Individual Subunits of the Ssn6-Tup11/12 Corepressor Are Selectively Required for Repression of Different Target Genes

ABSTRACT 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.

Ask10p Mediates the Oxidative Stress-Induced Destruction of the Saccharomyces cerevisiae C-Type Cyclin Ume3p/Srb11p

ABSTRACT Srb11p-Srb10p is the budding yeast C-type cyclin-cyclin-dependent kinase that is required for the repression of several stress response genes. To relieve this repression, Srb11p is destroyed in cells exposed to stressors, including heat shock and oxidative stress. In the present study, we identified Ask10p (for activator of Skn7) by two-hybrid analysis as an interactor with Srb11p. Coimmunoprecipitation studies confirmed this association, and we found that, similar to Srb11p-Srb10p, Ask10p is a component of the RNA polymerase II holoenzyme. Ask10p is required for Srb11p destruction in response to oxidative stress but not heat shock. Moreover, this destruction is important since the hypersensitivity of an ask10 mutant strain to oxidative stress is rescued by deleting SRB11. We further show that Ask10p is phosphorylated in response to oxidative stress but not heat shock. This modification requires the redundant mitogen-activated protein (MAP) kinase kinase Mkk1/2 but not their normal MAP kinase target Slt2p. Moreover, the other vegetative MAP kinases—Hog1p, Fus3p, or Kss1p—are not required for Ask10p phosphorylation, suggesting the existence of an alternative pathway for transducing the Pkc1p→Bck1→Mkk1/2 oxidative stress signal. In conclusion, Ask10p is a new component of the RNA polymerase II holoenzyme and an important regulator of the oxidative stress response. In addition, these results define a new role for the Pkc1p MAP kinase cascade (except the MAP kinase itself) in transducing the oxidative damage signal directly to the RNA polymerase II holoenzyme, thereby bypassing the stress-activated transcription factors.