Gene duplication is considered an important evolutionary mechanism. Unlike many characterized species, the fission yeast Schizosaccharomyces pombe contains two paralogous genes, tup11(+) and tup12(+), that encode transcriptional corepressors similar to the well-characterized budding yeast Tup1 protein. Previous reports have suggested that Tup11 and Tup12 proteins play redundant roles. Consistently, we show that the two Tup proteins can interact together when expressed at normal levels and that each can independently interact with the Ssn6 protein, as seen for Tup1 in budding yeast. However, tup11(-) and tup12(-) mutants have different phenotypes on media containing KCl and CaCl2. Consistent with the functional difference between tup11(-) and tup12- mutants, we identified a number of genes in genome-wide gene expression experiments that are differentially affected by mutations in the tup11(+) and tup12(+) genes. Many of these genes are differentially derepressed in tup11(-) mutants and are over-represented in genes that have previously been shown to respond to a range of different stress conditions. Genes specifically derepressed in tup12(-) mutants require the Ssn6 protein for their repression. As for Tupl.2, Ssn6 is also required for efficient adaptation to KCI- and CaCl2-mediated stress. We conclude that Tup11 and Tup12 are at least partly functionally diverged and suggest that the Tup12 and Ssn6 proteins have adopted a specific role in regulation of the stress response.

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 (CIr3) and III (Hst4 and Sir2) HDACs.

Expression profiling, ChiP-CHIP and phenotypic analysis were used to investigate the functional relationships of class III NAD(+)-dependent HDACs (Sirtuins) in fission yeast. We detected significant histone acetylation increases in Sirtuin mutants at their specific genomic binding targets and were thus able to identify an in vivo substrate preference for each Sirtuin. At heterochromatic loci, we demonstrate that although Hst2 is mainly cytoplasmic, a nuclear pool of Hst2 colocalizes with the other Sirtuins at silent regions (cen, mat, tel, rDNA), and that like the other Sirtuins, Hst2 is required for rDNA and centromeric silencing. Interestingly we found specific functions for the fission yeast Sirtuins Hst2 and Hst4 in gene regulation. Hst2 directly represses genes involved in transport and membrane function, whereas Hst4 represses amino-acid biosynthesis genes and Tf2 retrotransposons. A specific role for Hst4 in Tf2 50 mRNA processing was revealed. Thus, Sirtuins share functions at many genomic targets, but Hst2 and Hst4 have also evolved unique functions in gene regulation.