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PubMed1998

Molecular chaperones as HSF1-specific transcriptional repressors.

The rapid yet transient transcriptional activation of heat shock genes is mediated by the reversible conversion of HSF1 from an inert negatively regulated monomer to a transcriptionally active DNA-binding trimer. During attenuation of the heat shock response, transcription of heat shock genes returns to basal levels and HSF1 reverts to an inert monomer. These events coincide with elevated levels of Hsp70 and other heat shock proteins (molecular chaperones). Here, we show that the molecular chaperone Hsp70 and the cochaperone Hdj1 interact directly with the transactivation domain of HSF1 and repress heat shock gene transcription. Overexpression of either chaperone represses the transcriptional activity of a transfected GAL4-HSF1 activation domain fusion protein and endogenous HSF1. As neither the activation of HSF1 DNA binding nor inducible phosphorylation of HSF1 was affected, the primary autoregulatory role of Hsp70 is to negatively regulate HSF1 transcriptional activity. These results reveal that the repression of heat shock gene transcription, which occurs during attenuation, is due to the association of Hsp70 with the HSF1 transactivation domain, thus providing a plausible explanation for the role of molecular chaperones in at least one key step in the autoregulation of the heat shock response.

Animals↗
PubMed1997

Repression of p53-mediated transcription by MDM2: a dual mechanism.

The oncoprotein MDM2 binds to the activation domain of the tumor suppressor p53 and inhibits its ability to stimulate transcription. This same region of p53 is able to bind several basal transcription factors that appear to be important for the transactivation function of p53. It has therefore been suggested that MDM2 acts to inhibit p53 by concealing its activation domain from the basal machinery. Here we present data suggesting that MDM2 possesses an additional inhibitory function. Our experiments reveal that in addition to a p53-binding domain, MDM2 also contains an inhibitory domain that can directly repress basal transcription in the absence of p53. By fusing portions of MDM2 to a heterologous DNA-binding domain to allow p53-independent promoter recruitment, we have localized this inhibitory domain to a region encompassing amino acids 50-222 of MDM2. Furthermore, the function of this inhibitory domain does not require the presence of either TFIIA or the TAFs. Of the remaining basal factors, both the small subunit of TFIIE and monomeric TBP are bound by the MDM2 inhibitory domain. It is possible that MDM2 inhibits the ability of the preinitiation complex to synthesize RNA through one of these interactions. Our results are consistent with a model in which MDM2 represses p53-dependent transcription by a dual mechanism: a masking of the activation domain of p53 through a protein-protein interaction that additionally serves to recruit MDM2 to the promoter where it directly interferes with the basal transcription machinery.

Cell-Free System↗
PubMed2000

A regulatory cascade of the nuclear receptors FXR, SHP-1, and LRH-1 represses bile acid biosynthesis.

Bile acids repress the transcription of cytochrome P450 7A1 (CYP7A1), which catalyzes the rate-limiting step in bile acid biosynthesis. Although bile acids activate the farnesoid X receptor (FXR), the mechanism underlying bile acid-mediated repression of CYP7A1 remained unclear. We have used a potent, nonsteroidal FXR ligand to show that FXR induces expression of small heterodimer partner 1 (SHP-1), an atypical member of the nuclear receptor family that lacks a DNA-binding domain. SHP-1 represses expression of CYP7A1 by inhibiting the activity of liver receptor homolog 1 (LRH-1), an orphan nuclear receptor that is known to regulate CYP7A1 expression positively. This bile acid-activated regulatory cascade provides a molecular basis for the coordinate suppression of CYP7A1 and other genes involved in bile acid biosynthesis.

Animals↗
PubMed2002

Inhibition of the dihydrotestosterone-activated androgen receptor by nuclear receptor corepressor.

Nuclear receptor corepressor (NCoR) mediates transcriptional repression by unliganded nuclear receptors and certain steroid hormone receptors (SHRs) bound to nonphysiological antagonists, but has not been found to regulate SHRs bound to their natural ligands. This report demonstrates that NCoR interacts directly with the androgen receptor (AR) and represses dihydrotestosterone-stimulated AR transcriptional activity. The NCoR C terminus, containing the receptor interacting domains, was necessary for repression, which was ablated by mutations in the corepressor nuclear receptor (CoRNR) boxes. In contrast, the NCoR N terminus, containing domains that can recruit histone deacetylases, was not necessary for repression. Binding studies in vitro with a series of glutathione-S-transferase-NCoR and -AR fusion proteins demonstrated a direct interaction that was similarly dependent upon the NCoR corepressor nuclear receptor boxes and AR ligand binding domain and was independent of ligand and helix 12 in the AR ligand binding domain. This NCoR-AR interaction was further demonstrated in mammalian two-hybrid assays and by coimmunoprecipitation of the endogenous proteins from a prostate cancer cell line. Finally, AR transcriptional activity could be enhanced in vivo by sequestration of endogenous NCoR with unliganded thyroid hormone receptor. These results demonstrate that AR, in contrast to other SHRs, is regulated by NCoR and suggest the possibility of developing selective AR modulators that enhance this interaction.

Amino Acid Sequence↗
PubMed2006

A peptide motif in Raver1 mediates splicing repression by interaction with the PTB RRM2 domain.

Polypyrimidine tract-binding protein (PTB) is a regulatory splicing repressor. Raver1 acts as a PTB corepressor for splicing of alpha-tropomyosin (Tpm1) exon 3. Here we define a minimal region of Raver1 that acts as a repressor domain when recruited to RNA. A conserved [S/G][I/L]LGxxP motif is essential for splicing repressor activity and sufficient for interaction with PTB. An adjacent proline-rich region is also essential for repressor activity but not for PTB interaction. NMR analysis shows that LLGxxP peptides interact with a hydrophobic groove on the dorsal surface of the RRM2 domain of PTB, which constitutes part of the minimal repressor region of PTB. The requirement for the PTB-Raver1 interaction that we have characterized may serve to bring the additional repressor regions of both proteins into a configuration that allows them to synergistically effect exon skipping.

Amino Acid Motifs↗
PubMed1998

The C-terminal silencing domain of Rap1p is essential for the repression of ribosomal protein genes in response to a defect in the secretory pathway.

We have previously shown that a functional secretory pathway is essential for continued ribosome synthesis in Saccharomyces cerevisiae. When a temperature-sensitive mutant defective in the secretory pathway is transferred to the non-permissive temperature, transcription of both rRNA genes and ribosomal protein genes is nearly abolished. In order to define the cis -acting element(s) of ribosomal protein genes sensitive to a defect in the secretory pathway, we have constructed a series of fusion genes containing the CYH2 promoter region, with various deletions, fused to lacZ. Each fusion gene for which transcription is detected is subject to the repression. Rap1p is the transcriptional activator for most ribosomal protein genes, as well as having an important role in silencing in the vicinity of telomeres and at the silent mating-type loci. To assess its role in the repression of transcription by the defect in the secretory pathway, we have introduced rap1 mutations. The replacement of wild-type Rap1p by Rap1p truncated at the C-terminal region caused substantial attenuation of the repression. Furthermore, we have demonstrated that the Rap1p-truncation affects the repression of TCM1 , encoding ribosomal protein L3, which has no Rap1p-binding site in its upstream regulatory region. These results suggest that the repression of transcription of ribosomal protein genes by a secretory defect is mediated through Rap1p, but does not require a Rap1p-binding site within the UAS.

Artificial Gene Fusion↗
PubMed1996

A novel E1A domain mediates skeletal-muscle-specific enhancer repression independently of pRB and p300 binding.

The adenovirus E1A oncoprotein completely blocks muscle differentiation and specifically inhibits the transactivating function of myogenic basic helix-loop-helix (bHLH) transcription factors. This inhibition is dependent on the conserved region CR1 of E1A, which also constitutes part of the binding sites for the pocket proteins pRB, p107, and p130 and the transcriptional coactivators p300 and CBP. Here we report a detailed mutational analysis of E1A and the identification of a muscle inhibition motif within CR1. This motif encompasses amino acids 38 to 62 and inhibits Myf-5- or MyoD-mediated activation of myogenin and the muscle creatine kinase gene. Overexpression of this E1A region also inhibits the conversion of 10T1/2 fibroblasts to the myogenic lineage. The sequence motif EPDNEE (amino acids 55 to 60) within CR1 appears to be particularly important, because point mutations of this sequence diminish the E1A inhibitory activity. Interactions of E1A with pRB and with p300 do not seem to be necessary for the muscle-specific enhancer repression, because E1A mutants which lack these interactions still inhibit Myf-5- and MyoD-mediated transactivation. Moreover, overexpression of p300 fails to overcome muscle-specific inhibition by wild-type E1A and mutant E1A protein which lacks pRB binding. Since we have no evidence for direct E1A interaction with bHLH proteins, we propose that E1A may target a necessary cofactor of the muscle-specific bHLH transcription complex.

Adenovirus E1A Proteins↗
PubMed1996

p53 transactivation domain mutant Q22, S23 is impaired for repression of promoters and mediation of apoptosis.

p53 is multifunctional. To assess exactly what function is critical for the prevention of neoplastic transformation has proven difficult. Mutants with substitutions at positions 22 and 23 promised to address the relevance of transcription transactivation since they seemed to be defective specifically for this function. We report here that p53 mutant Q22, S23 [p53 (22,23)] is not only impaired for transactivation but for the repression of the fos promoter and SV40 early promoter. Furthermore, whereas p53 (22,23) fails to efficiently transactivate reporter genes in two p53-negative cell lines, it stimulates reporters and suppresses proliferation in two wild-type (wt) p53-positive cell lines strongly above the levels induced by the transfection procedure alone. This transactivation is refractory to inhibition by MDM-2. Finally, p53 (22,23) expressed from large plasmid quantity (1 microg) is crippled for the mediation of apoptosis in p53-negative Hep3B hepatocarcinoma cells. Nevertheless, at a quantity of only 10 ng, both mutant and wt p53 plasmids but not control plasmid, are able to induce some cell death which is not inhibitable by MDM-2. Thus, a correlation exists between p53's functions to regulate promoters and to efficiently mediate apoptosis in Hep3B cells.

Animals↗
PubMed2000

Modulation of thyroid hormone receptor silencing function by co-repressors and a synergizing transcription factor.

We have found that the thyroid hormone receptor (T3R) functionally synergizes with the CCCTC-binding factor (CTCF). CTCF is a highly conserved zinc-finger protein that has been connected with multiple functions in gene regulation including chromatin insulator activity, transcriptional enhancement and silencing as well as tumour suppression. A specific property of CTCF is that some of the binding sites are found in the vicinity of T3R-binding sites. Interestingly, both factors synergize in repression as well as in activation. T3R-mediated repression has been shown to involve co-repressors such as the silencing mediator for retinoic acid and thyroid hormone receptor (SMRT), N-CoR or Alien. These co-repressors in turn have been found to interact with Sin3A. Until now, the mechanisms by which CTCF synergizes with T3R in transcriptional repression has not been determined. Here we show that CTCF comprises autonomous silencing domains that mediate transcriptional repression when tethered to a promoter sequence. At least one of these domains, the zinc-finger region of CTCF, binds Sin3A without binding to SMRT or N-CoR and recruits histone deacetylation activity. For Sin3A we identified two different domains interacting independently with the CTCF zinc-finger cluster. The ability of regions of CTCF to retain deacetylase activity is correlated with the ability to bind to Sin3A and to repress transcription. Taking these results together, the synergy in repression mediated by T3R and CTCF might be achieved by the binding of multiple molecules of Sin3A to the T3R/CTCF-DNA complex, thus providing a large platform for the recruitment of histone deacetylases.

Animals↗
PubMed2004

How the Rgt1 transcription factor of Saccharomyces cerevisiae is regulated by glucose.

Rgt1 is a transcription factor that regulates expression of HXT genes encoding glucose transporters in the yeast Saccharomyces cerevisiae. Rgt1 represses HXT gene expression in the absence of glucose; high levels of glucose cause Rgt1 to activate expression of HXT1. We identified four functional domains of Rgt1. A domain required for transcriptional repression (amino acids 210-250) is required for interaction of Rgt1 with the Ssn6 corepressor. Another region of Rgt1 (320-380) is required for normal transcriptional activation, and sequences flanking this region (310-320 and 400-410) regulate this function. A central region (520-830) and a short sequence adjacent to the zinc cluster DNA-binding domain (80-90) inhibit transcriptional repression when glucose is present. We found that this middle region of Rgt1 physically interacts with the N-terminal portion of the protein that includes the DNA-binding domain. This interaction is inhibited by the Rgt1 regulator Mth1, which binds to Rgt1. Our results suggest that Mth1 promotes transcriptional repression by Rgt1 by binding to it and preventing the intramolecular interaction, probably by preventing phosphorylation of Rgt1, thereby enabling Rgt1 to bind to DNA. Glucose induces HXT1 gene expression by causing Mth1 degradation, allowing Rgt1 phosphorylation, and leading to the intramolecular interaction that inhibits DNA binding of Rgt1.

Adaptor Proteins, Signal Transducing↗
PubMed2003

A conformational switch between transcriptional repression and replication initiation in the RepA dimerization domain.

Plasmids are natural vectors for gene transfer. In Gram-negative bacteria, plasmid DNA replication is triggered when monomers of an initiator protein (Rep) bind to direct repeats at the origin sequence. Rep dimers, which are inactive as initiators, bind to an inverse repeat operator, repressing transcription of the rep gene. Rep proteins are composed of N-terminal dimerization and C-terminal DNA-binding domains. Activation of Rep is coupled to dimer dissociation, converting the dimerization domain into a second origin-binding module. Although the structure of the monomeric F plasmid initiator (mRepE) has been determined, the molecular nature of Rep activation remains unknown. Here we report the crystal structure of the dimeric N-terminal domain of the pPS10 plasmid initiator (dRepA). dRepA has a winged-helix fold, as does its homologous domain in mRepE. However, dimerization transforms an interdomain loop and beta-strand (monomeric RepE) into an alpha-helix (dimeric RepA). dRepA resemble the C terminus of eukaryotic and archaeal Cdc6, giving clues to the phylogeny of DNA replication initiators.

Amino Acid Sequence↗
PubMed2000

Cytoplasmic catalytic subunit of protein kinase A mediates cross-repression by NF-kappa B and the glucocorticoid receptor.

Negative transcriptional regulation or cross-coupling between NF-kappa B (RelA) and the glucocorticoid receptor (GR) is proposed to play a regulatory role in human physiology and disease. Despite previous advances, the biochemical basis of this phenomenon remains a subject of controversy. We show here that the inhibition of GR activity by RelA does not require the RelA DNA binding, transactivation, or nuclear localization domains. Surprisingly, RelA repression of GR is abolished by mutation of the conserved protein kinase A (PKA) site at amino acid residue 276 of RelA. We show that GR associates in vivo and in vitro with the catalytic subunit of PKA (PKAc) in a ligand-independent manner and that GR transcription depends on PKA signaling. Indeed, we demonstrated that GR-mediated inhibition of NF-kappa B transactivation is PKAc-dependent. In contrast to previous models, we suggest that the cross-coupling requires a cytoplasmic step and is regulated by a PKAc-associated signaling.

Catalytic Domain↗
PubMed2000

In-depth mutational analysis of the promyelocytic leukemia zinc finger BTB/POZ domain reveals motifs and residues required for biological and transcriptional functions.

The promyelocytic leukemia zinc finger (PLZF) protein is a transcription factor disrupted in patients with t(11;17)(q23;q21)-associated acute promyelocytic leukemia. PLZF contains an N-terminal BTB/POZ domain which is required for dimerization, transcriptional repression, formation of high-molecular-weight DNA-protein complexes, nuclear sublocalization, and growth suppression. X-ray crystallographic data show that the PLZF BTB/POZ domain forms an obligate homodimer via an extensive interface. In addition, the dimer possesses several highly conserved features, including a charged pocket, a hydrophobic monomer core, an exposed hydrophobic surface on the floor of the dimer, and two negatively charged surface patches. To determine the role of these structures, mutational analysis of the BTB/POZ domain was performed. We found that point mutations in conserved residues that disrupt the dimer interface or the monomer core result in a misfolded nonfunctional protein. Mutation of key residues from the exposed hydrophobic surface suggests that these are also important for the stability of PLZF complexes. The integrity of the charged-pocket region was crucial for proper folding of the BTB/POZ domain. In addition, the pocket was critical for the ability of the BTB/POZ domain to repress transcription. Alteration of charged-pocket residue arginine 49 to a glutamine (mutant R49Q) yields a domain that can still dimerize but activates rather than represses transcription. In the context of full-length PLZF, a properly folded BTB/POZ domain was required for all PLZF functions. However, PLZF with the single pocket mutation R49Q repressed transcription, while the double mutant D35N/R49Q could not, despite its ability to dimerize. These results indicate that PLZF requires the BTB/POZ domain for dimerization and the charged pocket for transcriptional repression.

Amino Acid Motifs↗
PubMed1996

Repression by HoxA7 is mediated by the homeodomain and the modulatory action of its N-terminal-arm residues.

Hox genes encode homeodomain-containing proteins that are presumed to control spatial patterning during murine embryogenesis through their actions as transcriptional regulatory proteins. In this study, we have investigated the transcriptional function of a prototypic member of this family, HoxA7. We demonstrate that HoxA7 function as a potent transcriptional repressor and that its action as such requires several domains, including both activator and repressor regions. The repressor regions are contained within the homeodomain and a C-terminal acidic region, both of which are well conserved among members of the Hox family. Accordingly, we show that two other members of this family also function as repressors, although they vary in their relative repressor potency. Finally, we explore the novel observation that the homeodomain of HoxA7 functions as a transcriptional repressor domain. We show that the homeodomain compared with two other DNA-binding domains, is unique in its ability to function as a repressor domain and that repression requires conserved residues, in helix III. We further show that residues in the N-terminal arm of the homeodomain contribute to the differential repressor actions of various Hox proteins. These findings demonstrate that the transcriptional function of HoxA7 and possibility of Hox proteins in general is determined by their unique combination of conserved and nonconserved regions as well as through the complex actions of their homeodomains.

3T3 Cells↗
PubMed1999

Three distinct domains in TEL-AML1 are required for transcriptional repression of the IL-3 promoter.

A cytogenetically cryptic (12;21) translocation is the most common molecular abnormality identified in childhood acute lymphoblastic leukemia (ALL), and it generates a chimeric TEL-AML1 protein. Fusion of the Helix-Loop-Helix (HLH) (also called the pointed) domain of TEL to AML1 has been suggested to convert AML1 from a transcriptional activator to a repressor. To define the structural features of this chimeric protein required for repression, we analysed the transcriptional activity of a series of TEL-AML1 mutants on the AML1-responsive interleukin-3 (IL-3) promoter, a potentially relevant gene target. Our results demonstrate that TEL-AML1 represses basal IL-3 promoter activity in lymphoid cells, and deletion mutant analysis identified three distinct domains of TEL-AML1 that are required for repression; the HLH (pointed) motif contained in the TEL portion of TEL-AML1, and both the runt homology domain (Rhd) and the 74 amino acids downstream of the Rhd that are present in the AML1 portion of the fusion protein. Although AML1B (and a shorter AML1 isoform, AML1A) have transcriptional activating activity on the IL-3 promoter, fusion of the AML1 gene to the TEL gene generates a repressor of IL-3 expression. Consistent with this activity, freshly isolated human ALL cells that contain TEL-AML1 do not express IL-3.

Child↗
PubMed2004

Epigenetic regulation of mammalian genomic imprinting.

Imprinted genes play important roles in development, and most are clustered in large domains. Their allelic repression is regulated by 'imprinting control regions' (ICRs), which are methylated on one of the two parental alleles. Non-histone proteins and nearby sequence elements influence the establishment of this differential methylation during gametogenesis. DNA methylation, histone modifications, and also polycomb group proteins are important for the somatic maintenance of imprinting. The way ICRs regulate imprinting differs between domains. At some, the ICR constitutes an insulator that prevents promoter-enhancer interactions, when unmethylated. At other domains, non-coding RNAs could be involved, possibly by attracting chromatin-modifying complexes. The latter silencing mechanism has similarities with X-chromosome inactivation.

Animals↗
PubMed1989

Mating-type control in Saccharomyces cerevisiae: isolation and characterization of mutants defective in repression by a1-alpha 2.

The alpha 2 protein, the product of the MAT alpha 2 cistron, represses various genes specific to the a mating type (alpha 2 repression), and when combined with the MATa1 gene product, it represses MAT alpha 1 and various haploid-specific genes (a1-alpha 2 repression). One target of a1-alpha 2 repression is RME1, which is a negative regulator of a/alpha-specific genes. We have isolated 13 recessive mutants whose a1-alpha 2 repression is defective but which retain alpha 2 repression in a genetic background of ho MATa HML alpha HMRa sir3 or ho MAT alpha HMRa HMRa sir3. These mutations can be divided into three different classes. One class contains a missense mutation, designated hml alpha 2-102, in the alpha 2 cistron of HML, and another class contains two mat alpha 2-202, in the MAT alpha locus. These three mutants each have an amino acid substitution of tyrosine or acid substitution of tyrosine or phenylalanine for cysteine at the 33rd codon from the translation initiation codon in the alpha 2 cistron of HML alpha or MAT alpha. The remaining 10 mutants make up the third class and form a single complementation group, having mutations designated aar1 (a1-alpha 2 repression), at a gene other than MAT, HML, HMR, RME1, or the four SIR genes. Although a diploid cell homozygous for the aarl and sir3 mutations and for the MATa, HML alpha, and HMRa alleles showed alpha mating type, it could sporulate and gave rise to asci containing four alpha mating-type spores. These facts indicate that the domain for alpha2 repression is separable from that for a1-alpha2 protein interaction or complex formation in the alpha2 protein and that an additional regulation gene, AAR1, is associated with the a1-alpha2 repression of the alpha1 cistron and haploid-specific genes.

Amino Acid Sequence↗
PubMed2006

CSPP and CSPP-L associate with centrosomes and microtubules and differently affect microtubule organization.

We recently described the identification of a centrosome/spindle pole associated protein, CSPP, involved in cell cycle progression. Here we report a CSPP isoform denoted CSPP-L, with a 294 amino acids longer N-terminus and a 51 amino acids insertion located in the coiled-coil mid-domain. Expression analysis indicates an inverse cell cycle dependent regulation. CSPP mRNA expression is highest in G1 whereas CSPP-L expression is highest in G2/M. Ectopic expression of CSPP-L impairs cell cycle progression weaker in G1 than CSPP. Furthermore, normal mitotic phenotypes were observed in CSPP-L but not in CSPP transfectants. CSPP-L relocates from spindle microtubules and poles in metaphase to the mid-spindle in anaphase and concentrates at the mid-body in telophase/cytokinesis. CSPP-L high-expressing mitotic cells were predominantly characterized by lagging chromosomes or monopolar spindles, in contrast to the predominant multipolar spindles observed with CSPP expression. The different effects of CSPP and CSPP-L on microtubule organization in mitosis depend on the coiled-coil mid-domain insertion. The common C-terminal domain is required to repress that activity until mitosis. Notably, this C-terminal domain alone can associate with centrosomes in a microtubule independent manner. Taken together, CSPP and CSPP-L interact with centrosomes and microtubules and can differently affect microtubule organization.

Cell Cycle↗