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At least 1,315 records · Page 73Linked to original sources
PubMed2000

Beta-cell differentiation factor Nkx6.1 contains distinct DNA binding interference and transcriptional repression domains.

beta-Cell differentiation factor Nkx6.1 is a homeodomain protein expressed in developing and mature beta-cells in the pancreatic islets of Langerhans. To understand how it contributes to beta-cell development and function, we characterized its DNA binding and transactivation properties. A single copy of the homeodomain of Nkx6. 1 binds to a strictly conserved 8-base pair DNA consensus sequence, TTAATTAC; even minor variations to this consensus reduce DNA binding affinity significantly. Full-length Nkx6.1, however, has markedly reduced DNA binding affinity due to an acidic domain at the carboxyl end of the molecule that functions as a mobile binding interference domain capable of interrupting the interaction between DNA and DNA binding domains of the helix-turn-helix type. When expressed in fibroblast cell lines, Nkx6.1 represses transcription through isolated Nkx6.1 binding sites; in beta-cell lines, Nkx6.1 specifically represses the intact insulin promoter through TAAT-containing sequences. In Gal4 one-hybrid fusion studies, transcriptional repression maps to a discreet region within the amino terminus. Our findings suggest a model in which Nkx6.1, regulated by interactions through its carboxyl terminus, directs the repression of specific genes in developing and mature beta-cells.

3T3 Cells↗
PubMed2000

The human tissue inhibitor of metalloproteinases (TIMP)-1 gene contains repressive elements within the promoter and intron 1.

Expression of the TIMP-1 (tissue inhibitor of metalloproteinases-1) gene is tightly controlled during embryonic development and in the adult animal. Previous studies have focused on elements within the gene promoter which activate transcription of the gene. Here, we identify two regions of the gene which repress transcription: An element upstream of the basal gene promoter at -1718/-1458, represses expression of a reporter gene by approximately 50%; addition of the first intron to any promoter-reporter construct also strongly represses gene expression. The TIMP-1 gene has a short first exon which is transcribed but not translated, with the translation start site located in exon 2. Deletion analysis through intron 1 reveals a number of potential regions which might mediate its effect. Protein binding studies and mutational analyses reveal that a repressive element at +684/+748 binds Sp1, Sp3, and an unidentified Ets-related factor to suppress transcription.

Base Sequence↗
PubMed2000

Regulation of Pap phase variation. Lrp is sufficient for the establishment of the phase off pap DNA methylation pattern and repression of pap transcription in vitro.

The pyelonephritis-associated pili (pap) operon in Escherichia coli is regulated by an epigenetic mechanism involving the formation of specific DNA methylation patterns characteristic of transcriptionally active (phase ON) and inactive (phase OFF) cells. The formation of pap DNA methylation patterns in vivo was previously shown to require the leucine-responsive regulatory protein (Lrp) and DNA adenine methylase (Dam). To monitor the binding of Lrp to pap DNA, an in vitro methylation protection assay was developed. Binding of Lrp to a Dam target site proximal to the papBA promoter (designated GATC(prox)) blocked methylation of this site and specifically repressed transcription. The DNA methylation pattern and transcription state are identical to those observed in vivo in phase OFF cells. To determine if binding of Lrp at GATC(prox) was necessary for repression of papBA transcription, we analyzed a pap mutation (pap-13) that reduced the affinity of Lrp for the GATC(prox) region. Binding of Lrp to pap-13 DNA was shifted to a promoter distal Dam target site (designated GATC(dist)). Lrp blocked methylation of GATC(dist) in the pap-13 mutant, but did not repress papBA transcription. Together, these results show that binding of Lrp to the GATC(prox) region is sufficient for the establishment of the phase OFF DNA methylation pattern and repression of papBA transcription.

Bacterial Adhesion↗
PubMed2003

Direct interaction of Ski with either Smad3 or Smad4 is necessary and sufficient for Ski-mediated repression of transforming growth factor-beta signaling.

The oncoprotein Ski represses transforming growth factor (TGF)-beta signaling in an N-CoR-independent manner. However, the molecular mechanism(s) underlying this event has not been elucidated. Here, we identify an additional domain in Ski that mediates interaction with Smad3 which is important for this repression. This domain is distinct from the previously reported N-terminal Smad3 binding domain in Ski. Individual alanine substitution of several residues in the domain significantly affected Ski-Smad3 interaction. Furthermore, combined mutations within this domain, together with those in the previously identified Smad3 binding domain, can completely abolish the interaction of Ski with Smad3, while mutation in each domain alone retained partial interaction. By introducing those mutations that abolish direct interaction with Smad3 or Smad4 individually, or in combination, we show that interaction of Ski with either Smad3 or Smad4 is sufficient for Ski-mediated repression of TGF-beta signaling. Furthermore our results clearly demonstrate that Ski does not disrupt Smad3-Smad4 heteromer formation, and recruitment of Ski to the Smad3/4 complex through binding to either Smad3 or Smad4 is both necessary and sufficient for repression.

Amino Acid Sequence↗
PubMed2000

The cyclin D1 gene is transcriptionally repressed by caveolin-1.

The cyclin D1 gene encodes the regulatory subunit of the holoenzyme that phosphorylates and inactivates the retinoblastoma pRB protein. Cyclin D1 protein levels are elevated by mitogenic and oncogenic signaling pathways, and antisense mRNA to cyclin D1 inhibits transformation by the ras, neu, and src oncogenes, thus linking cyclin D1 regulation to cellular transformation. Caveolins are the principal protein components of caveolae, vesicular plasma membrane invaginations that also function in signal transduction. We show here that caveolin-1 expression levels inversely correlate with cyclin D1 abundance levels in transformed cells. Expression of antisense caveolin-1 increased cyclin D1 levels, whereas caveolin-1 overexpression inhibited expression of the cyclin D1 gene. Cyclin D1 promoter activity was selectively repressed by caveolin-1, but not by caveolin-3, and this repression required the caveolin-1 N terminus. Maximal inhibition of the cyclin D1 gene promoter by caveolin-1 was dependent on the cyclin D1 promoter T-cell factor/lymphoid enhancer factor-1-binding site between -81 to -73. The T-cell factor/lymphoid enhancer factor sequence was sufficient for repression by caveolin-1. We suggest that transcriptional repression of the cyclin D1 gene may contribute to the inhibition of transformation by caveolin-1.

Amino Acid Sequence↗
PubMed2000

A functionally conserved N-terminal domain of the friend of GATA-2 (FOG-2) protein represses GATA4-dependent transcription.

GATA4 is a transcriptional activator of cardiac-restricted promoters and is required for normal cardiac morphogenesis. Friend of GATA-2 (FOG-2) is a multizinc finger protein that associates with GATA4 and represses GATA4-dependent transcription. To better understand the transcriptional repressor activity of FOG-2 we performed a functional analysis of the FOG-2 protein. The results demonstrated that 1) zinc fingers 1 and 6 of FOG-2 are each capable of interacting with evolutionarily conserved motifs within the N-terminal zinc finger of mammalian GATA proteins, 2) a nuclear localization signal (RKRRK) (amino acids 736-740) is required to program nuclear targeting of FOG-2, and 3) FOG-2 can interact with the transcriptional co-repressor, C-terminal-binding protein-2 via a conserved sequence motif in FOG-2 (PIDLS). Surprisingly, however, this interaction with C-terminal-binding protein-2 is not required for FOG-2-mediated repression of GATA4-dependent transcription. Instead, we have identified a novel N-terminal domain of FOG-2 (amino acids 1-247) that is both necessary and sufficient to repress GATA4-dependent transcription. This N-terminal repressor domain is functionally conserved in the related protein, Friend of GATA1. Taken together, these results define a set of evolutionarily conserved mechanisms by which FOG proteins repress GATA-dependent transcription and thereby form the foundation for genetic studies designed to elucidate the role of FOG-2 in cardiac development.

3T3 Cells↗
PubMed2000

Transcriptional activation by the Ewing's sarcoma (EWS) oncogene can be cis-repressed by the EWS RNA-binding domain.

The Ewing's sarcoma (EWS) oncogene contains an N-terminal transcriptional activation domain (EWS activation domain, EAD) and a C-terminal RNA-binding domain (RBD). Although it has been established that the EAD is a potent trans-activation domain that is required for the oncogenic activity of several EWS fusion proteins (EFPs), the precise function of the RBD and the normal role of intact EWS are poorly characterized. Here we show that a cis-linked RBD can strongly and specifically repress trans-activation by the EAD. Fusion proteins containing the RBD are expressed at normal levels, are nuclear-localized, and can bind to DNA both in vitro and in vivo, demonstrating that the RBD represses trans-activation directly at the promoter. The RNA recognition motif within the RBD is not required for repression, whereas regions of the RBD containing multiple RGG motifs play a critical role. The finding that the RBD can antagonize transcriptional activation by EWS provides the first direct evidence of a role for the RBD in transcription. Further studies of the repression phenomenon should illuminate key molecular interactions that distinguish EWS from EFPs and provide insights into the normal cellular function of EWS.

Cell Line↗
PubMed2000

Drosophila C-terminal binding protein functions as a context-dependent transcriptional co-factor and interferes with both mad and groucho transcriptional repression.

Drosophila C-terminal binding protein (dCtBP) and Groucho have been identified as Hairy-interacting proteins required for embryonic segmentation and Hairy-mediated transcriptional repression. While both dCtBP and Groucho are required for proper Hairy function, their properties are very different. As would be expected for a co-repressor, reduced Groucho activity enhances the hairy mutant phenotype. In contrast, reduced dCtBP activity suppresses it. We show here that dCtBP can function as either a co-activator or co-repressor of transcription in a context-dependent manner. The regions of dCtBP required for activation and repression are separable. We find that mSin3A-histone deacetylase complexes are altered in the presence of dCtBP and that dCtBP interferes with both Groucho and Mad transcriptional repression. Similar to CtBP's role in attenuating E1A's oncogenicity, we propose that dCtBP can interfere with corepressor-histone deacetylase complexes, thereby attenuating transcriptional repression. Hairy defines a new class of proteins that requires both CtBP and Groucho co-factors for proper function.

3T3 Cells↗
PubMed2000

Hepatocyte nuclear factor-3 homologue 1 (HFH-1) represses transcription of smooth muscle-specific genes.

Results show that smooth muscle-specific promoters represent novel downstream targets of the winged helix factor hepatocyte nuclear factor-3 homologue 1 (HFH-1). HFH-1 strongly represses telokin promoter activity when overexpressed in A10 vascular smooth muscle cells. HFH-1 was also found to repress transcription of several other smooth muscle-specific promoters, including the SM22alpha promoter. HFH-1 inhibits telokin promoter activity, by binding to a forkhead consensus site located within an AT-rich region of the telokin promoter. The DNA-binding domain alone was sufficient to mediate inhibition, suggesting that binding of HFH-1 blocks the binding of other positive-acting factors. HFH-1 does not disrupt serum response factor binding to an adjacent CArG box within the telokin promoter, implying that HFH-1 must compete with other unidentified trans-activators to mediate repression. The localization of HFH-1 mRNA to the epithelial cell layer of mouse bladder and stomach implicates HFH-1 in repressing telokin expression in epithelial cells. This suggests that cell-specific expression of telokin is likely mediated by both positive-acting factors in smooth muscle cells and negative-acting factors in nonmuscle cell types. We propose a model in which the smooth muscle specificity of the telokin promoter is regulated by interactions between positive- and negative-acting members of the hepatocyte nuclear factor-3/forkhead family of transcription factors.

Animals↗
PubMed2001

Heat shock factor-4 (HSF-4a) represses basal transcription through interaction with TFIIF.

The heat shock transcription factors (HSFs) regulate the expression of heat shock proteins (hsps), which are critical for normal cellular proliferation and differentiation. One of the HSFs, HSF-4, contains two alternative splice variants, one of which possesses transcriptional repressor properties in vivo. This repressor isoform inhibits basal transcription of hsps 27 and 90 in tissue culture cells. The molecular mechanisms of HSF-4a isoform-mediated transcriptional repression is unknown. Here, we present evidence that HSF-4a inhibits basal transcription in vivo when it is artificially targeted to basal promoters via the DNA-binding domain of the yeast transcription factor, GAL4. By using a highly purified, reconstituted in vitro transcription system, we show that HSF-4a represses basal transcription at an early step during preinitiation complex assembly, as pre-assembled preinitiation complexes are refractory to the inhibitory effect on transcription. This repression occurs by the HSF-4a isoform, but not by the HSF-4b isoform, which we show is capable of activating transcription from a heat shock element-driven promoter in vitro. The repression of basal transcription by HSF-4a occurs through interaction with the basal transcription factor TFIIF. TFIIF interacts with a segment of HSF-4a that is required for the trimerization of HSF-4a, and deletion of this segment no longer inhibits basal transcription. These studies suggest that HSF-4a inhibits basal transcription both in vivo and in vitro. Furthermore, this is the first report identifying an interaction between a transcriptional repressor with the basal transcription factor TFIIF.

Cell Line↗
PubMed2001

Translational repression and specific RNA binding by the coat protein of the Pseudomonas phage PP7.

PP7 is a single-strand RNA bacteriophage of Pseudomonas aeroginosa and a distant relative to coliphages like MS2 and Qbeta. Here we show that PP7 coat protein is a specific RNA-binding protein, capable of repressing the translation of sequences fused to the translation initiation region of PP7 replicase. Its RNA binding activity is specific since it represses the translational operator of PP7, but does not repress the operators of the MS2 or Qbeta phages. Conditions for the purification of coat protein and for the reconstitution of its RNA binding activity from disaggregated virus-like particles were established. Its dissociation constant for PP7 operator RNA in vitro was determined to be about 1 nm. Using a genetic system in which coat protein represses translation of a replicase-beta-galactosidase fusion protein, amino acid residues important for binding of PP7 RNA were identified.

Amino Acid Sequence↗
PubMed2001

Negative response elements in keratin genes mediate transcriptional repression and the cross-talk among nuclear receptors.

Very little is known about the mechanisms responsible for the findings that binding of nuclear receptors (NR) to some promoter elements leads to transcriptional activation, whereas binding to others leads to repression. Case in point is the group of epidermal keratin genes and their DNA sequences responsible for repression by NR. Keratin response elements (KREs) interact with receptors for retinoic acid, thyroid hormone, and glucocorticoids. KREs, by their structure and sequence, direct the binding of retinoic acid and thyroid hormone as homodimers and glucocorticoids as monomers. Such specific DNA-receptor interactions are crucial for the repression signal of transcription. In this paper we have analyzed the interactions between the KREs and NR that lead to such repression. We have found that KREs are promoter-independent. They not only provide a docking platform for the receptors, but also play a key role in directing the receptors to bind into particular configurations and coordinating the interactions among different receptors. Both an intact KRE and an intact receptor DNA-binding domain are necessary for the regulation to occur, which emphasizes the importance of interaction between the DNA and NR for proper signaling. Furthermore, KREs allow simultaneous binding of multiple receptors, thus providing fine-tuning of transcriptional regulation. The DNA/DNA-binding domain interactions in keratin promoters exemplify tissue and gene specificity of hormone action.

Base Sequence↗
PubMed2001

TGIF2 interacts with histone deacetylase 1 and represses transcription.

TG-interacting factor (TGIF) is a transcriptional repressor, which represses transcription by binding directly to DNA or interacts with transforming growth factor beta (TGF beta)-activated Smads, thereby repressing TGF beta-responsive gene expression. Mutation of TGIF in humans causes holoprosencephaly, a severe genetic disorder affecting craniofacial development. Searching human expressed sequence tag data bases revealed the presence of clones encoding a TGIF-related protein (TGIF2), which contains two regions of high sequence identity with TGIF. Here we show that, like TGIF, TGIF2 recruits histone deacetylase, but in contrast to TGIF, is unable to interact with the corepressor CtBP. TGIF2 and TGIF have very similar DNA-binding homeodomains, and TGIF2 represses transcription when bound to DNA via a TGIF binding site. TGIF2 interacts with TGF beta-activated Smads and represses TGF beta-responsive transcription. TGIF2 appears to be a context-independent transcriptional repressor, which can perform similar functions to TGIF and may play a role in processes, which, when disrupted by mutations in TGIF, cause holoprosencephaly.

Amino Acid Sequence↗
PubMed2001

p65-activated histone acetyltransferase activity is repressed by glucocorticoids: mifepristone fails to recruit HDAC2 to the p65-HAT complex.

Glucocorticoids acting through their specific receptor can either enhance or repress gene transcription. Dexamethasone represses interleukin-1beta-stimulated histone acetylation and granulocyte-macrophage colony-stimulating factor expression through a combination of direct inhibition of p65-associated histone acetyltransferase (HAT) activity and by recruiting histone deacetylase 2 (HDAC2) to the p65-HAT complex. Here we show that mifepristone, a glucocorticoid receptor partial agonist, has no ability to induce gene expression but represses interleukin-1beta-stimulated histone acetylation and granulocyte-macrophage colony-stimulating factor release by 50% maximally. Mifepristone was able to inhibit p65-associated HAT activity to the same extent as dexamethasone but failed to inhibit the natural promoter to an equal extent due to an inability to recruit HDAC2 to the p65-associated HAT complex. These data suggest that the maximal repressive actions of glucocorticoids require recruitment of HDAC2 to a p65-HAT complex. These data also suggest that pharmacological manipulation of specific histone acetylation status is a potentially useful approach for the treatment of inflammatory diseases.

Acetylation↗
PubMed2001

Transcriptional regulation of the human sterol 12alpha-hydroxylase gene (CYP8B1): roles of heaptocyte nuclear factor 4alpha in mediating bile acid repression.

Sterol 12alpha-hydroxylase catalyzes the synthesis of cholic acid and controls the ratio of cholic acid over chenodeoxycholic acid in the bile. Transcription of CYP8B1 is inhibited by bile acids, cholesterol, and insulin. To study the mechanism of CYP8B1 transcription by bile acids, we have cloned and determined 3389 base pairs of the 5'-upstream nucleotide sequences of the human CYP8B1. Deletion analysis of CYP8B1/luciferase reporter activity in HepG2 cells revealed that the sequences from -57 to +300 were important for basal and liver-specific promoter activities. Hepatocyte nuclear factor 4alpha (HNF4alpha) strongly activated human CYP8B1 promoter activities, whereas cholesterol 7alpha-hydroxylase promoter factor (CPF), an NR5A2 family of nuclear receptors, had much less effect. Electrophoretic mobility shift assay identified an overlapping HNF4alpha- and CPF-binding site in the +198/+227 region. The human CYP8B1 promoter activities were strongly repressed by bile acids, and the bile acid response element was localized between +137 and +220. Site-directed mutagenesis of the HNF4alpha-binding site markedly reduced promoter activity and its response to bile acid repression. On the other hand, mutation of the CPF-binding site had little effect on promoter activity and bile acid inhibition. A negative nuclear receptor, small heterodimer partner markedly inhibited transactivation of CYP8B1 by HNF4alpha. Mammalian two-hybrid assay confirmed that HNF4alpha interacted with small heterodimer partner. Furthermore, bile acids and farnesoid X receptor reduced the expression of nuclear HNF4alpha in HepG2 cells and rat livers and its binding to DNA. Bile acids and farnesoid X receptor also inhibited mouse HNF4alpha gene transcription. In summary, our data revealed the critical roles HNF4alpha play on CYP8B1 transcription and its repression by bile acids. Bile acids repress human CYP8B1 transcription by reducing the transactivation activity of HNF4alpha through interaction of HNF4alpha with SHP and reduction of HNF4alpha expression in the liver.

Base Sequence↗
PubMed2001

Dual mechanisms for repression of the monomeric orphan receptor liver receptor homologous protein-1 by the orphan small heterodimer partner.

The orphan nuclear hormone receptor liver receptor homologous protein-1 (LRH-1; NR5A2, also known as FTF), an unusual receptor that binds DNA as a monomer, is an essential regulator of expression of a rate-limiting enzyme in bile acid formation, cholesterol 7-alpha-hydroxylase. In a classic negative feedback loop that is a crucial component of the complex regulation of cholesterol metabolism, cholesterol 7-alpha-hydroxylase expression is decreased when bile acid levels are high. This repression is thought to be based on the bile acid-dependent induction of expression of the orphan receptor small heterodimer partner (SHP) NR0B2, which inhibits the activity of LRH-1. We have explored the molecular basis for this important regulatory effect by characterizing the mechanisms by which mouse and human SHP inhibit LRH-1-mediated transactivation. Both SHP proteins specifically interact with the AF-2 transactivation domain of LRH-1 both in vivo and in vitro. This domain is a common target for coactivator interaction, and the SHP proteins can compete with p160 coactivators for binding to LRH-1. In addition to the N-terminal receptor interaction domain, SHP includes a C-terminal domain with autonomous repression function. Neither a deletion nor a point mutation specifically affecting this domain blocked the ability to interact with LRH-1 to compete for coactivator binding or to repress LRH-1 transactivation. However, the relative ability of these mutants to inhibit LRH-1-mediated transactivation was markedly decreased. We conclude that the proposed central role of SHP in cholesterol metabolism is based on a two-step mechanism that is dependent on both coactivator competition and direct transcriptional repression.

Animals↗
PubMed2001

The retinoblastoma family of proteins directly represses transcription in Saccharomyces cerevisiae.

The retinoblastoma family of proteins are key cell cycle regulatory molecules important for the differentiation of various mammalian cell types. The retinoblastoma protein regulates transcription of a variety of genes either by blocking the activation domain of various activators or by active repression via recruitment to appropriate promoters. We show here that the retinoblastoma family of proteins functions as direct transcriptional repressors in a heterologous yeast system when fused to the DNA binding domain of Gal4. Mapping experiments indicate that either the A or the B domain of the pocket region is sufficient for repression in vivo. As is the case in mammalian cells, a phosphorylation site mutant of the retinoblastoma protein is a stronger transcriptional repressor than the wild type protein. We show that transcriptional repression by pRb is dependent on CLN3 in vivo. Furthermore, the yeast histone deacetylase components, RPD3 and SIN3, are required for transcriptional repression.

Binding Sites↗
PubMed2002

ATF3 represses 72-kDa type IV collagenase (MMP-2) expression by antagonizing p53-dependent trans-activation of the collagenase promoter.

The murine homologue of the ATF3 transcription factor increases tumor metastases but, surprisingly, represses 72-kDa type IV metalloproteinase (MMP-2) expression. The current study describes a novel mechanism by which ATF3 regulates transcription. Progressive deletions of the MMP-2 promoter indicated a 38-base pair region (-1659/-1622) necessary for the ATF3-mediated repression. This region lacked CREB/AP-1 motifs but contained a consensus p53 motif shown previously to regulate MMP-2 expression. The activity of a p53 response element-driven luciferase reporter was reduced in ATF3-expressing HT1080 clones. Although MMP-2 promoter activity was not repressed by ATF3 in p53-deficient Saos-2 cells, p53 re-expression increased MMP-2 promoter activity and restored the sensitivity to ATF3. The activity of a GAL4-driven reporter in HT1080 cells co-expressing the full-length p53 sequence fused to the GAL4 DNA binding domain was diminished by ATF3. p53-ATF3 protein-protein interactions were demonstrated both in vivo and in vitro. Cell cycle analysis, performed as an independent assay of p53 function, revealed that gamma-irradiation-induced slowed G(2)/M cell cycle progression (attributable to p53) was countered by ATF3. Thus, ATF3 represses MMP-2 expression by decreasing the trans-activation of this gene by p53.

Activating Transcription Factor 3↗