX∶MLL complex and by comparing wild type and mutant dynamics through MD simulations. The loop assumed MLL binding conformation similar to that observed in the KIX∶c-Myb state which disfavors the allosteric network. The coupling with c-Myb binding site faded, abolishing the positive cooperativity observed in the presence of MLL. Our major conclusion is that by eliciting a loop-mediated allosteric switch between the different states following the binding events, transcriptional activation can be regulated. The KIX system presents an example how nature makes use of conformational control in higher level regulation of transcriptional activity and thus cellular events.

DNA Binding by the Ribosomal DNA Transcription Factor Rrn3 Is Essential for Ribosomal DNA Transcription*

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Stepanchick, Ann; Zhi, Huijun; Cavanaugh, Alice H.; Rothblum, Katrina; Schneider, David A.; Rothblum, Lawrence I.

2013-01-01

The human homologue of yeast Rrn3 is an RNA polymerase I-associated transcription factor that is essential for ribosomal DNA (rDNA) transcription. The generally accepted model is that Rrn3 functions as a bridge between RNA polymerase I and the transcription factors bound to the committed template. In this model Rrn3 would mediate an interaction between the mammalian Rrn3-polymerase I complex and SL1, the rDNA transcription factor that binds to the core promoter element of the rDNA. In the course of studying the role of Rrn3 in recruitment, we found that Rrn3 was in fact a DNA-binding protein. Analysis of the sequence of Rrn3 identified a domain with sequence similarity to the DNA binding domain of heat shock transcription factor 2. Randomization, or deletion, of the amino acids in this region in Rrn3, amino acids 382–400, abrogated its ability to bind DNA, indicating that this domain was an important contributor to DNA binding by Rrn3. Control experiments demonstrated that these mutant Rrn3 constructs were capable of interacting with both rpa43 and SL1, two other activities demonstrated to be essential for Rrn3 function. However, neither of these Rrn3 mutants was capable of functioning in transcription in vitro. Moreover, although wild-type human Rrn3 complemented a yeast rrn3-ts mutant, the DNA-binding site mutant did not. These results demonstrate that DNA binding by Rrn3 is essential for transcription by RNA polymerase I. PMID:23393135

DNA binding by the ribosomal DNA transcription factor rrn3 is essential for ribosomal DNA transcription.

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Stepanchick, Ann; Zhi, Huijun; Cavanaugh, Alice H; Rothblum, Katrina; Schneider, David A; Rothblum, Lawrence I

2013-03-29

The human homologue of yeast Rrn3 is an RNA polymerase I-associated transcription factor that is essential for ribosomal DNA (rDNA) transcription. The generally accepted model is that Rrn3 functions as a bridge between RNA polymerase I and the transcription factors bound to the committed template. In this model Rrn3 would mediate an interaction between the mammalian Rrn3-polymerase I complex and SL1, the rDNA transcription factor that binds to the core promoter element of the rDNA. In the course of studying the role of Rrn3 in recruitment, we found that Rrn3 was in fact a DNA-binding protein. Analysis of the sequence of Rrn3 identified a domain with sequence similarity to the DNA binding domain of heat shock transcription factor 2. Randomization, or deletion, of the amino acids in this region in Rrn3, amino acids 382-400, abrogated its ability to bind DNA, indicating that this domain was an important contributor to DNA binding by Rrn3. Control experiments demonstrated that these mutant Rrn3 constructs were capable of interacting with both rpa43 and SL1, two other activities demonstrated to be essential for Rrn3 function. However, neither of these Rrn3 mutants was capable of functioning in transcription in vitro. Moreover, although wild-type human Rrn3 complemented a yeast rrn3-ts mutant, the DNA-binding site mutant did not. These results demonstrate that DNA binding by Rrn3 is essential for transcription by RNA polymerase I.

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

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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

Arg156 in the AP2-domain exhibits the highest binding activity among the 20 individuals to the GCC box in BnaERF-B3-hy15, a mutant ERF transcription factor from Brassica napus

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Jing Zhuang

2016-10-01

Full Text Available To develop mutants of the ERF factor with more binding activities to the GCC box, we performed in vitro directed evolution by using DNA shuffling and screened mutants through yeast one-hybrid assay. Here, a series of mutants were obtained and used to reveal key amino acids that induce changes in the DNA binding activity of the BnaERF-B3 protein. With the BnaERF-B3-hy15 as the template, we produced 12 mutants which host individual mutation of potential key residues. We found that amino acid 156 is the key site, and the other 18 mutants host the 18 corresponding individual amino acid residues at site 156. Among the 20 individuals comprising WT (Gly156, Mu3 (Arg156, and 18 mutants with other 18 amino acid residues, Arg156 in the AP2-domain is the amino acid residue with the highest binding activity to the GCC box. The structure of the α-helix in the AP2-domain affects the binding activity. Other residues within AP2-domain modulated binding activity of ERF protein, suggesting that these positions are important for binding activity. Comparison of the mutant and wild-type transcription factors revealed the relationship of protein function and sequence modification. Our result provides a potential useful resource for understanding the trans-activation of ERF proteins.

Radiation activation of transcription factors in mammalian cells

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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.)

Zinc coordination is required for and regulates transcription activation by Epstein-Barr nuclear antigen 1.

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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.

Environmental phthalate monoesters activate pregnane X receptor-mediated transcription

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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

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

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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.

Autoimmune regulator is acetylated by transcription coactivator CBP/p300

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Saare, Mario, E-mail: mario.saare@ut.ee [Molecular Pathology, Institute of General and Molecular Pathology, University of Tartu, 19th Ravila Str, Tartu (Estonia); Rebane, Ana [Molecular Pathology, Institute of General and Molecular Pathology, University of Tartu, 19th Ravila Str, Tartu (Estonia); SIAF, Swiss Institute of Allergy and Asthma Research, University of Zuerich, Davos (Switzerland); Rajashekar, Balaji; Vilo, Jaak [BIIT, Bioinformatics, Algorithmics and Data Mining group, Institute of Computer Science, University of Tartu, Tartu (Estonia); Peterson, Paert [Molecular Pathology, Institute of General and Molecular Pathology, University of Tartu, 19th Ravila Str, Tartu (Estonia)

2012-08-15

The Autoimmune Regulator (AIRE) is a regulator of transcription in the thymic medulla, where it controls the expression of a large set of peripheral-tissue specific genes. AIRE interacts with the transcriptional coactivator and acetyltransferase CBP and synergistically cooperates with it in transcriptional activation. Here, we aimed to study a possible role of AIRE acetylation in the modulation of its activity. We found that AIRE is acetylated in tissue culture cells and this acetylation is enhanced by overexpression of CBP and the CBP paralog p300. The acetylated lysines were located within nuclear localization signal and SAND domain. AIRE with mutations that mimicked acetylated K243 and K253 in the SAND domain had reduced transactivation activity and accumulated into fewer and larger nuclear bodies, whereas mutations that mimicked the unacetylated lysines were functionally similar to wild-type AIRE. Analogously to CBP, p300 localized to AIRE-containing nuclear bodies, however, the overexpression of p300 did not enhance the transcriptional activation of AIRE-regulated genes. Further studies showed that overexpression of p300 stabilized the AIRE protein. Interestingly, gene expression profiling revealed that AIRE, with mutations mimicking K243/K253 acetylation in SAND, was able to activate gene expression, although the affected genes were different and the activation level was lower from those regulated by wild-type AIRE. Our results suggest that the AIRE acetylation can influence the selection of AIRE activated genes. -- Highlights: Black-Right-Pointing-Pointer AIRE is acetylated by the acetyltransferases p300 and CBP. Black-Right-Pointing-Pointer Acetylation occurs between CARD and SAND domains and within the SAND domain. Black-Right-Pointing-Pointer Acetylation increases the size of AIRE nuclear dots. Black-Right-Pointing-Pointer Acetylation increases AIRE protein stability. Black-Right-Pointing-Pointer AIRE acetylation mimic regulates a different set of AIRE

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

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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.

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.

Potential Role of Activating Transcription Factor 5 during Osteogenesis.

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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.

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

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Henke, Sarah K; Cronan, John E

2016-11-01

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

RNA-guided transcriptional regulation

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Church, George M.; Mali, Prashant G.; Esvelt, Kevin M.

2016-02-23

Methods of modulating expression of a target nucleic acid in a cell are provided including introducing into the cell a first foreign nucleic acid encoding one or more RNAs complementary to DNA, wherein the DNA includes the target nucleic acid, introducing into the cell a second foreign nucleic acid encoding a nuclease-null Cas9 protein that binds to the DNA and is guided by the one or more RNAs, introducing into the cell a third foreign nucleic acid encoding a transcriptional regulator protein or domain, wherein the one or more RNAs, the nuclease-null Cas9 protein, and the transcriptional regulator protein or domain are expressed, wherein the one or more RNAs, the nuclease-null Cas9 protein and the transcriptional regulator protein or domain co-localize to the DNA and wherein the transcriptional regulator protein or domain regulates expression of the target nucleic acid.

The structure of the first representative of Pfam family PF09836 reveals a two-domain organization and suggests involvement in transcriptional regulation

International Nuclear Information System (INIS)

Das, Debanu; Grishin, Nick V.; Kumar, Abhinav; Carlton, Dennis; Bakolitsa, Constantina; Miller, Mitchell D.; Abdubek, Polat; Astakhova, Tamara; Axelrod, Herbert L.; Burra, Prasad; Chen, Connie; Chiu, Hsiu-Ju; Chiu, Michelle; Clayton, Thomas; Deller, Marc C.; Duan, Lian; Ellrott, Kyle; Ernst, Dustin; Farr, Carol L.; Feuerhelm, Julie; Grzechnik, Anna; Grzechnik, Slawomir K.; Grant, Joanna C.; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K.; Johnson, Hope A.; Klock, Heath E.; Knuth, Mark W.; Kozbial, Piotr; Krishna, S. Sri; Marciano, David; McMullan, Daniel; Morse, Andrew T.; Nigoghossian, Edward; Nopakun, Amanda; Okach, Linda; Oommachen, Silvya; Paulsen, Jessica; Puckett, Christina; Reyes, Ron; Rife, Christopher L.; Sefcovic, Natasha; Tien, Henry J.; Trame, Christine B.; Bedem, Henry van den; Weekes, Dana; Wooten, Tiffany; Xu, Qingping; Hodgson, Keith O.; Wooley, John; Elsliger, Marc-André; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Wilson, Ian A.

2009-01-01

The crystal structure of the NGO1945 gene product from N. gonorrhoeae (UniProt Q5F5IO) reveals that the N-terminal domain assigned as a domain of unknown function (DUF2063) is likely to bind DNA and that the protein may be involved in transcriptional regulation. Proteins with the DUF2063 domain constitute a new Pfam family, PF09836. The crystal structure of a member of this family, NGO1945 from Neisseria gonorrhoeae, has been determined and reveals that the N-terminal DUF2063 domain is likely to be a DNA-binding domain. In conjunction with the rest of the protein, NGO1945 is likely to be involved in transcriptional regulation, which is consistent with genomic neighborhood analysis. Of the 216 currently known proteins that contain a DUF2063 domain, the most significant sequence homologs of NGO1945 (∼40–99% sequence identity) are from various Neisseria and Haemophilus species. As these are important human pathogens, NGO1945 represents an interesting candidate for further exploration via biochemical studies and possible therapeutic intervention

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.

Neural Progenitors Adopt Specific Identities by Directly Repressing All Alternative Progenitor Transcriptional Programs.

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Kutejova, Eva; Sasai, Noriaki; Shah, Ankita; Gouti, Mina; Briscoe, James

2016-03-21

In the vertebrate neural tube, a morphogen-induced transcriptional network produces multiple molecularly distinct progenitor domains, each generating different neuronal subtypes. Using an in vitro differentiation system, we defined gene expression signatures of distinct progenitor populations and identified direct gene-regulatory inputs corresponding to locations of specific transcription factor binding. Combined with targeted perturbations of the network, this revealed a mechanism in which a progenitor identity is installed by active repression of the entire transcriptional programs of other neural progenitor fates. In the ventral neural tube, sonic hedgehog (Shh) signaling, together with broadly expressed transcriptional activators, concurrently activates the gene expression programs of several domains. The specific outcome is selected by repressive input provided by Shh-induced transcription factors that act as the key nodes in the network, enabling progenitors to adopt a single definitive identity from several initially permitted options. Together, the data suggest design principles relevant to many developing tissues. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Structure of a Novel DNA-binding Domain of Helicase-like Transcription Factor (HLTF) and Its Functional Implication in DNA Damage Tolerance.

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Hishiki, Asami; Hara, Kodai; Ikegaya, Yuzu; Yokoyama, Hideshi; Shimizu, Toshiyuki; Sato, Mamoru; Hashimoto, Hiroshi

2015-05-22

HLTF (helicase-like transcription factor) is a yeast RAD5 homolog found in mammals. HLTF has E3 ubiquitin ligase and DNA helicase activities, and plays a pivotal role in the template-switching pathway of DNA damage tolerance. HLTF has an N-terminal domain that has been designated the HIRAN (HIP116 and RAD5 N-terminal) domain. The HIRAN domain has been hypothesized to play a role in DNA binding; however, the structural basis of, and functional evidence for, the HIRAN domain in DNA binding has remained unclear. Here we show for the first time the crystal structure of the HIRAN domain of human HLTF in complex with DNA. The HIRAN domain is composed of six β-strands and two α-helices, forming an OB-fold structure frequently found in ssDNA-binding proteins, including in replication factor A (RPA). Interestingly, this study reveals that the HIRAN domain interacts with not only with a single-stranded DNA but also with a duplex DNA. Furthermore, the structure unexpectedly clarifies that the HIRAN domain specifically recognizes the 3'-end of DNA. These results suggest that the HIRAN domain functions as a sensor to the 3'-end of the primer strand at the stalled replication fork and that the domain facilitates fork regression. HLTF is recruited to a damaged site through the HIRAN domain at the stalled replication fork. Furthermore, our results have implications for the mechanism of template switching. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Common and distinct DNA-binding and regulatory activities of the BEN-solo transcription factor family.

Science.gov (United States)

Dai, Qi; Ren, Aiming; Westholm, Jakub O; Duan, Hong; Patel, Dinshaw J; Lai, Eric C

2015-01-01

Recently, the BEN (BANP, E5R, and NAC1) domain was recognized as a new class of conserved DNA-binding domain. The fly genome encodes three proteins that bear only a single BEN domain ("BEN-solo" factors); namely, Insensitive (Insv), Bsg25A (Elba1), and CG9883 (Elba2). Insv homodimers preferentially bind CCAATTGG palindromes throughout the genome to mediate transcriptional repression, whereas Bsg25A and Elba2 heterotrimerize with their obligate adaptor, Elba3 (i.e., the ELBA complex), to recognize a CCAATAAG motif in the Fab-7 insulator. While these data suggest distinct DNA-binding properties of BEN-solo proteins, we performed reporter assays that indicate that both Bsg25A and Elba2 can individually recognize Insv consensus sites efficiently. We confirmed this by solving the structure of Bsg25A complexed to the Insv site, which showed that key aspects of the BEN:DNA recognition strategy are similar between these proteins. We next show that both Insv and ELBA proteins are competent to mediate transcriptional repression via Insv consensus sequences but that the ELBA complex appears to be selective for the ELBA site. Reciprocally, genome-wide analysis reveals that Insv exhibits significant cobinding to class I insulator elements, indicating that it may also contribute to insulator function. Indeed, we observed abundant Insv binding within the Hox complexes with substantial overlaps with class I insulators, many of which bear Insv consensus sites. Moreover, Insv coimmunoprecipitates with the class I insulator factor CP190. Finally, we observed that Insv harbors exclusive activity among fly BEN-solo factors with respect to regulation of Notch-mediated cell fate choices in the peripheral nervous system. This in vivo activity is recapitulated by BEND6, a mammalian BEN-solo factor that conserves the Notch corepressor function of Insv but not its capacity to bind Insv consensus sites. Altogether, our data define an array of common and distinct biochemical and functional

A biophysical model for transcription factories

International Nuclear Information System (INIS)

Canals-Hamann, Ana Z; Neves, Ricardo Pires das; Reittie, Joyce E; Iñiguez, Carlos; Soneji, Shamit; Enver, Tariq; Buckle, Veronica J; Iborra, Francisco J

2013-01-01

Transcription factories are nuclear domains where gene transcription takes place although the molecular basis for their formation and maintenance are unknown. In this study, we explored how the properties of chromatin as a polymer may contribute to the structure of transcription factories. We found that transcriptional active chromatin contains modifications like histone H4 acetylated at Lysine 16 (H4K16ac). Single fibre analysis showed that this modification spans the entire body of the gene. Furthermore, H4K16ac genes cluster in regions up to 500 Kb alternating active and inactive chromatin. The introduction of H4K16ac in chromatin induces stiffness in the chromatin fibre. The result of this change in flexibility is that chromatin could behave like a multi-block copolymer with repetitions of stiff-flexible (active-inactive chromatin) components. Copolymers with such structure self-organize through spontaneous phase separation into microdomains. Consistent with such model H4K16ac chromatin form foci that associates with nascent transcripts. We propose that transcription factories are the result of the spontaneous concentration of H4K16ac chromatin that are in proximity, mainly in cis

Designed Transcriptional Regulation in Mammalian Cells Based on TALE- and CRISPR/dCas9.

Science.gov (United States)

Lebar, Tina; Jerala, Roman

2018-01-01

Transcriptional regulation lies at the center of many cellular processes and is the result of cellular response to different external and internal signals. Control of transcription of selected genes enables an unprecedented access to shape the cellular response. While orthogonal transcription factors from bacteria, yeast, plants, or other cells have been used to introduce new cellular logic into mammalian cells, the discovery of designable modular DNA binding domains, such as Transcription Activator-Like Effectors (TALEs) and the CRISPR system, enable targeting of almost any selected DNA sequence. Fusion or conditional association of DNA targeting domain with transcriptional effector domains enables controlled regulation of almost any endogenous or ectopic gene. Moreover, the designed regulators can be linked into genetic circuits to implement complex responses, such as different types of Boolean functions and switches. In this chapter, we describe the protocols for achieving efficient transcriptional regulation with TALE- and CRISPR-based designed transcription factors in mammalian cells.

APETALA 2-domain-containing transcription factors: focusing on abscisic acid and gibberellins antagonism.

Science.gov (United States)

Shu, Kai; Zhou, Wenguan; Yang, Wenyu

2018-02-01

The phytohormones abscisic acid (ABA) and gibberellin (GA) antagonistically mediate diverse plant developmental processes including seed dormancy and germination, root development, and flowering time control, and thus the optimal balance between ABA and GA is essential for plant growth and development. Although more than a half and one century have passed since the initial discoveries of ABA and GA, respectively, the precise mechanisms underlying ABA-GA antagonism still need further investigation. Emerging evidence indicates that two APETALA 2 (AP2)-domain-containing transcription factors (ATFs), ABI4 in Arabidopsis and OsAP2-39 in rice, play key roles in ABA and GA antagonism. These two transcription factors precisely regulate the transcription pattern of ABA and GA biosynthesis or inactivation genes, mediating ABA and GA levels. In this Viewpoint article, we try to shed light on the effects of ATFs on ABA-GA antagonism, and summarize the overlapping but distinct biological functions of these ATFs in the antagonism between ABA and GA. Finally, we strongly propose that further research is needed into the detailed roles of additional numerous ATFs in ABA and GA crosstalk, which will improve our understanding of the antagonism between these two phytohormones. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Identification of an estrogen receptor α non covalent ubiquitin-binding surface: role in 17β-estradiol-induced transcriptional activity.

Science.gov (United States)

Pesiri, Valeria; La Rosa, Piergiorgio; Stano, Pasquale; Acconcia, Filippo

2013-06-15

Ubiquitin (Ub)-binding domains (UBDs) located in Ub receptors decode the ubiquitination signal by non-covalently engaging the Ub modification on their binding partners and transduce the Ub signalling through Ub-based molecular interactions. In this way, inducible protein ubiquitination regulates diverse biological processes. The estrogen receptor alpha (ERα) is a ligand-activated transcription factor that mediates the pleiotropic effects of the sex hormone 17β-estradiol (E2). Fine regulation of E2 pleiotropic actions depends on E2-dependent ERα association with a plethora of binding partners and/or on the E2 modulation of receptor ubiquitination. Indeed, E2-induced ERα polyubiquitination triggers receptor degradation and transcriptional activity, and E2-dependent reduction in ERα monoubiquitination is crucial for E2 signalling. Monoubiquitinated proteins often contain UBDs, but whether non-covalent Ub-ERα binding could occur and play a role in E2-ERα signalling is unknown. Here, we report an Ub-binding surface within the ERα ligand binding domain that directs in vitro the receptor interaction with both ubiquitinated proteins and recombinant Ub chains. Mutational analysis reveals that ERα residues leucine 429 and alanine 430 are involved in Ub binding. Moreover, impairment of ERα association to ubiquitinated species strongly affects E2-induced ERα transcriptional activity. Considering the importance of UBDs in the Ub-based signalling network and the central role of different ERα binding partners in the modulation of E2-dependent effects, our discoveries provide novel insights into ERα activity that could also be relevant for ERα-dependent diseases.

Transcription factor Nrf1 is topologically repartitioned across membranes to enable target gene transactivation through its acidic glucose-responsive domains.

Science.gov (United States)

Zhang, Yiguo; Ren, Yonggang; Li, Shaojun; Hayes, John D

2014-01-01

The membrane-bound Nrf1 transcription factor regulates critical homeostatic and developmental genes. The conserved N-terminal homology box 1 (NHB1) sequence in Nrf1 targets the cap'n'collar (CNC) basic basic-region leucine zipper (bZIP) factor to the endoplasmic reticulum (ER), but it is unknown how its activity is controlled topologically within membranes. Herein, we report a hitherto unknown mechanism by which the transactivation activity of Nrf1 is controlled through its membrane-topology. Thus after Nrf1 is anchored within ER membranes, its acidic transactivation domains (TADs), including the Asn/Ser/Thr-rich (NST) glycodomain situated between acidic domain 1 (AD1) and AD2, are transiently translocated into the lumen of the ER, where NST is glycosylated in the presence of glucose to yield an inactive 120-kDa Nrf1 glycoprotein. Subsequently, portions of the TADs partially repartition across membranes into the cyto/nucleoplasmic compartments, whereupon an active 95-kDa form of Nrf1 accumulates, a process that is more obvious in glucose-deprived cells and may involve deglycosylation. The repartitioning of Nrf1 out of membranes is monitored within this protein by its acidic-hydrophobic amphipathic glucose-responsive domains, particularly the Neh5L subdomain within AD1. Therefore, the membrane-topological organization of Nrf1 dictates its post-translational modifications (i.e. glycosylation, the putative deglycosylation and selective proteolysis), which together control its ability to transactivate target genes.

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

Energy Technology Data Exchange (ETDEWEB)

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

2010-11-19

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

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

International Nuclear Information System (INIS)

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

2010-01-01

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

Novel Functions for TAF7, a Regulator of TAF1-independent Transcription

OpenAIRE

Devaiah, Ballachanda N.; Lu, Hanxin; Gegonne, Anne; Sercan, Zeynep; Zhang, Hongen; Clifford, Robert J.; Lee, Maxwell P.; Singer, Dinah S.

2010-01-01

The transcription factor TFIID components TAF7 and TAF1 regulate eukaryotic transcription initiation. TAF7 regulates transcription initiation of TAF1-dependent genes by binding to the acetyltransferase (AT) domain of TAF1 and inhibiting the enzymatic activity that is essential for transcription. TAF7 is released from the TAF1-TFIID complex upon completion of preinitiation complex assembly, allowing transcription to initiate. However, not all transcription is TAF1-dependent, and the role of TA...

Cdc15 Phosphorylates the C-terminal Domain of RNA Polymerase II for Transcription during Mitosis.

Science.gov (United States)

Singh, Amit Kumar; Rastogi, Shivangi; Shukla, Harish; Asalam, Mohd; Rath, Srikanta Kumar; Akhtar, Md Sohail

2017-03-31

In eukaryotes, the basal transcription in interphase is orchestrated through the regulation by kinases (Kin28, Bur1, and Ctk1) and phosphatases (Ssu72, Rtr1, and Fcp1), which act through the post-translational modification of the C-terminal domain (CTD) of the largest subunit of RNA polymerase II. The CTD comprises the repeated Tyr-Ser-Pro-Thr-Ser-Pro-Ser motif with potential epigenetic modification sites. Despite the observation of transcription and periodic expression of genes during mitosis with entailing CTD phosphorylation and dephosphorylation, the associated CTD specific kinase(s) and its role in transcription remains unknown. Here we have identified Cdc15 as a potential kinase phosphorylating Ser-2 and Ser-5 of CTD for transcription during mitosis in the budding yeast. The phosphorylation of CTD by Cdc15 is independent of any prior Ser phosphorylation(s). The inactivation of Cdc15 causes reduction of global CTD phosphorylation during mitosis and affects the expression of genes whose transcript levels peak during mitosis. Cdc15 also influences the complete transcription of clb2 gene and phosphorylates Ser-5 at the promoter and Ser-2 toward the 3' end of the gene. The observation that Cdc15 could phosphorylate Ser-5, as well as Ser-2, during transcription in mitosis is in contrast to the phosphorylation marks put by the kinases in interphase (G 1 , S, and G 2 ), where Cdck7/Kin28 phosphorylates Ser-5 at promoter and Bur1/Ctk1 phosphorylates Ser-2 at the 3' end of the genes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

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

A combinatorial approach to synthetic transcription factor-promoter combinations for yeast strain engineering

DEFF Research Database (Denmark)

Dossani, Zain Y.; Apel, Amanda Reider; Szmidt-Middleton, Heather

2018-01-01

regions, we have built a library of hybrid promoters that are regulated by a synthetic transcription factor. The hybrid promoters consist of native S. cerevisiae promoters, in which the operator regions have been replaced with sequences that are recognized by the bacterial LexA DNA binding protein....... Correspondingly, the synthetic transcription factor (TF) consists of the DNA binding domain of the LexA protein, fused with the human estrogen binding domain and the viral activator domain, VP16. The resulting system with a bacterial DNA binding domain avoids the transcription of native S. cerevisiae genes...... levels, using the same synthetic TF and a given estradiol. This set of promoters, in combination with our synthetic TF, has the potential to regulate numerous genes or pathways simultaneously, to multiple desired levels, in a single strain....

Interaction of the transactivation domain of B-Myb with the TAZ2 domain of the coactivator p300: molecular features and properties of the complex.

Directory of Open Access Journals (Sweden)

Ojore Oka

Full Text Available The transcription factor B-Myb is a key regulator of the cell cycle in vertebrates, with activation of transcription involving the recognition of specific DNA target sites and the recruitment of functional partner proteins, including the coactivators p300 and CBP. Here we report the results of detailed studies of the interaction between the transactivation domain of B-Myb (B-Myb TAD and the TAZ2 domain of p300. The B-Myb TAD was characterized using circular dichroism, fluorescence and NMR spectroscopy, which revealed that the isolated domain exists as a random coil polypeptide. Pull-down and spectroscopic experiments clearly showed that the B-Myb TAD binds to p300 TAZ2 to form a moderately tight (K(d ~1.0-10 µM complex, which results in at least partial folding of the B-Myb TAD. Significant changes in NMR spectra of p300 TAZ2 suggest that the B-Myb TAD binds to a relatively large patch on the surface of the domain (~1200 Å(2. The apparent B-Myb TAD binding site on p300 TAZ2 shows striking similarity to the surface of CBP TAZ2 involved in binding to the transactivation domain of the transcription factor signal transducer and activator of transcription 1 (STAT1, which suggests that the structure of the B-Myb TAD-p300 TAZ2 complex may share many features with that reported for STAT1 TAD-p300 TAZ2.

Preparation, crystallization and preliminary X-ray diffraction analysis of the DNA-binding domain of the Ets transcription factor in complex with target DNA

Energy Technology Data Exchange (ETDEWEB)

Suwa, Yoshiaki; Nakamura, Teruya; Toma, Sachiko; Ikemizu, Shinji; Kai, Hirofumi; Yamagata, Yuriko, E-mail: yamagata@gpo.kumamoto-u.ac.jp [Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto 862-0973 (Japan)

2008-03-01

The complex between the Ets domain of Ets2 and its target DNA has been crystallized. The crystals diffracted to 3.0 Å resolution. The Ets2 transcription factor is a member of the Ets transcription-factor family. Ets2 plays a role in the malignancy of cancer and in Down’s syndrome by regulating the transcription of various genes. The DNA-binding domain of Ets2 (Ets domain; ETSD), which contains residues that are highly conserved among Ets transcription-factor family members, was expressed as a GST-fusion protein. The aggregation of ETSD produced after thrombin cleavage could be prevented by treatment with NDSB-195 (nondetergent sulfobetaine 195). ETSD was crystallized in complex with DNA containing the Ets2 target sequence (GGAA) by the hanging-drop vapour-diffusion method. The best crystals were grown using 25% PEG 3350, 80 mM magnesium acetate, 50 mM sodium cacodylate pH 5.0/5.5 as the reservoir at 293 K. The crystals belonged to space group C2, with unit-cell parameters a = 85.89, b = 95.52, c = 71.89 Å, β = 101.7° and a V{sub M} value of 3.56 Å{sup 3} Da{sup −1}. Diffraction data were collected to a resolution of 3.0 Å.

Preparation, crystallization and preliminary X-ray diffraction analysis of the DNA-binding domain of the Ets transcription factor in complex with target DNA

International Nuclear Information System (INIS)

Suwa, Yoshiaki; Nakamura, Teruya; Toma, Sachiko; Ikemizu, Shinji; Kai, Hirofumi; Yamagata, Yuriko

2008-01-01

The complex between the Ets domain of Ets2 and its target DNA has been crystallized. The crystals diffracted to 3.0 Å resolution. The Ets2 transcription factor is a member of the Ets transcription-factor family. Ets2 plays a role in the malignancy of cancer and in Down’s syndrome by regulating the transcription of various genes. The DNA-binding domain of Ets2 (Ets domain; ETSD), which contains residues that are highly conserved among Ets transcription-factor family members, was expressed as a GST-fusion protein. The aggregation of ETSD produced after thrombin cleavage could be prevented by treatment with NDSB-195 (nondetergent sulfobetaine 195). ETSD was crystallized in complex with DNA containing the Ets2 target sequence (GGAA) by the hanging-drop vapour-diffusion method. The best crystals were grown using 25% PEG 3350, 80 mM magnesium acetate, 50 mM sodium cacodylate pH 5.0/5.5 as the reservoir at 293 K. The crystals belonged to space group C2, with unit-cell parameters a = 85.89, b = 95.52, c = 71.89 Å, β = 101.7° and a V M value of 3.56 Å 3 Da −1 . Diffraction data were collected to a resolution of 3.0 Å

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

International Nuclear Information System (INIS)

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

2004-01-01

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

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...

A-type nuclear lamins act as transcriptional repressors when targeted to promoters

International Nuclear Information System (INIS)

Lee, Damian C.; Welton, K. Linnea; Smith, Erica D.; Kennedy, Brian K.

2009-01-01

Regions of heterochromatin are often found at the periphery of the mammalian nucleus, juxtaposed to the nuclear lamina. Genes in these regions are likely maintained in a transcriptionally silent state, although other locations at the nuclear periphery associated with nuclear pores are sites of active transcription. As primary components of the nuclear lamina, A- and B-type nuclear lamins are intermediate filament proteins that interact with DNA, histones and known transcriptional repressors, leading to speculation that they may promote establishment of repressive domains. However, no direct evidence of a role for nuclear lamins in transcriptional repression has been reported. Here we find that human lamin A, when expressed in yeast and cultured human cells as a fusion protein to the Gal4 DNA-binding domain (DBD), can mediate robust transcriptional repression of promoters with Gal4 binding sites. Full repression by lamin A requires both the coiled-coil rod domain and the C-terminal tail domain. In human cells, other intermediate filament proteins such as lamin B and vimentin are unable to confer robust repression as Gal4-DBD fusions, indicating that this property is specific to A-type nuclear lamins. These findings indicate that A-type lamins can promote transcriptional repression when in proximity of a promoter

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...

Characterization of niphatenones that inhibit androgen receptor N-terminal domain.

Directory of Open Access Journals (Sweden)

Carmen A Banuelos

Full Text Available Androgen ablation therapy causes a temporary reduction in tumor burden in patients with advanced prostate cancer. Unfortunately the malignancy will return to form lethal castration-recurrent prostate cancer (CRPC. The androgen receptor (AR remains transcriptionally active in CRPC in spite of castrate levels of androgens in the blood. AR transcriptional activity resides in its N-terminal domain (NTD. Possible mechanisms of continued AR transcriptional activity may include, at least in part, expression of constitutively active splice variants of AR that lack the C-terminal ligand-binding domain (LBD. Current therapies that target the AR LBD, would not be effective against these AR variants. Currently no drugs are clinically available that target the AR NTD which should be effective against these AR variants as well as full-length AR. Niphatenones were originally isolated and identified in active extracts from Niphates digitalis marine sponge. Here we begin to characterize the mechanism of niphatenones in blocking AR transcriptional activity. Both enantiomers had similar IC50 values of 6 µM for inhibiting the full-length AR in a functional transcriptional assay. However, (S-niphatenone had significantly better activity against the AR NTD compared to (R-niphatenone. Consistent with niphatenones binding to and inhibiting transactivation of AR NTD, niphatenones inhibited AR splice variant. Niphatenone did not affect the transcriptional activity of the related progesterone receptor, but slightly decreased glucocorticoid receptor (GR activity and covalently bound to GR activation function-1 (AF-1 region. Niphatenone blocked N/C interactions of AR without altering either AR protein levels or its intracellular localization in response to androgen. Alkylation with glutathione suggests that niphatenones are not a feasible scaffold for further drug development.

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

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.

Regulating expressin of cell and tissue-specific genes by modifying transcription

Energy Technology Data Exchange (ETDEWEB)

Beachy, Roger N. [Donald Danforth Plant Science Center, St. Louis, MO (United States); Dai, Shunhong [Donald Danforth Plant Science Center, St. Louis, MO (United States)

2009-12-15

Transcriptional regulation is the primary step to control gene expression, therefore function. Such regulation is achieved primarily via a combination of the activities of the promoter cis regulatory DNA elements and trans regulatory proteins that function through binding to these DNA elements. Our research supported by this program has led to the identification of rice bZIP transcription factors RF2a, RF2b and RLP1 that play key roles in regulating the activity of a vascular tissue specific promoter isolated from Rice Tungro Bacilliform Virus (RTBV) through their interactions with the Box II essential cis element located in the promoter. RF2a, RF2b and RLP1 possess multiple regulatory domains. Functional characterization reveals that those domains can activate or repress the activity of the RTBV promoter. Studies of transcriptional regulation of the RTBV promoter by this group of bZIP proteins not only provide insights about gene expression in the vascular tissue, but also insights about general mechanisms of transcription activation and repression. The knowledge gained from this research will also enable us to develop a well-described set of tools that can be used to control expression of multiple genes in transgenic plants and to improve biofuel feedstock.

EWS/FLI mediates transcriptional repression via NKX2.2 during oncogenic transformation in Ewing's sarcoma.

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Leah A Owen

2008-04-01

Full Text Available EWS/FLI is a master regulator of Ewing's sarcoma formation. Gene expression studies in A673 Ewing's sarcoma cells have demonstrated that EWS/FLI downregulates more genes than it upregulates, suggesting that EWS/FLI, and/or its targets, function as transcriptional repressors. One critical EWS/FLI target, NKX2.2, is a transcription factor that contains both transcriptional activation and transcriptional repression domains, raising the possibility that it mediates portions of the EWS/FLI transcriptional signature. We now report that microarray analysis demonstrated that the transcriptional profile of NKX2.2 consists solely of downregulated genes, and overlaps with the EWS/FLI downregulated signature, suggesting that NKX2.2 mediates oncogenic transformation via transcriptional repression. Structure-function analysis revealed that the DNA binding and repressor domains in NKX2.2 are required for oncogenesis in Ewing's sarcoma cells, while the transcriptional activation domain is completely dispensable. Furthermore, blockade of TLE or HDAC function, two protein families thought to mediate the repressive function of NKX2.2, inhibited the transformed phenotype and reversed the NKX2.2 transcriptional profile in Ewing's sarcoma cells. Whole genome localization studies (ChIP-chip revealed that a significant portion of the NKX2.2-repressed gene expression signature was directly mediated by NKX2.2 binding. These data demonstrate that the transcriptional repressive function of NKX2.2 is necessary, and sufficient, for the oncogenic phenotype of Ewing's sarcoma, and suggest a therapeutic approach to this disease.

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

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.

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 ...

Targeted genome regulation via synthetic programmable transcriptional regulators

KAUST Repository

Piatek, Agnieszka Anna; Mahfouz, Magdy M.

2016-01-01

genes in linear and interacting pathways in a native context. Modular DNA-binding domains from zinc fingers (ZFs) and transcriptional activator-like proteins (TALE) are amenable to bioengineering to bind DNA target sequences of interest. As a result, ZF

Transcription initiation complex structures elucidate DNA opening.

Science.gov (United States)

Plaschka, C; Hantsche, M; Dienemann, C; Burzinski, C; Plitzko, J; Cramer, P

2016-05-19

Transcription of eukaryotic protein-coding genes begins with assembly of the RNA polymerase (Pol) II initiation complex and promoter DNA opening. Here we report cryo-electron microscopy (cryo-EM) structures of yeast initiation complexes containing closed and open DNA at resolutions of 8.8 Å and 3.6 Å, respectively. DNA is positioned and retained over the Pol II cleft by a network of interactions between the TATA-box-binding protein TBP and transcription factors TFIIA, TFIIB, TFIIE, and TFIIF. DNA opening occurs around the tip of the Pol II clamp and the TFIIE 'extended winged helix' domain, and can occur in the absence of TFIIH. Loading of the DNA template strand into the active centre may be facilitated by movements of obstructing protein elements triggered by allosteric binding of the TFIIE 'E-ribbon' domain. The results suggest a unified model for transcription initiation with a key event, the trapping of open promoter DNA by extended protein-protein and protein-DNA contacts.

Gene organization inside replication domains in mammalian genomes

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Zaghloul, Lamia; Baker, Antoine; Audit, Benjamin; Arneodo, Alain

2012-11-01

We investigate the large-scale organization of human genes with respect to "master" replication origins that were previously identified as bordering nucleotide compositional skew domains. We separate genes in two categories depending on their CpG enrichment at the promoter which can be considered as a marker of germline DNA methylation. Using expression data in mouse, we confirm that CpG-rich genes are highly expressed in germline whereas CpG-poor genes are in a silent state. We further show that, whether tissue-specific or broadly expressed (housekeeping genes), the CpG-rich genes are over-represented close to the replication skew domain borders suggesting some coordination of replication and transcription. We also reveal that the transcription of the longest CpG-rich genes is co-oriented with replication fork progression so that the promoter of these transcriptionally active genes be located into the accessible open chromatin environment surrounding the master replication origins that border the replication skew domains. The observation of a similar gene organization in the mouse genome confirms the interplay of replication, transcription and chromatin structure as the cornerstone of mammalian genome architecture.

Assembly of proteins and 5 S rRNA to transcripts of the major structural domains of 23 S rRNA

DEFF Research Database (Denmark)

Ostergaard, P; Phan, H; Johansen, L B

1998-01-01

The six major structural domains of 23 S rRNA from Escherichia coli, and all combinations thereof, were synthesized as separate T7 transcripts and reconstituted with total 50 S subunit proteins. Analysis by one and two-dimensional gel electrophoresis demonstrated the presence of at least one prim...... approach was used to map the putative binding regions on domain V of protein L9 and the 5 S RNA-L5-L18 complex....

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.

SH2 domains: modulators of nonreceptor tyrosine kinase activity.

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Filippakopoulos, Panagis; Müller, Susanne; Knapp, Stefan

2009-12-01

The Src homology 2 (SH2) domain is a sequence-specific phosphotyrosine-binding module present in many signaling molecules. In cytoplasmic tyrosine kinases, the SH2 domain is located N-terminally to the catalytic kinase domain (SH1) where it mediates cellular localization, substrate recruitment, and regulation of kinase activity. Initially, structural studies established a role of the SH2 domain stabilizing the inactive state of Src family members. However, biochemical characterization showed that the presence of the SH2 domain is frequently required for catalytic activity, suggesting a crucial function stabilizing the active state of many nonreceptor tyrosine kinases. Recently, the structure of the SH2-kinase domain of Fes revealed that the SH2 domain stabilizes the active kinase conformation by direct interactions with the regulatory helix alphaC. Stabilizing interactions between the SH2 and the kinase domains have also been observed in the structures of active Csk and Abl. Interestingly, mutations in the SH2 domain found in human disease can be explained by SH2 domain destabilization or incorrect positioning of the SH2. Here we summarize our understanding of mechanisms that lead to tyrosine kinase activation by direct interactions mediated by the SH2 domain and discuss how mutations in the SH2 domain trigger kinase inactivation.

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.

Directed evolution of the TALE N-terminal domain for recognition of all 5' bases.

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Lamb, Brian M; Mercer, Andrew C; Barbas, Carlos F

2013-11-01

Transcription activator-like effector (TALE) proteins can be designed to bind virtually any DNA sequence. General guidelines for design of TALE DNA-binding domains suggest that the 5'-most base of the DNA sequence bound by the TALE (the N0 base) should be a thymine. We quantified the N0 requirement by analysis of the activities of TALE transcription factors (TALE-TF), TALE recombinases (TALE-R) and TALE nucleases (TALENs) with each DNA base at this position. In the absence of a 5' T, we observed decreases in TALE activity up to >1000-fold in TALE-TF activity, up to 100-fold in TALE-R activity and up to 10-fold reduction in TALEN activity compared with target sequences containing a 5' T. To develop TALE architectures that recognize all possible N0 bases, we used structure-guided library design coupled with TALE-R activity selections to evolve novel TALE N-terminal domains to accommodate any N0 base. A G-selective domain and broadly reactive domains were isolated and characterized. The engineered TALE domains selected in the TALE-R format demonstrated modularity and were active in TALE-TF and TALEN architectures. Evolved N-terminal domains provide effective and unconstrained TALE-based targeting of any DNA sequence as TALE binding proteins and designer enzymes.

A transcript cleavage factor of Mycobacterium tuberculosis important for its survival.

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Arnab China

Full Text Available After initiation of transcription, a number of proteins participate during elongation and termination modifying the properties of the RNA polymerase (RNAP. Gre factors are one such group conserved across bacteria. They regulate transcription by projecting their N-terminal coiled-coil domain into the active center of RNAP through the secondary channel and stimulating hydrolysis of the newly synthesized RNA in backtracked elongation complexes. Rv1080c is a putative gre factor (MtbGre in the genome of Mycobacterium tuberculosis. The protein enhanced the efficiency of promoter clearance by lowering abortive transcription and also rescued arrested and paused elongation complexes on the GC rich mycobacterial template. Although MtbGre is similar in domain organization and shares key residues for catalysis and RNAP interaction with the Gre factors of Escherichia coli, it could not complement an E. coli gre deficient strain. Moreover, MtbGre failed to rescue E. coli RNAP stalled elongation complexes, indicating the importance of specific protein-protein interactions for transcript cleavage. Decrease in the level of MtbGre reduced the bacterial survival by several fold indicating its essential role in mycobacteria. Another Gre homolog, Rv3788 was not functional in transcript cleavage activity indicating that a single Gre is sufficient for efficient transcription of the M. tuberculosis genome.

Activation of a development-specific gene, dofA, by FruA, an essential transcription factor for development of Myxococcus xanthus.

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Ueki, Toshiyuki; Inouye, Sumiko

2005-12-01

FruA is an essential transcription factor for Myxococcus xanthus development. The expression of tps and dofA genes is fruA dependent. In this study, we show by gel shift and footprint assays with the C-terminal DNA-binding domain of FruA and by a lacZ fusion assay that FruA may directly activate dofA expression during development.

Activation of a Development-Specific Gene, dofA, by FruA, an Essential Transcription Factor for Development of Myxococcus xanthus

OpenAIRE

Ueki, Toshiyuki; Inouye, Sumiko

2005-01-01

FruA is an essential transcription factor for Myxococcus xanthus development. The expression of tps and dofA genes is fruA dependent. In this study, we show by gel shift and footprint assays with the C-terminal DNA-binding domain of FruA and by a lacZ fusion assay that FruA may directly activate dofA expression during development.

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

The LIM domain protein FHL2 interacts with the NR5A family of nuclear receptors and CREB to activate the inhibin-α subunit gene in ovarian granulosa cells.

Science.gov (United States)

Matulis, Christina K; Mayo, Kelly E

2012-08-01

Nuclear receptor transcriptional activity is enhanced by interaction with coactivators. The highly related nuclear receptor 5A (NR5A) subfamily members liver receptor homolog 1 and steroidogenic factor 1 bind to and activate several of the same genes, many of which are important for reproductive function. To better understand transcriptional activation by these nuclear receptors, we sought to identify interacting proteins that might function as coactivators. The LIM domain protein four and a half LIM domain 2 (FHL2) was identified as interacting with the NR5A receptors in a yeast two-hybrid screen of a human ovary cDNA library. FHL2, and the closely related FHL1, are both expressed in the rodent ovary and in granulosa cells. Small interfering RNA-mediated knockdown of FHL1 and FHL2 in primary mouse granulosa cells reduced expression of the NR5A target genes encoding inhibin-α and P450scc. In vitro assays confirmed the interaction between the FHL and NR5A proteins and revealed that a single LIM domain of FHL2 is sufficient for this interaction, whereas determinants in both the ligand binding domain and DNA binding domain of NR5A proteins are important. FHL2 enhances the ability of both liver receptor homolog 1 and steroidogenic factor 1 to activate the inhibin-α subunit gene promoter in granulosa cells and thus functions as a transcriptional coactivator. FHL2 also interacts with cAMP response element-binding protein and substantially augments activation of inhibin gene expression by the combination of NR5A receptors and forskolin, suggesting that FHL2 may facilitate integration of these two signals. Collectively these results identify FHL2 as a novel coactivator of NR5A nuclear receptors in ovarian granulosa cells and suggest its involvement in regulating target genes important for mammalian reproduction.

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.

The BEACH Domain Protein SPIRRIG Is Essential for Arabidopsis Salt Stress Tolerance and Functions as a Regulator of Transcript Stabilization and Localization.

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Alexandra Steffens

2015-07-01

Full Text Available Members of the highly conserved class of BEACH domain containing proteins (BDCPs have been established as broad facilitators of protein-protein interactions and membrane dynamics in the context of human diseases like albinism, bleeding diathesis, impaired cellular immunity, cancer predisposition, and neurological dysfunctions. Also, the Arabidopsis thaliana BDCP SPIRRIG (SPI is important for membrane integrity, as spi mutants exhibit split vacuoles. In this work, we report a novel molecular function of the BDCP SPI in ribonucleoprotein particle formation. We show that SPI interacts with the P-body core component DECAPPING PROTEIN 1 (DCP1, associates to mRNA processing bodies (P-bodies, and regulates their assembly upon salt stress. The finding that spi mutants exhibit salt hypersensitivity suggests that the local function of SPI at P-bodies is of biological relevance. Transcriptome-wide analysis revealed qualitative differences in the salt stress-regulated transcriptional response of Col-0 and spi. We show that SPI regulates the salt stress-dependent post-transcriptional stabilization, cytoplasmic agglomeration, and localization to P-bodies of a subset of salt stress-regulated mRNAs. Finally, we show that the PH-BEACH domains of SPI and its human homolog FAN (Factor Associated with Neutral sphingomyelinase activation interact with DCP1 isoforms from plants, mammals, and yeast, suggesting the evolutionary conservation of an association of BDCPs and P-bodies.

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.

clockwork orange encodes a transcriptional repressor important for circadian clock amplitude in Drosophila

OpenAIRE

Lim, Chunghun; Chung, Brian Y.; Pitman, Jena L.; McGill, Jermaine J.; Pradhan, Suraj; Lee, Jongbin; Keegan, Kevin P.; Choe, Joonho; Allada, Ravi

2007-01-01

Gene transcription is a central timekeeping process in animal clocks. In Drosophila, the basic helix-loop helix (bHLH)-PAS transcription factor heterodimer, CLOCK (CLK)/CYCLE(CYC) transcriptionally activates the clock components period (per), timeless (tim), Par domain protein 1 (Pdp1), and vrille (vri) that feedback and regulate distinct features of CLK/CYC function [1]. Microarray studies have identified numerous rhythmically expressed transcripts [2-7], some of which are potential direct C...

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.

Computational Studies of the Active and Inactive Regulatory Domains of Response Regulator PhoP Using Molecular Dynamics Simulations.

Science.gov (United States)

Qing, Xiao-Yu; Steenackers, Hans; Venken, Tom; De Maeyer, Marc; Voet, Arnout

2017-11-01

The response regulator PhoP is part of the PhoP/PhoQ two-component system, which is responsible for regulating the expression of multiple genes involved in controlling virulence, biofilm formation, and resistance to antimicrobial peptides. Therefore, modulating the transcriptional function of the PhoP protein is a promising strategy for developing new antimicrobial agents. There is evidence suggesting that phosphorylation-mediated dimerization in the regulatory domain of PhoP is essential for its transcriptional function. Disruption or stabilization of protein-protein interactions at the dimerization interface may inhibit or enhance the expression of PhoP-dependent genes. In this study, we performed molecular dynamics simulations on the active and inactive dimers and monomers of the PhoP regulatory domains, followed by pocket-detecting screenings and a quantitative hot-spot analysis in order to assess the druggability of the protein. Consistent with prior hypothesis, the calculation of the binding free energy shows that phosphorylation enhances dimerization of PhoP. Furthermore, we have identified two different putative binding sites at the dimerization active site (the α4-β5-α5 face) with energetic "hot-spot" areas, which could be used to search for modulators of protein-protein interactions. This study delivers insight into the dynamics and druggability of the dimerization interface of the PhoP regulatory domain, and may serve as a basis for the rational identification of new antimicrobial drugs. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

RNA-guided Transcriptional Regulation in Plants via dCas9 Chimeric Proteins

KAUST Repository

Baazim, Hatoon

2014-05-01

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

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...

Cellular corepressor TLE2 inhibits replication-and-transcription- activator-mediated transactivation and lytic reactivation of Kaposi's sarcoma-associated herpesvirus.

Science.gov (United States)

He, Zhiheng; Liu, Yunhua; Liang, Deguang; Wang, Zhuo; Robertson, Erle S; Lan, Ke

2010-02-01

Replication and transcription activator (RTA) encoded by open reading frame 50 (ORF50) of Kaposi's sarcoma-associated herpesvirus (KSHV) is essential and sufficient to initiate lytic reactivation. RTA activates its target genes through direct binding with high affinity to its responsive elements or by interaction with cellular factors, such as RBP-Jkappa, Ap-1, C/EBP-alpha, and Oct-1. In this study, we identified transducin-like enhancer of split 2 (TLE2) as a novel RTA binding protein by using yeast two-hybrid screening of a human spleen cDNA library. The interaction between TLE2 and RTA was confirmed by glutathione S-transferase (GST) binding and coimmunoprecipitation assays. Immunofluorescence analysis showed that TLE2 and RTA were colocalized in the same nuclear compartment in KSHV-infected cells. This interaction recruited TLE2 to RTA bound to its recognition sites on DNA and repressed RTA auto-activation and transactivation activity. Moreover, TLE2 also inhibited the induction of lytic replication and virion production driven by RTA. We further showed that the Q (Gln-rich), SP (Ser-Pro-rich), and WDR (Trp-Asp repeat) domains of TLE2 and the Pro-rich domain of RTA were essential for this interaction. RBP-Jkappa has been shown previously to bind to the same Pro-rich domain of RTA, and this binding can be subject to competition by TLE2. In addition, TLE2 can form a complex with RTA to access the cognate DNA sequence of the RTA-responsive element at different promoters. Intriguingly, the transcription level of TLE2 could be upregulated by RTA during the lytic reactivation process. In conclusion, we identified a new RTA binding protein, TLE2, and demonstrated that TLE2 inhibited replication and transactivation mediated by RTA. This provides another potentially important mechanism for maintenance of KSHV viral latency through interaction with a host protein.

Solution structure of a DNA mimicking motif of an RNA aptamer against transcription factor AML1 Runt domain.

Science.gov (United States)

Nomura, Yusuke; Tanaka, Yoichiro; Fukunaga, Jun-ichi; Fujiwara, Kazuya; Chiba, Manabu; Iibuchi, Hiroaki; Tanaka, Taku; Nakamura, Yoshikazu; Kawai, Gota; Kozu, Tomoko; Sakamoto, Taiichi

2013-12-01

AML1/RUNX1 is an essential transcription factor involved in the differentiation of hematopoietic cells. AML1 binds to the Runt-binding double-stranded DNA element (RDE) of target genes through its N-terminal Runt domain. In a previous study, we obtained RNA aptamers against the AML1 Runt domain by systematic evolution of ligands by exponential enrichment and revealed that RNA aptamers exhibit higher affinity for the Runt domain than that for RDE and possess the 5'-GCGMGNN-3' and 5'-N'N'CCAC-3' conserved motif (M: A or C; N and N' form Watson-Crick base pairs) that is important for Runt domain binding. In this study, to understand the structural basis of recognition of the Runt domain by the aptamer motif, the solution structure of a 22-mer RNA was determined using nuclear magnetic resonance. The motif contains the AH(+)-C mismatch and base triple and adopts an unusual backbone structure. Structural analysis of the aptamer motif indicated that the aptamer binds to the Runt domain by mimicking the RDE sequence and structure. Our data should enhance the understanding of the structural basis of DNA mimicry by RNA molecules.

Conserved TRAM Domain Functions as an Archaeal Cold Shock Protein via RNA Chaperone Activity

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Bo Zhang

2017-08-01

Full Text Available Cold shock proteins (Csps enable organisms to acclimate to and survive in cold environments and the bacterial CspA family exerts the cold protection via its RNA chaperone activity. However, most Archaea do not contain orthologs to the bacterial csp. TRAM, a conserved domain among RNA modification proteins ubiquitously distributed in organisms, occurs as an individual protein in most archaeal phyla and has a structural similarity to Csp proteins, yet its biological functions remain unknown. Through physiological and biochemical studies on four TRAM proteins from a cold adaptive archaeon Methanolobus psychrophilus R15, this work demonstrated that TRAM is an archaeal Csp and exhibits RNA chaperone activity. Three TRAM encoding genes (Mpsy_0643, Mpsy_3043, and Mpsy_3066 exhibited remarkable cold-shock induced transcription and were preferentially translated at lower temperature (18°C, while the fourth (Mpsy_2002 was constitutively expressed. They were all able to complement the cspABGE mutant of Escherichia coli BX04 that does not grow in cold temperatures and showed transcriptional antitermination. TRAM3066 (gene product of Mpsy_3066 and TRAM2002 (gene product of Mpsy_2002 displayed sequence-non-specific RNA but not DNA binding activity, and TRAM3066 assisted RNases in degradation of structured RNA, thus validating the RNA chaperone activity of TRAMs. Given the chaperone activity, TRAM is predicted to function beyond a Csp.

Barriers and facilitators to the implementation of a school-based physical activity policy in Canada: application of the theoretical domains framework.

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Weatherson, Katie A; McKay, Rhyann; Gainforth, Heather L; Jung, Mary E

2017-10-23

In British Columbia Canada, a Daily Physical Activity (DPA) policy was mandated that requires elementary school teachers to provide students with opportunities to achieve 30 min of physical activity during the school day. However, the implementation of school-based physical activity policies is influenced by many factors. A theoretical examination of the factors that impede and enhance teachers' implementation of physical activity policies is necessary in order to develop strategies to improve policy practice and achieve desired outcomes. This study used the Theoretical Domains Framework (TDF) to understand teachers' barriers and facilitators to the implementation of the DPA policy in one school district. Additionally, barriers and facilitators were examined and compared according to how the teacher implemented the DPA policy during the instructional school day. Interviews were conducted with thirteen teachers and transcribed verbatim. One researcher performed barrier and facilitator extraction, with double extraction occurring across a third of the interview transcripts by a second researcher. A deductive and inductive analytical approach in a two-stage process was employed whereby barriers and facilitators were deductively coded using TDF domains (content analysis) and analyzed for sub-themes within each domain. Two researchers performed coding. A total of 832 items were extracted from the interview transcripts. Some items were coded into multiple TDF domains, resulting in a total of 1422 observations. The most commonly coded TDF domains accounting for 75% of the total were Environmental context and resources (ECR; n = 250), Beliefs about consequences (n = 225), Social influences (n = 193), Knowledge (n = 100), and Intentions (n = 88). Teachers who implemented DPA during instructional time differed from those who relied on non-instructional time in relation to Goals, Behavioural regulation, Social/professional role and identity, Beliefs about

Barriers and facilitators to the implementation of a school-based physical activity policy in Canada: application of the theoretical domains framework

Directory of Open Access Journals (Sweden)

Katie A. Weatherson

2017-10-01

Full Text Available Abstract Background In British Columbia Canada, a Daily Physical Activity (DPA policy was mandated that requires elementary school teachers to provide students with opportunities to achieve 30 min of physical activity during the school day. However, the implementation of school-based physical activity policies is influenced by many factors. A theoretical examination of the factors that impede and enhance teachers’ implementation of physical activity policies is necessary in order to develop strategies to improve policy practice and achieve desired outcomes. This study used the Theoretical Domains Framework (TDF to understand teachers’ barriers and facilitators to the implementation of the DPA policy in one school district. Additionally, barriers and facilitators were examined and compared according to how the teacher implemented the DPA policy during the instructional school day. Methods Interviews were conducted with thirteen teachers and transcribed verbatim. One researcher performed barrier and facilitator extraction, with double extraction occurring across a third of the interview transcripts by a second researcher. A deductive and inductive analytical approach in a two-stage process was employed whereby barriers and facilitators were deductively coded using TDF domains (content analysis and analyzed for sub-themes within each domain. Two researchers performed coding. Results A total of 832 items were extracted from the interview transcripts. Some items were coded into multiple TDF domains, resulting in a total of 1422 observations. The most commonly coded TDF domains accounting for 75% of the total were Environmental context and resources (ECR; n = 250, Beliefs about consequences (n = 225, Social influences (n = 193, Knowledge (n = 100, and Intentions (n = 88. Teachers who implemented DPA during instructional time differed from those who relied on non-instructional time in relation to Goals, Behavioural regulation, Social

Growth hormone receptor C-terminal domains required for growth hormone-induced intracellular free Ca2+ oscillations and gene transcription

DEFF Research Database (Denmark)

Billestrup, N; Bouchelouche, P; Allevato, G

1995-01-01

of varying frequency and amplitude. GH-induced transcription of the serine protease inhibitor 2.1 gene required the same C-terminal 52-amino acid domain of the receptor as for Ca2+ signaling. Mutation of the four proline residues in the conserved box 1 region of the GHR, which is responsible for binding...

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

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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.

Rational Design of Mini-Cas9 for Transcriptional Activation.

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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.

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

Science.gov (United States)

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

2001-04-01

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

Global effects of the CSR-1 RNA interference pathway on transcriptional landscape

Science.gov (United States)

Cecere, Germano; Hoersch, Sebastian; O’Keeffe, Sean; Sachidanandam, Ravi; Grishok, Alla

2014-01-01

Argonaute proteins and their small RNA co-factors short interfering RNAs (siRNAs) are known to inhibit gene expression at the transcriptional and post-transcriptional levels. In Caenorhabditis elegans, the Argonaute CSR-1 binds thousands of endogenous siRNAs (endo-siRNAs) antisense to germline transcripts and associates with chromatin in a siRNA-dependent manner. However, its role in gene expression regulation remains controversial. Here, we used a genome-wide profiling of nascent RNA transcripts to demonstrate that the CSR-1 RNAi pathway promotes sense-oriented Pol II transcription. Moreover, a loss of CSR-1 function resulted in global increase in antisense transcription and ectopic transcription of silent chromatin domains, which led to reduced chromatin incorporation of centromere-specific histone H3. Based on these findings, we propose that the CSR-1 pathway has a role in maintaining the directionality of active transcription thereby propagating the distinction between transcriptionally active and silent genomic regions. PMID:24681887

Nuclear import of transcription factor BR-C is mediated by its interaction with RACK1.

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Cheng, Daojun; Qian, Wenliang; Wang, Yonghu; Meng, Meng; Wei, Ling; Li, Zhiqing; Kang, Lixia; Peng, Jian; Xia, Qingyou

2014-01-01

The transcription factor Broad Complex (BR-C) is an early ecdysone response gene in insects and contains two types of domains: two zinc finger domains for the activation of gene transcription and a Bric-a-brac/Tramtrack/Broad complex (BTB) domain for protein-protein interaction. Although the mechanism of zinc finger-mediated gene transcription is well studied, the partners interacting with the BTB domain of BR-C has not been elucidated until now. Here, we performed a yeast two-hybrid screen using the BTB domain of silkworm BR-C as bait and identified the receptor for activated C-kinase 1 (RACK1), a scaffolding/anchoring protein, as the novel partner capable of interacting with BR-C. The interaction between BR-C and RACK1 was further confirmed by far-western blotting and pull-down assays. Importantly, the disruption of this interaction, via RNAi against the endogenous RACK1 gene or deletion of the BTB domain, abolished the nuclear import of BR-C in BmN4 cells. In addition, RNAi against the endogenous PKC gene as well as phosphorylation-deficient mutation of the predicted PKC phosphorylation sites at either Ser373 or Thr406 in BR-C phenocopied RACK1 RNAi and altered the nuclear localization of BR-C. However, when BTB domain was deleted, phosphorylation mimics of either Ser373 or Thr406 had no effect on the nuclear import of BR-C. Moreover, mutating the PKC phosphorylation sites at Ser373 and Thr406 or deleting the BTB domain significantly decreased the transcriptional activation of a BR-C target gene. Given that RACK1 is necessary for recruiting PKC to close and phosphorylate target proteins, we suggest that the PKC-mediated phosphorylation and nuclear import of BR-C is determined by its interaction with RACK1. This novel finding will be helpful for further deciphering the mechanism underlying the role of BR-C proteins during insect development.

Nuclear import of transcription factor BR-C is mediated by its interaction with RACK1.

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Daojun Cheng

Full Text Available The transcription factor Broad Complex (BR-C is an early ecdysone response gene in insects and contains two types of domains: two zinc finger domains for the activation of gene transcription and a Bric-a-brac/Tramtrack/Broad complex (BTB domain for protein-protein interaction. Although the mechanism of zinc finger-mediated gene transcription is well studied, the partners interacting with the BTB domain of BR-C has not been elucidated until now. Here, we performed a yeast two-hybrid screen using the BTB domain of silkworm BR-C as bait and identified the receptor for activated C-kinase 1 (RACK1, a scaffolding/anchoring protein, as the novel partner capable of interacting with BR-C. The interaction between BR-C and RACK1 was further confirmed by far-western blotting and pull-down assays. Importantly, the disruption of this interaction, via RNAi against the endogenous RACK1 gene or deletion of the BTB domain, abolished the nuclear import of BR-C in BmN4 cells. In addition, RNAi against the endogenous PKC gene as well as phosphorylation-deficient mutation of the predicted PKC phosphorylation sites at either Ser373 or Thr406 in BR-C phenocopied RACK1 RNAi and altered the nuclear localization of BR-C. However, when BTB domain was deleted, phosphorylation mimics of either Ser373 or Thr406 had no effect on the nuclear import of BR-C. Moreover, mutating the PKC phosphorylation sites at Ser373 and Thr406 or deleting the BTB domain significantly decreased the transcriptional activation of a BR-C target gene. Given that RACK1 is necessary for recruiting PKC to close and phosphorylate target proteins, we suggest that the PKC-mediated phosphorylation and nuclear import of BR-C is determined by its interaction with RACK1. This novel finding will be helpful for further deciphering the mechanism underlying the role of BR-C proteins during insect development.

Mediator Recruitment to Heat Shock Genes Requires Dual Hsf1 Activation Domains and Mediator Tail Subunits Med15 and Med16*

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Kim, Sunyoung; Gross, David S.

2013-01-01

The evolutionarily conserved Mediator complex is central to the regulation of gene transcription in eukaryotes because it serves as a physical and functional interface between upstream regulators and the Pol II transcriptional machinery. Nonetheless, its role appears to be context-dependent, and the detailed mechanism by which it governs the expression of most genes remains unknown. Here we investigate Mediator involvement in HSP (heat shock protein) gene regulation in the yeast Saccharomyces cerevisiae. We find that in response to thermal upshift, subunits representative of each of the four Mediator modules (Head, Middle, Tail, and Kinase) are rapidly, robustly, and selectively recruited to the promoter regions of HSP genes. Their residence is transient, returning to near-background levels within 90 min. Hsf1 (heat shock factor 1) plays a central role in recruiting Mediator, as indicated by the fact that truncation of either its N- or C-terminal activation domain significantly reduces Mediator occupancy, whereas removal of both activation domains abolishes it. Likewise, ablation of either of two Mediator Tail subunits, Med15 or Med16, reduces Mediator recruitment to HSP promoters, whereas deletion of both abolishes it. Accompanying the loss of Mediator, recruitment of RNA polymerase II is substantially diminished. Interestingly, Mediator antagonizes Hsf1 occupancy of non-induced promoters yet facilitates enhanced Hsf1 association with activated ones. Collectively, our observations indicate that Hsf1, via its dual activation domains, recruits holo-Mediator to HSP promoters in response to acute heat stress through cooperative physical and/or functional interactions with the Tail module. PMID:23447536

Identification of tyrosine residues in the intracellular domain of the growth hormone receptor required for transcriptional signaling and Stat5 activation

DEFF Research Database (Denmark)

Hansen, L. H.; Wang, X.; Kopchick, J J

1996-01-01

The binding of growth hormone (GH) to its receptor results in its dimerization followed by activation of Jak2 kinase and tyrosine phosphorylation of the GH receptor itself, as well as Jak2 and the transcription factors Stat1, -3, and -5. In order to study the role of GH receptor tyrosine phosphor...

SH3 Domains Differentially Stimulate Distinct Dynamin I Assembly Modes and G Domain Activity.

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Sai Krishnan

Full Text Available Dynamin I is a highly regulated GTPase enzyme enriched in nerve terminals which mediates vesicle fission during synaptic vesicle endocytosis. One regulatory mechanism involves its interactions with proteins containing Src homology 3 (SH3 domains. At least 30 SH3 domain-containing proteins bind dynamin at its proline-rich domain (PRD. Those that stimulate dynamin activity act by promoting its oligomerisation. We undertook a systematic parallel screening of 13 glutathione-S-transferase (GST-tagged endocytosis-related SH3 domains on dynamin binding, GTPase activity and oligomerisation. No correlation was found between dynamin binding and their potency to stimulate GTPase activity. There was limited correlation between the extent of their ability to stimulate dynamin activity and the level of oligomerisation, indicating an as yet uncharacterised allosteric coupling of the PRD and G domain. We examined the two variants, dynamin Iab and Ibb, which differ in the alternately splice middle domain α2 helix. They responded differently to the panel of SH3s, with the extent of stimulation between the splice variants varying greatly between the SH3s. This study reveals that SH3 binding can act as a heterotropic allosteric regulator of the G domain via the middle domain α2 helix, suggesting an involvement of this helix in communicating the PRD-mediated allostery. This indicates that SH3 binding both stabilises multiple conformations of the tetrameric building block of dynamin, and promotes assembly of dynamin-SH3 complexes with distinct rates of GTP hydrolysis.

Global effects of the CSR-1 RNA interference pathway on the transcriptional landscape.

Science.gov (United States)

Cecere, Germano; Hoersch, Sebastian; O'Keeffe, Sean; Sachidanandam, Ravi; Grishok, Alla

2014-04-01

Argonaute proteins and their small RNA cofactors short interfering RNAs are known to inhibit gene expression at the transcriptional and post-transcriptional levels. In Caenorhabditis elegans, the Argonaute CSR-1 binds thousands of endogenous siRNAs (endo-siRNAs) that are antisense to germline transcripts. However, its role in gene expression regulation remains controversial. Here we used genome-wide profiling of nascent RNA transcripts and found that the CSR-1 RNA interference pathway promoted sense-oriented RNA polymerase II transcription. Moreover, a loss of CSR-1 function resulted in global increase in antisense transcription and ectopic transcription of silent chromatin domains, which led to reduced chromatin incorporation of centromere-specific histone H3. On the basis of these findings, we propose that the CSR-1 pathway helps maintain the directionality of active transcription, thereby propagating the distinction between transcriptionally active and silent genomic regions.

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

Structural Studies of the SET Domain from RIZ1 Tumor Suppressor

Energy Technology Data Exchange (ETDEWEB)

Briknarova, Klara; Zhou, Xinliang; Satterthwait, Arnold C.; Hoyt, David W.; Ely, Kathryn R.; Huang, Shi

2008-02-15

Histone lysine methyltransferases (HKMTs) are involved in regulation of chromatin structure, and, as such, are important for longterm gene activation and repression that is associated with cell memory and establishment of cell-type specific transcriptional programs. Most HKMTs contain a SET domain, which is responsible for their catalytic activity. RIZ1 is a transcription regulator and tumor suppressor that catalyzes methylation of lysine 9 of histone H3 and contains a rather distinct SET domain. Similar SET domains, sometimes refererred to as PR (PRDI-BF1 and RIZ1 homology) domains, are also found in other proteins including Blimp-1/PRDI-BF1, MDS1-EVI1 and Meisetz. We determined the solution structure of the PR domain from RIZ1 and characterized its interaction with S-adenosyl homocysteine (SAH) and a peptide from histone H3. Despite low sequence identity with canonical SET domains, the PR domain displays a typical SET fold including a pseudo-knot at the C-terminus. The N-flanking sequence of RIZ1 PR domain adopts a novel conformation and interacts closely with the SET fold. The C-flanking sequence contains an α-helix that exhibits higher mobility than the SET fold and points away from the protein face that harbors active site in other SET domains. Residues that interact with the methylation cofactor in SET domains are not conserved in RIZ1 or other PR domains, and the SET fold of RIZ1 does not bind SAH. However, the PR domain of RIZ1 interacts specifically with a synthetic peptide comprising residues 1-20 of histone H3.

The DOF transcription factor Dof5.1 influences leaf axial patterning by promoting Revoluta transcription in Arabidopsis

KAUST Repository

Kim, Hyungsae

2010-10-05

Dof proteins are transcription factors that have a conserved single zinc finger DNA-binding domain. In this study, we isolated an activation tagging mutant Dof5.1-D exhibiting an upward-curling leaf phenotype due to enhanced expression of the REV gene that is required for establishing adaxialabaxial polarity. Dof5.1-D plants also had reduced transcript levels for IAA6 and IAA19 genes, indicating an altered auxin biosynthesis in Dof5.1-D. An electrophoretic mobility shift assay using the Dof5.1 DNA-binding motif and the REV promoter region indicated that the DNA-binding domain of Dof5.1 binds to a TAAAGT motif located in the 5′-distal promoter region of the REV promoter. Further, transient and chromatin immunoprecipitation assays verified binding activity of the Dof5.1 DNA-binding motif with the REV promoter. Consistent with binding assays, constitutive over-expression of the Dof5.1 DNA-binding domain in wild-type plants caused a downward-curling phenotype, whereas crossing Dof5.1-D to a rev mutant reverted the upward-curling phenotype of the Dof5.1-D mutant leaf to the wild-type. These results suggest that the Dof5.1 protein directly binds to the REV promoter and thereby regulates adaxialabaxial polarity. © 2010 Blackwell Publishing Ltd.

The DOF transcription factor Dof5.1 influences leaf axial patterning by promoting Revoluta transcription in Arabidopsis

KAUST Repository

Kim, Hyungsae; Kim, Sungjin; Abbasi, Nazia; Bressan, Ray Anthony; Yun, Daejin; Yoo, Sangdong; Kwon, SukYun; Choi, Sangbong

2010-01-01

Dof proteins are transcription factors that have a conserved single zinc finger DNA-binding domain. In this study, we isolated an activation tagging mutant Dof5.1-D exhibiting an upward-curling leaf phenotype due to enhanced expression of the REV gene that is required for establishing adaxialabaxial polarity. Dof5.1-D plants also had reduced transcript levels for IAA6 and IAA19 genes, indicating an altered auxin biosynthesis in Dof5.1-D. An electrophoretic mobility shift assay using the Dof5.1 DNA-binding motif and the REV promoter region indicated that the DNA-binding domain of Dof5.1 binds to a TAAAGT motif located in the 5′-distal promoter region of the REV promoter. Further, transient and chromatin immunoprecipitation assays verified binding activity of the Dof5.1 DNA-binding motif with the REV promoter. Consistent with binding assays, constitutive over-expression of the Dof5.1 DNA-binding domain in wild-type plants caused a downward-curling phenotype, whereas crossing Dof5.1-D to a rev mutant reverted the upward-curling phenotype of the Dof5.1-D mutant leaf to the wild-type. These results suggest that the Dof5.1 protein directly binds to the REV promoter and thereby regulates adaxialabaxial polarity. © 2010 Blackwell Publishing Ltd.

Antimicrobial activity of a novel hypervariable immunoglobulin domain-containing receptor Dscam in Cherax quadricarinatus.

Science.gov (United States)

Li, Dan; Yu, Ai-Qing; Li, Xue-Jie; Zhu, You-Ting; Jin, Xing-Kun; Li, Wei-Wei; Wang, Qun

2015-12-01

Down syndrome cell adhesion molecule (Dscam) mediates innate immunity against pathogens in arthropods. Here, a novel Dscam from red claw crayfish Cherax quadricarinatus (CqDscam) was isolated. The CqDscam protein contains one signal peptide, ten immunoglobulin domains, six fibronectin type III domains, one transmembrane domain and cytoplasmic tail. CqDscam phylogenetically clustered with other invertebrate Dscams. Variable regions of CqDscam in N-terminal halves of Ig2 and Ig3 domains, complete Ig7 domain and TM domain can be reshuffled after transcription to produce a deluge of >37,620 potential alternative splice forms. CqDscam was detected in all tissues tested and abundantly expressed in immune system and nerve system. Upon lipopolysaccharides (LPS) and b-1, 3-glucans (Glu) challenged, the expression of CqDscam was up-regulated, while no response in expression occurred after injection with peptidoglycans (PG). Membrane-bound and secreted types of CqDscam were separated on the protein level, and were both extensively induced post LPS challenge. Membrane-bound CqDscam protein was not detected in the serum, but localized to the hemocyte surface by immuno-localization assay. In the antimicrobial assays, the recombinant LPS-induced isoform of CqDscam protein displayed bacterial binding and growth inhibitory activities, especially with Escherichia coli. These results suggested that CqDscam, as one of pattern-recognition receptors (PRRs), involved in innate immune recognition and defense mechanisms in C. quadricarinatus, possibly through alternative splicing. Copyright © 2015 Elsevier Ltd. All rights reserved.

Ubiquitin fusion constructs allow the expression and purification of multi-KOW domain complexes of the Saccharomyces cerevisiae transcription elongation factor Spt4/5.

Science.gov (United States)

Blythe, Amanda; Gunasekara, Sanjika; Walshe, James; Mackay, Joel P; Hartzog, Grant A; Vrielink, Alice

2014-08-01

Spt4/5 is a hetero-dimeric transcription elongation factor that can both inhibit and promote transcription elongation by RNA polymerase II (RNAPII). However, Spt4/5's mechanism of action remains elusive. Spt5 is an essential protein and the only universally-conserved RNAP-associated transcription elongation factor. The protein contains multiple Kyrpides, Ouzounis and Woese (KOW) domains. These domains, in other proteins, are thought to bind RNA although there is little direct evidence in the literature to support such a function in Spt5. This could be due, at least in part, to difficulties in expressing and purifying recombinant Spt5. When expressed in Escherichia coli (E. coli), Spt5 is innately insoluble. Here we report a new approach for the successful expression and purification of milligram quantities of three different multi-KOW domain complexes of Saccharomyces cerevisiae Spt4/5 for use in future functional studies. Using the E. coli strain Rosetta2 (DE3) we have developed strategies for co-expression of Spt4 and multi-KOW domain Spt5 complexes from the bi-cistronic pET-Duet vector. In a second strategy, Spt4/5 was expressed via co-transformation of Spt4 in the vector pET-M11 with Spt5 ubiquitin fusion constructs in the vector pHUE. We characterized the multi-KOW domain Spt4/5 complexes by Western blot, limited proteolysis, circular dichroism, SDS-PAGE and size exclusion chromatography-multiangle light scattering and found that the proteins are folded with a Spt4:Spt5 hetero-dimeric stoichiometry of 1:1. These expression constructs encompass a larger region of Spt5 than has previously been reported, and will provide the opportunity to elucidate the biological function of the multi-KOW containing Spt5. Copyright © 2014 Elsevier Inc. All rights reserved.

Directed evolution of the TALE N-terminal domain for recognition of all 5′ bases

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Lamb, Brian M.; Mercer, Andrew C.; Barbas, Carlos F.

2013-01-01

Transcription activator-like effector (TALE) proteins can be designed to bind virtually any DNA sequence. General guidelines for design of TALE DNA-binding domains suggest that the 5′-most base of the DNA sequence bound by the TALE (the N0 base) should be a thymine. We quantified the N0 requirement by analysis of the activities of TALE transcription factors (TALE-TF), TALE recombinases (TALE-R) and TALE nucleases (TALENs) with each DNA base at this position. In the absence of a 5′ T, we observed decreases in TALE activity up to >1000-fold in TALE-TF activity, up to 100-fold in TALE-R activity and up to 10-fold reduction in TALEN activity compared with target sequences containing a 5′ T. To develop TALE architectures that recognize all possible N0 bases, we used structure-guided library design coupled with TALE-R activity selections to evolve novel TALE N-terminal domains to accommodate any N0 base. A G-selective domain and broadly reactive domains were isolated and characterized. The engineered TALE domains selected in the TALE-R format demonstrated modularity and were active in TALE-TF and TALEN architectures. Evolved N-terminal domains provide effective and unconstrained TALE-based targeting of any DNA sequence as TALE binding proteins and designer enzymes. PMID:23980031

Identification of the kinase that activates a nonmetazoan STAT gives insights into the evolution of phosphotyrosine-SH2 domain signaling.

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Araki, Tsuyoshi; Kawata, Takefumi; Williams, Jeffrey G

2012-07-10

SH2 domains are integral to many animal signaling pathways. By interacting with specific phosphotyrosine residues, they provide regulatable protein-protein interaction domains. Dictyostelium is the only nonmetazoan with functionally characterized SH2 domains, but the cognate tyrosine kinases are unknown. There are no orthologs of the animal tyrosine kinases, but there are very many tyrosine kinase-like kinases (TKLs), a group of kinases which, despite their family name, are classified mainly as serine-threonine kinases. STATs are transcription factors that dimerize via phosphotyrosine-SH2 domain interactions. STATc is activated by phosphorylation on Tyr922 when cells are exposed to the prestalk inducer differentiation inducing factor (DIF-1), a chlorinated hexaphenone. We show that in a null mutant for Pyk2, a tyrosine-specific TKL, exposure to DIF-1 does not activate STATc. Conversely, overexpression of Pyk2 causes constitutive STATc activation. Pyk2 phosphorylates STATc on Tyr922 in vitro and complexes with STATc both in vitro and in vivo. This demonstration that a TKL directly activates a STAT has significant implications for understanding the evolutionary origins of SH2 domain-phosphotyrosine signaling. It also has mechanistic implications. Our previous work suggested that a predicted constitutive STATc tyrosine kinase activity is counterbalanced in vivo by the DIF-1-regulated activity of PTP3, a Tyr922 phosphatase. Here we show that the STATc-Pyk2 complex is formed constitutively by an interaction between the STATc SH2 domain and phosphotyrosine residues on Pyk2 that are generated by autophosphorylation. Also, as predicted, Pyk2 is constitutively active as a STATc kinase. This observation provides further evidence for this highly atypical, possibly ancestral, STAT regulation mechanism.

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

DEFF Research Database (Denmark)

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

2008-01-01

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

A nanobody modulates the p53 transcriptional program without perturbing its functional architecture

Science.gov (United States)

Bethuyne, Jonas; De Gieter, Steven; Zwaenepoel, Olivier; Garcia-Pino, Abel; Durinck, Kaat; Verhelle, Adriaan; Hassanzadeh-Ghassabeh, Gholamreza; Speleman, Frank; Loris, Remy; Gettemans, Jan

2014-01-01

The p53 transcription factor plays an important role in genome integrity. To perform this task, p53 regulates the transcription of genes promoting various cellular outcomes including cell cycle arrest, apoptosis or senescence. The precise regulation of this activity remains elusive as numerous mechanisms, e.g. posttranslational modifications of p53 and (non-)covalent p53 binding partners, influence the p53 transcriptional program. We developed a novel, non-invasive tool to manipulate endogenous p53. Nanobodies (Nb), raised against the DNA-binding domain of p53, allow us to distinctively target both wild type and mutant p53 with great specificity. Nb3 preferentially binds ‘structural’ mutant p53, i.e. R175H and R282W, while a second but distinct nanobody, Nb139, binds both mutant and wild type p53. The co-crystal structure of the p53 DNA-binding domain in complex with Nb139 (1.9 Å resolution) reveals that Nb139 binds opposite the DNA-binding surface. Furthermore, we demonstrate that Nb139 does not disturb the functional architecture of the p53 DNA-binding domain using conformation-specific p53 antibody immunoprecipitations, glutaraldehyde crosslinking assays and chromatin immunoprecipitation. Functionally, the binding of Nb139 to p53 allows us to perturb the transactivation of p53 target genes. We propose that reduced recruitment of transcriptional co-activators or modulation of selected post-transcriptional modifications account for these observations. PMID:25324313

Structure of Thermotoga maritima TM0439: implications for the mechanism of bacterial GntR transcription regulators with Zn2+-binding FCD domains

International Nuclear Information System (INIS)

Zheng, Meiying; Cooper, David R.; Grossoehme, Nickolas E.; Yu, Minmin; Hung, Li-Wei; Cieslik, Marcin; Derewenda, Urszula; Lesley, Scott A.; Wilson, Ian A.; Giedroc, David P.; Derewenda, Zygmunt S.

2009-01-01

Here, the crystal structure of TM0439, a GntR regulator with an FCD domain found in the Thermotoga maritima genome, is described. The GntR superfamily of dimeric transcription factors, with more than 6200 members encoded in bacterial genomes, are characterized by N-terminal winged-helix DNA-binding domains and diverse C-terminal regulatory domains which provide a basis for the classification of the constituent families. The largest of these families, FadR, contains nearly 3000 proteins with all-α-helical regulatory domains classified into two related Pfam families: FadR-C and FCD. Only two crystal structures of FadR-family members, those of Escherichia coli FadR protein and LldR from Corynebacterium glutamicum, have been described to date in the literature. Here, the crystal structure of TM0439, a GntR regulator with an FCD domain found in the Thermotoga maritima genome, is described. The FCD domain is similar to that of the LldR regulator and contains a buried metal-binding site. Using atomic absorption spectroscopy and Trp fluorescence, it is shown that the recombinant protein contains bound Ni 2+ ions but that it is able to bind Zn 2+ with K d < 70 nM. It is concluded that Zn 2+ is the likely physiological metal and that it may perform either structural or regulatory roles or both. Finally, the TM0439 structure is compared with two other FadR-family structures recently deposited by structural genomics consortia. The results call for a revision in the classification of the FadR family of transcription factors

Modulation of catalytic activity in multi-domain protein tyrosine phosphatases.

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Lalima L Madan

Full Text Available Signaling mechanisms involving protein tyrosine phosphatases govern several cellular and developmental processes. These enzymes are regulated by several mechanisms which include variation in the catalytic turnover rate based on redox stimuli, subcellular localization or protein-protein interactions. In the case of Receptor Protein Tyrosine Phosphatases (RPTPs containing two PTP domains, phosphatase activity is localized in their membrane-proximal (D1 domains, while the membrane-distal (D2 domain is believed to play a modulatory role. Here we report our analysis of the influence of the D2 domain on the catalytic activity and substrate specificity of the D1 domain using two Drosophila melanogaster RPTPs as a model system. Biochemical studies reveal contrasting roles for the D2 domain of Drosophila Leukocyte antigen Related (DLAR and Protein Tyrosine Phosphatase on Drosophila chromosome band 99A (PTP99A. While D2 lowers the catalytic activity of the D1 domain in DLAR, the D2 domain of PTP99A leads to an increase in the catalytic activity of its D1 domain. Substrate specificity, on the other hand, is cumulative, whereby the individual specificities of the D1 and D2 domains contribute to the substrate specificity of these two-domain enzymes. Molecular dynamics simulations on structural models of DLAR and PTP99A reveal a conformational rationale for the experimental observations. These studies reveal that concerted structural changes mediate inter-domain communication resulting in either inhibitory or activating effects of the membrane distal PTP domain on the catalytic activity of the membrane proximal PTP domain.

Regulation of endogenous human gene expression by ligand-inducible TALE transcription factors.

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Mercer, Andrew C; Gaj, Thomas; Sirk, Shannon J; Lamb, Brian M; Barbas, Carlos F

2014-10-17

The construction of increasingly sophisticated synthetic biological circuits is dependent on the development of extensible tools capable of providing specific control of gene expression in eukaryotic cells. Here, we describe a new class of synthetic transcription factors that activate gene expression in response to extracellular chemical stimuli. These inducible activators consist of customizable transcription activator-like effector (TALE) proteins combined with steroid hormone receptor ligand-binding domains. We demonstrate that these ligand-responsive TALE transcription factors allow for tunable and conditional control of gene activation and can be used to regulate the expression of endogenous genes in human cells. Since TALEs can be designed to recognize any contiguous DNA sequence, the conditional gene regulatory system described herein will enable the design of advanced synthetic gene networks.

Inhibition of FoxO1 acetylation by INHAT subunit SET/TAF-Iβ induces p21 transcription.

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Chae, Yun-Cheol; Kim, Kee-Beom; Kang, Joo-Young; Kim, Se-Ryeon; Jung, Hyeon-Soo; Seo, Sang-Beom

2014-08-25

Post-translational modification of forkhead family transcription factor, FoxO1, is an important regulatory mode for its diverse activities. FoxO1 is acetylated by HAT coactivators and its transcriptional activity is decreased via reduced DNA binding affinity. Here, we report that SET/TAF-Iβ inhibited p300-mediated FoxO1 acetylation in an INHAT domain-dependent manner. SET/TAF-Iβ interacted with FoxO1 and activated transcription of FoxO1 target gene, p21. Moreover, SET/TAF-Iβ inhibited acetylation of FoxO1 and increased p21 transcription induced by oxidative stress. Our results suggest that SET/TAF-Iβ inhibits FoxO1 acetylation and activates its transcriptional activity toward p21. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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...

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.

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.

A Synthetic Biology Framework for Programming Eukaryotic Transcription Functions

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Khalil, Ahmad S.; Lu, Timothy K.; Bashor, Caleb J.; Ramirez, Cherie L.; Pyenson, Nora C.; Joung, J. Keith; Collins, James J.

2013-01-01

SUMMARY Eukaryotic transcription factors (TFs) perform complex and combinatorial functions within transcriptional networks. Here, we present a synthetic framework for systematically constructing eukaryotic transcription functions using artificial zinc fingers, modular DNA-binding domains found within many eukaryotic TFs. Utilizing this platform, we construct a library of orthogonal synthetic transcription factors (sTFs) and use these to wire synthetic transcriptional circuits in yeast. We engineer complex functions, such as tunable output strength and transcriptional cooperativity, by rationally adjusting a decomposed set of key component properties, e.g., DNA specificity, affinity, promoter design, protein-protein interactions. We show that subtle perturbations to these properties can transform an individual sTF between distinct roles (activator, cooperative factor, inhibitory factor) within a transcriptional complex, thus drastically altering the signal processing behavior of multi-input systems. This platform provides new genetic components for synthetic biology and enables bottom-up approaches to understanding the design principles of eukaryotic transcriptional complexes and networks. PMID:22863014

Activation of the Arabidopsis membrane-bound transcription factor bZIP28 is mediated by site-2 protease, but not site-1 protease.

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Iwata, Yuji; Ashida, Makoto; Hasegawa, Chisa; Tabara, Kazuki; Mishiba, Kei-Ichiro; Koizumi, Nozomu

2017-08-01

The unfolded protein response (UPR) is a homeostatic cellular response conserved in eukaryotic cells to alleviate the accumulation of unfolded proteins in the endoplasmic reticulum (ER). Arabidopsis bZIP28 is a membrane-bound transcription factor activated by proteolytic cleavage in response to ER stress, thereby releasing its cytosolic portion containing the bZIP domain from the membrane to translocate into the nucleus where it induces the transcription of genes encoding ER-resident molecular chaperones and folding enzymes. It has been widely recognized that the proteolytic activation of bZIP28 is mediated by the sequential cleavage of site-1 protease (S1P) and site-2 protease (S2P). In the present study we provide evidence that bZIP28 protein is cleaved by S2P, but not by S1P. We demonstrated that wild-type and s1p mutant plants produce the active, nuclear form of bZIP28 in response to the ER stress inducer tunicamycin. In contrast, tunicamycin-treated s2p mutants do not accumulate the active, nuclear form of bZIP28. Consistent with these observations, s2p mutants, but not s1p mutants, exhibited a defective transcriptional response of ER stress-responsive genes and significantly higher sensitivity to tunicamycin. Interestingly, s2p mutants accumulate two membrane-bound bZIP28 fragments with a shorter ER lumen-facing C-terminal domain. Importantly, the predicted cleavage sites are located far from the canonical S1P recognition motif previously described. We propose that ER stress-induced proteolytic activation of bZIP28 is mediated by the sequential actions of as-yet-unidentified protease(s) and S2P, and does not require S1P. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

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.

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.

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

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Oyoshi Takanori

2012-01-01

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

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

International Nuclear Information System (INIS)

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

2005-01-01

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

Structure of the C-terminal effector-binding domain of AhrC bound to its corepressor l-arginine

International Nuclear Information System (INIS)

Garnett, James A.; Baumberg, Simon; Stockley, Peter G.; Phillips, Simon E. V.

2007-01-01

The crystal structure of the C-terminal domain hexameric core of AhrC, with bound corepressor (l-arginine), has been solved at 1.95 Å resolution. Binding of l-arginine results in a rotation between the two trimers of the hexamer, leading to the activation of the DNA-binding state. The arginine repressor/activator protein (AhrC) from Bacillus subtilis belongs to a large family of multifunctional transcription factors that are involved in the regulation of bacterial arginine metabolism. AhrC interacts with operator sites in the promoters of arginine biosynthetic and catabolic operons, acting as a transcriptional repressor at biosynthetic sites and an activator of transcription at catabolic sites. AhrC is a hexamer of identical subunits, each having two domains. The C-terminal domains form the core of the protein and are involved in oligomerization and l-arginine binding. The N-terminal domains lie on the outside of the compact core and play a role in binding to 18 bp DNA operators called ARG boxes. The C-terminal domain of AhrC has been expressed, purified and characterized, and also crystallized as a hexamer with the bound corepressor l-arginine. Here, the crystal structure refined to 1.95 Å is presented

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

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Gao Chen

2012-02-01

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

Transcription instability in high-risk neuroblastoma is associated with a global perturbation of chromatin domains.

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Zanon, Carlo; Tonini, Gian Paolo

2017-11-01

Chromosome instability has a pivotal role among the hallmarks of cancer, but its transcriptional counterpart is rarely considered a relevant factor in cell destabilization. To examine transcription instability (TIN), we first devised a metric we named TIN index and used it to evaluate TIN on a dataset containing more than 500 neuroblastoma samples. We found that metastatic tumors from high-risk (HR) patients are characterized by significantly different TIN index values compared to low/intermediate-risk patients. Our results indicate that the TIN index is a good predictor of neuroblastoma patient's outcome, and a related TIN index gene signature (TIN-signature) is also able to predict the neuroblastoma patient's outcome with high confidence. Interestingly, we find that TIN-signature genes have a strong positional association with superenhancers in neuroblastoma tumors. Finally, we show that TIN is linked to chromatin structural domains and interferes with their integrity in HR neuroblastoma patients. This novel approach to gene expression analysis broadens the perspective of genome instability investigations to include functional aspects. © 2017 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

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

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Granier Thierry

2011-08-01

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

Transcription Factor Functional Protein-Protein Interactions in Plant Defense Responses

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Murilo S. Alves

2014-03-01

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

The transcription factor DREAM represses A20 and mediates inflammation

OpenAIRE

Tiruppathi, Chinnaswamy; Soni, Dheeraj; Wang, Dong-Mei; Xue, Jiaping; Singh, Vandana; Thippegowda, Prabhakar B.; Cheppudira, Bopaiah P.; Mishra, Rakesh K.; DebRoy, Auditi; Qian, Zhijian; Bachmaier, Kurt; Zhao, Youyang; Christman, John W.; Vogel, Stephen M.; Ma, Averil

2014-01-01

Here we show that the transcription-repressor DREAM binds to the A20 promoter to repress the expression of A20, the deubiquitinase suppressing inflammatory NF-κB signaling. DREAM-deficient (Dream−/− ) mice displayed persistent and unchecked A20 expression in response to endotoxin. DREAM functioned by transcriptionally repressing A20 through binding to downstream regulatory elements (DREs). In contrast, USF1 binding to the DRE-associated E-box domain activated A20 expression in response to inf...

SOXE transcription factors form selective dimers on non-compact DNA motifs through multifaceted interactions between dimerization and high-mobility group domains.

Science.gov (United States)

Huang, Yong-Heng; Jankowski, Aleksander; Cheah, Kathryn S E; Prabhakar, Shyam; Jauch, Ralf

2015-05-27

The SOXE transcription factors SOX8, SOX9 and SOX10 are master regulators of mammalian development directing sex determination, gliogenesis, pancreas specification and neural crest development. We identified a set of palindromic SOX binding sites specifically enriched in regulatory regions of melanoma cells. SOXE proteins homodimerize on these sequences with high cooperativity. In contrast to other transcription factor dimers, which are typically rigidly spaced, SOXE group proteins can bind cooperatively at a wide range of dimer spacings. Using truncated forms of SOXE proteins, we show that a single dimerization (DIM) domain, that precedes the DNA binding high mobility group (HMG) domain, is sufficient for dimer formation, suggesting that DIM : HMG rather than DIM:DIM interactions mediate the dimerization. All SOXE members can also heterodimerize in this fashion, whereas SOXE heterodimers with SOX2, SOX4, SOX6 and SOX18 are not supported. We propose a structural model where SOXE-specific intramolecular DIM:HMG interactions are allosterically communicated to the HMG of juxtaposed molecules. Collectively, SOXE factors evolved a unique mode to combinatorially regulate their target genes that relies on a multifaceted interplay between the HMG and DIM domains. This property potentially extends further the diversity of target genes and cell-specific functions that are regulated by SOXE proteins.

SOXE transcription factors form selective dimers on non-compact DNA motifs through multifaceted interactions between dimerization and high-mobility group domains

Science.gov (United States)

Huang, Yong-Heng; Jankowski, Aleksander; Cheah, Kathryn S. E.; Prabhakar, Shyam; Jauch, Ralf

2015-01-01

The SOXE transcription factors SOX8, SOX9 and SOX10 are master regulators of mammalian development directing sex determination, gliogenesis, pancreas specification and neural crest development. We identified a set of palindromic SOX binding sites specifically enriched in regulatory regions of melanoma cells. SOXE proteins homodimerize on these sequences with high cooperativity. In contrast to other transcription factor dimers, which are typically rigidly spaced, SOXE group proteins can bind cooperatively at a wide range of dimer spacings. Using truncated forms of SOXE proteins, we show that a single dimerization (DIM) domain, that precedes the DNA binding high mobility group (HMG) domain, is sufficient for dimer formation, suggesting that DIM : HMG rather than DIM:DIM interactions mediate the dimerization. All SOXE members can also heterodimerize in this fashion, whereas SOXE heterodimers with SOX2, SOX4, SOX6 and SOX18 are not supported. We propose a structural model where SOXE-specific intramolecular DIM:HMG interactions are allosterically communicated to the HMG of juxtaposed molecules. Collectively, SOXE factors evolved a unique mode to combinatorially regulate their target genes that relies on a multifaceted interplay between the HMG and DIM domains. This property potentially extends further the diversity of target genes and cell-specific functions that are regulated by SOXE proteins. PMID:26013289

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.

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.

Functional interactions of the AF-2 activation domain core region of the human androgen receptor with the amino-terminal domain and with the transcriptional coactivator TIF2 (transcriptional intermediary factor2)

NARCIS (Netherlands)

C.A. Berrevoets (Cor); P. Doesburg (Paul); K. Steketee (Karine); J. Trapman (Jan); A.O. Brinkmann (Albert)

1998-01-01

textabstractPrevious studies in yeast and mammalian cells showed a functional interaction between the amino-terminal domain and the carboxy-terminal, ligand-binding domain (LBD) of the human androgen receptor (AR). In the present study, the AR subdomains involved in

Benchmarking of TALE- and CRISPR/dCas9-Based Transcriptional Regulators in Mammalian Cells for the Construction of Synthetic Genetic Circuits.

Science.gov (United States)

Lebar, Tina; Jerala, Roman

2016-10-21

Transcriptional activator-like effector (TALE)- and CRISPR/Cas9-based designable recognition domains represent a technological breakthrough not only for genome editing but also for building designed genetic circuits. Both platforms are able to target rarely occurring DNA segments, even within complex genomes. TALE and dCas9 domains, genetically fused to transcriptional regulatory domains, can be used for the construction of engineered logic circuits. Here we benchmarked the performance of the two platforms, targeting the same DNA sequences, to compare their advantages for the construction of designed circuits in mammalian cells. Optimal targeting strands for repression and activation of dCas9-based designed transcription factors were identified; both platforms exhibited good orthogonality and were used to construct functionally complete NOR gates. Although the CRISPR/dCas9 system is clearly easier to construct, TALE-based activators were significantly stronger, and the TALE-based platform performed better, especially for the construction of layered circuits.

Crystal structure of Thermotoga maritima TM0439: implications for the mechanism of bacterial GntR transcription regulators with Zn2+-binding FCD domains

Energy Technology Data Exchange (ETDEWEB)

Zheng, Meiying; Cooper, David; Grossoehmerb, Nickolas; Yu, Minmin; Hung, Li-Wei; Cieslik, Murcin; Derewendaro, Urszula; Lesley, Scott; Wilson, Ian; Giedrocb, David; Derewenda, Zygmunt

2009-06-06

The GntR superfamily of dimeric transcription factors, with more than 6200 members encoded in bacterial genomes, are characterized by N-terminal winged helix (WH) DNA-binding domains and diverse C-terminal, regulatory domains, which provide a basis for the classification of the constituent families. The largest of these families, FadR, contains nearly 3000 proteins with all a-helical regulatory domains classified into two related Pfam families: FadR{_}C and FCD. Only two crystal structures of the FadR family members, i.e. the E. coli FadR protein and the LldR from C. glutamicum, have been described to date in literature. Here we describe the crystal structure of TM0439, a GntR regulator with an FCD domain, found in the Thermotoga maritima genome. The FCD domain is similar to that of the LldR regulator, and contains a buried metal binding site. Using atomic absorption spectroscopy and Trp fluorescence, we show that the recombinant protein contains bound Ni{sup 2+} ions, but it is able to bind Zn{sup 2+} with K{sub D} < 70 nM . We conclude that Zn{sup 2+} is the likely physiological metal, where it may perform either or both structural and regulatory roles. Finally, we compare the TM0439 structure to two other FadR family structures recently deposited by Structural Genomics consortia. The results call for a revision in the classification of the FadR family of transcription factors.

TAL effectors target the C-terminal domain of RNA polymerase II (CTD by inhibiting the prolyl-isomerase activity of a CTD-associated cyclophilin.

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Mariane Noronha Domingues

Full Text Available Transcriptional activator-like (TAL effectors of plant pathogenic bacteria function as transcription factors in plant cells. However, how TAL effectors control transcription in the host is presently unknown. Previously, we showed that TAL effectors of the citrus canker pathogen Xanthomonas citri, named PthAs, targeted the citrus protein complex comprising the thioredoxin CsTdx, ubiquitin-conjugating enzymes CsUev/Ubc13 and cyclophilin CsCyp. Here we show that CsCyp complements the function of Cpr1 and Ess1, two yeast cyclophilins that regulate transcription by the isomerization of proline residues of the regulatory C-terminal domain (CTD of RNA polymerase II. We also demonstrate that CsCyp, CsTdx, CsUev and four PthA variants interact with the citrus CTD and that CsCyp co-immunoprecipitate with the CTD in citrus cell extracts and with PthA2 transiently expressed in sweet orange epicotyls. The interactions of CsCyp with the CTD and PthA2 were inhibited by cyclosporin A (CsA, a cyclophilin inhibitor. Moreover, we present evidence that PthA2 inhibits the peptidyl-prolyl cis-trans isomerase (PPIase activity of CsCyp in a similar fashion as CsA, and that silencing of CsCyp, as well as treatments with CsA, enhance canker lesions in X. citri-infected leaves. Given that CsCyp appears to function as a negative regulator of cell growth and that Ess1 negatively regulates transcription elongation in yeast, we propose that PthAs activate host transcription by inhibiting the PPIase activity of CsCyp on the CTD.

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

DEFF Research Database (Denmark)

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

2004-01-01

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

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

Science.gov (United States)

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

2012-09-01

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

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.

Characterization of the CrbS/R Two-Component System in Pseudomonas fluorescens Reveals a New Set of Genes under Its Control and a DNA Motif Required for CrbR-Mediated Transcriptional Activation

Directory of Open Access Journals (Sweden)

Edgardo Sepulveda

2017-11-01

Full Text Available The CrbS/R system is a two-component signal transduction system that regulates acetate utilization in Vibrio cholerae, P. aeruginosa, and P. entomophila. CrbS is a hybrid histidine kinase that belongs to a recently identified family, in which the signaling domain is fused to an SLC5 solute symporter domain through aSTAC domain. Upon activation by CrbS, CrbR activates transcription of the acs gene, which encodes an acetyl-CoA synthase (ACS, and the actP gene, which encodes an acetate/solute symporter. In this work, we characterized the CrbS/R system in Pseudomonas fluorescens SBW25. Through the quantitative proteome analysis of different mutants, we were able to identify a new set of genes under its control, which play an important role during growth on acetate. These results led us to the identification of a conserved DNA motif in the putative promoter region of acetate-utilization genes in the Gammaproteobacteria that is essential for the CrbR-mediated transcriptional activation of genes under acetate-utilizing conditions. Finally, we took advantage of the existence of a second SLC5-containing two-component signal transduction system in P. fluorescens, CbrA/B, to demonstrate that the activation of the response regulator by the histidine kinase is not dependent on substrate transport through the SLC5 domain.

NMR assignments of SPOC domain of the human transcriptional corepressor SHARP in complex with a C-terminal SMRT peptide.

Science.gov (United States)

Mikami, Suzuka; Kanaba, Teppei; Ito, Yutaka; Mishima, Masaki

2013-10-01

The transcriptional corepressor SMRT/HDAC1-associated repressor protein (SHARP) recruits histone deacetylases. Human SHARP protein is thought to function in processes involving steroid hormone responses and the Notch signaling pathway. SHARP consists of RNA recognition motifs (RRMs) in the N-terminal region and the spen paralog and ortholog C-terminal (SPOC) domain in the C-terminal region. It is known that the SPOC domain binds the LSD motif in the C-terminal tail of corepressors silencing mediator for retinoid and thyroid receptor (SMRT)/nuclear receptor corepressor (NcoR). We are interested in delineating the mechanism by which the SPOC domain recognizes the LSD motif of the C-terminal tail of SMRT/NcoR. To this end, we are investigating the tertiary structure of the SPOC/SMRT peptide using NMR. Herein, we report on the (1)H, (13)C and (15)N resonance assignments of the SPOC domain in complex with a SMRT peptide, which contributes towards a structural understanding of the SPOC/SMRT peptide and its molecular recognition.

Plant-Derived Transcription Factors for Orthologous Regulation of Gene Expression in the Yeast Saccharomyces cerevisiae.

Science.gov (United States)

Naseri, Gita; Balazadeh, Salma; Machens, Fabian; Kamranfar, Iman; Messerschmidt, Katrin; Mueller-Roeber, Bernd

2017-09-15

Control of gene expression by transcription factors (TFs) is central in many synthetic biology projects for which a tailored expression of one or multiple genes is often needed. As TFs from evolutionary distant organisms are unlikely to affect gene expression in a host of choice, they represent excellent candidates for establishing orthogonal control systems. To establish orthogonal regulators for use in yeast (Saccharomyces cerevisiae), we chose TFs from the plant Arabidopsis thaliana. We established a library of 106 different combinations of chromosomally integrated TFs, activation domains (yeast GAL4 AD, herpes simplex virus VP64, and plant EDLL) and synthetic promoters harboring cognate cis-regulatory motifs driving a yEGFP reporter. Transcriptional output of the different driver/reporter combinations varied over a wide spectrum, with EDLL being a considerably stronger transcription activation domain in yeast than the GAL4 activation domain, in particular when fused to Arabidopsis NAC TFs. Notably, the strength of several NAC-EDLL fusions exceeded that of the strong yeast TDH3 promoter by 6- to 10-fold. We furthermore show that plant TFs can be used to build regulatory systems encoded by centromeric or episomal plasmids. Our library of TF-DNA binding site combinations offers an excellent tool for diverse synthetic biology applications in yeast.

Transcription facilitated genome-wide recruitment of topoisomerase I and DNA gyrase.

Science.gov (United States)

Ahmed, Wareed; Sala, Claudia; Hegde, Shubhada R; Jha, Rajiv Kumar; Cole, Stewart T; Nagaraja, Valakunja

2017-05-01

Movement of the transcription machinery along a template alters DNA topology resulting in the accumulation of supercoils in DNA. The positive supercoils generated ahead of transcribing RNA polymerase (RNAP) and the negative supercoils accumulating behind impose severe topological constraints impeding transcription process. Previous studies have implied the role of topoisomerases in the removal of torsional stress and the maintenance of template topology but the in vivo interaction of functionally distinct topoisomerases with heterogeneous chromosomal territories is not deciphered. Moreover, how the transcription-induced supercoils influence the genome-wide recruitment of DNA topoisomerases remains to be explored in bacteria. Using ChIP-Seq, we show the genome-wide occupancy profile of both topoisomerase I and DNA gyrase in conjunction with RNAP in Mycobacterium tuberculosis taking advantage of minimal topoisomerase representation in the organism. The study unveils the first in vivo genome-wide interaction of both the topoisomerases with the genomic regions and establishes that transcription-induced supercoils govern their recruitment at genomic sites. Distribution profiles revealed co-localization of RNAP and the two topoisomerases on the active transcriptional units (TUs). At a given locus, topoisomerase I and DNA gyrase were localized behind and ahead of RNAP, respectively, correlating with the twin-supercoiled domains generated. The recruitment of topoisomerases was higher at the genomic loci with higher transcriptional activity and/or at regions under high torsional stress compared to silent genomic loci. Importantly, the occupancy of DNA gyrase, sole type II topoisomerase in Mtb, near the Ter domain of the Mtb chromosome validates its function as a decatenase.

Hydrophobic interaction between the SH2 domain and the kinase domain is required for the activation of Csk.

Science.gov (United States)

Mikkola, Esa T; Gahmberg, Carl G

2010-06-18

The protein tyrosine kinase C-terminal Src kinase (Csk) is activated by the engagement of its Src homology (SH) 2 domain. However, the molecular mechanism required for this is not completely understood. The crystal structure of the active Csk indicates that Csk could be activated by contact between the SH2 domain and the beta3-alphaC loop in the N-terminal lobe of the kinase domain. To study the importance of this interaction for the SH2-domain-mediated activation of Csk, we mutated the amino acid residues forming the contacts between the SH2 domain and the beta3-alphaC loop. The mutation of the beta3-alphaC loop Ala228 to glycine and of the SH2 domain Tyr116, Tyr133, Leu138, and Leu149 to alanine resulted in the inability of the SH2 domain ligand to activate Csk. Furthermore, the overexpressed Csk mutants A228G, Y133A/Y116A, L138A, and L149A were unable to efficiently inactivate endogenous Src in human embryonic kidney 293 cells. The results suggest that the SH2-domain-mediated activation of Csk is dependent on the binding of the beta3-alphaC loop Ala228 to the hydrophobic pocket formed by the side chains of Tyr116, Tyr133, Leu138, and Leu149 on the surface of the SH2 domain. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

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...

Identification of the functional domains of ANT-1, a novel coactivator of the androgen receptor

International Nuclear Information System (INIS)

Fan Shuli; Goto, Kiminobu; Chen Guangchun; Morinaga, Hidetaka; Nomura, Masatoshi; Okabe, Taijiro; Nawata, Hajime; Yanase, Toshihiko

2006-01-01

Previously, we identified a transcriptional coactivator for the activation function-1 (AF-1) domain of the human androgen receptor (AR) and designated it androgen receptor N-terminal domain transactivating protein-1 (ANT-1). This coactivator, which contains multiple tetratricopeptide repeat (TPR) motifs from amino acid (aa) 294, is identical to a component of U5 small nuclear ribonucleoprotein particles and binds specifically to the AR or glucocorticoid receptor. Here, we identified four distinct functional domains. The AR-AF-1-binding domain, which bound to either aa 180-360 or 360-532 in AR-AF-1, clearly overlapped with TAU-1 and TAU-5. This domain and the subnuclear speckle formation domain in ANT-1 were assigned within the TPR motifs, while the transactivating and nuclear localization signal domains resided within the N-terminal sequence. The existence of these functional domains may further support the idea that ANT-1 can function as an AR-AF-1-specific coactivator while mediating a transcription-splicing coupling

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.

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.

Cloning and characterization of GETS-1, a goldfish Ets family member that functions as a transcriptional repressor in muscle.

Science.gov (United States)

Goldman, D; Sapru, M K; Stewart, S; Plotkin, J; Libermann, T A; Wasylyk, B; Guan, K

1998-10-15

An Ets transcription factor family member, GETS-1, was cloned from a goldfish retina cDNA library. GETS-1 contains a conserved Ets DNA-binding domain at its N-terminus and is most similar to ternary complex factor (TCF) serum-response-factor protein-1a (SAP-1a). GETS-1 is expressed in many tissues, but is enriched in retina and brain. As with the TCFs SAP-1a and ets-related protein (ERP), overexpression of the GETS-1 promoter suppresses nicotinic acetylcholine receptor epsilon-subunit gene expression in cultured muscle cells. A consensus Ets binding site sequence in the promoter of the epsilon-subunit gene is required for GETS-1-mediated repression. GETS-1 repressor activity is abrogated by overexpression of an activated Ras/mitogen-activated protein kinase (MAP kinase) or by mutation of Ser-405, a MAP kinase phosphorylation site in GETS-1. Fusion proteins created between GETS-1 and the Gal4 DNA-binding domain show that, like other TCFs, GETS-1 contains a C-terminal activation domain that is activated by a Ras/MAP kinase signalling cascade. Interestingly, mutation of Ser-405 located within this activation domain abrogated transcriptional activation of the fusion protein.

PML-associated repressor of transcription (PAROT), a novel KRAB-zinc finger repressor, is regulated through association with PML nuclear bodies

International Nuclear Information System (INIS)

Fleischer, Sandra; Wiemann, Stefan; Will, Hans; Hofmann, Thomas G.

2006-01-01

Promyelocytic leukemia nuclear bodies (PML-NBs) are implicated in transcriptional regulation. Here we identify a novel transcriptional repressor, PML-associated repressor of transcription (PAROT), which is regulated in its repressor activity through recruitment to PML-NBs. PAROT is a Krueppel-associated box ( KRAB) zinc-finger (ZNF) protein, which comprises an amino terminal KRAB-A and KRAB-B box, a linker domain and 8 tandemly repeated C 2 H 2 -ZNF motifs at its carboxy terminus. Consistent with its domain structure, when tethered to DNA, PAROT represses transcription, and this is partially released by the HDAC inhibitor trichostatin A. PAROT colocalizes with members of the heterochromatin protein 1 (HP1) family and with transcriptional intermediary factor-1β/KRAB-associated protein 1 (TIF-1β/KAP1), a transcriptional corepressor for the KRAB-ZNF family. Interestingly, PML isoform IV, in contrast to PML-III, efficiently recruits PAROT and TIF-1β from heterochromatin to PML-NBs. PML-NB recruitment of PAROT partially releases its transcriptional repressor activity, indicating that PAROT can be regulated through subnuclear compartmentalization. Taken together, our data identify a novel transcriptional repressor and provide evidence for its regulation through association with PML-NBs

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

In silico engineering and optimization of Transcription Activator-Like Effectors and their derivatives for improved DNA binding predictions.

KAUST Repository

Piatek, Marek J.

2015-12-01

Transcription Activator-Like Effectors (TALEs) can be used as adaptable DNAbinding modules to create site-specific chimeric nucleases or synthetic transcriptional regulators. The central repeat domain mediates specific DNA binding via hypervariable repeat di-residues (RVDs). This DNA-Binding Domain can be engineered to bind preferentially to any user-selected DNA sequence if engineered appropriately. Therefore, TALEs and their derivatives have become indispensable molecular tools in site-specific manipulation of genes and genomes. This thesis revolves around two problems: in silico design and improved binding site prediction of TALEs. In the first part, a study is shown where TALEs are successfully designed in silico and validated in laboratory to yield the anticipated effects on selected genes. Software is developed to accompany the process of designing and prediction of binding sites. I expanded the functionality of the software to be used as a more generic set of tools for the design, target and offtarget searching. Part two contributes a method and associated toolkit developed to allow users to design in silico optimized synthetic TALEs with user-defined specificities for various experimental purposes. This method is based on a mutual relationship of three consecutive tandem repeats in the DNA-binding domain. This approach revealed positional and compositional bias behind the binding of TALEs to DNA. In conclusion, I developed methods, approaches, and software to enhance the functionality of synthetic TALEs, which should improve understanding of TALEs biology and will further advance genome-engineering applications in various organisms and cell types.

Structural studies of bacterial transcriptional regulatory proteins by multidimensional heteronuclear NMR

Energy Technology Data Exchange (ETDEWEB)

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

1995-02-01

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

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

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Rouet Fabien

2009-10-01

Full Text Available Abstract Background Commercially available microarrays have been used in many settings to generate expression profiles for a variety of applications, including target selection for disease detection, classification, profiling for pharmacogenomic response to therapeutics, and potential disease staging. However, many commercially available microarray platforms fail to capture transcript diversity produced by alternative splicing, a major mechanism for driving proteomic diversity through transcript heterogeneity. Results The human Genome-Wide SpliceArray™ (GWSA, a novel microarray platform, utilizes an existing probe design concept to monitor such transcript diversity on a genome scale. The human GWSA allows the detection of alternatively spliced events within the human genome through the use of exon body and exon junction probes to provide a direct measure of each transcript, through simple calculations derived from expression data. This report focuses on the performance and validation of the array when measured against standards recently published by the Microarray Quality Control (MAQC Project. The array was shown to be highly quantitative, and displayed greater than 85% correlation with the HG-U133 Plus 2.0 array at the gene level while providing more extensive coverage of each gene. Almost 60% of splice events among genes demonstrating differential expression of greater than 3 fold also contained extensive splicing alterations. Importantly, almost 10% of splice events within the gene set displaying constant overall expression values had evidence of transcript diversity. Two examples illustrate the types of events identified: LIM domain 7 showed no differential expression at the gene level, but demonstrated deregulation of an exon skip event, while erythrocyte membrane protein band 4.1 -like 3 was differentially expressed and also displayed deregulation of a skipped exon isoform. Conclusion Significant changes were detected independent of

A conserved motif in the linker domain of STAT1 transcription factor is required for both recognition and release from high-affinity DNA-binding sites.

Science.gov (United States)

Hüntelmann, Bettina; Staab, Julia; Herrmann-Lingen, Christoph; Meyer, Thomas

2014-01-01

Binding to specific palindromic sequences termed gamma-activated sites (GAS) is a hallmark of gene activation by members of the STAT (signal transducer and activator of transcription) family of cytokine-inducible transcription factors. However, the precise molecular mechanisms involved in the signal-dependent finding of target genes by STAT dimers have not yet been very well studied. In this study, we have characterized a sequence motif in the STAT1 linker domain which is highly conserved among the seven human STAT proteins and includes surface-exposed residues in close proximity to the bound DNA. Using site-directed mutagenesis, we have demonstrated that a lysine residue in position 567 of the full-length molecule is required for GAS recognition. The substitution of alanine for this residue completely abolished both binding to high-affinity GAS elements and transcriptional activation of endogenous target genes in cells stimulated with interferon-γ (IFNγ), while the time course of transient nuclear accumulation and tyrosine phosphorylation were virtually unchanged. In contrast, two glutamic acid residues (E559 and E563) on each monomer are important for the dissociation of dimeric STAT1 from DNA and, when mutated to alanine, result in elevated levels of tyrosine-phosphorylated STAT1 as well as prolonged IFNγ-stimulated nuclear accumulation. In conclusion, our data indicate that the kinetics of signal-dependent GAS binding is determined by an array of glutamic acid residues located at the interior surface of the STAT1 dimer. These negatively charged residues appear to align the long axis of the STAT1 dimer in a position perpendicular to the DNA, thereby facilitating the interaction between lysine 567 and the phosphodiester backbone of a bound GAS element, which is a prerequisite for transient gene induction.

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

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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.

Interaction between C/EBPβ and Tax down-regulates human T-cell leukemia virus type I transcription

International Nuclear Information System (INIS)

Hivin, P.; Gaudray, G.; Devaux, C.; Mesnard, J.-M.

2004-01-01

The human T-cell leukemia virus type I (HTLV-I) Tax protein trans-activates viral transcription through three imperfect tandem repeats of a 21-bp sequence called Tax-responsive element (TxRE). Tax regulates transcription via direct interaction with some members of the activating transcription factor/CRE-binding protein (ATF/CREB) family including CREM, CREB, and CREB-2. By interacting with their ZIP domain, Tax stimulates the binding of these cellular factors to the CRE-like sequence present in the TxREs. Recent observations have shown that CCAAT/enhancer binding protein β (C/EBPβ) forms stable complexes on the CRE site in the presence of CREB-2. Given that C/EBPβ has also been found to interact with Tax, we analyzed the effects of C/EBPβ on viral Tax-dependent transcription. We show here that C/EBPβ represses viral transcription and that Tax is no more able to form a stable complex with CREB-2 on the TxRE site in the presence of C/EBPβ. We also analyzed the physical interactions between Tax and C/EBPβ and found that the central region of C/EBPβ, excluding its ZIP domain, is required for direct interaction with Tax. It is the first time that Tax is described to interact with a basic leucine-zipper (bZIP) factor without recognizing its ZIP domain. Although unexpected, this result explains why C/EBPβ would be unable to form a stable complex with Tax on the TxRE site and could then down-regulate viral transcription. Lastly, we found that C/EBPβ was able to inhibit Tax expression in vivo from an infectious HTLV-I molecular clone. In conclusion, we propose that during cell activation events, which stimulate the Tax synthesis, C/EBPβ may down-regulate the level of HTLV-I expression to escape the cytotoxic-T-lymphocyte response

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

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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.

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

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...

Effect of ARA9 on dioxin receptor mediated transcription

International Nuclear Information System (INIS)

Lees, M.J.; Whitelaw, M.L.

2002-01-01

The dioxin (Aryl hydrocarbon) receptor (DR) is a unique bHLH transcription factor which is activated by binding of planar aromatic hydrocarbons typified by dioxin (TCDD). The active receptor is key to metabolism of aryl hydrocarbon xenobiotics by being a potent inducer of CYP1A1 gene activity. Chlorinated dioxins are inert to metabolism and initiate multifarious toxicities, including potent tumour promotion. These ill-effects are mediated by the activated DR and we are studying the mechanisms by which the ligand binding domain of the DR controls activity of the protein. The DR ligand binding domain resides within a PAS (Per/Arnt/Sim homology) region which is contiguous with the bHLH. The latent bHLH/PAS dioxin receptor (DR) is found in the cytoplasm of most mammalian cell types in a complex with heat shock protein 90, a novel immunophilin like protein termed ARA9/XAP2/AIP, and the co-chaperone p23. Here we use antisense ARA9 constructs to reveal that in the absence of ARA9, the DR is unable to form a transcriptionally active complex. Co-expression of antisense ARA9 with a form of the DR which is constitutively targeted to the nucleus leads to dramatically decreased levels of the nuclear DR protein, implying that ARA9 may function beyond its currently proposed role in cytoplasmic retention of the latent DR

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.

Targeted genome regulation via synthetic programmable transcriptional regulators

KAUST Repository

Piatek, Agnieszka Anna

2016-04-19

Regulation of gene transcription controls cellular functions and coordinates responses to developmental, physiological and environmental cues. Precise and efficient molecular tools are needed to characterize the functions of single and multiple genes in linear and interacting pathways in a native context. Modular DNA-binding domains from zinc fingers (ZFs) and transcriptional activator-like proteins (TALE) are amenable to bioengineering to bind DNA target sequences of interest. As a result, ZF and TALE proteins were used to develop synthetic programmable transcription factors. However, these systems are limited by the requirement to re-engineer proteins for each new target sequence. The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR associated 9 (Cas9) genome editing tool was recently repurposed for targeted transcriptional regulation by inactivation of the nuclease activity of Cas9. Due to the facile engineering, simplicity, precision and amenability to library construction, the CRISPR/Cas9 system is poised to revolutionize the functional genomics field across diverse eukaryotic species. In this review, we discuss the development of synthetic customizable transcriptional regulators and provide insights into their current and potential applications, with special emphasis on plant systems, in characterization of gene functions, elucidation of molecular mechanisms and their biotechnological applications. © 2016 Informa UK Limited, trading as Taylor & Francis Group

The Arabidopsis thaliana NAC transcription factor family: structure-function relationships and determinants of ANAC019 stress signalling

DEFF Research Database (Denmark)

Jensen, Michael K; Kjaersgaard, Trine; Nielsen, Michael M.

2010-01-01

-termini. Nine of the ten NAC domains analysed bind a previously identified conserved DNA target sequence with a CGT[GA] core, although with different affinities. Likewise, all but one of the NAC proteins analysed is dependent on the C-terminal region for transactivational activity. In silico analyses show......TFs (transcription factors) are modular proteins minimally containing a DBD (DNA-binding domain) and a TRD (transcription regulatory domain). NAC [for NAM (no apical meristem), ATAF, CUC (cup-shaped cotyledon)] proteins comprise one of the largest plant TF families. They are key regulators...... of stress perception and developmental programmes, and most share an N-terminal NAC domain. On the basis of analyses of gene expression data and the phylogeny of Arabidopsis thaliana NAC TFs we systematically decipher structural and functional specificities of the conserved NAC domains and the divergent C...

The forkhead transcription factor FoxY regulates Nanos.

Science.gov (United States)

Song, Jia L; Wessel, Gary M

2012-10-01

FoxY is a member of the forkhead transcription factor family that appeared enriched in the presumptive germ line of sea urchins (Ransick et al. Dev Biol 2002;246:132). Here, we test the hypothesis that FoxY is involved in germ line determination in this animal. We found two splice forms of FoxY that share the same DNA-binding domain, but vary in the carboxy-terminal trans-activation/repression domain. Both forms of the FoxY protein are present in the egg and in the early embryo, and their mRNAs accumulate to their highest levels in the small micromeres and adjacent non-skeletogenic mesoderm. Knockdown of FoxY resulted in a dramatic decrease in Nanos mRNA and protein levels as well as a loss of coelomic pouches in 2-week-old larvae. Our results indicate that FoxY positively regulates Nanos at the transcriptional level and is essential for reproductive potential in this organism. Copyright © 2012 Wiley Periodicals, Inc.

Unexpected role of the L-domain of calpastatin during the autoproteolytic activation of human erythrocyte calpain.

Science.gov (United States)

De Tullio, Roberta; Franchi, Alice; Martines, Antonino; Averna, Monica; Pedrazzi, Marco; Melloni, Edon; Sparatore, Bianca

2018-04-26

Autoproteolysis of human erythrocyte calpain-1 proceeds in vitro at high [Ca 2+ ], through the conversion of the 80-kDa catalytic subunit into a 75-kDa activated enzyme that requires lower [Ca 2+ ] for catalysis. Importantly, here we detect a similar 75 kDa calpain-1 form also in vivo , in human meningiomas. Although calpastatin is so far considered the specific inhibitor of calpains, we have previously identified in rat brain a calpastatin transcript truncated at the end of the L-domain (cast110, L-DOM), coding for a protein lacking the inhibitory units. Aim of the present study was to characterize the possible biochemical role of the L-DOM during calpain-1 autoproteolysis in vitro , at high (100 µM) and low (5 µM) [Ca 2+ ]. Here we demonstrate that the L-DOM binds the 80 kDa proenzyme in the absence of Ca 2+ Consequently, we have explored the ability of the 75 kDa activated protease to catalyze at 5 µM Ca 2+ the intermolecular activation of native calpain-1 associated with the L-DOM. Notably, this [Ca 2+ ] is too low to promote the autoproteolytic activation of calpain-1 but enough to support the catalysis of the 75 kDa calpain. We show for the first time that the L-DOM preserves native calpain-1 from the degradation mediated by the 75 kDa form. Taken together, our data suggest that the free L-domain of calpastatin is a novel member of the calpain/calpastatin system endowed with a function alternative to calpain inhibition. For this reason, it will be crucial to define the intracellular relevance of the L-domain in controlling calpain activation/activity in physiopathological conditions having altered Ca 2+ homeostasis. © 2018 The Author(s).

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.

Transcriptional regulation by competing transcription factor modules.

Directory of Open Access Journals (Sweden)

Rutger Hermsen

2006-12-01

Full Text Available Gene regulatory networks lie at the heart of cellular computation. In these networks, intracellular and extracellular signals are integrated by transcription factors, which control the expression of transcription units by binding to cis-regulatory regions on the DNA. The designs of both eukaryotic and prokaryotic cis-regulatory regions are usually highly complex. They frequently consist of both repetitive and overlapping transcription factor binding sites. To unravel the design principles of these promoter architectures, we have designed in silico prokaryotic transcriptional logic gates with predefined input-output relations using an evolutionary algorithm. The resulting cis-regulatory designs are often composed of modules that consist of tandem arrays of binding sites to which the transcription factors bind cooperatively. Moreover, these modules often overlap with each other, leading to competition between them. Our analysis thus identifies a new signal integration motif that is based upon the interplay between intramodular cooperativity and intermodular competition. We show that this signal integration mechanism drastically enhances the capacity of cis-regulatory domains to integrate signals. Our results provide a possible explanation for the complexity of promoter architectures and could be used for the rational design of synthetic gene circuits.

Evaluation of transcriptional activity of the oestrogen receptor with sodium iodide symporter as an imaging reporter gene.

Science.gov (United States)

Kang, Joo Hyun; Chung, June-Key; Lee, Yong Jin; Kim, Kwang Il; Jeong, Jae Min; Lee, Dong Soo; Lee, Myung Chul

2006-10-01

Oestrogen receptors are ligand-dependent transcription factors whose activity is modulated either by oestrogens or by an alternative signalling pathway. Oestrogen receptors interact via a specific DNA-binding domain, the oestrogen responsive element (ERE), in the promoter region of sensitive genes. This binding leads to an initiation of gene expression and hormonal effects. To determine the transcriptional activity of the oestrogen receptor, we developed a molecular imaging system using sodium iodide symporter (NIS) as a reporter gene. The NIS reporter gene was placed under the control of an artificial ERE derived from pERE-TA-SEAP and named as pERE-NIS. pERE-NIS was transferred to MCF-7, human breast cancer cells, which highly expressed oestrogen receptor-alpha with lipofectamine. Stably expressing cells were generated by selection with G418 for 14 days. After treatment of 17beta-oestradiol and tamoxifen with serial doses, the (125)I uptake was measured for the determination of NIS expression. The inhibition of NIS activity was performed with 50 micromol x l(-1) potassium perchlorate. The MCF7/pERE-NIS treated with 17beta-oestradiol accumulated (125)I up to 70-80% higher than did non-treated cells. NIS expression was increased according to increasing doses of 17beta-oestradiol. MCF7/pERE-NIS treated with tamoxifen also accumulated (125)I up to 50% higher than did non-treated cells. Potassium perchlorate completely inhibited (125)I uptake. When MDA-MB231 cells, the oestrogen receptor-negative breast cancer cells, were transfected with pERE-NIS, (125)I uptake of MDA-MB-231/pERE-NIS did not increase. This pERE-NIS reporter system is sufficiently sensitive for monitoring transcriptional activity of the oestrogen receptor. Therefore, cis-enhancer reporter systems with ERE will be applicable to the development of a novel selective oestrogen receptor modulator with low toxicity and high efficacy.

T cell activation and differentiation is modulated by a CD6 domain 1 antibody Itolizumab.

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Usha Bughani

Full Text Available CD6 is associated with T-cell modulation and is implicated in several autoimmune diseases. We previously demonstrated that Itolizumab, a CD6 domain 1 (CD6D1 specific humanized monoclonal antibody, inhibited the proliferation and cytokine production by T lymphocytes stimulated with anti-CD3 antibody or when co-stimulated with ALCAM. Aberrant IL-17 producing CD4+ helper T-cells (Th17 have been identified as pivotal for the pathogenesis of certain inflammatory autoimmune disorders, including psoriasis. Itolizumab has demonstrated efficacy in human diseases known to have an IL-17 driven pathogenesis. Here, in in vitro experiments we show that by day 3 of human PBMC activation using anti-CD3 and anti-CD28 co-stimulation in a Th17 polarizing milieu, 15-35% of CD4+ T-cells overexpress CD6 and there is an establishment of differentiated Th17 cells. Addition of Itolizumab reduces the activation and differentiation of T cells to Th17 cells and decreases production of IL-17. These effects are associated with the reduction of key transcription factors pSTAT3 and RORγT. Further, transcription analysis studies in these conditions indicate that Itolizumab suppressed T cell activation by primarily reducing cell cycle, DNA transcription and translation associated genes. To understand the mechanism of this inhibition, we evaluated the effect of this anti-human CD6D1 mAb on ALCAM-CD6 as well as TCR-mediated T cell activation. We show that Itolizumab but not its F(ab'2 fragment directly inhibits CD6 receptor hyper-phosphorylation and leads to subsequent decrease in associated ZAP70 kinase and docking protein SLP76. Since Itolizumab binds to CD6 expressed only on human and chimpanzee, we developed an antibody binding specifically to mouse CD6D1. This antibody successfully ameliorated the incidence of experimental autoimmune encephalitis in the mice model. These results position CD6 as a key molecule in sustaining the activation and differentiation of T cells and an

Inhibiting fungal multidrug resistance by disrupting an activator-Mediator interaction.

Science.gov (United States)

Nishikawa, Joy L; Boeszoermenyi, Andras; Vale-Silva, Luis A; Torelli, Riccardo; Posteraro, Brunella; Sohn, Yoo-Jin; Ji, Fei; Gelev, Vladimir; Sanglard, Dominique; Sanguinetti, Maurizio; Sadreyev, Ruslan I; Mukherjee, Goutam; Bhyravabhotla, Jayaram; Buhrlage, Sara J; Gray, Nathanael S; Wagner, Gerhard; Näär, Anders M; Arthanari, Haribabu

2016-02-25

Eukaryotic transcription activators stimulate the expression of specific sets of target genes through recruitment of co-activators such as the RNA polymerase II-interacting Mediator complex. Aberrant function of transcription activators has been implicated in several diseases. However, therapeutic targeting efforts have been hampered by a lack of detailed molecular knowledge of the mechanisms of gene activation by disease-associated transcription activators. We previously identified an activator-targeted three-helix bundle KIX domain in the human MED15 Mediator subunit that is structurally conserved in Gal11/Med15 Mediator subunits in fungi. The Gal11/Med15 KIX domain engages pleiotropic drug resistance transcription factor (Pdr1) orthologues, which are key regulators of the multidrug resistance pathway in Saccharomyces cerevisiae and in the clinically important human pathogen Candida glabrata. The prevalence of C. glabrata is rising, partly owing to its low intrinsic susceptibility to azoles, the most widely used antifungal agent. Drug-resistant clinical isolates of C. glabrata most commonly contain point mutations in Pdr1 that render it constitutively active, suggesting that this transcriptional activation pathway represents a linchpin in C. glabrata multidrug resistance. Here we perform sequential biochemical and in vivo high-throughput screens to identify small-molecule inhibitors of the interaction of the C. glabrata Pdr1 activation domain with the C. glabrata Gal11A KIX domain. The lead compound (iKIX1) inhibits Pdr1-dependent gene activation and re-sensitizes drug-resistant C. glabrata to azole antifungals in vitro and in animal models for disseminated and urinary tract C. glabrata infection. Determining the NMR structure of the C. glabrata Gal11A KIX domain provides a detailed understanding of the molecular mechanism of Pdr1 gene activation and multidrug resistance inhibition by iKIX1. We have demonstrated the feasibility of small-molecule targeting of a

CARF and WYL domains: ligand-binding regulators of prokaryotic defense systems

Directory of Open Access Journals (Sweden)

Kira eMakarova

2014-04-01

Full Text Available CRISPR-Cas adaptive immunity systems of bacteria and archaea insert fragments of virus or plasmid DNA as spacer sequences into CRISPR repeat loci. Processed transcripts encompassing these spacers guide the cleavage of the cognate foreign DNA or RNA. Most CRISPR-Cas loci, in addition to recognized cas genes, also include genes that are not directly implicated in spacer acquisition, CRISPR transcript processing or interference. Here we comprehensively analyze sequences, structures and genomic neighborhoods of one of the most widespread groups of such genes that encode proteins containing a predicted nucleotide-binding domain with a Rossmann-like fold, which we denote CARF (CRISPR-associated Rossmann fold. Several CARF protein structures have been determined but functional characterization of these proteins is lacking. The CARF domain is most frequently combined with a C-terminal winged helix-turn-helix DNA-binding domain and effector domains most of which are predicted to possess DNase or RNase activity. Divergent CARF domains are also found in RtcR proteins, sigma-54 dependent regulators of the rtc RNA repair operon. CARF genes frequently co-occur with those coding for proteins containing the WYL domain with the Sm-like SH3 β-barrel fold, which is also predicted to bind ligands. CRISPR-Cas and possibly other defense systems are predicted to be transcriptionally regulated by multiple ligand-binding proteins containing WYL and CARF domains which sense modified nucleotides and nucleotide derivatives generated during virus infection. We hypothesize that CARF domains also transmit the signal from the bound ligand to the fused effector domains which attack either alien or self nucleic acids, resulting, respectively, in immunity complementing the CRISPR-Cas action or in dormancy/programmed cell death.

Interaction of GlnK with the GAF domain of Herbaspirillum seropedicae NifA mediates NH₄⁺-regulation.

Science.gov (United States)

Oliveira, Marco A S; Aquino, Bruno; Bonatto, Ana Claudia; Huergo, Luciano F; Chubatsu, Leda S; Pedrosa, Fábio O; Souza, Emanuel M; Dixon, Ray; Monteiro, Rose A

2012-04-01

Nitrogen fixation in Herbaspirillum seropedicae is transcriptionally regulated by NifA, a σ(54) transcriptional activator with three structural domains: an N-terminal GAF domain, a catalytic AAA+ domain and a C-terminal DNA-binding domain. NifA is only active in H. seropedicae when cultures are grown in the absence of fixed nitrogen and at low oxygen tensions. There is evidence that the inactivation of NifA in response to fixed nitrogen is mediated by the regulatory GAF domain. However, the mechanism of NifA repression by the GAF domain, as well as the transduction of nitrogen status to NifA, is not understood. In order to study the regulation of NifA activity by fixed nitrogen independently of oxygen regulation, we constructed a chimeric protein containing the GAF domain of H. seropedicae NifA fused to the AAA+ and C-terminal domains of Azotobacter vinelandii NifA. This chimeric protein (NifAQ1) lacks the cysteine motif found in oxygen sensitive NifA proteins and is not oxygen responsive in vivo. Our results demonstrate that NifAQ1 responds to fixed nitrogen and requires GlnK protein for activity, a behavior similar to H. seropedicae NifA. In addition, protein footprinting analysis indicates that this response probably involves a protein-protein contact between the GAF domain and the GlnK protein. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Structural basis of transcriptional gene silencing mediated by Arabidopsis MOM1.

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Nishimura, Taisuke; Molinard, Guillaume; Petty, Tom J; Broger, Larissa; Gabus, Caroline; Halazonetis, Thanos D; Thore, Stéphane; Paszkowski, Jerzy

2012-02-01

Shifts between epigenetic states of transcriptional activity are typically correlated with changes in epigenetic marks. However, exceptions to this rule suggest the existence of additional, as yet uncharacterized, layers of epigenetic regulation. MOM1, a protein of 2,001 amino acids that acts as a transcriptional silencer, represents such an exception. Here we define the 82 amino acid domain called CMM2 (Conserved MOM1 Motif 2) as a minimal MOM1 fragment capable of transcriptional regulation. As determined by X-ray crystallography, this motif folds into an unusual hendecad-based coiled-coil. Structure-based mutagenesis followed by transgenic complementation tests in plants demonstrate that CMM2 and its dimerization are effective for transcriptional suppression at chromosomal loci co-regulated by MOM1 and the siRNA pathway but not at loci controlled by MOM1 in an siRNA-independent fashion. These results reveal a surprising separation of epigenetic activities that enable the single, large MOM1 protein to coordinate cooperating mechanisms of epigenetic regulation.

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.

Alanine-scanning mutagenesis of human signal transducer and activator of transcription 1 to estimate loss- or gain-of-function variants.

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Kagawa, Reiko; Fujiki, Ryoji; Tsumura, Miyuki; Sakata, Sonoko; Nishimura, Shiho; Itan, Yuval; Kong, Xiao-Fei; Kato, Zenichiro; Ohnishi, Hidenori; Hirata, Osamu; Saito, Satoshi; Ikeda, Maiko; El Baghdadi, Jamila; Bousfiha, Aziz; Fujiwara, Kaori; Oleastro, Matias; Yancoski, Judith; Perez, Laura; Danielian, Silvia; Ailal, Fatima; Takada, Hidetoshi; Hara, Toshiro; Puel, Anne; Boisson-Dupuis, Stéphanie; Bustamante, Jacinta; Casanova, Jean-Laurent; Ohara, Osamu; Okada, Satoshi; Kobayashi, Masao

2017-07-01

Germline heterozygous mutations in human signal transducer and activator of transcription 1 (STAT1) can cause loss of function (LOF), as in patients with Mendelian susceptibility to mycobacterial diseases, or gain of function (GOF), as in patients with chronic mucocutaneous candidiasis. LOF and GOF mutations are equally rare and can affect the same domains of STAT1, especially the coiled-coil domain (CCD) and DNA-binding domain (DBD). Moreover, 6% of patients with chronic mucocutaneous candidiasis with a GOF STAT1 mutation have mycobacterial disease, obscuring the functional significance of the identified STAT1 mutations. Current computational approaches, such as combined annotation-dependent depletion, do not distinguish LOF and GOF variants. We estimated variations in the CCD/DBD of STAT1. We mutagenized 342 individual wild-type amino acids in the CCD/DBD (45.6% of full-length STAT1) to alanine and tested the mutants for STAT1 transcriptional activity. Of these 342 mutants, 201 were neutral, 30 were LOF, and 111 were GOF mutations in a luciferase assay. This assay system correctly estimated all previously reported LOF mutations (100%) and slightly fewer GOF mutations (78.1%) in the CCD/DBD of STAT1. We found that GOF alanine mutants occurred at the interface of the antiparallel STAT1 dimer, suggesting that they destabilize this dimer. This assay also precisely predicted the effect of 2 hypomorphic and dominant negative mutations, E157K and G250E, in the CCD of STAT1 that we found in 2 unrelated patients with Mendelian susceptibility to mycobacterial diseases. The systematic alanine-scanning assay is a useful tool to estimate the GOF or LOF status and the effect of heterozygous missense mutations in STAT1 identified in patients with severe infectious diseases, including mycobacterial and fungal diseases. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

O-GlcNAc transferase regulates transcriptional activity of human Oct4.

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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.

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

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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.

Reconstitution of the yeast RNA polymerase III transcription system with all recombinant factors.

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Ducrot, Cécile; Lefebvre, Olivier; Landrieux, Emilie; Guirouilh-Barbat, Josée; Sentenac, André; Acker, Joel

2006-04-28

Transcription factor TFIIIC is a multisubunit complex required for promoter recognition and transcriptional activation of class III genes. We describe here the reconstitution of complete recombinant yeast TFIIIC and the molecular characterization of its two DNA-binding domains, tauA and tauB, using the baculovirus expression system. The B block-binding module, rtauB, was reconstituted with rtau138, rtau91, and rtau60 subunits. rtau131, rtau95, and rtau55 formed also a stable complex, rtauA, that displayed nonspecific DNA binding activity. Recombinant rTFIIIC was functionally equivalent to purified yeast TFIIIC, suggesting that the six recombinant subunits are necessary and sufficient to reconstitute a transcriptionally active TFIIIC complex. The formation and the properties of rTFIIIC-DNA complexes were affected by dephosphorylation treatments. The combination of complete recombinant rTFIIIC and rTFIIIB directed a low level of basal transcription, much weaker than with the crude B'' fraction, suggesting the existence of auxiliary factors that could modulate the yeast RNA polymerase III transcription system.

AguR, a Transmembrane Transcription Activator of the Putrescine Biosynthesis Operon in Lactococcus lactis, Acts in Response to the Agmatine Concentration.

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Linares, Daniel M; Del Rio, Beatriz; Redruello, Begoña; Ladero, Victor; Martin, M Cruz; de Jong, Anne; Kuipers, Oscar P; Fernandez, Maria; Alvarez, Miguel A

2015-09-01

Dairy industry fermentative processes mostly use Lactococcus lactis as a starter. However, some dairy L. lactis strains produce putrescine, a biogenic amine that raises food safety and spoilage concerns, via the agmatine deiminase (AGDI) pathway. The enzymatic activities responsible for putrescine biosynthesis in this bacterium are encoded by the AGDI gene cluster. The role of the catabolic genes aguB, aguD, aguA, and aguC has been studied, but knowledge regarding the role of aguR (the first gene in the cluster) remains limited. In the present work, aguR was found to be a very low level constitutively expressed gene that is essential for putrescine biosynthesis and is transcribed independently of the polycistronic mRNA encoding the catabolic genes (aguBDAC). In response to agmatine, AguR acts as a transcriptional activator of the aguB promoter (PaguB), which drives the transcription of the aguBDAC operon. Inverted sequences required for PaguB activity were identified by deletion analysis. Further work indicated that AguR is a transmembrane protein which might function as a one-component signal transduction system that senses the agmatine concentration of the medium and, accordingly, regulates the transcription of the aguBDAC operon through a C-terminal cytoplasmic DNA-binding domain typically found in LuxR-like proteins. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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...

MED14 tethers mediator to the N-terminal domain of peroxisome proliferator-activated receptor gamma and is required for full transcriptional activity and adipogenesis

DEFF Research Database (Denmark)

Grøntved, Lars; Madsen, Maria S; Boergesen, Michael

2010-01-01

and proximal promoter of the PPARgamma target gene Fabp4 is also independent of MED1. Using a small interfering RNA (siRNA)-based approach, we identify MED14 as a novel critical Mediator component for PPARgamma-dependent transactivation, and we demonstrate that MED14 interacts directly with the N terminus...... of PPARgamma in a ligand-independent manner. Interestingly, MED14 knockdown does not affect the recruitment of PPARgamma, MED6, and MED8 to the Fabp4 enhancer but does reduce their occupancy of the Fabp4 proximal promoter. In agreement with the necessity of MED14 for PPARgamma transcriptional activity, we show...

Post-translational regulation of Oct4 transcriptional activity.

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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.

Global RNA association with the transcriptionally active chromosome of chloroplasts.

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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.

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.

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.

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

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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.

Phosphorylation of both nucleoplasmin domains is required for activation of its chromatin decondensation activity

DEFF Research Database (Denmark)

Bañuelos, Sonia; Omaetxebarria, Miren J; Ramos, Isbaal

2007-01-01

Nucleoplasmin (NP) is a histone chaperone involved in nucleosome assembly, chromatin decondensation at fertilization, and apoptosis. To carry out these activities NP has to interact with different types of histones, an interaction that is regulated by phosphorylation. Here we have identified...... are found at the tail domain, flanking the nuclear localization signal. Phosphorylation-mimicking mutations render a recombinant protein as active in chromatin decondensation as hyperphosphorylated NP isolated from Xenopus laevis eggs. Comparison of mutants in which the core and tail domains of the protein...... were independently or simultaneously "activated" indicates that activation or phosphorylation of both protein domains is required for NP to efficiently extract linker-type histones from chromatin....

The WRKY transcription factor family in Brachypodium distachyon.

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Tripathi, Prateek; Rabara, Roel C; Langum, Tanner J; Boken, Ashley K; Rushton, Deena L; Boomsma, Darius D; Rinerson, Charles I; Rabara, Jennifer; Reese, R Neil; Chen, Xianfeng; Rohila, Jai S; Rushton, Paul J

2012-06-22

A complete assembled genome sequence of wheat is not yet available. Therefore, model plant systems for wheat are very valuable. Brachypodium distachyon (Brachypodium) is such a system. The WRKY family of transcription factors is one of the most important families of plant transcriptional regulators with members regulating important agronomic traits. Studies of WRKY transcription factors in Brachypodium and wheat therefore promise to lead to new strategies for wheat improvement. We have identified and manually curated the WRKY transcription factor family from Brachypodium using a pipeline designed to identify all potential WRKY genes. 86 WRKY transcription factors were found, a total higher than all other current databases. We therefore propose that our numbering system (BdWRKY1-BdWRKY86) becomes the standard nomenclature. In the JGI v1.0 assembly of Brachypodium with the MIPS/JGI v1.0 annotation, nine of the transcription factors have no gene model and eleven gene models are probably incorrectly predicted. In total, twenty WRKY transcription factors (23.3%) do not appear to have accurate gene models. To facilitate use of our data, we have produced The Database of Brachypodium distachyon WRKY Transcription Factors. Each WRKY transcription factor has a gene page that includes predicted protein domains from MEME analyses. These conserved protein domains reflect possible input and output domains in signaling. The database also contains a BLAST search function where a large dataset of WRKY transcription factors, published genes, and an extensive set of wheat ESTs can be searched. We also produced a phylogram containing the WRKY transcription factor families from Brachypodium, rice, Arabidopsis, soybean, and Physcomitrella patens, together with published WRKY transcription factors from wheat. This phylogenetic tree provides evidence for orthologues, co-orthologues, and paralogues of Brachypodium WRKY transcription factors. The description of the WRKY transcription factor

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.

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.

Large ethnic variations in recommended physical activity according to activity domains in amsterdam, the netherlands

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Kunst Anton E

2010-11-01

Full Text Available Abstract Purpose The level of recommended physical activity (PA is met less frequently by people from some ethnic minorities than others. We explored whether these differences in recommended PA between ethnic minority groups and the general population varied by domain and type of culturally-specific activity. Methods Participants were sampled from the population based SUNSET study and were from ethnic Dutch (n = 567, Hindustani-Surinamese (n = 370 and African-Surinamese (n = 689 descent. The validated SQUASH-questionnaire measured PA for the following domains: commuting, occupation, household, leisure time. Culturally-specific activities were added as extra question within the leisure time domain. The effect of each domain on ethnic differences in recommended PA prevalence was examined by odds-ratio (OR analysis through recalculating recommended PA, while, in turn, excluding the contribution of each domain. Results In the ethnic Dutch population, more vigorous PA in commuting and leisure time was reported compared to the Surinamese groups. The Hindustani-Surinamese and African-Surinamese reported more walking as commuting activity, while the Dutch group reported cycling more frequently. Ethnic differences in recommended PA became smaller in both Surinamese groups compared with the Dutch after removing commuting activity, for example, in Hindustani-Surinamese men (OR = 0.92, 95%CI: 0.62-1.37 vs. OR = 1.33, 0.89-2.00 and women (OR = 1.61, 1.12-2.32 vs. OR = 2.03, 1.41-2.92. Removing occupational activity resulted in larger ethnic differences in both groups compared with the Dutch. Smaller effects were found for yoga and dancing, leisure time and household activities. Conclusion This study shows that differences in PA between ethnic minority groups and the general population vary according to the activity domain. The results indicate that including all relevant domains and activities is essential for assessment of ethnic differences in recommended

An engineered tale-transcription factor rescues transcription of factor VII impaired by promoter mutations and enhances its endogenous expression in hepatocytes.

Science.gov (United States)

Barbon, Elena; Pignani, Silvia; Branchini, Alessio; Bernardi, Francesco; Pinotti, Mirko; Bovolenta, Matteo

2016-06-24

Tailored approaches to restore defective transcription responsible for severe diseases have been poorly explored. We tested transcription activator-like effectors fused to an activation domain (TALE-TFs) in a coagulation factor VII (FVII) deficiency model. In this model, the deficiency is caused by the -94C > G or -61T > G mutation, which abrogate the binding of Sp1 or HNF-4 transcription factors. Reporter assays in hepatoma HepG2 cells naturally expressing FVII identified a single TALE-TF (TF4) that, by targeting the region between mutations, specifically trans-activated both the variant (>100-fold) and wild-type (20-40-fold) F7 promoters. Importantly, in the genomic context of transfected HepG2 and transduced primary hepatocytes, TF4 increased F7 mRNA and protein levels (2- to 3-fold) without detectable off-target effects, even for the homologous F10 gene. The ectopic F7 expression in renal HEK293 cells was modestly affected by TF4 or by TALE-TF combinations. These results provide experimental evidence for TALE-TFs as gene-specific tools useful to counteract disease-causing promoter mutations.

Crystallization and preliminary crystal structure analysis of the ligand-binding domain of PqsR (MvfR), the Pseudomonas quinolone signal (PQS) responsive quorum-sensing transcription factor of Pseudomonas aeruginosa

International Nuclear Information System (INIS)

Xu, Ningna; Yu, Shen; Moniot, Sébastien; Weyand, Michael; Blankenfeldt, Wulf

2012-01-01

The ligand-binding domain of the transcription factor PqsR from P. aeruginosa has been crystallized and initial phases have been obtained using SAD data from seleno-l-methionine-labelled crystals. The opportunistic bacterial pathogen Pseudomonas aeruginosa employs three transcriptional regulators, LasR, RhlR and PqsR, to control the transcription of a large subset of its genes in a cell-density-dependent process known as quorum sensing. Here, the recombinant production, crystallization and structure solution of the ligand-binding domain of PqsR (MvfR), the LysR-type transcription factor that responds to the Pseudomonas quinolone signal (PQS), a quinolone-based quorum-sensing signal that is unique to P. aeruginosa and possibly a small number of other bacteria, is reported. PqsR regulates the expression of many virulence genes and may therefore be an interesting drug target. The ligand-binding domain (residues 91–319) was produced as a fusion with SUMO, and hexagonal-shaped crystals of purified PqsR-91–319 were obtained using the vapour-diffusion method. Crystallization in the presence of a PQS precursor allowed data collection to 3.25 Å resolution on a synchrotron beamline, and initial phases have been obtained using single-wavelength anomalous diffraction data from seleno-l-methionine-labelled crystals, revealing the space group to be P6 5 22, with unit-cell parameters a = b = 116–120, c = 115–117 Å

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.

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.

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

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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.

SH2 domains: modulators of nonreceptor tyrosine kinase activity

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Filippakopoulos, Panagis; Müller, Susanne; Knapp, Stefan

2009-01-01

The Src homology 2 (SH2) domain is a sequence-specific phosphotyrosine-binding module present in many signaling molecules. In cytoplasmic tyrosine kinases, the SH2 domain is located N-terminally to the catalytic kinase domain (SH1) where it mediates cellular localization, substrate recruitment, and regulation of kinase activity. Initially, structural studies established a role of the SH2 domain stabilizing the inactive state of Src family members. However, biochemical characterization showed ...

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.

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

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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.

Regulation of expression of two LY-6 family genes by intron retention and transcription induced chimerism

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Mallya Meera

2008-09-01

Full Text Available Abstract Background Regulation of the expression of particular genes can rely on mechanisms that are different from classical transcriptional and translational control. The LY6G5B and LY6G6D genes encode LY-6 domain proteins, whose expression seems to be regulated in an original fashion, consisting of an intron retention event which generates, through an early premature stop codon, a non-coding transcript, preventing expression in most cell lines and tissues. Results The MHC LY-6 non-coding transcripts have shown to be stable and very abundant in the cell, and not subject to Nonsense Mediated Decay (NMD. This retention event appears not to be solely dependent on intron features, because in the case of LY6G5B, when the intron is inserted in the artificial context of a luciferase expression plasmid, it is fully spliced but strongly stabilises the resulting luciferase transcript. In addition, by quantitative PCR we found that the retained and spliced forms are differentially expressed in tissues indicating an active regulation of the non-coding transcript. EST database analysis revealed that these genes have an alternative expression pathway with the formation of Transcription Induced Chimeras (TIC. This data was confirmed by RT-PCR, revealing the presence of different transcripts that would encode the chimeric proteins CSNKβ-LY6G5B and G6F-LY6G6D, in which the LY-6 domain would join to a kinase domain and an Ig-like domain, respectively. Conclusion In conclusion, the LY6G5B and LY6G6D intron-retained transcripts are not subjected to NMD and are more abundant than the properly spliced forms. In addition, these genes form chimeric transcripts with their neighbouring same orientation 5' genes. Of interest is the fact that the 5' genes (CSNKβ or G6F undergo differential splicing only in the context of the chimera (CSNKβ-LY6G5B or G6F-LY6G6C and not on their own.

Insights into Hox protein function from a large scale combinatorial analysis of protein domains.

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Samir Merabet

2011-10-01

Full Text Available Protein function is encoded within protein sequence and protein domains. However, how protein domains cooperate within a protein to modulate overall activity and how this impacts functional diversification at the molecular and organism levels remains largely unaddressed. Focusing on three domains of the central class Drosophila Hox transcription factor AbdominalA (AbdA, we used combinatorial domain mutations and most known AbdA developmental functions as biological readouts to investigate how protein domains collectively shape protein activity. The results uncover redundancy, interactivity, and multifunctionality of protein domains as salient features underlying overall AbdA protein activity, providing means to apprehend functional diversity and accounting for the robustness of Hox-controlled developmental programs. Importantly, the results highlight context-dependency in protein domain usage and interaction, allowing major modifications in domains to be tolerated without general functional loss. The non-pleoitropic effect of domain mutation suggests that protein modification may contribute more broadly to molecular changes underlying morphological diversification during evolution, so far thought to rely largely on modification in gene cis-regulatory sequences.

Effect of point mutations on Herbaspirillum seropedicae NifA activity

International Nuclear Information System (INIS)

Aquino, B.; Stefanello, A.A.; Oliveira, M.A.S.; Pedrosa, F.O.; Souza, E.M.; Monteiro, R.A.; Chubatsu, L.S.

2015-01-01

NifA is the transcriptional activator of the nif genes in Proteobacteria. It is usually regulated by nitrogen and oxygen, allowing biological nitrogen fixation to occur under appropriate conditions. NifA proteins have a typical three-domain structure, including a regulatory N-terminal GAF domain, which is involved in control by fixed nitrogen and not strictly required for activity, a catalytic AAA+ central domain, which catalyzes open complex formation, and a C-terminal domain involved in DNA-binding. In Herbaspirillum seropedicae, a β-proteobacterium capable of colonizing Graminae of agricultural importance, NifA regulation by ammonium involves its N-terminal GAF domain and the signal transduction protein GlnK. When the GAF domain is removed, the protein can still activate nif genes transcription; however, ammonium regulation is lost. In this work, we generated eight constructs resulting in point mutations in H. seropedicae NifA and analyzed their effect on nifH transcription in Escherichia coli and H. seropedicae. Mutations K22V, T160E, M161V, L172R, and A215D resulted in inactive proteins. Mutations Q216I and S220I produced partially active proteins with activity control similar to wild-type NifA. However, mutation G25E, located in the GAF domain, resulted in an active protein that did not require GlnK for activity and was partially sensitive to ammonium. This suggested that G25E may affect the negative interaction between the N-terminal GAF domain and the catalytic central domain under high ammonium concentrations, thus rendering the protein constitutively active, or that G25E could lead to a conformational change comparable with that when GlnK interacts with the GAF domain

Effect of point mutations on Herbaspirillum seropedicae NifA activity

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B. Aquino

2015-08-01

Full Text Available NifA is the transcriptional activator of the nif genes in Proteobacteria. It is usually regulated by nitrogen and oxygen, allowing biological nitrogen fixation to occur under appropriate conditions. NifA proteins have a typical three-domain structure, including a regulatory N-terminal GAF domain, which is involved in control by fixed nitrogen and not strictly required for activity, a catalytic AAA+ central domain, which catalyzes open complex formation, and a C-terminal domain involved in DNA-binding. In Herbaspirillum seropedicae, a β-proteobacterium capable of colonizing Graminae of agricultural importance, NifA regulation by ammonium involves its N-terminal GAF domain and the signal transduction protein GlnK. When the GAF domain is removed, the protein can still activate nif genes transcription; however, ammonium regulation is lost. In this work, we generated eight constructs resulting in point mutations in H. seropedicae NifA and analyzed their effect on nifH transcription in Escherichia coli and H. seropedicae. Mutations K22V, T160E, M161V, L172R, and A215D resulted in inactive proteins. Mutations Q216I and S220I produced partially active proteins with activity control similar to wild-type NifA. However, mutation G25E, located in the GAF domain, resulted in an active protein that did not require GlnK for activity and was partially sensitive to ammonium. This suggested that G25E may affect the negative interaction between the N-terminal GAF domain and the catalytic central domain under high ammonium concentrations, thus rendering the protein constitutively active, or that G25E could lead to a conformational change comparable with that when GlnK interacts with the GAF domain.

Effect of point mutations on Herbaspirillum seropedicae NifA activity.

Science.gov (United States)

Aquino, B; Stefanello, A A; Oliveira, M A S; Pedrosa, F O; Souza, E M; Monteiro, R A; Chubatsu, L S

2015-08-01

NifA is the transcriptional activator of the nif genes in Proteobacteria. It is usually regulated by nitrogen and oxygen, allowing biological nitrogen fixation to occur under appropriate conditions. NifA proteins have a typical three-domain structure, including a regulatory N-terminal GAF domain, which is involved in control by fixed nitrogen and not strictly required for activity, a catalytic AAA+ central domain, which catalyzes open complex formation, and a C-terminal domain involved in DNA-binding. In Herbaspirillum seropedicae, a β-proteobacterium capable of colonizing Graminae of agricultural importance, NifA regulation by ammonium involves its N-terminal GAF domain and the signal transduction protein GlnK. When the GAF domain is removed, the protein can still activate nif genes transcription; however, ammonium regulation is lost. In this work, we generated eight constructs resulting in point mutations in H. seropedicae NifA and analyzed their effect on nifH transcription in Escherichia coli and H. seropedicae. Mutations K22V, T160E, M161V, L172R, and A215D resulted in inactive proteins. Mutations Q216I and S220I produced partially active proteins with activity control similar to wild-type NifA. However, mutation G25E, located in the GAF domain, resulted in an active protein that did not require GlnK for activity and was partially sensitive to ammonium. This suggested that G25E may affect the negative interaction between the N-terminal GAF domain and the catalytic central domain under high ammonium concentrations, thus rendering the protein constitutively active, or that G25E could lead to a conformational change comparable with that when GlnK interacts with the GAF domain.

Effect of point mutations on Herbaspirillum seropedicae NifA activity

Energy Technology Data Exchange (ETDEWEB)

Aquino, B.; Stefanello, A.A.; Oliveira, M.A.S.; Pedrosa, F.O.; Souza, E.M.; Monteiro, R.A.; Chubatsu, L.S. [Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR (Brazil)

2015-07-10

NifA is the transcriptional activator of the nif genes in Proteobacteria. It is usually regulated by nitrogen and oxygen, allowing biological nitrogen fixation to occur under appropriate conditions. NifA proteins have a typical three-domain structure, including a regulatory N-terminal GAF domain, which is involved in control by fixed nitrogen and not strictly required for activity, a catalytic AAA+ central domain, which catalyzes open complex formation, and a C-terminal domain involved in DNA-binding. In Herbaspirillum seropedicae, a β-proteobacterium capable of colonizing Graminae of agricultural importance, NifA regulation by ammonium involves its N-terminal GAF domain and the signal transduction protein GlnK. When the GAF domain is removed, the protein can still activate nif genes transcription; however, ammonium regulation is lost. In this work, we generated eight constructs resulting in point mutations in H. seropedicae NifA and analyzed their effect on nifH transcription in Escherichia coli and H. seropedicae. Mutations K22V, T160E, M161V, L172R, and A215D resulted in inactive proteins. Mutations Q216I and S220I produced partially active proteins with activity control similar to wild-type NifA. However, mutation G25E, located in the GAF domain, resulted in an active protein that did not require GlnK for activity and was partially sensitive to ammonium. This suggested that G25E may affect the negative interaction between the N-terminal GAF domain and the catalytic central domain under high ammonium concentrations, thus rendering the protein constitutively active, or that G25E could lead to a conformational change comparable with that when GlnK interacts with the GAF domain.

Mediator Undergoes a Compositional Change during Transcriptional Activation.

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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.

Four novel FBN1 mutations: Significance for mutant transcript level and EGF-like domain calcium binding in the pathogenesis of Marfan syndrome

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Dietz, H.C.; McIntosh, I.; Pyeritz, R.E.; Francomano, C.A. (Johns Hopkins Univ. School of Medicine, Baltimore, MD (United States)); Sakai, L.Y.; Corson, G.M.; Chalberg, S.C. (Oregon Health Sciences Univ., Portland (United States))

1993-08-01

Defects of fibrillin (FBN1), a glycoprotein component of the extracellular microfibril, cause Marfan syndrome. This disorder is characterized by marked inter- and intrafamilial variation in phenotypic severity. To understand the molecular basis for this clinical observation, the authors have screened the fibrillin gene (FBN1) on chromosome 15, including the newly cloned 5[prime] coding sequence, for disease-producing alterations in a panel of patients with a wide range of manifestations and clinical severity. All the missense mutations identified to date, including two novel mutations discussed here, are associated with classic and moderate to severe disease and occur at residues with putative significance for calcium binding to epidermal growth factor (EGF)-like domains. In contrast, two new mutations that create premature signals for termination of translation of mRNA and are associated with reduction in the amount of mutant allele transcript produce a range of phenotypic severity. The patient with the lowest amount of mutant transcript has the mildest disease. These data support a role for altered calcium binding to EGF-like domains in the pathogenesis of Marfan syndrome and suggest a dominant negative mechanism for the pathogenesis of this disorder. 26 refs., 6 figs., 1 tab.

Progress towards the development of SH2 domain inhibitors.

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Kraskouskaya, Dziyana; Duodu, Eugenia; Arpin, Carolynn C; Gunning, Patrick T

2013-04-21

Src homology 2 (SH2) domains are 100 amino acid modular units, which recognize and bind to tyrosyl-phosphorylated peptide sequences on their target proteins, and thereby mediate intracellular protein-protein interactions. This review summarizes the progress towards the development of synthetic agents that disrupt the function of the SH2 domains in different proteins as well as the clinical relevance of targeting a specific SH2 domain. Since 1986, SH2 domains have been identified in over 110 human proteins, including kinases, transcription factors, and adaptor proteins. A number of these proteins are over-activated in many diseases, including cancer, and their function is highly dependent on their SH2 domain. Thus, inhibition of a protein's function through disrupting that of its SH2 domain has emerged as a promising approach towards the development of novel therapeutic modalities. Although targeting the SH2 domain is a challenging task in molecular recognition, the progress reported here demonstrates the feasibility of such an approach.

Regulation of pokemon 1 activity by sumoylation.

Science.gov (United States)

Roh, Hee-Eun; Lee, Min-Nyung; Jeon, Bu-Nam; Choi, Won-Il; Kim, Yoo-Jin; Yu, Mi-Young; Hur, Man-Wook

2007-01-01

Pokemon 1 is a proto-oncogenic transcriptional regulator that contains a POZ domain at the N-terminus and four Kruppel-like zinc fingers at the C-terminus. Pokemon 1 plays an important role in adipogenesis, osteogenesis, oncogenesis, and transcription of NF-kB responsive genes. Recent reports have shown that biological activities of transcription factors are regulated by sumolylation. We investigated whether Pokemon 1 is post-translationally modified by sumoylation and whether the modification affects Pokemon 1's transcriptional properties. We found that Pokemon 1 is sumoylated in vitro and in vivo. Upon careful analysis of the amino acid sequence of Pokemon 1, we found ten potential sumoylation sites located at lysines 61, 354, 371, 379, 383, 396, 486, 487, 536 and 539. We mutated each of these amino acids into arginine and tested whether the mutation could affect the transcriptional properties of Pokemon 1 on the Pokemon 1 responsive genes, such as ADH5/FDH and pG5-FRE-Luc. Wild-type Pokemon 1 potently represses transcription of ADH5/FDH. Most of the mutants, however, were weaker transcription repressors and repressed transcription 1.3-3.3 fold less effective. Although potential sumoylation sites were located close to the DNA binding domain or the nuclear localization sequence, the mutations did not alter nuclear localization or DNA binding activity. In addition, on the pG5-FRE-Luc test promoter construct, ectopic SUMO-1 repressed transcription in the presence of Pokemon 1. The sumoylation target lysine residue at amino acid 61, which is located in the middle of the POZ-domain, is important because K61R mutation resulted in a much weaker molecular interaction with corepressors. Our data suggest that Pokemon 1's activity as a transcription factor may involve sumoylation, and that sumoylation might be important in the regulation of transcription by Pokemon 1.

An excited state underlies gene regulation of a transcriptional riboswitch

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Zhao, Bo; Guffy, Sharon L.; Williams, Benfeard; Zhang, Qi

2017-01-01

Riboswitches control gene expression through ligand-dependent structural rearrangements of the sensing aptamer domain. However, we found that the Bacillus cereus fluoride riboswitch aptamer adopts identical tertiary structures in solution with and without ligand. Using chemical exchange saturation transfer (CEST) NMR spectroscopy, we revealed that the structured ligand-free aptamer transiently accesses a low-populated (~1%) and short-lived (~3 ms) excited conformational state that unravels a conserved ‘linchpin’ base pair to signal transcription termination. Upon fluoride binding, this highly localized fleeting process is allosterically suppressed to activate transcription. We demonstrated that this mechanism confers effective fluoride-dependent gene activation over a wide range of transcription rates, which is essential for robust toxicity response across diverse cellular conditions. These results unveil a novel switching mechanism that employs ligand-dependent suppression of an aptamer excited state to coordinate regulatory conformational transitions rather than adopting distinct aptamer ground-state tertiary architectures, exemplifying a new mode of ligand-dependent RNA regulation. PMID:28719589

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

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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.

Transcript Profiling Identifies NAC-Domain Genes Involved in Regulating Wall Ingrowth Deposition in Phloem Parenchyma Transfer Cells of Arabidopsis thaliana

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Yuzhou Wu

2018-03-01

Full Text Available Transfer cells (TCs play important roles in facilitating enhanced rates of nutrient transport at key apoplasmic/symplasmic junctions along the nutrient acquisition and transport pathways in plants. TCs achieve this capacity by developing elaborate wall ingrowth networks which serve to increase plasma membrane surface area thus increasing the cell's surface area-to-volume ratio to achieve increased flux of nutrients across the plasma membrane. Phloem parenchyma (PP cells of Arabidopsis leaf veins trans-differentiate to become PP TCs which likely function in a two-step phloem loading mechanism by facilitating unloading of photoassimilates into the apoplasm for subsequent energy-dependent uptake into the sieve element/companion cell (SE/CC complex. We are using PP TCs in Arabidopsis as a genetic model to identify transcription factors involved in coordinating deposition of the wall ingrowth network. Confocal imaging of pseudo-Schiff propidium iodide-stained tissue revealed different profiles of temporal development of wall ingrowth deposition across maturing cotyledons and juvenile leaves, and a basipetal gradient of deposition across mature adult leaves. RNA-Seq analysis was undertaken to identify differentially expressed genes common to these three different profiles of wall ingrowth deposition. This analysis identified 68 transcription factors up-regulated two-fold or more in at least two of the three experimental comparisons, with six of these transcription factors belonging to Clade III of the NAC-domain family. Phenotypic analysis of these NAC genes using insertional mutants revealed significant reductions in levels of wall ingrowth deposition, particularly in a double mutant of NAC056 and NAC018, as well as compromised sucrose-dependent root growth, indicating impaired capacity for phloem loading. Collectively, these results support the proposition that Clade III members of the NAC-domain family in Arabidopsis play important roles in

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

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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

Inducible, tunable and multiplex human gene regulation using CRISPR-Cpf1-based transcription factors | Office of Cancer Genomics

Science.gov (United States)

Targeted and inducible regulation of mammalian gene expression is a broadly important research capability that may also enable development of novel therapeutics for treating human diseases. Here we demonstrate that a catalytically inactive RNA-guided CRISPR-Cpf1 nuclease fused to transcriptional activation domains can up-regulate endogenous human gene expression. We engineered drug-inducible Cpf1-based activators and show how this system can be used to tune the regulation of endogenous gene transcription in human cells.

Structural basis of transcriptional gene silencing mediated by Arabidopsis MOM1.

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Taisuke Nishimura

2012-02-01

Full Text Available Shifts between epigenetic states of transcriptional activity are typically correlated with changes in epigenetic marks. However, exceptions to this rule suggest the existence of additional, as yet uncharacterized, layers of epigenetic regulation. MOM1, a protein of 2,001 amino acids that acts as a transcriptional silencer, represents such an exception. Here we define the 82 amino acid domain called CMM2 (Conserved MOM1 Motif 2 as a minimal MOM1 fragment capable of transcriptional regulation. As determined by X-ray crystallography, this motif folds into an unusual hendecad-based coiled-coil. Structure-based mutagenesis followed by transgenic complementation tests in plants demonstrate that CMM2 and its dimerization are effective for transcriptional suppression at chromosomal loci co-regulated by MOM1 and the siRNA pathway but not at loci controlled by MOM1 in an siRNA-independent fashion. These results reveal a surprising separation of epigenetic activities that enable the single, large MOM1 protein to coordinate cooperating mechanisms of epigenetic regulation.

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.

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

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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.

A TAD further: exogenous control of gene activation.

Science.gov (United States)

Mapp, Anna K; Ansari, Aseem Z

2007-01-23

Designer molecules that can be used to impose exogenous control on gene transcription, artificial transcription factors (ATFs), are highly desirable as mechanistic probes of gene regulation, as potential therapeutic agents, and as components of cell-based devices. Recently, several advances have been made in the design of ATFs that activate gene transcription (activator ATFs), including reports of small-molecule-based systems and ATFs that exhibit potent activity. However, the many open mechanistic questions about transcriptional activators, in particular, the structure and function of the transcriptional activation domain (TAD), have hindered rapid development of synthetic ATFs. A compelling need thus exists for chemical tools and insights toward a more detailed portrait of the dynamic process of gene activation.

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.

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

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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.

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...

Structural basis of antifreeze activity of a bacterial multi-domain antifreeze protein.

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Chen Wang

Full Text Available Antifreeze proteins (AFPs enhance the survival of organisms inhabiting cold environments by affecting the formation and/or structure of ice. We report the crystal structure of the first multi-domain AFP that has been characterized. The two ice binding domains are structurally similar. Each consists of an irregular β-helix with a triangular cross-section and a long α-helix that runs parallel on one side of the β-helix. Both domains are stabilized by hydrophobic interactions. A flat plane on the same face of each domain's β-helix was identified as the ice binding site. Mutating any of the smaller residues on the ice binding site to bulkier ones decreased the antifreeze activity. The bulky side chain of Leu174 in domain A sterically hinders the binding of water molecules to the protein backbone, partially explaining why antifreeze activity by domain A is inferior to that of domain B. Our data provide a molecular basis for understanding differences in antifreeze activity between the two domains of this protein and general insight on how structural differences in the ice-binding sites affect the activity of AFPs.

Synergistic cooperation of MDM2 and E2F1 contributes to TAp73 transcriptional activity

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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.

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

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.

Elevated expression and potential roles of human Sp5, a member of Sp transcription factor family, in human cancers

International Nuclear Information System (INIS)

Chen Yongxin; Guo Yingqiu; Ge Xijin; Itoh, Hirotaka; Watanabe, Akira; Fujiwara, Takeshi; Kodama, Tatsuhiko; Aburatani, Hiroyuki

2006-01-01

In this report, we describe the expression and function of human Sp5, a member of the Sp family of zinc finger transcription factors. Like other family members, the Sp5 protein contains a Cys2His2 zinc finger DNA binding domain at the C-terminus. Our experiments employing Gal4-Sp5 fusion proteins reveal multiple transcriptional domains, including a N-terminal activity domain, an intrinsic repressive element, and a C-terminal synergistic domain. Elevated expression of Sp5 was noted in several human tumors including hepatocellular carcinoma, gastric cancer, and colon cancer. To study the effects of the Sp5 protein on growth properties of human cancer cells and facilitate the identification of its downstream genes, we combined an inducible gene expression system with microarray analysis to screen for its transcriptional targets. Transfer of Sp5 into MCF-7 cells that expressed no detectable endogenous Sp5 protein elicited significant growth promotion activity. Several of the constitutively deregulated genes have been associated with tumorigenesis (CDC25C, CEACAM6, TMPRSS2, XBP1, MYBL1, ABHD2, and CXCL12) and Wnt/β-Catenin signaling pathways (BAMBI, SIX1, IGFBP5, AES, and p21 WAF1 ). This information could be utilized for further mechanistic research and for devising optimized therapeutic strategies against human cancers

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

Crystallization and preliminary crystallographic data of the PAS domain of the NifL protein from Azotobacter vinelandii.

NARCIS (Netherlands)

Hefti, M.H.; Hendle, J.; Enroth, C.; Vervoort, J.J.M.; Tucker, P.A.

2001-01-01

The Azotobacter vinelandii NifL protein is a redox-sensing flavoprotein which inhibits the activity of the nitrogen-specific transcriptional activator NifA. The N-terminal PAS domain has been overexpressed in Escherichia coli and crystallized by the hanging-drop vapour-diffusion method. The crystal

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.

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.

Interaction of Arabidopsis Trihelix-Domain Transcription Factors VFP3 and VFP5 with Agrobacterium Virulence Protein VirF.

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Elena García-Cano

Full Text Available Agrobacterium is a natural genetic engineer of plants that exports several virulence proteins into host cells in order to take advantage of the cell machinery to facilitate transformation and support bacterial growth. One of these effectors is the F-box protein VirF, which presumably uses the host ubiquitin/proteasome system (UPS to uncoat the packaging proteins from the invading bacterial T-DNA. By analogy to several other bacterial effectors, VirF most likely has several functions in the host cell and, therefore, several interacting partners among host proteins. Here we identify one such interactor, an Arabidopsis trihelix-domain transcription factor VFP3, and further show that its very close homolog VFP5 also interacted with VirF. Interestingly, interactions of VirF with either VFP3 or VFP5 did not activate the host UPS, suggesting that VirF might play other UPS-independent roles in bacterial infection. To better understand the potential scope of VFP3 function, we used RNAi to reduce expression of the VFP3 gene. Transcriptome profiling of these VFP3-silenced plants using high-throughput cDNA sequencing (RNA-seq revealed that VFP3 substantially affected plant gene expression; specifically, 1,118 genes representing approximately 5% of all expressed genes were significantly either up- or down-regulated in the VFP3 RNAi line compared to wild-type Col-0 plants. Among the 507 up-regulated genes were genes implicated in the regulation of transcription, protein degradation, calcium signaling, and hormone metabolism, whereas the 611 down-regulated genes included those involved in redox regulation, light reactions of photosynthesis, and metabolism of lipids, amino acids, and cell wall. Overall, this pattern of changes in gene expression is characteristic of plants under stress. Thus, VFP3 likely plays an important role in controlling plant homeostasis.

Interaction of Arabidopsis Trihelix-Domain Transcription Factors VFP3 and VFP5 with Agrobacterium Virulence Protein VirF

Science.gov (United States)

García-Cano, Elena; Magori, Shimpei; Sun, Qi; Ding, Zehong; Lazarowitz, Sondra G.; Citovsky, Vitaly

2015-01-01

Agrobacterium is a natural genetic engineer of plants that exports several virulence proteins into host cells in order to take advantage of the cell machinery to facilitate transformation and support bacterial growth. One of these effectors is the F-box protein VirF, which presumably uses the host ubiquitin/proteasome system (UPS) to uncoat the packaging proteins from the invading bacterial T-DNA. By analogy to several other bacterial effectors, VirF most likely has several functions in the host cell and, therefore, several interacting partners among host proteins. Here we identify one such interactor, an Arabidopsis trihelix-domain transcription factor VFP3, and further show that its very close homolog VFP5 also interacted with VirF. Interestingly, interactions of VirF with either VFP3 or VFP5 did not activate the host UPS, suggesting that VirF might play other UPS-independent roles in bacterial infection. To better understand the potential scope of VFP3 function, we used RNAi to reduce expression of the VFP3 gene. Transcriptome profiling of these VFP3-silenced plants using high-throughput cDNA sequencing (RNA-seq) revealed that VFP3 substantially affected plant gene expression; specifically, 1,118 genes representing approximately 5% of all expressed genes were significantly either up- or down-regulated in the VFP3 RNAi line compared to wild-type Col-0 plants. Among the 507 up-regulated genes were genes implicated in the regulation of transcription, protein degradation, calcium signaling, and hormone metabolism, whereas the 611 down-regulated genes included those involved in redox regulation, light reactions of photosynthesis, and metabolism of lipids, amino acids, and cell wall. Overall, this pattern of changes in gene expression is characteristic of plants under stress. Thus, VFP3 likely plays an important role in controlling plant homeostasis. PMID:26571494

Cooperation of three WRKY-domain transcription factors WRKY18, WRKY40, and WRKY60 in repressing two ABA-responsive genes ABI4 and ABI5 in Arabidopsis

OpenAIRE

Liu, Zhi-Qiang; Yan, Lu; Wu, Zhen; Mei, Chao; Lu, Kai; Yu, Yong-Tao; Liang, Shan; Zhang, Xiao-Feng; Wang, Xiao-Fang; Zhang, Da-Peng

2012-01-01

Three evolutionarily closely related WRKY-domain transcription factors WRKY18, WRKY40, and WRKY60 in Arabidopsis were previously identified as negative abscisic acid (ABA) signalling regulators, of which WRKY40 regulates ABI4 and ABI5 expression, but it remains unclear whether and how the three transcription factors cooperate to regulate expression of ABI4 and ABI5. In the present experiments, it was shown that WRKY18 and WRKY60, like WRKY40, interact with the W-box in the promoters of ABI4 a...

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.

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.

Highly acidic C-terminal domain of pp32 is required for the interaction with histone chaperone, TAF-Ibeta.

Science.gov (United States)

Lee, In-Seon; Oh, Sang-Min; Kim, Sung-Mi; Lee, Dong-Seok; Seo, Sang-Beom

2006-12-01

We have previously reported that INHAT (inhibitor of acetyltransferases) complex subunits, TAF (template activating factor)-Ialpha, TAF-Ibeta and pp32 can inhibit histone acetylation and HAT (histone acetyltransferase)-dependent transcription by binding to histones. Evidences are accumulating that INHAT complex subunits have important regulatory roles in various cellular activities such as replication, transcription, and apoptosis etc. However, how these subunits interact each other remains largely unknown. Using immunoprecipitation (IP) and protein-protein interaction assays with TAF-Ibeta and pp32 deletion mutant proteins, we identify INHAT complex subunits, TAF-Ibeta and pp32 interaction requires highly acidic C-terminal domain of pp32. We also show that the interaction between the INHAT complex subunits is stronger in the presence of histones. In this study, we report that the synergistic inhibition of HAT-mediated transcription by TAF-Ibeta and pp32 is dependent on the highly acidic C-terminal domain of pp32.

Design of potentially active ligands for SH2 domains by molecular modeling methods

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Hurmach V. V.

2014-07-01

Full Text Available Search for new chemical structures possessing specific biological activity is a complex problem that needs the use of the latest achievements of molecular modeling technologies. It is well known that SH2 domains play a major role in ontogenesis as intermediaries of specific protein-protein interactions. Aim. Developing an algorithm to investigate the properties of SH2 domain binding, search for new potential active compounds for the whole SH2 domains class. Methods. In this paper, we utilize a complex of computer modeling methods to create a generic set of potentially active compounds targeting universally at the whole class of SH2 domains. A cluster analysis of all available three-dimensional structures of SH2 domains was performed and general pharmacophore models were formulated. The models were used for virtual screening of collection of drug-like compounds provided by Enamine Ltd. Results. The design technique for library of potentially active compounds for SH2 domains class was proposed. Conclusions. The original algorithm of SH2 domains research with molecular docking method was developed. Using our algorithm, the active compounds for SH2 domains were found.

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.

Identification of novel small molecules that inhibit STAT3-dependent transcription and function.

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Iryna Kolosenko

Full Text Available Activation of Signal Transducer and Activator of Transcription 3 (STAT3 has been linked to several processes that are critical for oncogenic transformation, cancer progression, cancer cell proliferation, survival, drug resistance and metastasis. Inhibition of STAT3 signaling has shown a striking ability to inhibit cancer cell growth and therefore, STAT3 has become a promising target for anti-cancer drug development. The aim of this study was to identify novel inhibitors of STAT-dependent gene transcription. A cellular reporter-based system for monitoring STAT3 transcriptional activity was developed which was suitable for high-throughput screening (Z' = 0,8. This system was used to screen a library of 28,000 compounds (the ENAMINE Drug-Like Diversity Set. Following counter-screenings and toxicity studies, we identified four hit compounds that were subjected to detailed biological characterization. Of the four hits, KI16 stood out as the most promising compound, inhibiting STAT3 phosphorylation and transcriptional activity in response to IL6 stimulation. In silico docking studies showed that KI16 had favorable interactions with the STAT3 SH2 domain, however, no inhibitory activity could be observed in the STAT3 fluorescence polarization assay. KI16 inhibited cell viability preferentially in STAT3-dependent cell lines. Taken together, using a targeted, cell-based approach, novel inhibitors of STAT-driven transcriptional activity were discovered which are interesting leads to pursue further for the development of anti-cancer therapeutic agents.

Distinct properties of hexameric but functionally conserved Mycobacterium tuberculosis transcription-repair coupling factor.

Science.gov (United States)

Prabha, Swayam; Rao, Desirazu N; Nagaraja, Valakunja

2011-04-29

Transcription coupled nucleotide excision repair (TC-NER) is involved in correcting UV-induced damage and other road-blocks encountered in the transcribed strand. Mutation frequency decline (Mfd) is a transcription repair coupling factor, involved in repair of template strand during transcription. Mfd from M. tuberculosis (MtbMfd) is 1234 amino-acids long harboring characteristic modules for different activities. Mtbmfd complemented Escherichia coli mfd (Ecomfd) deficient strain, enhanced survival of UV irradiated cells and increased the road-block repression in vivo. The protein exhibited ATPase activity, which was stimulated ∼1.5-fold in the presence of DNA. While the C-terminal domain (CTD) comprising amino acids 630 to 1234 showed ∼2-fold elevated ATPase activity than MtbMfd, the N-terminal domain (NTD) containing the first 433 amino acid residues was able to bind ATP but deficient in hydrolysis. Overexpression of NTD of MtbMfd led to growth defect and hypersensitivity to UV light. Deletion of 184 amino acids from the C-terminal end of MtbMfd (MfdΔC) increased the ATPase activity by ∼10-fold and correspondingly exhibited efficient translocation along DNA as compared to the MtbMfd and CTD. Surprisingly, MtbMfd was found to be distributed in monomer and hexamer forms both in vivo and in vitro and the monomer showed increased susceptibility to proteases compared to the hexamer. MfdΔC, on the other hand, was predominantly monomeric in solution implicating the extreme C-terminal region in oligomerization of the protein. Thus, although the MtbMfd resembles EcoMfd in many of its reaction characteristics, some of its hitherto unknown distinct properties hint at its species specific role in mycobacteria during transcription-coupled repair.

Distinct properties of hexameric but functionally conserved Mycobacterium tuberculosis transcription-repair coupling factor.

Directory of Open Access Journals (Sweden)

Swayam Prabha

Full Text Available Transcription coupled nucleotide excision repair (TC-NER is involved in correcting UV-induced damage and other road-blocks encountered in the transcribed strand. Mutation frequency decline (Mfd is a transcription repair coupling factor, involved in repair of template strand during transcription. Mfd from M. tuberculosis (MtbMfd is 1234 amino-acids long harboring characteristic modules for different activities. Mtbmfd complemented Escherichia coli mfd (Ecomfd deficient strain, enhanced survival of UV irradiated cells and increased the road-block repression in vivo. The protein exhibited ATPase activity, which was stimulated ∼1.5-fold in the presence of DNA. While the C-terminal domain (CTD comprising amino acids 630 to 1234 showed ∼2-fold elevated ATPase activity than MtbMfd, the N-terminal domain (NTD containing the first 433 amino acid residues was able to bind ATP but deficient in hydrolysis. Overexpression of NTD of MtbMfd led to growth defect and hypersensitivity to UV light. Deletion of 184 amino acids from the C-terminal end of MtbMfd (MfdΔC increased the ATPase activity by ∼10-fold and correspondingly exhibited efficient translocation along DNA as compared to the MtbMfd and CTD. Surprisingly, MtbMfd was found to be distributed in monomer and hexamer forms both in vivo and in vitro and the monomer showed increased susceptibility to proteases compared to the hexamer. MfdΔC, on the other hand, was predominantly monomeric in solution implicating the extreme C-terminal region in oligomerization of the protein. Thus, although the MtbMfd resembles EcoMfd in many of its reaction characteristics, some of its hitherto unknown distinct properties hint at its species specific role in mycobacteria during transcription-coupled repair.

Natural variation in monoterpene synthesis in kiwifruit: transcriptional regulation of terpene synthases by NAC and ETHYLENE-INSENSITIVE3-like transcription factors.

Science.gov (United States)

Nieuwenhuizen, Niels J; Chen, Xiuyin; Wang, Mindy Y; Matich, Adam J; Perez, Ramon Lopez; Allan, Andrew C; Green, Sol A; Atkinson, Ross G

2015-04-01

Two kiwifruit (Actinidia) species with contrasting terpene profiles were compared to understand the regulation of fruit monoterpene production. High rates of terpinolene production in ripe Actinidia arguta fruit were correlated with increasing gene and protein expression of A. arguta terpene synthase1 (AaTPS1) and correlated with an increase in transcript levels of the 2-C-methyl-D-erythritol 4-phosphate pathway enzyme 1-deoxy-D-xylulose-5-phosphate synthase (DXS). Actinidia chinensis terpene synthase1 (AcTPS1) was identified as part of an array of eight tandemly duplicated genes, and AcTPS1 expression and terpene production were observed only at low levels in developing fruit. Transient overexpression of DXS in Nicotiana benthamiana leaves elevated monoterpene synthesis by AaTPS1 more than 100-fold, indicating that DXS is likely to be the key step in regulating 2-C-methyl-D-erythritol 4-phosphate substrate flux in kiwifruit. Comparative promoter analysis identified potential NAC (for no apical meristem [NAM], Arabidopsis transcription activation factor [ATAF], and cup-shaped cotyledon [CUC])-domain transcription factor) and ETHYLENE-INSENSITIVE3-like transcription factor (TF) binding sites in the AaTPS1 promoter, and cloned members of both TF classes were able to activate the AaTPS1 promoter in transient assays. Electrophoretic mobility shift assays showed that AaNAC2, AaNAC3, and AaNAC4 bind a 28-bp fragment of the proximal NAC binding site in the AaTPS1 promoter but not the A. chinensis AcTPS1 promoter, where the NAC binding site was mutated. Activation could be restored by reintroducing multiple repeats of the 12-bp NAC core-binding motif. The absence of NAC transcriptional activation in ripe A. chinensis fruit can account for the low accumulation of AcTPS1 transcript, protein, and monoterpene volatiles in this species. These results indicate the importance of NAC TFs in controlling monoterpene production and other traits in ripening fruits. © 2015 American

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.

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

International Nuclear Information System (INIS)

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

2013-01-01

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

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

Energy Technology Data Exchange (ETDEWEB)

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

2013-01-18

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

Constitutive NADPH-dependent electron transferase activity of the Nox4 dehydrogenase domain.

Science.gov (United States)

Nisimoto, Yukio; Jackson, Heather M; Ogawa, Hisamitsu; Kawahara, Tsukasa; Lambeth, J David

2010-03-23

NADPH oxidase 4 (Nox4) is constitutively active, while Nox2 requires the cytosolic regulatory subunits p47(phox) and p67(phox) and activated Rac with activation by phorbol 12-myristate 13-acetate (PMA). This study was undertaken to identify the domain on Nox4 that confers constitutive activity. Lysates from Nox4-expressing cells exhibited constitutive NADPH- but not NADH-dependent hydrogen peroxide production with a K(m) for NADPH of 55 +/- 10 microM. The concentration of Nox4 in cell lysates was estimated using Western blotting and allowed calculation of a turnover of approximately 200 mol of H(2)O(2) min(-1) (mol of Nox4)(-1). A chimeric protein (Nox2/4) consisting of the Nox2 transmembrane (TM) domain and the Nox4 dehydrogenase (DH) domain showed H(2)O(2) production in the absence of cytosolic regulatory subunits. In contrast, chimera Nox4/2, consisting of the Nox4 TM and Nox2 DH domains, exhibited PMA-dependent activation that required coexpression of regulatory subunits. Nox DH domains from several Nox isoforms were purified and evaluated for their electron transferase activities. Nox1 DH, Nox2 DH, and Nox5 DH domains exhibited barely detectable activities toward artificial electron acceptors, while the Nox4 DH domain exhibited significant rates of reduction of cytochrome c (160 min(-1), largely superoxide dismutase-independent), ferricyanide (470 min(-1)), and other electron acceptors (artificial dyes and cytochrome b(5)). Rates were similar to those observed for H(2)O(2) production by the Nox4 holoenzyme in cell lysates. The activity required added FAD and was seen with NADPH but not NADH. These results indicate that the Nox4 DH domain exists in an intrinsically activated state and that electron transfer from NADPH to FAD is likely to be rate-limiting in the NADPH-dependent reduction of oxygen by holo-Nox4.

Autoregulation of Co-Chaperone BAG3 Gene Transcription

OpenAIRE

Gentilella, Antonio; Khalili, Kamel

2009-01-01

The Bcl-2-associated athanogene, BAG, protein family through their BAG domain associates with the heat shock protein 70 (HSP-70) and modulates its chaperone activity. One member of this family, BAG3, appears to play an important role in protein homeostasis, as its expression promotes cell survival by preventing polyubiquitination of Hsp-70 client proteins. Expression of BAG3 is enhanced by a variety of stress-inducing agents. Here we describe a role for BAG3 to modulate transcription of its o...

The WW domain protein Kibra acts upstream of Hippo in Drosophila

DEFF Research Database (Denmark)

Baumgartner, Roland; Poernbacher, Ingrid; Buser, Nathalie

2010-01-01

inactivating the transcriptional coactivator Yorkie is well established, much less is known about the upstream events that regulate Hippo signaling activity. The FERM domain proteins Expanded and Merlin appear to represent two different signaling branches that feed into the Hippo pathway. Signaling...... by the atypical cadherin Fat may act via Expanded, but how Merlin is regulated has remained elusive. Here, we show that the WW domain protein Kibra is a Hippo signaling component upstream of Hippo and Merlin. Kibra acts synergistically with Expanded, and it physically interacts with Merlin. Thus, Kibra...

Cloning and functional characterisation of avian transcription factor E2A

Directory of Open Access Journals (Sweden)

Meyer Kerstin B

2004-06-01

Full Text Available Abstract Background During B lymphocyte development the E2A gene is a critical regulator of cell proliferation and differentiation. With regards to the immunoglobulin genes the E2A proteins contribute to the regulation of gene rearrangement, expression and class switch recombination. We are now using the chicken cell line DT40 as a model system to further analyse the function of E2A. Results Here we report the cloning and functional analysis of the transcription factor E2A from chicken. Using RACE PCR on the chicken lymphoma cell line DT40 we have isolated full-length clones for the two E2A splice variants E12 and E47. Sequence conservation between the human and chicken proteins is extensive: the basic-helix-loop-helix DNA binding domain of human and chicken E47 and E12 are 93% and 92% identical, respectively. In addition high levels of conservation are seen in activation domain I, the potential NLS and the ubiquitin ligase interaction domain. E2A is expressed in a variety of tissues in chicken, with higher levels of expression in organs rich in immune cells. We demonstrate that chicken E12 and E47 proteins are strong transcriptional activators whose function depends on the presence of activation domain I. As in mammals, the dominant negative proteins Id1 and Id3 can inhibit the function of chicken E47. Conclusions The potential for homologous recombination in DT40 allows the genetic dissection of biochemical pathways in somatic cells. With the cloning of avian E2A and the recent description of an in vitro somatic hypermutation assay in this cell line, it should now be possible to dissect the potential role of E2A in the regulation of somatic hypermutation and gene conversion.

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

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

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.

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

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

Science.gov (United States)

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

2003-10-03

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

The nucleotide-binding domain of NLRC5 is critical for nuclear import and transactivation activity

International Nuclear Information System (INIS)

Meissner, Torsten B.; Li, Amy; Liu, Yuen-Joyce; Gagnon, Etienne; Kobayashi, Koichi S.

2012-01-01

Highlights: ► NLRC5 requires an intact NLS for its function as MHC class I transactivator. ► Nuclear presence of NLRC5 is required for MHC class I induction. ► Nucleotide-binding controls nuclear import and transactivation activity of NLRC5. -- Abstract: Major histocompatibility complex (MHC) class I and class II are crucial for the function of the human adaptive immune system. A member of the NLR (nucleotide-binding domain, leucine-rich repeat) protein family, NLRC5, has recently been identified as a transcriptional regulator of MHC class I and related genes. While a ‘master regulator’ of MHC class II genes, CIITA, has long been known, NLRC5 specifically associates with and transactivates the proximal promoters of MHC class I genes. In this study, we analyzed the molecular requirements of NLRC5 nuclear import and transactivation activity. We show that NLRC5-mediated MHC class I gene induction requires an intact nuclear localization signal and nuclear distribution of NLRC5. In addition, we find that the nucleotide-binding domain (NBD) of NLRC5 is critical not only for nuclear translocation but also for the transactivation of MHC class I genes. Changing the cellular localization of NLRC5 is likely to immediately impact MHC class I expression as well as MHC class I-mediated antigen presentation. NLRC5 may thus provide a promising target for the modulation of MHC class I antigen presentation, especially in the setting of transplant medicine.

Transcription Activator-Like Effectors (TALEs) Hybrid Nucleases for Genome Engineering Application

KAUST Repository

Wibowo, Anjar

2011-06-06

Gene targeting is a powerful genome engineering tool that can be used for a variety of biotechnological applications. Genomic double-strand DNA breaks generated by engineered site-specific nucleases can stimulate gene targeting. Hybrid nucleases are composed of DNA binding module and DNA cleavage module. Zinc Finger Nucleases were used to generate double-strand DNA breaks but it suffers from failures and lack of reproducibility. The transcription activator–like effectors (TALEs) from plant pathogenic Xanthomonas contain a unique type of DNA-binding domain that bind specific DNA targets. The purpose of this study is to generate novel sequence specific nucleases by fusing a de novo engineered Hax3 TALE-based DNA binding domain to a FokI cleavage domain. Our data show that the de novo engineered TALE nuclease can bind to its target sequence and create double-strand DNA breaks in vitro. We also show that the de novo engineered TALE nuclease is capable of generating double-strand DNA breaks in its target sequence in vivo, when transiently expressed in Nicotiana benthamiana leaves. In conclusion, our data demonstrate that TALE-based hybrid nucleases can be tailored to bind a user-selected DNA sequence and generate site-specific genomic double-strand DNA breaks. TALE-based hybrid nucleases hold much promise as powerful molecular tools for gene targeting applications.

Identification of a novel and unique transcription factor in the intraerythrocytic stage of Plasmodium falciparum.

Directory of Open Access Journals (Sweden)

Kanako Komaki-Yasuda

Full Text Available The mechanisms of stage-specific gene regulation in the malaria parasite Plasmodium falciparum are largely unclear, with only a small number of specific regulatory transcription factors (AP2 family having been identified. In particular, the transcription factors that function in the intraerythrocytic stage remain to be elucidated. Previously, as a model case for stage-specific transcription in the P. falciparum intraerythrocytic stage, we analyzed the transcriptional regulation of pf1-cys-prx, a trophozoite/schizont-specific gene, and suggested that some nuclear factors bind specifically to the cis-element of pf1-cys-prx and enhance transcription. In the present study, we purified nuclear factors from parasite nuclear extract by 5 steps of chromatography, and identified a factor termed PREBP. PREBP is not included in the AP2 family, and is a novel protein with four K-homology (KH domains. The KH domain is known to be found in RNA-binding or single-stranded DNA-binding proteins. PREBP is well conserved in Plasmodium species and partially conserved in phylum Apicomplexa. To evaluate the effects of PREBP overexpression, we used a transient overexpression and luciferase assay combined approach. Overexpression of PREBP markedly enhanced luciferase expression under the control of the pf1-cys-prx cis-element. These results provide the first evidence of a novel transcription factor that activates the gene expression in the malaria parasite intraerythrocytic stage. These findings enhance our understanding of the evolution of specific transcription machinery in Plasmodium and other eukaryotes.

Natural Variation in Monoterpene Synthesis in Kiwifruit: Transcriptional Regulation of Terpene Synthases by NAC and ETHYLENE-INSENSITIVE3-Like Transcription Factors1

Science.gov (United States)

Nieuwenhuizen, Niels J.; Chen, Xiuyin; Wang, Mindy Y.; Matich, Adam J.; Perez, Ramon Lopez; Allan, Andrew C.; Green, Sol A.; Atkinson, Ross G.

2015-01-01

Two kiwifruit (Actinidia) species with contrasting terpene profiles were compared to understand the regulation of fruit monoterpene production. High rates of terpinolene production in ripe Actinidia arguta fruit were correlated with increasing gene and protein expression of A. arguta terpene synthase1 (AaTPS1) and correlated with an increase in transcript levels of the 2-C-methyl-d-erythritol 4-phosphate pathway enzyme 1-deoxy-d-xylulose-5-phosphate synthase (DXS). Actinidia chinensis terpene synthase1 (AcTPS1) was identified as part of an array of eight tandemly duplicated genes, and AcTPS1 expression and terpene production were observed only at low levels in developing fruit. Transient overexpression of DXS in Nicotiana benthamiana leaves elevated monoterpene synthesis by AaTPS1 more than 100-fold, indicating that DXS is likely to be the key step in regulating 2-C-methyl-d-erythritol 4-phosphate substrate flux in kiwifruit. Comparative promoter analysis identified potential NAC (for no apical meristem [NAM], Arabidopsis transcription activation factor [ATAF], and cup-shaped cotyledon [CUC])-domain transcription factor) and ETHYLENE-INSENSITIVE3-like transcription factor (TF) binding sites in the AaTPS1 promoter, and cloned members of both TF classes were able to activate the AaTPS1 promoter in transient assays. Electrophoretic mobility shift assays showed that AaNAC2, AaNAC3, and AaNAC4 bind a 28-bp fragment of the proximal NAC binding site in the AaTPS1 promoter but not the A. chinensis AcTPS1 promoter, where the NAC binding site was mutated. Activation could be restored by reintroducing multiple repeats of the 12-bp NAC core-binding motif. The absence of NAC transcriptional activation in ripe A. chinensis fruit can account for the low accumulation of AcTPS1 transcript, protein, and monoterpene volatiles in this species. These results indicate the importance of NAC TFs in controlling monoterpene production and other traits in ripening fruits. PMID:25649633

A phospho-sugar binding domain homologous to NagB enzymes regulates the activity of the central glycolytic genes repressor.

Science.gov (United States)

Doan, Thierry; Martin, Laetitia; Zorrilla, Silvia; Chaix, Denis; Aymerich, Stéphane; Labesse, Gilles; Declerck, Nathalie

2008-06-01

CggR belongs to the SorC family of bacterial transcriptional regulators which control the expression of genes and operons involved in carbohydrate catabolism. CggR was first identified in Bacillus subtilis where it represses the gapA operon encoding the five enzymes that catalyze the central part of glycolysis. Here we present a structure/function study demonstrating that the C-terminal region of CggR regulates the DNA binding activity of this repressor in response to binding of a phosphorylated sugar. Molecular modeling of CggR revealed a winged-helix DNA-binding motif followed by a C-terminal domain presenting weak but significant homology with glucosamine-6-phosphate deaminases from the NagB family. In silico ligand screening suggested that the CggR C-terminal domain would bind preferentially bi-phosphorylated compounds, in agreement with previous studies that proposed fructuose-1,6-biphosphate (FBP) as the inducer metabolite. In vitro, FBP was the only sugar compound capable of interfering with CggR cooperative binding to DNA. FBP was also found to protect CggR against trypsin degradation at two arginine residues predicted to reside in a mobile loop forming the active site lid of the NagB enzymes. Replacement of residues predicted to interact with FBP led to mutant CggR with altered repressor activity in vivo but retaining their structural integrity and DNA binding activity in vitro. Interestingly, some of the mutant repressors responded with different specificity towards mono- and di-phospho-fructosides. Based on these results, we propose that the activity of the CggR-like repressors is controlled by a phospho-sugar binding (PSB) domain presenting structural and functional homology with NagB enzymes. (c) 2008 Wiley-Liss, Inc.

Acetylation within the N- and C-Terminal Domains of Src Regulates Distinct Roles of STAT3-Mediated Tumorigenesis.

Science.gov (United States)

Huang, Chao; Zhang, Zhe; Chen, Lihan; Lee, Hank W; Ayrapetov, Marina K; Zhao, Ting C; Hao, Yimei; Gao, Jinsong; Yang, Chunzhang; Mehta, Gautam U; Zhuang, Zhengping; Zhang, Xiaoren; Hu, Guohong; Chin, Y Eugene

2018-06-01

Posttranslational modifications of mammalian c-Src N-terminal and C-terminal domains regulate distinct functions. Myristoylation of G 2 controls its cell membrane association and phosphorylation of Y419/Y527 controls its activation or inactivation, respectively. We provide evidence that Src-cell membrane association-dissociation and catalytic activation-inactivation are both regulated by acetylation. In EGF-treated cells, CREB binding protein (CBP) acetylates an N-terminal lysine cluster (K5, K7, and K9) of c-Src to promote dissociation from the cell membrane. CBP also acetylates the C-terminal K401, K423, and K427 of c-Src to activate intrinsic kinase activity for STAT3 recruitment and activation. N-terminal domain phosphorylation (Y14, Y45, and Y68) of STAT3 by c-Src activates transcriptionally active dimers of STAT3. Moreover, acetyl-Src translocates into nuclei, where it forms the Src-STAT3 enhanceosome for gene regulation and cancer cell proliferation. Thus, c-Src acetylation in the N-terminal and C-terminal domains play distinct roles in Src activity and regulation. Significance: CBP-mediated acetylation of lysine clusters in both the N-terminal and C-terminal regions of c-Src provides additional levels of control over STAT3 transcriptional activity. Cancer Res; 78(11); 2825-38. ©2018 AACR . ©2018 American Association for Cancer Research.

Identification of functional domains of the IR2 protein of equine herpesvirus 1 required for inhibition of viral gene expression and replication

International Nuclear Information System (INIS)

Kim, Seong K.; Kim, Seongman; Dai Gan; Zhang Yunfei; Ahn, Byung C.; O'Callaghan, Dennis J.

2011-01-01

The equine herpesvirus 1 (EHV-1) negative regulatory IR2 protein (IR2P), an early 1,165-amino acid (aa) truncated form of the 1487-aa immediate-early protein (IEP), lacks the trans-activation domain essential for IEP activation functions but retains domains for binding DNA, TFIIB, and TBP and the nuclear localization signal. IR2P mutants of the N-terminal region which lack either DNA-binding activity or TFIIB-binding activity were unable to down-regulate EHV-1 promoters. In EHV-1-infected cells expressing full-length IR2P, transcription and protein expression of viral regulatory IE, early EICP0, IR4, and UL5, and late ETIF genes were dramatically inhibited. Viral DNA levels were reduced to 2.1% of control infected cells, but were vey weakly affected in cells that express the N-terminal 706 residues of IR2P. These results suggest that IR2P function requires the two N-terminal domains for binding DNA and TFIIB as well as the C-terminal residues 707 to 1116 containing the TBP-binding domain. - Highlights: → We examine the functional domains of IR2P that mediates negative regulation. → IR2P inhibits at the transcriptional level. → DNA-binding mutant or TFIIB-binding mutant fails to inhibit. → C-terminal aa 707 to 1116 are required for full inhibition. → Inhibition requires the DNA-binding domain, TFIIB-binding domain, and C-terminus.

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.

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

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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.

Membrane Localization is Critical for Activation of the PICK1 BAR Domain

OpenAIRE

Madsen, Kenneth L.; Eriksen, Jacob; Milan-Lobo, Laura; Han, Daniel S.; Niv, Masha Y.; Ammendrup-Johnsen, Ina; Henriksen, Ulla; Bhatia, Vikram K.; Stamou, Dimitrios; Sitte, Harald H.; McMahon, Harvey T.; Weinstein, Harel; Gether, Ulrik

2008-01-01

The PSD-95/Discs-large/ZO-1 homology (PDZ) domain protein, protein interacting with C kinase 1 (PICK1) contains a C-terminal Bin/amphiphysin/Rvs (BAR) domain mediating recognition of curved membranes; however, the molecular mechanisms controlling the activity of this domain are poorly understood. In agreement with negative regulation of the BAR domain by the N-terminal PDZ domain, PICK1 distributed evenly in the cytoplasm, whereas truncation of the PDZ domain caused BAR domain-dependent redis...

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

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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.

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.

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...

Senescence-associated barley NAC (NAM, ATAF1,2, CUC) transcription factor interacts with radical-induced cell death 1 through a disordered regulatory domain

DEFF Research Database (Denmark)

Kjaersgaard, Trine; Jensen, Michael K; Christiansen, Michael W

2011-01-01

as a transcriptional activator suggesting that an involvement of HvNAC013 and HvNAC005 in senescence will be different. HvNAC013 interacted with barley radical-induced cell death 1 (RCD1) via the very C-terminal part of its TRD, outside of the region containing the LP motif. No significant secondary structure...... (NAM, ATAF1,2, CUC) TF family are up-regulated during senescence in barley (Hordeum vulgare). Both HvNAC005 and HvNAC013 bound the conserved NAC DNA target sequence. Computational and biophysical analyses showed that both proteins are intrinsically disordered in their large C-terminal domains, which...... was induced in the HvNAC013 TRD upon interaction with RCD1. RCD1 also interacted with regions dominated by intrinsic disorder in TFs of the MYB and basic helix-loop-helix families. We propose that RCD1 is a regulatory protein capable of interacting with many different TFs by exploiting their intrinsic...

Three distinct domains contribute to nuclear transport of murine Foxp3.

Directory of Open Access Journals (Sweden)

Wayne W Hancock

2009-11-01

Full Text Available Foxp3, a 47-kDa transcription factor, is necessary for the function of CD4+CD25+ regulatory T cells (Tregs, with an essential role in the control of self-reactive T cells and in preventing autoimmunity. Activation of Tregs by TCR engagement results in upregulation of Foxp3 expression, followed by its rapid nuclear transport and binding to chromatin. Here, we identify three distinct Foxp3 domains that contribute to nuclear transport. The first domain (Domain 1 comprises the C-terminal 12 amino acids. The second domain (Domain 2 is located immediately N-terminal to the forkhead domain (FHD, recently reported to be a binding site for the runt-related transcription factor 1/acute myeloid leukemia 1 (Runx1/AML1. The third domain (Domain 3 is located within the N-terminal first 51 amino acids. Unlike the known nuclear localization signals (NLSs, none of these three regions are rich in basic residues and do not bear any similarity to known monopartite or bipartite NLSs that have one or more clusters of basic amino acids. The basic arginine-lysine-lysine-arginine (RKKR sequence, located 12-aa from the C-terminal end of Foxp3 was previously reported to be a nuclear localization signal (NLS for several proteins, including for a GFP-Foxp3 hybrid. Evidence is provided here that in the full-length native Foxp3 RKKR does not function as an NLS. The data reported in this study indicates that Foxp3 achieves nuclear transport by binding to other nuclear factors and co-transporting with them to the nucleus.

Small-Molecule Inhibitors of the SOX18 Transcription Factor.

Science.gov (United States)

Fontaine, Frank; Overman, Jeroen; Moustaqil, Mehdi; Mamidyala, Sreeman; Salim, Angela; Narasimhan, Kamesh; Prokoph, Nina; Robertson, Avril A B; Lua, Linda; Alexandrov, Kirill; Koopman, Peter; Capon, Robert J; Sierecki, Emma; Gambin, Yann; Jauch, Ralf; Cooper, Matthew A; Zuegg, Johannes; Francois, Mathias

2017-03-16

Pharmacological modulation of transcription factors (TFs) has only met little success over the past four decades. This is mostly due to standard drug discovery approaches centered on blocking protein/DNA binding or interfering with post-translational modifications. Recent advances in the field of TF biology have revealed a central role of protein-protein interaction in their mode of action. In an attempt to modulate the activity of SOX18 TF, a known regulator of vascular growth in development and disease, we screened a marine extract library for potential small-molecule inhibitors. We identified two compounds, which inspired a series of synthetic SOX18 inhibitors, able to interfere with the SOX18 HMG DNA-binding domain, and to disrupt HMG-dependent protein-protein interaction with RBPJ. These compounds also perturbed SOX18 transcriptional activity in a cell-based reporter gene system. This approach may prove useful in developing a new class of anti-angiogenic compounds based on the inhibition of TF activity. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

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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.

The impact of the human DNA topoisomerase II C-terminal domain on activity.

Directory of Open Access Journals (Sweden)

Emma L Meczes

2008-03-01

Full Text Available Type II DNA topoisomerases (topos are essential enzymes needed for the resolution of topological problems that occur during DNA metabolic processes. Topos carry out an ATP-dependent strand passage reaction whereby one double helix is passed through a transient break in another. Humans have two topoII isoforms, alpha and beta, which while enzymatically similar are differentially expressed and regulated, and are thought to have different cellular roles. The C-terminal domain (CTD of the enzyme has the most diversity, and has been implicated in regulation. We sought to investigate the impact of the CTD domain on activity.We have investigated the role of the human topoII C-terminal domain by creating constructs encoding C-terminally truncated recombinant topoIIalpha and beta and topoIIalpha+beta-tail and topoIIbeta+alpha-tail chimeric proteins. We then investigated function in vivo in a yeast system, and in vitro in activity assays. We find that the C-terminal domain of human topoII isoforms is needed for in vivo function of the enzyme, but not needed for cleavage activity. C-terminally truncated enzymes had similar strand passage activity to full length enzymes, but the presence of the opposite C-terminal domain had a large effect, with the topoIIalpha-CTD increasing activity, and the topoIIbeta-CTD decreasing activity.In vivo complementation data show that the topoIIalpha C-terminal domain is needed for growth, but the topoIIbeta isoform is able to support low levels of growth without a C-terminal domain. This may indicate that topoIIbeta has an additional localisation signal. In vitro data suggest that, while the lack of any C-terminal domain has little effect on activity, the presence of either the topoIIalpha or beta C-terminal domain can affect strand passage activity. Data indicates that the topoIIbeta-CTD may be a negative regulator. This is the first report of in vitro data with chimeric human topoIIs.

The C-type lectin of the aggrecan G3 domain activates complement.

Directory of Open Access Journals (Sweden)

Camilla Melin Fürst

Full Text Available Excessive complement activation contributes to joint diseases such as rheumatoid arthritis and osteoarthritis during which cartilage proteins are fragmented and released into the synovial fluid. Some of these proteins and fragments activate complement, which may sustain inflammation. The G3 domain of large cartilage proteoglycan aggrecan interacts with other extracellular matrix proteins, fibulins and tenascins, via its C-type lectin domain (CLD and has important functions in matrix organization. Fragments containing G3 domain are released during normal aggrecan turnover, but increasingly so in disease. We now show that the aggrecan CLD part of the G3 domain activates the classical and to a lesser extent the alternative pathway of complement, via binding of C1q and C3, respectively. The complement control protein (CCP domain adjacent to the CLD showed no effect on complement initiation. The binding of C1q to G3 depended on ionic interactions and was decreased in D2267N mutant G3. However, the observed complement activation was attenuated due to binding of complement inhibitor factor H to CLD and CCP domains. This was most apparent at the level of deposition of terminal complement components. Taken together our observations indicate aggrecan CLD as one factor involved in the sustained inflammation of the joint.

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

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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.

The evolution of WRKY transcription factors.

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Rinerson, Charles I; Rabara, Roel C; Tripathi, Prateek; Shen, Qingxi J; Rushton, Paul J

2015-02-27

The availability of increasing numbers of sequenced genomes has necessitated a re-evaluation of the evolution of the WRKY transcription factor family. Modern day plants descended from a charophyte green alga that colonized the land between 430 and 470 million years ago. The first charophyte genome sequence from Klebsormidium flaccidum filled a gap in the available genome sequences in the plant kingdom between unicellular green algae that typically have 1-3 WRKY genes and mosses that contain 30-40. WRKY genes have been previously found in non-plant species but their occurrence has been difficult to explain. Only two WRKY genes are present in the Klebsormidium flaccidum genome and the presence of a Group IIb gene was unexpected because it had previously been thought that Group IIb WRKY genes first appeared in mosses. We found WRKY transcription factor genes outside of the plant lineage in some diplomonads, social amoebae, fungi incertae sedis, and amoebozoa. This patchy distribution suggests that lateral gene transfer is responsible. These lateral gene transfer events appear to pre-date the formation of the WRKY groups in flowering plants. Flowering plants contain proteins with domains typical for both resistance (R) proteins and WRKY transcription factors. R protein-WRKY genes have evolved numerous times in flowering plants, each type being restricted to specific flowering plant lineages. These chimeric proteins contain not only novel combinations of protein domains but also novel combinations and numbers of WRKY domains. Once formed, R protein WRKY genes may combine different components of signalling pathways that may either create new diversity in signalling or accelerate signalling by short circuiting signalling pathways. We propose that the evolution of WRKY transcription factors includes early lateral gene transfers to non-plant organisms and the occurrence of algal WRKY genes that have no counterparts in flowering plants. We propose two alternative hypotheses

Exploring end user adoption and maintenance of a telephone-based physical activity counseling service for individuals with physical disabilities using the Theoretical Domains Framework.

Science.gov (United States)

Tomasone, Jennifer R; Arbour-Nicitopoulos, Kelly P; Pila, Eva; Lamontagne, Marie-Eve; Cummings, Isabelle; Latimer-Cheung, Amy E; Routhier, François

2017-06-01

In Canada, two counseling services are offered to facilitate physical activity participation among persons with physical disabilities, yet both have encountered concerns related to the recruitment and retainment of clients. The purpose of this paper is to explore factors related to service adoption among nonusers, and the barriers and facilitators to maintaining service participation among adopters. Individuals who had never enrolled in the services (nonusers, n = 13) as well as current/previous service clients (adopters, n = 26) participated in interviews based on the Theoretical Domains Framework. Transcripts were subjected to deductive thematic analysis according to participant group. Fifteen themes relating to service adoption within 10 of the 12 theoretical domains were identified for nonusers, while 23 themes relating to maintenence of service participation were identified across all 12 theoretical domains for adopters. The findings provide strategies to improve recruitment, adoption, and retention of clients in counseling services and to enhance the experiences of targeted service users. Implications for Rehabiliation Peer support and education for equipment use should be built into physical activity programs to encourage participation among persons with physical disabilities. Programs that encourage physical activity among individuals with disabilities should be designed by practitioners to be responsive to a variety of needs, which are addressed in the program's advertisements and offerings. The Theoretical Domains Framework is a useful framework for providing valuable insight about clients' experiences of adoption and maintenance of a behavior change service, suggesting merit in other rehabilitation settings.

Activity-Centered Domain Characterization for Problem-Driven Scientific Visualization.

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Marai, G Elisabeta

2018-01-01

Although visualization design models exist in the literature in the form of higher-level methodological frameworks, these models do not present a clear methodological prescription for the domain characterization step. This work presents a framework and end-to-end model for requirements engineering in problem-driven visualization application design. The framework and model are based on the activity-centered design paradigm, which is an enhancement of human-centered design. The proposed activity-centered approach focuses on user tasks and activities, and allows an explicit link between the requirements engineering process with the abstraction stage-and its evaluation-of existing, higher-level visualization design models. In a departure from existing visualization design models, the resulting model: assigns value to a visualization based on user activities; ranks user tasks before the user data; partitions requirements in activity-related capabilities and nonfunctional characteristics and constraints; and explicitly incorporates the user workflows into the requirements process. A further merit of this model is its explicit integration of functional specifications, a concept this work adapts from the software engineering literature, into the visualization design nested model. A quantitative evaluation using two sets of interdisciplinary projects supports the merits of the activity-centered model. The result is a practical roadmap to the domain characterization step of visualization design for problem-driven data visualization. Following this domain characterization model can help remove a number of pitfalls that have been identified multiple times in the visualization design literature.

Control of plant phosphate homeostasis by inositol pyrophosphates and the SPX domain.

Science.gov (United States)

Jung, Ji-Yul; Ried, Martina K; Hothorn, Michael; Poirier, Yves

2018-02-01

Proteins containing a SPX domain are involved in phosphate (Pi) homeostasis, including Pi transport and adaptation to Pi deficiency. The SPX domain harbors a basic surface binding Pi at low affinity and inositol pyrophosphates (PP-InsPs) at high affinity. Genetic and biochemical studies revealed that PP-InsPs serve as ligands for the SPX domain. Residues in the PHO1 SPX domain involved in PP-InsPs binding are critical for its Pi export activity, and the interaction between SPX proteins and the PHR1 transcription factor, which results in PHR1 inactivation, is promoted by PP-InsPs. Changes in PP-InsPs levels in response to Pi deficiency may thus contribute to the adaptation of plants to stress via the modulation of the activity of SPX-containing proteins and their interactors. Modulating PP-InsP levels or the affinity/specificity of the SPX domain for PP-InsP could potentially be used to engineer crops to maintain high yield under reduced Pi fertilizer input. Copyright © 2017 Elsevier Ltd. All rights reserved.

ERG induces epigenetic activation of Tudor domain-containing protein 1 (TDRD1) in ERG rearrangement-positive prostate cancer.

Science.gov (United States)

Kacprzyk, Lukasz A; Laible, Mark; Andrasiuk, Tatjana; Brase, Jan C; Börno, Stefan T; Fälth, Maria; Kuner, Ruprecht; Lehrach, Hans; Schweiger, Michal R; Sültmann, Holger

2013-01-01

Overexpression of ERG transcription factor due to genomic ERG-rearrangements defines a separate molecular subtype of prostate tumors. One of the consequences of ERG accumulation is modulation of the cell's gene expression profile. Tudor domain-containing protein 1 gene (TDRD1) was reported to be differentially expressed between TMPRSS2:ERG-negative and TMPRSS2:ERG-positive prostate cancer. The aim of our study was to provide a mechanistic explanation for the transcriptional activation of TDRD1 in ERG rearrangement-positive prostate tumors. Gene expression measurements by real-time quantitative PCR revealed a remarkable co-expression of TDRD1 and ERG (r(2) = 0.77) but not ETV1 (r(2)prostate cancer in vivo. DNA methylation analysis by MeDIP-Seq and bisulfite sequencing showed that TDRD1 expression is inversely correlated with DNA methylation at the TDRD1 promoter in vitro and in vivo (ρ = -0.57). Accordingly, demethylation of the TDRD1 promoter in TMPRSS2:ERG-negative prostate cancer cells by DNA methyltransferase inhibitors resulted in TDRD1 induction. By manipulation of ERG dosage through gene silencing and forced expression we show that ERG governs loss of DNA methylation at the TDRD1 promoter-associated CpG island, leading to TDRD1 overexpression. We demonstrate that ERG is capable of disrupting a tissue-specific DNA methylation pattern at the TDRD1 promoter. As a result, TDRD1 becomes transcriptionally activated in TMPRSS2:ERG-positive prostate cancer. Given the prevalence of ERG fusions, TDRD1 overexpression is a common alteration in human prostate cancer which may be exploited for diagnostic or therapeutic procedures.

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...

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

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