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PubMed1996

A mechanism for repression of class II gene transcription through specific binding of NC2 to TBP-promoter complexes via heterodimeric histone fold domains.

Negative co-factor 2 (NC2) regulates transcription of the class II genes through binding to TFIID and inhibition of pre-initiation complex formation. We have isolated and cloned NC2, and investigated the molecular mechanism underlying repression of transcription. NC2 consists of two subunits, termed NC2alpha and NC2beta, the latter of which is identical to Dr1. The NC2 subunits dimerize and bind to TATA binding protein (TBP)-promoter complexes via histone fold domains of the H2A-H2B type. Repression of basal transcription requires the histone fold and carboxy-terminal domains of the NC2 subunits. Several mechanisms probably contribute to transcriptional repression. Binding of NC2 inhibits association of TFIIB with TBP-promoter complexes. NC2 binds directly to DNA, and binding of NC2 to TBP-promoter complexes affects the conformation of DNA, which could be one cause for the inhibition of TFIIB. In addition, multimerization of repressor-TBP complexes on DNA might inhibit the assembly of the pre-initiation complex. We suggest that binding of the repressor to TRP-promoter complexes establishes a mechanism that controls the rate of transcription by RNA polymerase II.

Amino Acid Sequence↗

PubMed2004

Distinct mechanisms for repression of RNA polymerase III transcription by the retinoblastoma tumor suppressor protein.

The retinoblastoma (RB) protein represses global RNA polymerase III transcription of genes that encode nontranslated RNAs, potentially to control cell growth. However, RNA polymerase III-transcribed genes exhibit diverse promoter structures and factor requirements for transcription, and a universal mechanism explaining global repression is uncertain. We show that RB represses different classes of RNA polymerase III-transcribed genes via distinct mechanisms. Repression of human U6 snRNA (class 3) gene transcription occurs through stable promoter occupancy by RB, whereas repression of adenovirus VAI (class 2) gene transcription occurs in the absence of detectable RB-promoter association. Endogenous RB binds to a human U6 snRNA gene in both normal and cancer cells that maintain functional RB but not in HeLa cells whose RB function is disrupted by the papillomavirus E7 protein. Both U6 promoter association and transcriptional repression require the A/B pocket domain and C region of RB. These regions of RB contribute to U6 promoter targeting through numerous interactions with components of the U6 general transcription machinery, including SNAP(C) and TFIIIB. Importantly, RB also concurrently occupies a U6 promoter with RNA polymerase III during repression. These observations suggest a novel mechanism for RB function wherein RB can repress U6 transcription at critical steps subsequent to RNA polymerase III recruitment.

Binding Sites↗

PubMed1995

Adenovirus E1A represses cardiac gene transcription and reactivates DNA synthesis in ventricular myocytes, via alternative pocket protein- and p300-binding domains.

To examine the potential impact of disrupting "pocket" protein function on cardiac differentiation and growth, we introduced 12 S E1A genes into neonatal ventricular myocytes, by adenoviral gene transfer. In the absence of E1B, E1A was cytotoxic, with features typical of apoptosis. In the presence of E1B, E1A preferentially inhibited transcription of cardiac-restricted alpha-actin promoters, and reactivated DNA synthesis in cardiac myocytes, without cell death. Mutations that abrogate known activities of the amino terminus of E1A, versus conserved region 2, demonstrate that the "pocket" protein- and p300-binding domains each suffice, in the absence of the other, for transcriptional repression and re-entry into S phase.

Adenovirus E1A Proteins↗

PubMed1992

The zonal expression of alpha-fetoprotein transgenes in the livers of adult mice.

The developmental regulation of the alpha-fetoprotein (AFP) gene in liver results in high-level expression in the fetus, followed by dramatic transcriptional repression after birth. We have examined the mouse AFP gene for transcriptional control sequences that may be involved in its postnatal repression in liver. We showed previously that removal of a DNA region between positions -250 base pairs (bp) and -838 bp of the AFP gene resulted in the persistence of expression of an AFP minigene in the postpartum liver of transgenic mice (Vacher and Tilghman, Science 250:1732-1735, 1990). This study examines the distribution of these transgene transcripts in liver using in situ hybridization. We show that there is a zonal distribution of minigene transcripts in the adult livers of these animals. Hepatocytes surrounding the central veins express high levels of minigene transcripts, while hepatocytes in the intermediate and portal areas contain few, if any, transcripts. Quantitative RNAse protection analysis shows a decrease in transgene RNA levels after birth, consistent with repression in all but a small subset of hepatocytes. These results indicate that repression in the pericentral hepatocytes is dependent upon the presence of a cis-acting, negative-regulatory domain, which is located between the enhancers and the proximal promoter of the AFP gene. In contrast, this domain is not essential for complete repression of AFP transgenes in the intermediate zone and periportal hepatocytes.

Animals↗

PubMed1997

Mapping and characterization of the functional domains responsible for the differential activity of the A and B isoforms of the human progesterone receptor.

In humans, the biological response to progesterone is mediated by two distinct forms of the progesterone receptor (human (h) PR-A, 94 kDa and hPR-B, 114 kDa). These two isoforms are transcribed from distinct estrogen-inducible promoters within a single copy PR gene; the only difference between them is that the first 164 amino acids of hPR-B (B-upstream sequence) are absent in hPR-A. In most cell lines such as MCF-7 (human breast cancer cells), CV-1 (monkey kidney fibroblasts), and HeLa (human cervical carcinoma cells), hPR-A functions as a transcriptional repressor, whereas hPR-B functions as a transcriptional activator of progesterone-responsive genes. Interestingly, in these cell contexts, hPR-A also acts as a trans-dominant repressor of the transcriptional activity of other steroid hormone receptors. In contrast to hPR-A, which functions predominantly as a ligand-dependent transcriptional repressor, we show in this study that the A isoform of the chicken PR (cPR-A) lacks this trans-dominant repressor function and is a transcriptional activator in all contexts examined. By constructing chimeras between the N-terminal domains of the chicken and human PR, we mapped the trans-dominant repressor function of hPR-A to the first 140 amino acids of the protein. Notably, when this 140-amino acid "repressor" domain is placed onto chicken PR-A, the activity of the latter changes from a transcriptional activator to a repressor. Interestingly, however, this "repressor domain" is necessary, but not sufficient, for trans-repression as it is inactive when it is tethered to a heterologous protein. This suggests that the trans-repression function is comprised not only of the repressor domain of hPR-A but also requires the context of the receptor to function. The identification of a discrete inhibitory region within hPR-A which is transferable to another receptor implies that this region interacts with a set of transcription factors or adaptors that are distinct from those recognized by hPR-B, the identification of which will be required to define the mechanism by which hPR-A modulates steroid hormone receptor transcriptional activity. Thus, although chickens and humans both produce two very similar forms of the progesterone receptor, it is clear from these studies that the mechanism of action of progesterone in these two systems is quite different.

Animals↗

PubMed2000

MdmX protects p53 from Mdm2-mediated degradation.

The p53 tumor suppressor protein is stabilized in response to cellular stress, resulting in activation of genes responsible for either cell cycle arrest or apoptosis. The cellular pathway for releasing normal cells from p53-dependent cell cycle arrest involves the Mdm2 protein. Recently, a p53-binding protein with homology to Mdm2 was identified and called MdmX. Like Mdm2, MdmX is able to bind p53 and inhibit p53 transactivation; however, the ability of MdmX to degrade p53 has yet to be examined. We report here that MdmX is capable of associating with p53 yet is unable to facilitate nuclear export or induce p53 degradation. In addition, expression of MdmX can reverse Mdm2-targeted degradation of p53 while maintaining suppression of p53 transactivation. Using a series of MdmX deletions, we have determined that there are two distinct domains of the MdmX protein that can stabilize p53 in the presence of Mdm2. One domain requires MdmX interaction with p53 and results in the retention of both proteins within the nucleus and repression of p53 transactivation. The second domain involves the MdmX ring finger and results in stabilization of p53 and an increase in p53 transactivation. The potential basis for stabilization and increased p53 transactivation by the MdmX ring finger domain is discussed. Based on these observations, we propose that the MdmX protein may function to maintain a nuclear pool of p53 protein in undamaged cells.

3T3 Cells↗

PubMed1991

Transcriptional regulation by Fos and Jun in vitro: interaction among multiple activator and regulatory domains.

The proteins encoded by the proto-oncogenes c-fos and c-jun (Fos and Jun, respectively) form a heterodimeric complex that regulates transcription by interacting with the DNA-regulatory element known as the activator protein 1 (AP-1) binding site. Fos and Jun are members of a family of related transcription factors that dimerize via a leucine zipper structure and interact with DNA through a bipartite domain formed between regions of each protein that are rich in basic amino acids. Here we have defined other domains in the Fos-Jun heterodimer that contribute to transcriptional function in vitro. Although DNA-binding specificity is mediated by the leucine zipper and basic regions, Jun also contains a proline- and glutamine-rich region that functions as an ancillary DNA-binding domain but does not contribute directly to transcriptional activation. Transcriptional stimulation in vitro was associated with two regions in Fos and a single N-terminal activation domain in Jun. These activator regions were capable of operating independently; however, they appear to function cooperatively in the heterodimeric complex. The activity of these domains was modulated by inhibitory regions in Fos and Jun that repressed transcription in vitro. In the context of the heterodimer, the Jun activation domain was the major contributor to transcriptional stimulation and the inhibitory regions in Fos were the major contributors to transcriptional repression in vitro. Potentially, the inhibitory domains could serve a regulatory function in vivo. Thus, transcriptional regulation by the Fos-Jun heterodimer results from a complex integration of multiple activator and regulatory domains.

Binding Sites↗

PubMed2006

Selective repression of translation by the brome mosaic virus 1a RNA replication protein.

Differential expression of viral replication proteins is essential for successful infection. We report here that overexpression of the brome mosaic virus (BMV) 1a protein can repress viral RNA replication in a dosage-dependent manner. Using RNA replication-incompetent reporter constructs, repression of translation from BMV RNA1 and RNA2 was observed, suggesting that the effect on translation of the BMV RNA replication proteins is responsible for the decrease in RNA levels. Furthermore, repression of translation by 1a required the B box in the 5'-untranslated region (5' UTR); BMV RNA3 that lacks a B box in its 5' UTR is not subject to 1a-mediated translational inhibition. Mutations in either the methyltransferase or the helicase-like domains of 1a reduced the repression of replication and translation. These results suggest that in addition to its known functions in BMV RNA synthesis, 1a also regulates viral gene expression.

5' Untranslated Regions↗

PubMed2005

Cyclin D1 represses p300 transactivation through a cyclin-dependent kinase-independent mechanism.

Cyclin D1 encodes a regulatory subunit, which with its cyclin-dependent kinase (Cdk)-binding partner forms a holoenzyme that phosphorylates and inactivates the retinoblastoma protein. In addition to its Cdk binding-dependent functions, cyclin D1 regulates cellular differentiation in part by modifying several transcription factors and nuclear receptors. The molecular mechanism through which cyclin D1 regulates the function of transcription factors involved in cellular differentiation remains to be clarified. The histone acetyltransferase protein p300 is a co-integrator required for regulation of multiple transcription factors. Here we show that cyclin D1 physically interacts with p300 and represses p300 transactivation. We demonstrated further that the interaction of the two proteins occurs at the peroxisome proliferator-activated receptor gamma-responsive element of the lipoprotein lipase promoter in the context of the local chromatin structure. We have mapped the domains in p300 and cyclin D1 involved in this interaction. The bromo domain and cysteine- and histidine-rich domains of p300 were required for repression by cyclin D1. Cyclin D1 repression of p300 was independent of the Cdk- and retinoblastoma protein-binding domains of cyclin D1. Cyclin D1 inhibits histone acetyltransferase activity of p300 in vitro. Microarray analysis identified a signature of genes repressed by cyclin D1 and induced by p300 that promotes cellular differentiation and induces cell cycle arrest. Together, our results suggest that cyclin D1 plays an important role in cellular proliferation and differentiation through regulation of p300.

Cyclin D1↗

PubMed2005

Transcriptional repression and heterochromatin formation by MBD1 and MCAF/AM family proteins.

DNA methylation cooperates with methylation at lysine 9 of histone H3 (H3-K9), a modified histone molecule that is targeted by heterochromatin protein 1, to form a transcriptionally silent chromatin. Methyl CpG-binding protein MBD1 recognizes methylated CpG dinucleotide and recruits H3-K9 methyltransferases such as SETDB1 to genomic regions. Here we show that MBD1-containing chromatin-associated factor (MCAF) 1, also known as the human homologue of murine ATFa-associated modulator (AM), is required for transcriptional repression and heterochromatin formation by MBD1, together with the involvement of SETDB1. Moreover, the amino acid sequence of MCAF1 shows similarity to a number of sequences of the MCAF/AM-related proteins, resulting in the identification of a new member of the protein family, termed MCAF2. Immunoprecipitation and in vitro binding analyses reveal that both MCAF proteins interact with MBD1, SETDB1, and Sp1 via two evolutionarily conserved distinct domains. Furthermore, MCAF1 enhances transcriptional repression by MBD1 together with SETDB1, and exogenous expression of MCAF2 partly compensates for the repressive activity in MCAF1 knockdown HeLa cells. The expression of MBD1 mutant, which lacks interaction with MCAF proteins, perturbs heterochromatin protein 1-enriched heterochromatin formation at the MBD1-containing chromosomal loci. These data suggest that MBD1.MCAF1.SETDB1 complex facilitates the formation of heterochromatic domains, emphasizing the role of MCAF/AM family proteins in epigenetic control.

Amino Acid Sequence↗

PubMed2002

Pleiohomeotic can link polycomb to DNA and mediate transcriptional repression.

Polycomb group (PcG) proteins function through cis-acting DNA elements called PcG response elements (PREs) to stably silence developmental regulators, including the homeotic genes. However, the mechanism by which they are targeted to PREs remains largely unclear. Pleiohomeotic (PHO) is a sequence-specific DNA-binding PcG protein and therefore may function to tether other PcG proteins to the DNA. Here, we show that PHO can directly bind to a Polycomb (PC)-containing complex as well as the Brahma (BRM) chromatin-remodeling complex. PHO contacts the BRM complex through its zinc finger DNA-binding domain and a short N-terminal region. A distinct domain of PHO containing a conserved motif contacts the PcG proteins PC and Polyhomeotic (PH). With mobility shift assays and DNA pulldown experiments, we demonstrated that PHO is able to link PC, which lacks sequence-specific DNA-binding activity, to the DNA. Importantly, we found that the PC-binding domain of PHO can mediate transcriptional repression in transfected Drosophila Schneider cells. Concomitant overexpression of PC resulted in stronger PHO-directed repression that was dependent on its PC-binding domain. Together, these results suggest that PHO can contribute to PRE-mediated silencing by direct recruitment of a PC complex to repress transcription.

Animals↗

PubMed1997

Overexpression of nreB, a new GATA factor-encoding gene of Penicillium chrysogenum, leads to repression of the nitrate assimilatory gene cluster.

To investigate the mechanism of nitrogen metabolite repression in the biotechnologically important fungus Penicillium chrysogenum a polymerase chain reaction approach was employed to identify transcription factors involved in this regulatory circuit, leading to the isolation of a new gene (nreB) encoding a 298 amino acid protein. Despite a low overall amino acid sequence identity of approximately 30%, it shares several features with Dal80p/Uga43p and Gzf3p/Nil2p, both repressors in nitrogen metabolism in Saccharomyces cerevisiae. All three proteins contain an N-terminal GATA-type zinc finger motif, displaying 86% amino acid sequence identity, and a putative leucine zipper motif in the C terminus. Northern blot analysis revealed the presence of two nreB transcripts, 1.8 and 1.5 kilobases in length, that differ in polyadenylation sites. The steady state level of both transcripts is subject to nitrogen metabolite repression. The putative DNA binding domain of NREB, expressed as a fusion protein in Escherichia coli, binds in vitro to GATA sites of its own 5'-upstream region as well as in the promoter of the nitrate assimilation gene cluster. Consistent with a role in the regulation of nitrogen metabolism, overexpression of nreB leads to repression of nitrate assimilatory genes. Hence, the simple view of nitrogen regulation by four GATA factors in yeast, but only one key regulator in filamentous ascomycetes seems no longer valid.

Amino Acid Sequence↗

PubMed2000

Signal-dependent activation of the MEF2 transcription factor by dissociation from histone deacetylases.

Myocyte enhancer factor-2 (MEF2) transcription factors control muscle-specific and growth factor-inducible genes. We show that hypertrophic growth of cardiomyocytes in response to phenylephrine and serum is accompanied by activation of MEF2 through a posttranslational mechanism mediated by calcium, calmodulin-dependent protein kinase (CaMK), and mitogen-activated protein kinase (MAPK) signaling. CaMK stimulates MEF2 activity by dissociating class II histone deacetylases (HDACs) from the DNA-binding domain. MAPKs, which activate MEF2 by phosphorylation of the transcription activation domain, maximally stimulate MEF2 activity only when repression by HDACs is relieved by CaMK signaling to the DNA-binding domain. These findings identify MEF2 as an endpoint for hypertrophic stimuli in cardiomyocytes and demonstrate that MEF2 mediates synergistic transcriptional responses to the CaMK and MAPK signaling pathways by signal-dependent dissociation from HDACs.

Animals↗

PubMed1995

Identification of a sequence-specific single-stranded DNA binding protein that suppresses transcription of the mouse myelin basic protein gene.

The myelin basic protein (MBP) gene is expressed only in oligodendrocytes and Schwann cells, and expression follows a tightly regulated developmental time course. Cell type- and developmental stage-specific expression of the MBP gene appears to be regulated by a series of cis-acting elements located upstream of the transcription start site. The proximal element of the MBP regulatory region (MB1), located between nucleotides -14 and -50, is one of several elements participating in the programmed expression of MBP. In this report, we describe the molecular cloning and characterization of myelin gene expression factor-2 (Myef-2), a protein isolated from mouse brain that binds specifically to single-stranded DNA derived from the MB1 element and represses transcription of the MBP gene in transient transfection assay. Myef-2 mRNA is developmentally regulated in mouse brain; its peak expression occurs at postnatal day 7, prior to the onset of MBP expression. The developmental pattern of Myef-2 mRNA expression coincides with that previously described for SCIP, a POU domain transcription factor that also represses myelin basic protein expression. The myef-2 gene maps to mouse chromosome 2. The relevance of these findings for regulation of MBP gene expression and oligodendrocyte differentiation is discussed.

Aging↗

PubMed1998

Chromatin domain boundaries in the Bithorax complex.

Eukaryotic chromosomes are thought to be organized into a series of discrete higher-order chromatin domains. This organization is believed to be important not only in the compaction of the chromatin fibre, but also in the utilization of genetic information. Critical to this model are the domain boundaries that delimit and segregate the chromosomes into units of independent gene activity. In Drosophila, such domain boundaries have been identified through two different approaches. On the one hand, elements like scs/scs' and the reiterated binding site for the SU(HW) protein have been characterized through their activity of impeding enhancer-promoter interactions when intercalated between them. Their role of chromatin insulators can protect transgenes from genomic position effects, thereby establishing independent functional domains within the chromosome. On the other hand, domain boundaries of the Bithorax complex (BX-C) like Fab-7 and Mcp have been identified through mutational analysis. Mcp and Fab-7, however, may represent a specific class of boundary elements; instead of separating adjacent domains that contain separate structural genes. Mcp and Fab-7 delimit adjacent cis-regulatory domains, each of which interacts independently with their target promoters. In this article, we review the genetic and molecular characteristics of the domain boundaries of the BX-C. We describe how Fab-7 functions to confine activating as well as repressive signals to the flanking regulatory domains. Although the mechanisms by which Fab-7 works as a domain boundary remain an open issue, we provide preliminary evidence that Fab-7 is not a mere insulator like scs or the reiterated binding site for the SU(HW) protein.

Animals↗

PubMed1994

LFB1/HNF1 acts as a repressor of its own transcription.

LFB1/HNF1 is a hepatocyte-enriched trans-activator involved in the regulation of many liver-specific genes. We report the cloning and characterization of a rat genomic DNA fragment containing about 3.5 kb of the LFB1/HNF1 gene 5'-flanking region. This DNA segment is capable of directing the liver-specific expression of a reporter gene in transfection assays. More interestingly, the basal activity of the LFB1/HNF1 promoter in cultured hepatoma cell lines is down-regulated by exogenously added LFB1/HNF1 protein itself. The ability to repress transcription starting from its own promoter requires the integrity of the N-terminal LFB1/HNF1 DNA-binding domain. Contrary to the expectations, in vitro binding experiments failed to demonstrate any specific and functional interaction of purified LFB1/HNF1 with the -3.5 kb promoter sequence. In addition to the DNA-binding domain, a 60 aa region contained in the C-terminus of the protein and distinct from the previously characterized activation domains, is also required for the repressing function.

Animals↗

PubMed2001

HERP, a novel heterodimer partner of HES/E(spl) in Notch signaling.

HERP1 and -2 are members of a new basic helix-loop-helix (bHLH) protein family closely related to HES/E(spl), the only previously known Notch effector. Like that of HES, HERP mRNA expression is directly up-regulated by Notch ligand binding without de novo protein synthesis. HES and HERP are individually expressed in certain cells, but they are also coexpressed within single cells after Notch stimulation. Here, we show that HERP has intrinsic transcriptional repression activity. Transcriptional repression by HES/E(spl) entails the recruitment of the corepressor TLE/Groucho via a conserved WRPW motif, whereas unexpectedly the corresponding-but modified-tetrapeptide motif in HERP confers marginal repression. Rather, HERP uses its bHLH domain to recruit the mSin3 complex containing histone deacetylase HDAC1 and an additional corepressor, N-CoR, to mediate repression. HES and HERP homodimers bind similar DNA sequences, but with distinct sequence preferences, and they repress transcription from specific DNA binding sites. Importantly, HES and HERP associate with each other in solution and form a stable HES-HERP heterodimer upon DNA binding. HES-HERP heterodimers have both a greater DNA binding activity and a stronger repression activity than do the respective homodimers. Thus, Notch signaling relies on cooperation between HES and HERP, two transcriptional repressors with distinctive repression mechanisms which, either as homo- or as heterodimers, regulate target gene expression.

3T3 Cells↗

PubMed2005

[Identification of a highly conserved domain that suppresses the DNA-binding domain-DNA interactions in the androgen receptor].

OBJECTIVE: To determine the gene regulation of androgen receptor (AR). METHODS: Gel shift assay, protein-protein pull down assay, western blot and technique of site-directed mutagenesis were used to study the gene regulation of AR. RESULTS: The N terminal of AR contained an inhibitory domain located in an 81-amino acid segment lying upstream of the DNA-binding domain (DBD). The inhibitory domain interacted directly with DBD and repressed DBD binding to the ARE. Mutations of the conserved amino acid residues (K520E and R538E) within the inhibitory domain decreased its inhibiting ability in vitro and increased AR trans-activation in vivo. CONCLUSION: These data demonstrated the existence of a novel inhibitory domain in the N-terminal part of AR, which might play important role in the regulation of AR trans-activation.

Adult↗