Individual subunits of the Ssn6-Tup11/12 corepressor are selectively required for repression of different target genes

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.     

The Set2 methyltransferase associates with Ssn6 yet Tup1-Ssn6 repression is independent of histone methylation

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.     

Pleiotropic corepressors Sin3 and Ssn6 interact with repressor Opi1 and negatively regulate transcription of genes required for phospholipid biosynthesis in the yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Repressor protein Opi1 is required to negatively regulate yeast structural genes of phospholipid biosynthesis in the presence of precursor molecules inositol and choline (IC). Opi1 interacts with the paired amphipathic helix 1 (PAH1) of pleiotropic corepressor Sin3, leading to recruitment of histone deacetylases (HDACs). Mutational analysis of the Opi1–Sin3 interaction domain (OSID) revealed that hydrophobic OSID residues L56, V59 and V67 of Opi1 are indispensable for gene repression. Our results also suggested that repression is not executed entirely via Sin3. Indeed, we could show that OSID contacts a second pleiotropic corepressor, Ssn6 (=Cyc8), which together with Tup1 is also able to recruit HDACs. Interestingly, mutations sin3 and ssn6 turned out as synthetically lethal. Our analysis further revealed that OSID not only binds to PAH1 but also interacts with tetratricopeptide repeats (TPR) of Ssn6. This interaction could no longer be observed with Opi1 OSID variants. To trigger gene repression, Opi1 must also interact with activator Ino2, using its activator interaction domain (AID). AID contains a hydrophobic structural motif reminiscent of a leucine zipper. Our mutational analysis of selected positions indeed confirmed that residues L333, L340, V343, V350, L354 and V361 are necessary for repression of Opi1 target genes.     

Recruitment of Tup1-Ssn6 by yeast hypoxic genes and chromatin-independent exclusion of TATA binding protein

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.