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Recruitment of Tup1-Ssn6 by yeast hypoxic genes and chromatin-independent exclusion of TATA binding protein 总被引:2,自引:0,他引:2
The Tup1-Ssn6 general repression complex in Saccharomyces cerevisiae represses a wide variety of regulons. Regulon-specific DNA binding proteins recruit the repression complex, and their synthesis, activity, or localization controls the conditions for repression. Rox1 is the hypoxic regulon-specific protein, and a second DNA binding protein, Mot3, augments repression at tightly controlled genes. We addressed the requirements for Tup1-Ssn6 recruitment to two hypoxic genes, ANB1 and HEM13, by using chromatin immunoprecipitation assays. Either Rox1 or Mot3 could recruit Ssn6, but Tup1 recruitment required Ssn6 and Rox1. We also monitored events during derepression. Rox1 and Mot3 dissociated from DNA quickly, accounting for the rapid accumulation of ANB1 and HEM13 RNAs, suggesting a simple explanation for induction. However, Tup1 remained associated with these genes, suggesting that the localization of Tup1-Ssn6 is not the sole determinant of repression. We could not reproduce the observation that deletion of the Tup1-Ssn6-interacting protein Cti6 was required for induction. Finally, Tup1 is capable of repression through a chromatin-dependent mechanism, the positioning of a nucleosome over the TATA box, or a chromatin-independent mechanism. We found that the rate of derepression was independent of the positioned nucleosome and that the TATA binding protein was excluded from ANB1 even in the absence of the positioned nucleosome. The mediator factor Srb7 has been shown to interact with Tup1 and to play a role in repression at several regulons, but we found that significant levels of repression remained in srb7 mutants even when the chromatin-dependent repression mechanism was eliminated. These findings suggest that the repression of different regulons or genes may invoke different mechanisms. 相似文献
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The Rox1 repressor of the Saccharomyces cerevisiae hypoxic genes is a specific DNA-binding protein with a high-mobility-group motif. 总被引:11,自引:5,他引:6
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The ROX1 gene encodes a repressor of the hypoxic functions of the yeast Saccharomyces cerevisiae. The DNA sequence of the gene was determined and found to encode a protein of 368 amino acids. The amino-terminal third of the protein contains a high-mobility-group motif characteristic of DNA-binding proteins. To determine whether the Rox1 repressor bound DNA, the gene was expressed in Escherichia coli cells as a fusion to the maltose-binding protein and this fusion was partially purified by amylose affinity chromatography. By using a gel retardation assay, both the fusion protein and Rox1 itself were found to bind specifically to a synthetic 32-bp DNA containing the hypoxic consensus sequence. We assessed the role of the general repressor Ssn6 in ANB1 repression. An ANB1-lacZ fusion was expressed constitutively in an ssn6 deletion strain, and deletion of the Rox1 binding sites in the ANB1 upstream region did not increase the level of derepression, suggesting that Ssn6 exerts its effect through Rox1. Finally, ROX1 was mapped to yeast chromosome XVI, near the ARO7-OSM2 locus. 相似文献
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We have identified a repressor of hyphal growth in the pathogenic yeast Candida albicans. The gene was originally cloned in an attempt to characterize the homologue of the Saccharomyces cerevisiae Rox1, a repressor of hypoxic genes. Rox1 is an HMG-domain, DNA binding protein with a repression domain that recruits the Tup1/Ssn6 general repression complex to achieve repression. The C. albicans clone also encoded an HMG protein that was capable of repression of a hypoxic gene in a S. cerevisiae rox1 deletion strain. Gel retardation experiments using the purified HMG domain of this protein demonstrated that it was capable of binding specifically to a S. cerevisiae hypoxic operator DNA sequence. These data seemed to indicate that this gene encoded a hypoxic repressor. However, surprisingly, when a homozygous deletion was generated in C. albicans, the cells became constitutive for hyphal growth. This phenotype was rescued by the reintroduction of the wild-type gene on a plasmid, proving that the hyphal growth phenotype was due to the deletion and not a secondary mutation. Furthermore, oxygen repression of the hypoxic HEM13 gene was not affected by the deletion nor was this putative ROX1 gene regulated positively by oxygen as is the case for the S. cerevisiae gene. All these data indicate that this gene, now designated RFG1 for Repressor of Filamentous Growth, is a repressor of genes required for hyphal growth and not a hypoxic repressor. 相似文献
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Synergistic repression of anaerobic genes by Mot3 and Rox1 in Saccharomyces cerevisiae 总被引:3,自引:0,他引:3
Two groups of anaerobic genes (genes induced in anaerobic cells and repressed in aerobic cells) are negatively regulated by heme, a metabolite present only in aerobic cells. Members of both groups, the hypoxic genes and the DAN/TIR/ERG genes, are jointly repressed under aerobic conditions by two factors. One is Rox1, an HMG protein, and the second, originally designated Rox7, is shown here to be Mot3, a global C2H2 zinc finger regulator. Repression of anaerobic genes results from co-induction of Mot3 and Rox1 in aerobic cells. Repressor synthesis is triggered by heme, which de-represses a mechanism controlling expression of both MOT3 and ROX1 in anaerobic cells; it includes Hap1, Tup1, Ssn6 and a fourth unidentified factor. The constitutive expression of various anaerobic genes in aerobic rox1Δ or mot3Δ cells directly implies that neither factor can repress by itself at endogenous levels and that stringent aerobic repression results from the concerted action of both. Mot3 and Rox1 are not essential components of a single complex, since each can repress independently in the absence of the other, when artificially induced at high levels. Moreover, the two repression mechanisms appear to be distinct: as shown here repression of ANB1 by Rox1 alone requires Tup1–Ssn6, whereas repression by Mot3 does not. Though artificially high levels of either factor can repress well, the absolute efficiency observed in normal cells when both are present—at much lower levels—demonstrates a novel inhibitory synergy. Evidently, expression levels for the two mutually dependent repressors are calibrated to permit a range of variation in basal aerobic expression at different promoters with differing operator site combinations. 相似文献
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The DAN/TIR genes of Saccharomyces cerevisiae encode homologous mannoproteins, some of which are essential for anaerobic growth. Expression of these genes is induced during anaerobiosis and in some cases during cold shock. We show that several heme-responsive mechanisms combine to regulate DAN/TIR gene expression. The first mechanism employs two repression factors, Mox1 and Mox2, and an activation factor, Mox4 (for mannoprotein regulation by oxygen). The genes encoding these proteins were identified by selecting for recessive mutants with altered regulation of a dan1::ura3 fusion. MOX4 is identical to UPC2, encoding a binucleate zinc cluster protein controlling expression of an anaerobic sterol transport system. Mox4/Upc2 is required for expression of all the DAN/TIR genes. It appears to act through a consensus sequence termed the AR1 site, as does Mox2. The noninducible mox4Delta allele was epistatic to the constitutive mox1 and mox2 mutations, suggesting that Mox1 and Mox2 modulate activation by Mox4 in a heme-dependent fashion. Mutations in a putative repression domain in Mox4 caused constitutive expression of the DAN/TIR genes, indicating a role for this domain in heme repression. MOX4 expression is induced both in anaerobic and cold-shocked cells, so heme may also regulate DAN/TIR expression through inhibition of expression of MOX4. Indeed, ectopic expression of MOX4 in aerobic cells resulted in partially constitutive expression of DAN1. Heme also regulates expression of some of the DAN/TIR genes through the Rox7 repressor, which also controls expression of the hypoxic gene ANB1. In addition Rox1, another heme-responsive repressor, and the global repressors Tup1 and Ssn6 are also required for full aerobic repression of these genes. 相似文献
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Multiple Elements and Auto-Repression Regulate Rox1, a Repressor of Hypoxic Genes in Saccharomyces Cerevisiae 总被引:3,自引:0,他引:3
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The ROX1 gene encodes a heme-induced repressor of hypoxic genes in yeast. Using RNA blot analysis and a ROX1/lacZ fusion construct that included the ROX1 upstream region and only the first codon, we discovered that Rox1 represses its own expression. Gel-retardation experiments indicated that Rox1 was capable of binding to its own upstream region. Overexpression of Rox1 from the inducible GAL1 promoter was found to be inhibitory to cell growth. Also, we found that, as reported previously, Hap1 is partially responsible for heme-induction of ROX1, but, in addition, it also may play a role in ROX1 repression in the absence of heme. There is a second repressor of anaerobic ROX1 expression that requires the general repressor Tup1/Ssn6 for its function. 相似文献
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Tripic T Edmondson DG Davie JK Strahl BD Dent SY 《Biochemical and biophysical research communications》2006,339(3):905-914
The Tup1-Ssn6 corepressor regulates the expression of diverse classes of genes in Saccharomyces cerevisiae. Chromatin is an important component of Tup1-Ssn6-mediated repression. Tup1 binds to underacetylated tails of histones H3 and H4, and requires multiple histone deacetylases for the repression. Here we examine if histone methylation, in addition to histone deacetylation, plays a role in Tup1-Ssn6 repression. We found that like other genes, Tup1-Ssn6 target genes exhibit increased levels of histone H3 lysine 4 trimethylation upon activation. However, deletion of individual or multiple histone methyltransferases and other SET-domain containing genes has no apparent effect on Tup1-Ssn6-mediated repression of a number of well-defined targets. Interestingly, we discovered that Ssn6 interacts with Set2. Although deletion of SET2 does not affect Tup1-Ssn6 repression of a number of target genes, Ssn6 may utilize Set2 in specific contexts to regulate gene repression. 相似文献
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Fagerström-Billai F Durand-Dubief M Ekwall K Wright AP 《Molecular and cellular biology》2007,27(3):1069-1082
The Saccharomyces cerevisiae Ssn6 and Tup1 proteins form a corepressor complex that is recruited to target genes by DNA-bound repressor proteins. Repression occurs via several mechanisms, including interaction with hypoacetylated N termini of histones, recruitment of histone deacetylases (HDACs), and interactions with the RNA polymerase II holoenzyme. The distantly related fission yeast, Schizosaccharomyces pombe, has two partially redundant Tup1-like proteins that are dispensable during normal growth. In contrast, we show that Ssn6 is an essential protein in S. pombe, suggesting a function that is independent of Tup11 and Tup12. Consistently, the group of genes that requires Ssn6 for their regulation overlaps but is distinct from the group of genes that depend on Tup11 or Tup12. Global chip-on-chip analysis shows that Ssn6 is almost invariably found in the same genomic locations as Tup11 and/or Tup12. All three corepressor subunits are generally bound to genes that are selectively regulated by Ssn6 or Tup11/12, and thus, the subunit specificity is probably manifested in the context of a corepressor complex containing all three subunits. The corepressor binds to both the intergenic and coding regions of genes, but differential localization of the corepressor within genes does not appear to account for the selective dependence of target genes on the Ssn6 or Tup11/12 subunits. Ssn6, Tup11, and Tup12 are preferentially found at genomic locations at which histones are deacetylated, primarily by the Clr6 class I HDAC. Clr6 is also important for the repression of corepressor target genes. Interestingly, a subset of corepressor target genes, including direct target genes affected by Ssn6 overexpression, is associated with the function of class II (Clr3) and III (Hst4 and Sir2) HDACs. 相似文献
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