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At least 487 records · Page 27Linked to original sources
PubMed2003

The roles of auxin response factor domains in auxin-responsive transcription.

Auxin response factors (ARFs) are transcription factors that bind to TGTCTC auxin response elements in promoters of early auxin response genes. ARFs have a conserved N-terminal DNA binding domain (DBD) and in most cases a conserved C-terminal dimerization domain (CTD). The ARF CTD is related in amino acid sequence to motifs III and IV found in Aux/IAA proteins. Just C terminal to the DBD, ARFs contain a nonconserved region referred to as the middle region (MR), which has been proposed to function as a transcriptional repression or activation domain. Results with transfected protoplasts reported here show that ARFs with Q-rich MRs function as activators, whereas most, if not all other ARFs, function as repressors. ARF DBDs alone are sufficient to recruit ARFs to their DNA target sites, and auxin does not influence this recruitment. ARF MRs alone function as activation or repression domains when targeted to reporter genes via a yeast Gal4 DBD, and auxin does not influence the potency of activation or repression. ARF CTDs, along with a Q-rich MR, are required for an auxin response whether the MRs plus CTDs are recruited to a promoter by an ARF DBD or by a Gal4 DBD. The auxin response is mediated by the recruitment of Aux/IAA proteins to promoters that contain a DNA binding protein with a Q-rich MR and an attached CTD.

Arabidopsis Proteins↗
PubMed1998

A domain necessary for the transforming activity of SnoN is required for specific DNA binding, transcriptional repression and interaction with TAF(II)110.

sno is a member of the ski oncogene family and shares ski's ability to transform avian fibroblasts and induce muscle differentiation. Ski and Sno are nuclear proteins that form homodimers and heterodimers. Ski activates transcription of cellular and viral enhancers and we have identified a DNA binding site (GTCTAGAC) through which it represses transcription. In this work, we show that SnoN binds this site and represses transcription of reporters with this binding site as an upstream element. Using fusions with the Gal4-DNA binding domain in a heterologous reporter assay, we identify a tripartite repression domain in SnoN. A 107 amino acid stretch of the SnoN repression domain, that contains two of the subdomains, is closely related to the minimal region of Ski required for transformation. The third subdomain is unique to SnoN. By analysing deletions involving each of the subdomains, we show that subdomains II and III are also required for DNA binding and cellular transformation. We provide evidence for a quenching mechanism of transcriptional repression by which subdomain II binds to TAF(II)110.

Animals↗
PubMed1997

Repression of the heat shock factor 1 transcriptional activation domain is modulated by constitutive phosphorylation.

Heat shock transcription factor 1 (HSF1) is constitutively expressed in mammalian cells and negatively regulated for DNA binding and transcriptional activity. Upon exposure to heat shock and other forms of chemical and physiological stress, these activities of HSF1 are rapidly induced. In this report, we demonstrate that constitutive phosphorylation of HSF1 at serine residues distal to the transcriptional activation domain functions to repress transactivation. Tryptic phosphopeptide analysis of a collection of chimeric GAL4-HSF1 deletion and point mutants identified a region of constitutive phosphorylation encompassing serine residues 303 and 307. The significance of phosphorylation at serines 303 and 307 in the regulation of HSF1 transcriptional activity was demonstrated by transient transfection and assay of a chloramphenicol acetyltransferase reporter construct. Whereas the transfected wild-type GAL4-HSF1 chimera is repressed for transcriptional activity and derepressed by heat shock, mutation of serines 303 and 307 to alanine results in derepression to a high level of constitutive activity. Similar results were obtained with mutation of these serine residues in the context of full-length HSF1. These data reveal that constitutive phosphorylation of serines 303 and 307 has an important role in the negative regulation of HSF1 transcriptional activity at control temperatures.

3T3 Cells↗
PubMed2004

SMAR1 and Cux/CDP modulate chromatin and act as negative regulators of the TCRbeta enhancer (Ebeta).

Chromatin modulation at various cis-acting elements is critical for V(D)J recombination during T and B cell development. MARbeta, a matrix-associated region (MAR) located upstream of the T cell receptor beta (TCRbeta) enhancer (Ebeta), serves a crucial role in silencing Ebeta-mediated TCR activation. By DNaseI hypersensitivity assays, we show here that overexpression of the MAR binding proteins SMAR1 and Cux/CDP modulate the chromatin structure at MARbeta. We further demonstrate that the silencer function of MARbeta is mediated independently by SMAR1 and Cux/CDP as judged by their ability to repress Ebeta-dependent reporter gene expression. Moreover, the repressor activity of SMAR1 is strongly enhanced in the presence of Cux/CDP. These two proteins physically interact with each other and colocalize within the perinuclear region through a SMAR1 domain required for repression. The repression domain of SMAR1 is separate from the MARbeta binding domain and contains a nuclear localization signal and an arginine-serine (RS)-rich domain, characteristic of pre-mRNA splicing regulators. Our data suggest that at the double positive stage of T cell development, cis-acting MARbeta elements recruit the strong negative regulators Cux and SMAR1 to control Ebeta-mediated recombination and transcription.

Amino Acid Sequence↗
PubMed2004

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

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.

DNA-Binding Proteins↗
PubMed1999

A novel paired domain DNA recognition motif can mediate Pax2 repression of gene transcription.

The paired domain (PD) is an evolutionarily conserved DNA-binding domain encoded by the Pax gene family of developmental regulators. The Pax proteins are transcription factors and are involved in a variety of processes such as brain development, patterning of the central nervous system (CNS), and B-cell development. In this report we demonstrate that the zebrafish Pax2 PD can interact with a novel type of DNA sequences in vitro, the triple-A motif, consisting of a heptameric nucleotide sequence G/CAAACA/TC with an invariant core of three adjacent adenosines. This recognition sequence was found to be conserved in known natural Pax5 repressor elements involved in controlling the expression of the p53 and J-chain genes. By identifying similar high affinity binding sites in potential target genes of the Pax2 protein, including the pax2 gene itself, we obtained further evidence that the triple-A sites are biologically significant. The putative natural target sites also provide a basis for defining an extended consensus recognition sequence. In addition, we observed in transformation assays a direct correlation between Pax2 repressor activity and the presence of triple-A sites. The results suggest that a transcriptional regulatory function of Pax proteins can be modulated by PD binding to different categories of target sequences.

5' Untranslated Regions↗
PubMed1999

Net, a negative Ras-switchable TCF, contains a second inhibition domain, the CID, that mediates repression through interactions with CtBP and de-acetylation.

Signalling cascades are integrated at the transcriptional level by the interplay between factors such as the ternary complex factors (TCFs) that interact with serum response factor (SRF) and the serum response element (SRE) of the fos promoter. Net is a negative TCF that is switched to a positive regulator by the Ras signal. To understand the mechanisms of repression by Net, we used a yeast two-hybrid screen to identify factors that interact with its inhibitory domain. We isolated mCtBP1, the murine homologue of huCtBP1, a factor implicated in negative regulation of transformation by E1A plus Ras. We show that mCtBP1 interacts strongly with Net both in vitro and in vivo. The CtBP interaction domain of Net, the CID, mediates repression independently of the previously identified negative element, the NID. The CID inhibits by recruiting the co-repressor mCtBP1. The CID and mCtBP1 need to use de-acetylase activity for repression, whereas the NID apparently represses by other mechanisms. Finally, we provide evidence that CtBP and de-acetylation repress the c-fos SRE in low serum when it is inactive, but not in high serum when it is active. These results provide insights into the cross-talk between pathways that inhibit and stimulate transformation at the level of Net, a regulator of gene expression.

Acetylation↗
PubMed2004

Negative cross-talk between the human orphan nuclear receptor Nur77/NAK-1/TR3 and nuclear factor-kappaB.

The effect of orphan nuclear receptor Nur77 overexpression on activation of an interleukin-2 (IL-2) promoter-luciferase construct was analyzed in the human leukemic cell line Jurkat. Cotransfection of the IL-2 promoter construct together with the Nur77 expression plasmid resulted in a significant repression of IL-2 promoter activation compared to control cells. The repression by Nur77 requires the N-terminal activation function-1 domain. The repressive effect of Nur77 on IL-2 promoter activation is mediated through inhibition of the transcription factor complex nuclear factor-kappaB (NF-kappaB), since blocking or alteration of the IL-2 NF-kappaB binding sites resulted in abrogation of the repressive effect of Nur77. Moreover, further examination of a reporter gene construct containing multiple copies of the IL-2 CD28 response element (CD28RE) showed that Nur77 can inhibit transactivation mediated by the NF-kappaB components p65 and c-Rel. However, no effect of Nur77 was seen on p65-mediated transactivation of a construct containing multiple NF-kappaB binding sites of the HIV LTR. Our data suggest that Nur77 is able to block activation through NF-kappaB when bound to low-affinity NF-kappaB binding sites, such as those located in the IL-2 promoter.

Amino Acid Sequence↗
PubMed2004

The p75NTR-interacting protein SC1 inhibits cell cycle progression by transcriptional repression of cyclin E.

Schwann cell factor 1 (SC1), a p75 neurotrophin receptor-interacting protein, is a member of the positive regulatory/suppressor of variegation, enhancer of zeste, trithorax (PR/SET) domain-containing zinc finger protein family, and it has been shown to be regulated by serum and neurotrophins. SC1 shows a differential cytoplasmic and nuclear distribution, and its presence in the nucleus correlates strongly with the absence of bromodeoxyuridine (BrdU) in these nuclei. Here, we investigated potential transcriptional activities of SC1 and analyzed the function of its various domains. We show that SC1 acts as a transcriptional repressor when it is tethered to Gal4 DNA-binding domain. The repressive activity requires a trichostatin A-sensitive histone deacetylase (HDAC) activity, and SC1 is found in a complex with HDACs 1, 2, and 3. Transcriptional repression exerted by SC1 requires the presence of its zinc finger domains and the PR domain. Additionally, these two domains are involved in the efficient block of BrdU incorporation by SC1. The zinc finger domains are also necessary to direct SC1's nuclear localization. Lastly, SC1 represses the promoter of a promitotic gene, cyclin E, suggesting a mechanism for how growth arrest is regulated by SC1.

3T3 Cells↗
PubMed2003

Protein inhibitor of activated signal transducer and activator of transcription 1 interacts with the N-terminal domain of mineralocorticoid receptor and represses its transcriptional activity: implication of small ubiquitin-related modifier 1 modification.

Molecular mechanisms underlying mineralocorticoid receptor (MR)-mediated gene expression are not fully understood but seem to largely depend upon interactions with specific coregulators. To identify novel human MR (hMR) molecular partners, yeast two-hybrid screenings performed using the N-terminal domain as bait, allowed us to isolate protein inhibitor of activated signal transducer and activator of transcription (PIAS)1 and PIASxbeta, described as SUMO (small ubiquitin-related modifier) E3-ligases. Specific interaction between PIAS1 and hMR was confirmed by glutathione-S-transferase pull-down experiments and N-terminal subdomains responsible for physical contacts were delineated. Transient transfections demonstrated that PIAS1 is a corepressor of aldosterone-activated MR transactivation but has no significant effect on human glucocorticoid receptor transactivation. The agonist or antagonist nature of the bound ligand also determines PIAS1 corepressive action. We provided evidence that PIAS1 conjugated SUMO-1 to hMR both in vitro and in vivo. Deciphering the unique sumoylation pattern of hMR, which possesses five consensus SUMO-1 binding sites, by combinatorial lysine substitutions, revealed a major impact of sumoylation on hMR properties. Using a murine mammary tumor virus promoter, PIAS1 action was independent of sumoylation whereas with glucocorticoid response element promoter, PIAS1 corepressive action depended on hMR sumoylation status. Taken together, our results identify a novel function for PIAS1 which interacts with the N-terminal domain of hMR and represses its ligand-dependent transcriptional activity, at least in part, through SUMO modifications.

Base Sequence↗
PubMed2003

Regulation of MSV and WDV virion-sense promoters by WDV nonstructural proteins: a role for their retinoblastoma protein-binding motifs.

In this work we demonstrate that wheat dwarf virus (WDV) RepA can activate WDV and maize streak virus (MSV) virion (V)-sense expression in plant tissues. Rep alone does not have any effect on the silent WDV promoter and it represses the basal MSV promoter activity. MSV promoter activation by RepA depends on an intact RepA retinoblastoma protein (RB)-binding domain. Promoter repression by Rep also depends on this domain to some extent. Mutation of the RepA RB-binding domain has no effect on WDV promoter activation. The WDV promoter contains two sites that fit the consensus E2F-binding site. One, WDV1, binds human E2F-1 in one-hybrid assays in yeast. It also binds specifically to maize and wheat proteins in vitro and, when fused to a minimal 35S promoter, it confers responsiveness to RepA only when the RepA RB-binding domain and the WDV1 site are intact. In the whole WDV V-sense promoter context, mutations of this sequence have no effect, suggesting that additional sequences are important for RepA-mediated promoter activation.

Amino Acid Motifs↗
PubMed2001

Identification of SCAN dimerization domains in four gene families.

Zinc-finger transcription factors are often accompanied by modular sequence motifs such as the Kruppel-associated box (KRAB) and the SCAN domain. The KRAB domain mediates transcriptional repression while the SCAN domain mediates selective protein dimerization. The hypoalphalipoproteinemia susceptibility gene ZNF202 encodes a SCAN box and a KRAB domain followed by eight Cys2-His2 zinc-finger motifs. In order to identify the existence of genes which encode proteins of structural homology to ZNF202, a mouse lambda library was screened with a human ZNF202 cDNA probe. The isolated cDNA clones represented three SCAN-domain-encoding gene families. We purified three novel cDNAs that encode a SCAN-KRAB-(Cys2-His2)x domain alignment and one cDNA that encodes a SCAN-(Cys2-His2)x domain alignment. In addition, we identified one cDNA sequence with a predicted protein sequence containing a KRAB-SCAN-KRAB-(Cys2-His2)x domain alignment. Therefore, when combined with the recently discovered family of isolated SCAN-domain-encoding genes, four SCAN domain gene families can be distinguished. The consensus sequences for the murine SCAN and KRAB domains are highly conserved within the mammalian phylogenetic tree which may be useful in elucidating the biological function of these protein modules and the crucial residues responsible for their binding specificity.

Amino Acid Sequence↗
PubMed1998

Aromatic ligand binding and intramolecular signalling of the phenol-responsive sigma54-dependent regulator DmpR.

The Pseudomonas-derived sigma54-dependent regulator DmpR has an amino-terminal A-domain controlling the specificity of activation by aromatic effectors, a central C-domain mediating an ATPase activity essential for transcriptional activation and a carboxy-terminal D-domain involved in DNA binding. In the presence of aromatic effectors, the DmpR protein promotes transcription from the -24, -12 Po promoter controlling the expression of specialized (methyl)phenol catabolic enzymes. Previous analysis of DmpR has led to a model in which the A-domain acts as an interdomain repressor of DmpR's ATPase and transcriptional promoting property until specific aromatic effectors are bound. Here, the autonomous nature of the A-domain in exerting its biological functions has been dissected by expressing portions of DmpR as independent polypeptides. The A-domain of DmpR is shown to be both necessary and sufficient to bind phenol. Analysis of phenol binding suggests one binding site per monomer of DmpR, with a dissociation constant of 16 microM. The A-domain is also shown to have specific affinity for the C-domain and to repress the C-domain mediated ATPase activity in vitro autonomously. However, physical uncoupling of the A-domain from the remainder of the regulator results in a system that does not respond to aromatics by its normal derepression mechanism. The mechanistic implications of aromatic non-responsiveness of autonomously expressed A-domain, despite its demonstrated ability to bind phenol, are discussed.

Adenosine Triphosphatases↗
PubMed2003

Yeast Ume6p repressor permits activator binding but restricts TBP binding at the HOP1 promoter.

Ume6p plays essential roles in the regulation of early meiotic genes in Saccharomyces cerevisiae. Ume6p exerts repression via recruitment of the Sin3p-Rpd3p histone deacetylase and Isw2p chromatin remodeling complexes. The transcriptional step that is ultimately inhibited by Ume6p is unknown. Here, in vivo footprinting shows that transcriptional activators Hap1p and Abf1p occupy upstream sites in repressed and derepressed promoters. In contrast, chromatin immunoprecipitation shows that TATA box-binding protein (TBP)- promoter binding is reduced upon repression of HOP1. Fusion of TBP to a zinc cluster DNA binding domain relieves repression at a HOP1 promoter modified to include the zinc cluster target site. We suggest that TBP binding is inhibited through chromatin modification by the Sin3p-Rpd3p and Isw2p complexes recruited by Ume6p.

Binding Sites↗
PubMed2003

Neurogenin3 activates the islet differentiation program while repressing its own expression.

Expression of the proendocrine factor Neurogenin3 determines which progenitor cells in the developing pancreas will differentiate into the endocrine cells of the islets of Langerhans. To better understand how Neurogenin3 directs endocrine differentiation, we examined the mechanisms by which Neurogenin3 regulates the promoters of three transcription factor genes expressed in endocrine precursor cells: the nkx2.2 gene, the PAX4 gene, and the NEUROG3 gene, the human gene encoding Neurogenin3 itself. The function of all three promoters depends on at least one critical E box, a common DNA sequence that forms a binding site for basic helix-loop-helix proteins like Neurogenin3. Neurogenin3 bound to and effectively activated transcription through the nkx2.2 and PAX4 E boxes. In contrast, Neurogenin3 strongly repressed the NEUROG3 promoter, although a proximal E box was required for activity in the absence of Neurogenin3, suggesting that a ubiquitous transcriptional activator may bind to this site, and that Neurogenin3 could act as a competitive inhibitor of this activator. This hypothesis was supported by the lack of evidence for significant intrinsic transcriptional repression capacity in the Neurogenin3 protein, and by the ability of isolated DNA-binding basic helix-loop-helix domains to repress the NEUROG3 promoter. Neurogenin3 produced additional repression, however, when the protein included an intact transcriptional activation domain, suggesting that it may also induce the expression of a downstream transcriptional repressor. In summary, while Neurogenin3 orchestrates islet cell differentiation by activating islet cell transcription factor genes, it simultaneously represses its own promoter.

3T3 Cells↗
PubMed2001

Transcriptional repression mediated by the KRAB domain of the human C2H2 zinc finger protein Kox1/ZNF10 does not require histone deacetylation.

The KRAB domain of human Kox1, a member of the KRAB C2H2 zinc finger family, confers strong transcriptional repressor activities even to remote promoter positions. Here, HDAC inhibitors were used to demonstrate that histone deacetylation is not required for mediating transcriptional repression of KRAB zinc finger proteins. Two reporter systems with either stably integrated or transiently transfected templates, both under control of strong viral promoters, were analyzed. Under all circumstances, HDAC inhibition did not alter the repression potential of the KRAB domain. In case of the stably integrated luciferase reporter gene system, neither expression levels of the KRAB fusion protein nor complex formation with its putative co-repressor TIF1beta were significantly changed. Furthermore, the TIF1beta/KRAB complex was devoid of mSin3A and HDAC1. In the transient transfection system, the transcriptional repression induced by TIF1beta and HP1alpha was not diminished by HDAC inhibitors, whereas the repressory activity of TIF1alpha was significantly affected. Thus, KRAB, TIF1beta and HP1alpha are likely to be functionally linked. In conclusion, HDAC activity is not essential for the strong transcriptional repressor activity mediated by the KRAB domain of Kox1 in particular and, presumably, by KRAB domains in general. This feature might be helpful in identifying and characterizing target genes under the control of

Chromobox Protein Homolog 5↗
PubMed1996

Transcriptional repression by the proto-oncogene BCL-6.

In up to 45% of reported cases of the non-Hodgkin's lymphoma, diffuse large cell lymphoma, there are translocations of the BCL-6 gene, which are presumed to deregulate its expression. The BCL-6 protein, which is unmutated in these lymphomas, contains six Krüppel-like zinc fingers at its carboxy terminus and a 121 amino acid domain at its amino terminus, termed the POZ domain, which bears homology with amino terminal domains in a subset zinc finger transcription factors. In this study, we tested whether BCL-6 regulates transcription and if the POZ domain has a role in this function. The BCL-6 POZ domain, when fused to the GAL4 DNA binding domain, strongly repressed transcriptional activation initiated from several different promoters including the SV40 enhancer/promoter. Repression was also observed when the fusion protein was bound at a distance of 200 bp 5' of the promoter. When the GAL4/BCL6 POZ domain fusion protein was expressed in yeast, it was able to homodimerize in the nucleus. Nevertheless, in contrast with mammalian cells, the fusion protein did not repress transcription. To test the ability of the full length BC1-6 protein to repress transcription when bound to DNA through its zinc finger DNA binding domain, high affinity BCL-6 binding sites were selected from a pool of random oligonucleotides. Full length BCL-6 was able to strongly repress transcription when bound to its cognate site cloned upstream of the thymidine kinase promoter. This repression was mediated, in large measure, by the POZ domain, although a variant of BCL-6 lacking the POZ domain was able to repress transcription modestly. The ability of BCL-6 to function as a transcriptional repressor may contribute to its ability to transform B lymphocytes in diffuse large cell lymphoma.

Base Sequence↗
PubMed1993

A structure-function analysis of transcriptional repression mediated by the WT1, Wilms' tumor suppressor protein.

The chromosome 11p13 Wilms' tumor locus (wt1) encodes a zinc finger-containing transcription factor (WT1). WT1 binds to the consensus sequence (5'-GCGGGGGCG-3') and represses transcription when bound to this site in vivo. The mechanism of repression is not yet defined. To investigate the mechanisms of transcriptional repression and map the domains of WT1 responsible, we constructed hybrid proteins between the yeast GAL4 1-147 DNA binding domain and WT1. Fusion of a 298 amino acid glutamine-proline-rich N-terminal segment of WT1 to the GAL4 DNA binding domain created a potent transcriptional repressor. The use of N- and C-terminal truncations of this segment demonstrated that as few as 96 amino acids were required for active repression by GAL4-WT1 hybrid proteins in NIH3T3 fibroblasts. However, the truncated GAL4-WT1 fusion proteins functioned poorly as repressors in embryonic kidney-derived 293 cells, suggesting cell type-specific requirements for transcriptional repression. Site-directed mutagenesis of the WT1 repression domain revealed that deletion of homopolymeric proline and glycine regions, as well as single amino acid changes, partially inactivated the repression function. Single repressor binding sites placed upstream of the transcription start site conferred WT1-mediated repression to a heterologous promoter, whereas multiple sites resulted in additive (non-synergistic) increases in transcriptional repression. Significant repression of transcription was observed when binding sites were placed 760 base pairs upstream or 1000 base pairs downstream relative to the site of transcription initiation. We conclude that the transcriptional repression function of WT1 is contained in the N-terminal, non-DNA binding domain of the protein and that repression can be functionally transferred to a heterologous DNA binding domain.

3T3 Cells↗