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At least 811 records · Page 45Linked to original sources
PubMed1996

Inhibition of NFkappaB activity through maintenance of IkappaBalpha levels contributes to dihydrotestosterone-mediated repression of the interleukin-6 promoter.

Androgens repress expression of many genes, yet the mechanism of this activity has remained elusive. The cytokine, interleukin-6, is active in a variety of biological systems, and its expression is repressed by androgens. Accordingly we dissected the mechanism of androgen's ability to inhibit interleukin-6 expression at the molecular level. In a series of co-transfection assays, we found that 5alpha-dihydrotestosterone, through the androgen receptor, repressed activation of the interleukin-6 promoter, in part, by inhibiting NFkappaB activity. It did not appear that 5alpha-dihydrotestosterone inhibited NFkappaB by activating the androgen receptor to compete for the NFkappaB response element as we could not detect androgen receptor binding to the IL-6 promoter by DNase I footprinting assay. However, by electrophoretic mobility shift assay we found that 5alpha-dihydrotestosterone repressed formation of NFkappaB middle dotNFkappaB response element complex formation. In LNCaP prostate carcinoma cells, 5alpha-dihydrotestosterone achieved this effect through maintenance of IkappaBalpha protein levels in the face of phorbol ester, a stimulus that results in IkappaBalpha degradation. Finally, we confirmed that IkappaBalpha inhibits NFkappaB-mediated activation of the interleukin-6 promoter. These data suggest that maintenance of IkappaBalpha levels may represent the first identified mechanism for androgen-mediated repression of a natural androgen-regulated gene.

Cell Line↗
PubMed1997

Overexpression of the G1-cyclin gene CLN2 represses the mating pathway in Saccharomyces cerevisiae at the level of the MEKK Ste11.

Basal and induced transcription of pheromone-dependent genes is regulated in a cell cycle-dependent way. FUS1, a gene strongly induced after pheromone treatment, shows high mRNA levels in mitosis and early G1 phase of the cell cycle, a decrease in G1 after START and again an increase in S phase. Overexpression of CLN2 was shown to repress the transcript number of pheromone-dependent genes (1). We asked whether the activities of components of the mating pathway fluctuate during the cell cycle. We were also interested in determining at what level Cln2 represses the signal transduction machinery. Here we show that the activity of the mitogen-activated protein kinase Fus3 indeed fluctuates during the cell cycle, reflecting the oscillations of the gene transcripts. CLN2 overexpression represses Fus3 kinase activity, independently of the phosphatase Msg5. Additionally, we show that the activity of the MEK Ste7 also fluctuates during the cell cycle. Increased Cln2 levels repress the ability of hyperactive STE11 alleles to induce the pathway. G protein-independent activation of Ste11 caused by an rga1 pbs2 mutation is resistant to high levels of Cln2 kinase. Therefore our results suggest that Cln2-dependent repression of the mating pathway occurs at the level of Ste11.

Cell Cycle↗
PubMed1997

Copper-specific transcriptional repression of yeast genes encoding critical components in the copper transport pathway.

Copper is an essential micronutrient that is toxic in excess. To maintain an adequate yet non-toxic concentration of copper, cells possess several modes of control. One involves copper uptake mediated by genes encoding proteins that play key roles in high affinity copper transport. These include the FRE1-encoded Cu2+/Fe3+ reductase and the CTR1 and CTR3-encoded membrane-associated copper transport proteins. Each of these genes is transcriptionally regulated as a function of copper availability: repressed when cells are grown in the presence of copper and highly activated during copper starvation. Our data demonstrate that repression of CTR3 transcription is exquisitely copper-sensitive and specific. Although copper represses CTR3 gene expression at picomolar metal concentrations, cadmium and mercury down-regulate CTR3 expression only at concentrations 3 orders magnitude greater. Furthermore, copper-starvation rapidly and potently induces CTR3 gene expression. We demonstrate that the CTR1, CTR3, and FRE1 genes involved in high affinity copper uptake share a common promoter element, TTTGCTC, which is necessary for both copper repression and copper-starvation activation of gene expression. Furthermore, the Mac1p is essential for down- or up-regulation of the copper-transport genes. In vivo footprinting studies reveal that the cis-acting element, termed CuRE (copper-response element), is occupied under copper-starvation and accessible to DNA modifying agents in response to copper repression, and that this regulated occupancy requires a functional MAC1 gene. Therefore, yeast cells coordinately express genes involved in high affinity copper transport through the action of a common signaling pathway.

Antiporters↗
PubMed1997

A new function for the C-terminal zinc finger of the glucocorticoid receptor. Repression of RelA transactivation.

Glucocorticoids inhibit NF-kappaB signaling by interfering with the NF-kappaB transcription factor RelA. Previous studies have identified the DNA-binding domain (DBD) in the glucocorticoid receptor (GR) as the major region responsible for this repressive activity. Using GR mutants with chimeric DBDs the repressive function was found to be located in the C-terminal zinc finger. As predicted from these results the mineralocorticoid receptor that contains a C-terminal zinc finger identical to that of the GR was also able to repress RelA-dependent transcription. Mutation of a conserved arginine or a lysine in the second zinc finger of the GR DBD (Arg-488 or Lys-490 in the rat GR) abolished the ability of GR to inhibit RelA activity. In contrast, C-terminal zinc finger GR mutants with mutations in the dimerization box or mutations necessary for full transcriptional GR activity were still able to repress RelA-dependent transcription. In addition, we found that the steroid analog ZK98299 known to induce GR transrepression of AP-1 had no inhibitory effect on RelA activity. In summary, these results demonstrate that the inhibition of NF-kappaB by glucocorticoids involves two critical amino acids in the C-terminal zinc finger of the GR. Furthermore, the results from the use of mineralocorticoid receptor and anti-glucocorticoids suggest that the mechanisms for GR-mediated repression of NF-kappaB and AP-1 are different.

Amino Acid Sequence↗
PubMed1997

A weak TATA box is a prerequisite for glucocorticoid-dependent repression of the osteocalcin gene.

The TATA box element is not only important for establishing basal levels of transcription, but it can also be used to modulate cell type or stage specific gene activity. In the case of the human osteocalcin gene, which is transcriptionally repressed by glucocorticoids, a specific binding element for the glucocorticoid receptor (GR) overlaps a noncanonical TATA box. In the present study, the relevance and function of the TATA element in glucocorticoid-mediated repression of the human osteocalcin gene was characterized. Mutating this noncanonical TATA box into a consensus TATA box within the context of the osteocalcin promoter greatly decreased hormone-dependent transcriptional repression by GR. TATA-binding protein (TBP) bound this mutated element much more strongly suggesting a physiologically relevant role for the weak osteocalcin TATA element in the regulation of this bone specific gene. The optimization of the putative transcription factor IIB recognition site did not affect the level of GR-mediated repression. Our results support a model wherein competitive DNA binding of GR and TBP for their overlapping sites explains conditional repression of the osteocalcin gene by glucocorticoids.

Binding, Competitive↗
PubMed1998

Identification of transcriptional activation and repression domains in human CCAAT/enhancer-binding protein epsilon.

Human CCAAT/enhancer-binding protein epsilon (C/EBPepsilon), a new member of the C/EBP family, significantly up-regulates both the mim-1 and human myeloperoxidase promoters, suggesting an important role for C/EBPepsilon in the transcriptional regulation of a subset of myeloid-specific genes. To elucidate the structure and function of C/EBPepsilon in transcriptional activation, amino acid residues 1-115, 147-249, or 1-249 of C/EBPepsilon were fused to the yeast GAL4 DNA binding domain. These expression vectors were cotransfected with a chloramphenicol acetyltransferase reporter gene and, in all cell lines tested, only the GAL-C/EBPepsilon-(1-115) fusion protein significantly activated expression from the chloramphenicol acetyltransferase reporter gene. Sixteen deletion mutants of C/EBPepsilon mapped the transactivation domain to amino acids 1-18 at the N terminus and revealed the presence of a transcription repression element between amino acid residues 116 and 162. Expression vectors containing the repression domain of C/EBPepsilon strongly inhibited gene transcription from TK, SV40, and adenoviral major late promoters bearing GAL4 binding sites. Fusion of this repression domain to the VP16 activation domain inhibited the transactivation function of VP16. Deletion of this repression domain increased gene transcription from a neutrophil elastase promoter-luciferase reporter. Taken together, these data suggest that C/EBPepsilon regulates transcription by utilizing both activation and repression functions.

CCAAT-Enhancer-Binding Proteins↗
PubMed1998

Central role for phosphatidylinositide 3-kinase in the repression of glucose-6-phosphatase gene transcription by insulin.

Transcription of the gene encoding the catalytic subunit of glucose-6-phosphatase (G6Pase) is stimulated by glucocorticoids and strongly repressed by insulin. We have explored the signaling pathways by which insulin mediates the repression of G6Pase transcription in H4IIE cells. Wortmannin, a phosphatidylinositide 3-kinase (PtdIns 3-kinase) inhibitor blocked the repression of G6Pase mRNA expression by insulin. However, both rapamycin, which inhibits p70S6 kinase activation, and PD98059, an inhibitor of mitogen-activated protein kinase activation, were without effect. Insulin inhibited dexamethasone-induced luciferase expression from a transiently transfected plasmid that places the luciferase gene under the control of the G6Pase promoter. This effect of insulin was mimicked by the overexpression of a constitutively active PtdIns 3-kinase but not by a constitutively active protein kinase B. Taken together, these data demonstrate that PtdIns 3-kinase activation is both necessary and at least partly sufficient for the repression of G6Pase expression by insulin, but neither mitogen-activated protein kinase nor p70S6 kinase are involved. In addition, activation of protein kinase B alone is not sufficient for repression of the G6Pase gene. These results imply the existence of a novel signaling pathway downstream of PtdIns 3 kinase that is involved in the regulation of G6Pase expression by insulin.

Amino Acid Sequence↗
PubMed1999

Cloning and characterization of a novel zinc finger transcriptional repressor. A direct role of the zinc finger motif in repression.

We have identified a novel transcriptional repressor, AEBP2, that binds to a regulatory sequence (termed AE-1) located in the proximal promoter region of the aP2 gene that encodes the adipose fatty acid-binding protein. Sequence analysis of AEBP2 cDNA revealed that it encodes a protein containing three Gli-Krüppel (Cys2-His2)-type zinc fingers. Northern blot analysis revealed two transcripts (4.5 and 3.5 kilobases) which were ubiquitously expressed in every mouse tissue examined. In co-transfection assays, AEBP2 repressed transcription from the homologous aP2 promoter containing multiple copies of the AE-1 sequence. Moreover, a chimeric construct encoding a fusion AEBP2 protein with the Gal4 DNA-binding domain was able to repress the transcriptional activity of a heterologous promoter containing the Gal4-binding sequence. The transcriptional repression function of AEBP2 was completely abolished when one of the conserved histidine residues and a flanking serine residue in the middle zinc finger were replaced with an arginine residue. The defective transcriptional repression function of the mutant derivative was due neither to lack of expression nor to a failure to localize to the nucleus. Moreover, both the wild-type and mutant derivative of either the histidine-tagged recombinant AEBP2 proteins or the in vitro translated Gal4-AEBP2 fusion proteins were equally able to bind to the target DNA. These results suggest that a portion of the zinc finger structure may play a direct role in transcriptional repression function, but not in DNA binding.

Amino Acid Sequence↗
PubMed1999

Transcriptional anti-repression. Thyroid hormone receptor beta-2 recruits SMRT corepressor but interferes with subsequent assembly of a functional corepressor complex.

Thyroid hormone receptors (T3Rs) are hormone-regulated transcription factors. Different T3R isoforms are expressed in a tissue-specific and developmentally regulated manner. The T3Ralpha-1, beta-0, and beta-1 isoforms typically repress target gene expression in the absence of hormone and activate transcription in the presence of hormone. Intriguingly, however, the T3Rbeta-2 isoform fails to repress, and instead is able to activate transcription in both the absence and presence of hormone. We investigated the molecular mechanism behind this absence of repression by T3Rbeta-2. Repression by T3Ralpha-1, beta-0, and beta-1 is mediated by the ability of these isoforms to physically recruit a SMRT/N-CoR corepressor complex. We determined that the unliganded T3Rbeta-2 also recruits the SMRT corepressor; in contrast to the alpha-1, beta-0, and beta-1 isoforms, however, the T3Rbeta-2 protein interacts not only with the C-terminal "receptor-interaction domain" of SMRT, but also makes additional contacts with the N-terminal "silencing domain" of the SMRT corepressor. These additional, T3Rbeta-2-specific contacts interfere with the subsequent association of SMRT with mSin3, a crucial second subunit of the corepressor holo-complex. Our results suggest that T3Rbeta-2 regulates transcription through a novel anti-repression mechanism, recruiting SMRT, but preventing the subsequent formation of a functional corepressor complex.

Animals↗
PubMed1999

The Wilms' tumor 1 tumor suppressor gene represses transcription of the human telomerase reverse transcriptase gene.

Regulation of the human telomerase reverse transcriptase (hTERT) gene is the primary determinant for telomerase enzyme activity, which is found in tumor cells but is largely absent from normal somatic cells. Recent studies have shown that Myc protein can transcriptionally activate the hTERT gene. However, little is known about the repression mechanism of the hTERT gene and telomerase enzyme. Here, we developed an expression cloning strategy to identify cDNAs whose products can repress hTERT promoter activity in telomerase-positive immortal cells. Using this screen, we isolated the Wilms' tumor 1 suppressor gene (WT1). WT1 can repress hTERT promoter activity in 293 kidney cells. The WT1 binding site on the hTERT promoter was identified by deletional analysis. Alteration of the WT1 binding site markedly derepresses transcription from an isolated hTERT promoter by inhibiting interaction of WT1 with DNA. These specific repression effects of WT1 were not observed in HeLa cells, which express no endogenous WT1. Furthermore, we show that WT1 can repress the endogenous hTERT promoter and telomerase enzyme activities. These results suggest that WT1 may be a transcriptional repressor of the hTERT gene, at least in some specific cells.

DNA-Binding Proteins↗
PubMed1999

Viral ski inhibits retinoblastoma protein (Rb)-mediated transcriptional repression in a dominant negative fashion.

The mechanism by which the viral oncogene ski (v-ski) transforms chicken embryo fibroblasts is currently unknown. Recently, the c-ski gene product (c-Ski) was found to bind to N-CoR (nuclear hormone receptor co-repressor), an element implicated in transcriptional repression mediated by multiple transcriptional repressors including the nuclear hormone receptors and Mad. c-Ski is required for transcriptional repression mediated by Mad involved in negative regulation of cellular proliferation. v-Ski abrogates Mad-induced transcriptional repression in a dominant negative fashion. Here we report that v-Ski also inhibits transcriptional repression mediated by Rb, another tumor suppressor gene product. Rb forms a complex with c-Ski, Sin3A, and histone deacetylase (HDAC) via direct binding to c-Ski and HDAC. c-Ski is required for the transcriptional repression mediated by Rb. These results suggest that inhibition of Rb activity contributes, at least partly, to transformation by v-Ski.

Animals↗
PubMed2000

A mechanism of repression by acute myeloid leukemia-1, the target of multiple chromosomal translocations in acute leukemia.

AML1 is one of the most frequently translocated genes in human leukemia. Here we demonstrate that acute myeloid leukemia-1 (AML-1) (Runx-1) represses transcription from a native promoter, p21(Waf1/Cip1). Unexpectedly, this repression did not require interactions with the Groucho co-repressor. To define the mechanism of repression, we asked whether other co-repressors could interact with AML-1. We demonstrate that AML-1 interacts with the mSin3 co-repressors. Moreover, endogenous AML-1 associated with endogenous mSin3A in mammalian cells. A deletion mutant of AML-1 that did not interact with mSin3A failed to repress transcription. The AML-1/mSin3 association suggests a mechanism of repression for the chromosomal translocation fusion proteins that disrupt AML-1.

Acute Disease↗
PubMed2001

Transcriptional repression by p53 through direct binding to a novel DNA element.

The tumor suppressor protein p53 has been well documented as a transcriptional activator involved in the regulation of a number of critical genes involved in the cell cycle, response to DNA damage, and apoptosis. Activation by p53 requires the interaction of the protein with a consensus binding site consisting of two half-sites, each comprising two copies of the sequence PuPuPuC(A/T) arranged head-to-head and separated by 0-13 base pairs. In addition to activation, p53 has been shown to be a potent repressor of transcription. However, the basis for p53-mediated repression is not well understood and has been proposed to occur indirectly through interactions with other promoter-bound transcription factors. In the present study, we show that p53 can repress transcription directly by binding to a novel head-to-tail (HT) site within the MDR1 promoter. A mutation that disrupted p53 binding to the MDR1 HT site blocked p53-mediated repression of the MDR1 promoter in transfection assays. Replacement of the HT site with a head-to-head (HH) site converted the activity of p53 from repression to activation, indicating that simple recruitment of p53 to the promoter is not sufficient for repression and that the orientation of the binding element determines the fate of p53-regulated promoters.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

Association of COOH-terminal-binding protein (CtBP) and MEF2-interacting transcription repressor (MITR) contributes to transcriptional repression of the MEF2 transcription factor.

The class II histone deacetylases (HDACs) 4, 5, and 7 share a common structural organization, with a carboxyl-terminal catalytic domain and an amino-terminal extension that mediates interactions with members of the myocyte enhancer factor-2 (MEF2) family of transcription factors. Association of these HDACs with MEF2 factors represses transcription of MEF2 target genes. MEF2-interacting transcription repressor (MITR) shares homology with the amino-terminal extensions of class II HDACs and also acts as a transcriptional repressor, but lacks a histone deacetylase catalytic domain. This suggests that MITR represses transcription by recruiting other corepressors. We show that the amino-terminal regions of MITR and class II HDACs interact with the transcriptional corepressor, COOH-terminal-binding protein (CtBP), through a CtBP-binding motif (P-X-D-L-R) conserved in MITR and HDACs 4, 5, and 7. Mutation of this sequence in MITR abolishes interaction with CtBP and impairs, but does not eliminate, the ability of MITR to inhibit MEF2-dependent transcription. The residual repressive activity of MITR mutants that fail to bind CtBP can be attributed to association with other HDAC family members. These findings reveal CtBP-dependent and -independent mechanisms for transcriptional repression by MITR and show that MITR represses MEF2 activity through recruitment of multicomponent corepressor complexes that include CtBP and HDACs.

Alcohol Oxidoreductases↗
PubMed2001

Multiple regions of ETO cooperate in transcriptional repression.

In acute myeloid leukemias (AMLs) with t(8;21), the transcription factor AML1 is juxtaposed to the zinc finger nuclear protein ETO (Eight-Twenty-One), resulting in transcriptional repression of AML1 target genes. ETO has been shown to interact with corepressors, such as N-CoR and mSin3A to form complexes containing histone deacetylases. To define regions of ETO required for maximal repressor activity, we analyzed amino-terminal deletions in a transcriptional repression assay. We found that ETO mutants lacking the first 236 amino acids were not affected in their repressor activity, whereas a further deletion of 85 amino acids drastically reduced repressor function and high molecular weight complex formation. This latter mutant can still homodimerize and bind to N-CoR but shows only weak binding to mSin3A. Furthermore, we could show that a "core repressor domain" comprising nervy homology region 2 and its amino- and carboxyl-terminal flanking sequences recruits mSin3A and induces transcriptional repression. These results suggest that mSin3A and N-CoR bind to ETO independently and that both binding sites cooperate to maximize ETO-mediated transcriptional repression. Thus, ETO has a modular structure, and the interaction between the individual elements is essential for the formation of a stable repressor complex and efficient transcriptional repression.

Amino Acids↗
PubMed2001

The basic helix-loop-helix protein, sharp-1, represses transcription by a histone deacetylase-dependent and histone deacetylase-independent mechanism.

Many aspects of neurogenesis and neuronal differentiation are controlled by basic helix-loop-helix (bHLH) proteins. One such factor is SHARP-1, initially identified on the basis of its sequence similarity to hairy. Unlike hairy, and atypically for bHLHs, SHARP-1 is expressed late in development, suggestive of a role in terminal aspects of differentiation. Nevertheless, the role of SHARP-1 and the identity of its target genes remain unknown. During the course of a one-hybrid screen for transcription factors that bind to regulatory domains of the M1 muscarinic acetylcholine receptor gene, we isolated the bHLH transcription factor SHARP-1. In this study, we investigated the functional role of SHARP-1 in regulating transcription. Fusion proteins of SHARP-1 tethered to the gal4 DNA binding domain repress both basal and activated transcription when recruited to either a TATA-containing or a TATAless promoter. Furthermore, we identified two independent repression domains that operate via distinct mechanisms. Repression by a domain in the C terminus is sensitive to the histone deacetylase inhibitor trichostatin A, whereas repression by the bHLH domain is insensitive to TSA. Furthermore, overexpression of SHARP-1 represses transcription from the M(1) promoter. This study represents the first report to assign a function to, and to identify a target gene for, the bHLH transcription factor SHARP-1.

Amino Acid Sequence↗
PubMed2001

Human T-cell leukemia virus type I tax repression of p73beta is mediated through competition for the C/H1 domain of CBP.

The Tax protein, encoded by the human T-cell leukemia virus type I (HTLV-I), is required for high level viral transcription and HTLV-I-associated malignant transformation. Although the precise mechanism of malignant transformation by Tax is unclear, it is well established that Tax represses the transcription function of the tumor suppressor p53, possibly accelerating the accumulation of genetic mutations that are critical in HTLV-I-mediated malignant transformation. Tax repression of p53 transcription function appears to occur, at least in part, through competition for the cellular coactivator CBP/p300. In this study, we characterize the effect of Tax on the p53 family member, p73. We demonstrate that Tax also represses the transcription function of p73beta and that the repression is reciprocal in vivo, consistent with the idea that both transcription factors may compete for CBP/p300 in vivo. We provide evidence showing that both Tax and p73 interact strongly with the C/H1 domain of CBP and that their binding to this region is mutually exclusive in vitro. This finding provides evidence supporting the idea that reciprocal transcriptional repression between Tax and p73 is mediated through coactivator competition.

Animals↗
PubMed2001

Huntingtin is present in the nucleus, interacts with the transcriptional corepressor C-terminal binding protein, and represses transcription.

Huntingtin is a protein of unknown function that contains a polyglutamine tract, which is expanded in patients with Huntington's disease (HD). We investigated the localization and a potential function for huntingtin in the nucleus. In human fibroblasts from normal and HD patients, huntingtin localized diffusely in the nucleus and in subnuclear compartments identified as speckles, promyelocytic leukemia protein bodies, and nucleoli. Huntingtin-positive nuclear bodies redistributed after treatment with sodium butyrate. By Western blot, purified nuclei had low levels of full-length huntingtin compared with the cytoplasm but contained high levels of N- and C-terminal huntingtin fragments, which tightly bound the nuclear matrix. Full-length huntingtin co-immunoprecipitated with the transcriptional corepressor C-terminal binding protein, and polyglutamine expansion in huntingtin reduced this interaction. Full-length wild-type and mutant huntingtin repressed transcription when targeted to DNA. Truncated N-terminal mutant huntingtin repressed transcription, whereas the corresponding wild-type fragment did not repress transcription. We speculate that wild-type huntingtin may function in the nucleus in the assembly of nuclear matrix-bound protein complexes involved with transcriptional repression and RNA processing. Proteolysis of mutant huntingtin may alter nuclear functions by disrupting protein complexes and inappropriately repressing transcription in HD.

Alcohol Oxidoreductases↗