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PubMed1998

HIV-1 tat binds TAFII250 and represses TAFII250-dependent transcription of major histocompatibility class I genes.

HIV Tat, a transactivator of viral transcription, represses transcription of major histocompatibility (MHC) class I genes. Repression depends exclusively on the C-terminal domain of Tat, although the mechanism of this repression has not been known. We now show that repression results from the interaction of Tat with the TAFII250 component of the general transcription factor, TFIID. The C-terminal domain of Tat binds to a site on TAFII250 that overlaps the histone acetyl transferase domain, inhibiting TAFII250 histone acetyl transferase activity. Furthermore, promoters repressed by Tat, including the MHC class I promoter, are dependent on TAFII250 whereas those that are not repressed by Tat, such as SV40 and MuLV promoters, are independent of functional TAFII250. Thus, Tat repression of MHC class I transcription would be one mechanism by which HIV avoids immune surveillance.

Acetyltransferases↗
PubMed1999

Inhibition of hTAFII32-binding implicated in the transcriptional repression by central regions of mutant p53 proteins.

We previously identified a movable and regulable inactivation function within the central region (CRts247) of a temperature-sensitive p53 (p53(ts)) mutant, p53(N247I). Here we showed that central regions from several p53(ts) mutants behaved similarly, i.e. they repressed a neighboring activation domain only when existing in the mutant status. Using chimeric protein GAL4VP16-CRts247 as an example, we demonstrated that de novo protein synthesis was not required for the reactivation of the chimeric protein, indicating that a post-translational mechanism was involved in the control of CRts247 activity. The CRts247-conferred thermo-regulability did not work via a mechanism demanding either an alteration of the subcellular compartmentalization of or the inactivation of DNA-binding activity of the GAL4 chimera. Further, CRts247 did not function in trans, eliminating the possibility that the observed repression was because of the competition for a putative factor(s) by the mutant p53 domain. Rather, CRts247 bestowed temperature-dependent interaction with hTAFII32 to the VP16 activation domain. In a parallel experiment, CRts247 also caused a large reduction in the affinity of hTAFII32 to the p53 activation domain at the nonpermissive temperature. These results strongly suggested that inhibition of hTAFII32 binding could be one of the mechanisms responsible for the transcriptional repression by mutant p53 central regions.

Mutation↗
PubMed1997

Two domains of MyoD mediate transcriptional activation of genes in repressive chromatin: a mechanism for lineage determination in myogenesis.

Genetic studies have demonstrated that MyoD and Myf5 establish the skeletal muscle lineage, whereas myogenin mediates terminal differentiation, yet the molecular basis for this distinction is not understood. We show that MyoD can remodel chromatin at binding sites in muscle gene enhancers and activate transcription at previously silent loci. TGF-beta, basic-FGF, and sodium butyrate blocked MyoD-mediated chromatin reorganization and the initiation of transcription. In contrast, TGF-beta and sodium butyrate did not block transcription when added after chromatin remodeling had occurred. MyoD and Myf-5 were 10-fold more efficient than myogenin at activating genes in regions of transcriptionally silent chromatin. Deletion mutagenesis of the MyoD protein demonstrated that the ability to activate endogenous genes depended on two regions: a region rich in cysteine and histidine residues between the acidic activation domain and the bHLH domain, and a second region in the carboxyl terminus of the protein. Neither region has been shown previously to regulate gene transcription and both have domains that are conserved in the Myf5 protein. Our results establish a mechanism for chromatin modeling in the skeletal muscle lineage and define domains of MyoD, independent of the activation domain, that participate in chromatin reorganization.

3T3 Cells↗
PubMed1997

Transcriptional repression of p53 promoter by hepatitis C virus core protein.

Our previous results have suggested that the putative core protein of hepatitis C virus (HCV) transcriptionally regulates cellular and viral genes, inhibits cisplatin and c-myc-mediated apoptotic cell death under certain conditions, and transforms primary rat embryo fibroblast cells with a cooperative oncogene. Because HCV appears to cause hepatocellular carcinoma, we evaluated the regulatory role of the HCV core protein on p53, a well known tumor suppressor gene, by an in vitro transfection assay. HCV core protein repressed transcriptional activity of the p53 promoter when tested separately in COS7 and HeLa cells. Deletion mutational analysis of the HCV core gene indicated that the regulatory domain involved in the repression of p53 transcriptional activity is located around amino acid residues 80-122 encompassing a putative DNA binding motif and two major phosphorylation sites. Results from this study suggest that the putative core protein may have an important biological role in the promotion of cell growth by repressing p53 transcription, and this appears to be consistent with certain earlier observations about HCV core moving into the nucleus.

Animals↗
PubMed2003

Specific induction and carbon/nitrogen repression of arginine catabolism gene of Aspergillus nidulans--functional in vivo analysis of the otaA promoter.

The arginine catabolism gene otaA encoding ornithine transaminase (OTAse) is specifically induced by arginine and is under the control of the broad-domain carbon and nitrogen repression systems. Arginine induction is mediated by a product of arcA gene coding for Zn(2)C(6) activator. We have identified a region responsible for arginine induction in the otaA promoter (AnUAS(arg)). Deletions within this region result in non-inducibility of OTAse by arginine, whether in an arcA(+) strain or in the presence of the arcA(d)47 gain of function allele. AnUAS(arg) is very similar to the Saccharomyces cerevisiae UAS(arg), a sequence bound by the Zn(2)C(6) activator (ArgRIIp), acting in a complex with two MADS-box proteins (McmIp and ArgRIp). We demonstrate here that two CREA in vitro binding sites in the otaA promoter are functional in vivo. CREA is directly involved in carbon repression of the otaA gene and it also reduces its basal level of expression. Although AREA binds to the otaA promoter in vitro, it probably does not participate in nitrogen metabolite repression of the gene in vivo. We show here that another putative negatively acting GATA factor AREB participates directly or indirectly in otaA nitrogen repression. We also demonstrate that the high levels of OTAse activity are an important factor in the suppression of proline auxotrophic mutations. This suppression can be achieved neither by growing of the proline auxotroph under carbon/nitrogen derepressing conditions nor by introducing of a creA(d) mutation.

Arginine↗
PubMed1998

Repressive effect on oligonucleosome transcription of the core histone tail domains.

Histone-DNA templates for bacteriophage T7 RNA polymerase were assembled from histone octamers and three different DNA species, two circular (pGEMEX-1 and pT207-18) and one linear (T7-207-18). pGEMEX is devoid of nucleosome positioning sequences, while in pT207-18 and T7-207-18 the region downstream of the promoter contains 18 tandem repeats of a 207 bp positioning sequence derived from the 5S RNA gene of the sea urchin Lytechinus variegatus. Elimination of the histone tails in the assembled oligonucleosomes by trypsin digestion is accompanied, in all three DNA species, by substantial increases in transcription efficiency, assayed at different KCl and MgCl2 concentrations, after allowing for the aggregation observed under certain conditions. In the absence of KCl and at low MgCl2 concentration, the presence of 2 mM spermidine causes substantial aggregation of the intact oligonucleosomes but has a much smaller effect on those trypsin digested. The untreated histone-DNA templates, assembled on pGEMEX-1 and T7-207-18, give transcription products significantly shorter than those obtained with the corresponding free DNA. With oligonucleosome templates lacking histone tails, the transcripts have an average length intermediate between those corresponding to free DNA and intact histone-DNA, which indicates a partial elimination of the elongation restrictions imposed by intact histone octamers. The absence of histone terminal domains facilitates both transcriptional initiation and elongation. Apparently, the interaction of the histone tails with DNA at the nucleosomal level is responsible, at least in part, for their repressive effect on transcription.

Bacteriophage T7↗
PubMed2003

Nuclear receptor corepressors.

The ability of NR LBDs to transfer repression function to a heterologous DNA binding domain, and the cross-squelching of repression by untethered LBDs, has suggested that repression is mediated by interactions with putative cellular corepressor proteins. The yeast-two hybrid screen for protein interactors has proven to be the key to the isolation and characterization of corepressors. This short review will focus on N-CoR and SMRT.

Journal Article↗
PubMed2001

Sp1 acts as a repressor of the human adenine nucleotide translocase-2 (ANT2) promoter.

The human adenine nucleotide translocator-2 promoter is activated by adjacent Sp1 activation elements centered at nucleotides -79 and -68 (Abox and Bbox, respectively), and is repressed by Sp1 bound to a GC element (Cbox) that lies adjacent to transcription start. Here, we address the mechanism of this unique Sp1-mediated repression using transfected Drosophila SL2 and mammalian cell lines. We show that repression is not due to steric interference with assembly of the transcription machinery, as Sp1 bound to the Cbox can, under certain conditions, activate the promoter. Furthermore, ectopic expression of Sp1 deletion mutants in SL2 cells demonstrates that both the Sp1-mediated repression and activation require the D transactivation domain of Sp1 bound to the Cbox. In addition, repression of ABbox-mediated activation is eliminated by separating the Abox and Bbox. Thus, for Cbox-bound Sp1 to repress, Sp1 must be precisely positioned at the region of the ABboxes. Together, these data suggest that the D transactivation domain mediates interactions by Sp1 complexes on separate GC elements that results in repression of the activating Sp1 species.

Animals↗
PubMed1999

Regulation of the Wilms' tumour suppressor protein transcriptional activation domain.

The Wilms' tumour suppressor protein WT1 contains a transcriptional regulatory domain that can either activate or repress transcription depending upon its cellular environment. The mechanistic basis for this dichotomy is unclear however. Here, we dissect the transcriptional regulatory domains of WT1. We find that a region within the domain of WT1 attributed to transcriptional repression is a potent suppressor of the activation domain at several promoters and in different cell types. In vitro transcription analysis suggests that the mechanism of suppression of the activation domain occurs at the level of transcription initiation. Furthermore we find that the WT1 suppression domain is able to inhibit a heterologous activation domain when fused in cis. Dissection of this domain resulted in the delineation of a 30 amino acid region that was sufficient to confer suppression of a transcriptional activation domain both in vivo and in vitro. Additionally, we find that the WT1 transcriptional activation domain interacts with the general transcription factor TFIIB and that this interaction is not affected by the suppression domain. Taken together, these studies suggest that the suppression domain of WT1 interacts with a cosuppressor protein to mediate inhibition of the WT1 transcriptional activation domain.

3T3 Cells↗
PubMed1998

Retinoblastoma protein tethered to promoter DNA represses TBP-mediated transcription.

The retinoblastoma (RB) tumour suppressor protein negatively regulates cell proliferation by modulating transcription of growth-regulatory genes. Recruitment of Rb to promoters, by association with E2F complex or by fusion with heterologous DNA-binding domains, demonstrated that Rb represses directly transcription. Recent studies also suggest that the RB protein is able to repress gene transcription mediated by the RNA polymerase I and III. Since the TATA-binding protein (TBP) is an important component for transcription mediated by all three RNA polymerases, we have analysed the functional interaction between Rb and TBP in vivo in the context of RNA pol II-driven transcription. We demonstrated that in mammalian cells Rb tethered to promoter represses TBP-mediated activation in vivo, and Rb-mediated repression is reversed in the presence of the inhibition of histone deacetylase activity by trichostatin A (TSA).

Cell Line↗
PubMed2003

Repression of E2F1-mediated transcription by the ErbB3 binding protein Ebp1 involves histone deacetylases.

Ebp1, an ErbB3 binding protein that is a member of the proliferation-associated PA2G4 family, inhibits the proliferation and induces the differentiation of human ErbB positive breast and prostate cancer cell lines. Ebp1 binds the tumor suppressor retinoblastoma protein (Rb) both in vivo and in vitro, and Rb and Ebp1 cooperate to inhibit the transcription of the E2F1-regulated cyclin E promoter. We show here that Ebp1 can inhibit the transcription of other E2F-regulated reporter genes and of several endogenous E2F-regulated genes important in cell cycle progression in both Rb positive and Rb null cells. The Ebp1-mediated transcriptional repression depended on the presence of an E2F1 consensus element in the promoters. A fusion of Ebp1 with the GAL4 DNA binding domain protein had independent transcriptional repression activity that mapped to the C-terminal region of Ebp1. This C-terminal region of Ebp1 bound functional histone deacetylase (HDAC) activity and inhibitors of HDAC significantly reduced Ebp1-mediated repression. Ebp1 bound HDAC2, but not HDAC1, in vitro. An Ebp1 mutant lacking the HDAC binding domain failed to inhibit transcription. Our results suggest that Ebp1 can repress transcription of some E2F-regulated promoters and that one mechanism of Ebp1- mediated transcriptional repression is via its ability to recruit HDAC activity.

Adaptor Proteins, Signal Transducing↗
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↗
PubMed2005

FOG-1 recruits the NuRD repressor complex to mediate transcriptional repression by GATA-1.

Transcription factor GATA-1 and its cofactor FOG-1 coordinate erythroid cell maturation by activating erythroid-specific genes and repressing genes associated with the undifferentiated state. Here we show that FOG-1 binds to the NuRD corepressor complex in vitro and in vivo. The interaction is mediated by a small conserved domain at the extreme N-terminus of FOG-1 that is necessary and sufficient for NuRD binding. This domain defines a novel repression module found in diverse transcriptional repressors. NuRD is present at GATA-1/FOG-1-repressed genes in erythroid cells in vivo. Point mutations near the N-terminus of FOG-1 that abrogate NuRD binding block gene repression by FOG-1. Finally, the ability of GATA-1 to repress transcription was impaired in erythroid cells expressing mutant forms of FOG-1 that are defective for NuRD binding. Together, these studies show that FOG-1 and likely other FOG-like proteins are corepressors that link GATA factors to histone deacetylation and nucleosome remodeling.

Amino Acid Motifs↗
PubMed2005

Analysis of RovA, a transcriptional regulator of Yersinia pseudotuberculosis virulence that acts through antirepression and direct transcriptional activation.

The transcription factor RovA of Yersinia pseudotuberculosis and analogous proteins in other Enterobacteriaceae activate the expression of virulence genes that play a crucial role in stress adaptation and pathogenesis. In this study, we demonstrate that the RovA protein forms dimers independent of DNA binding, stimulates RNA polymerase, most likely via its C-terminal domain, and counteracts transcriptional repression by the histone-like protein H-NS. As the molecular function of the RovA family is largely uncharacterized, random mutagenesis and terminal deletions were used to identify functionally important domains. Our analysis showed that a winged-helix motif in the center of the molecule is essential and directly involved in DNA binding. Terminal deletions and amino acid changes within both termini also abrogate RovA activation and DNA-binding functions, most likely due to their implication in dimer formation. Finally, we show that the last four amino acids of RovA are crucial for activation of gene transcription. Successive deletions of these residues result in a continuous loss of RovA activity. Their removal reduced the capacity of RovA to activate RNA polymerase and abolished transcription of RovA-activated promoters in the presence of H-NS, although dimerization and DNA binding functions were retained. Our structural model implies that the final amino acids of RovA play a role in protein-protein interactions, adjusting RovA activity.

Bacterial Proteins↗
PubMed1999

A 13-amino acid amphipathic alpha-helix is required for the functional interaction between the transcriptional repressor Mad1 and mSin3A.

Members of the Mad family of bHLHZip proteins heterodimerize with Max and function to repress the transcriptional and transforming activities of the Myc proto-oncogene. Mad:Max heterodimers repress transcription by recruiting a large multi-protein complex containing the histone deacetylases, HDAC1 and HDAC2, to DNA. The interaction between Mad proteins and HDAC1/2 is mediated by the corepressor mSin3A and requires sequences at the amino terminus of the Mad proteins, termed the SID, for Sin3 interaction domain, and the second of four paired amphipathic alpha-helices (PAH2) in mSin3A. To better understand the requirements for the interaction between the SID and PAH2, we have performed mutagenesis and structural studies on the SID. These studies show that amino acids 8-20 of Mad1 are sufficient for SID:PAH2 interaction. Further, this minimal 13-residue SID peptide forms an amphipathic alpha-helix in solution, and residues on the hydrophobic face of the SID helix are required for interaction with PAH2. Finally, the minimal SID can function as an autonomous and portable repression domain, demonstrating that it is sufficient to target a functional mSin3A/HDAC corepressor complex.

Amino Acid Sequence↗
PubMed2004

The proline-rich homeodomain protein recruits members of the Groucho/Transducin-like enhancer of split protein family to co-repress transcription in hematopoietic cells.

The proline-rich homeodomain protein (PRH/Hex) is important in the control of cell proliferation and differentiation and in the regulation of multiple processes in embryonic development. We have shown previously that PRH contains two domains that can independently bring about transcriptional repression. The PRH homeodomain represses transcription by binding to TATA box sequences, whereas the proline-rich N-terminal domain of PRH can repress transcription when attached to a heterologous DNA-binding domain. The Groucho/transducin-like enhancer of split (TLE) family of proteins are transcriptional co-repressors that interact with a number of DNA-bound transcription factors and play multiple roles in development. Here we demonstrate that the proline-rich N-terminal domain of PRH binds to TLE1 in vitro and in yeast two-hybrid assays. We show that PRH and TLE proteins are co-expressed in hematopoietic cells and interact in co-immunoprecipitation assays. We demonstrate that TLE1 increases repression by PRH in transient transfection assays and that titration of endogenous TLE proteins by co-expression of Grg5, a natural trans-dominant negative protein, alleviates transcriptional repression by PRH. Finally, we show that a mutation in the PRH N-terminal domain that blocks the PRH-TLE1 interaction in vitro eliminates co-repression. We discuss these results in terms of the roles of PRH and TLE in cell differentiation and development.

Amino Acid Motifs↗
PubMed2001

Sequence-specific transcriptional repression by KS1, a multiple-zinc-finger-Krüppel-associated box protein.

The vertebrate genome contains a large number of Krüppel-associated box-zinc finger genes that encode 10 or more C(2)-H(2) zinc finger motifs. Members of this gene family have been proposed to function as transcription factors by binding DNA through their zinc finger region and repressing gene expression via the KRAB domain. To date, however, no Krüppel-associated box-zinc finger protein (KRAB-ZFP) and few proteins with 10 or more zinc finger motifs have been shown to bind DNA in a sequence-specific manner. Our laboratory has recently identified KS1, a member of the KRAB-ZFP family that contains 10 different C(2)-H(2) zinc finger motifs, 9 clustered at the C terminus with an additional zinc finger separated by a short linker region. In this study, we used a random oligonucleotide binding assay to identify a 27-bp KS1 binding element (KBE). Reporter assays demonstrate that KS1 represses the expression of promoters containing this DNA sequence. Deletion and site-directed mutagenesis reveal that KS1 requires nine C-terminal zinc fingers and the KRAB domain for transcriptional repression through the KBE site, whereas the isolated zinc finger and linker region are dispensable for this function. Additional biochemical assays demonstrate that the KS1 KRAB domain interacts with the KAP-1 corepressor, and mutations that abolish this interaction alleviate KS1-mediated transcriptional repression. Thus, this study provides the first direct evidence that a KRAB-ZFP binds DNA to regulate gene expression and provides insight into the mechanisms used by multiple-zinc-finger proteins to recognize DNA sequences.

Animals↗
PubMed2000

Silencing subdomains of v-ErbA interact cooperatively with corepressors: involvement of helices 5/6.

Members of the thyroid hormone receptor (TR) family act on vertebrate development and homeostasis by activating or repressing transcription of specific target genes in a ligand-dependent way. Repression by TR in the absence of ligand is mediated by an active silencing mechanism. The oncogene v-ErbA is a variant form of TR unable to bind hormone and thus acts as a constitutive repressor. Functional studies and mutation analysis revealed that the TR/v-ErbA silencing domain is composed of three silencing subdomains (SSD1-3) which, although nonfunctional individually, synergize such that silencing activity is restored when they are combined in a heteromeric complex. Here we demonstrate, using protein interaction assays in vitro and in vivo, that the inactive v-ErbA point mutant L489R within helix 5/6 in SSD2 fails to interact with the two corepressors N-CoR (nuclear receptor corepressor) or SMRT (silencing mediator of retinoic acid and thyroid hormone receptor). Furthermore, mutants in SSD1 and SSD3 exhibit a reduced corepressor recruitment corresponding to their weak residual silencing activity. In mammalian two-hybrid assays, only the combination of all three silencing subdomains, SSD1-3, leads to a cooperative binding to the corepressors N-CoR or SMRT comparable to that of the full-length v-ErbA repression domain. In conclusion, full silencing activity requires corepressor interaction with all three silencing subdomains, SSD1-3. Among these, SSD2 is a new target for N-CoR and SMRT and is essential for corepressor binding and function.

Animals↗