The Sin3p PAH domains provide separate functions repressing meiotic gene transcription in Saccharomyces cerevisiae

Meiotic genes in budding yeast are repressed during vegetative growth but are transiently induced during specific stages of meiosis. Sin3p represses the early meiotic gene (EMG) by bridging the DNA binding protein Ume6p to the histone deacetylase Rpd3p. Sin3p contains four paired amphipathic helix (PAH) domains, one of which (PAH3) is required for repressing several genes expressed during mitotic cell division. This report examines the roles of the PAH domains in mediating EMG repression during mitotic cell division and following meiotic induction. PAH2 and PAH3 are required for mitotic EMG repression, while electrophoretic mobility shift assays indicate that only PAH2 is required for stable Ume6p-promoter interaction. Unlike mitotic repression, reestablishing EMG repression following transient meiotic induction requires PAH3 and PAH4. In addition, the role of Sin3p in reestablishing repression is expanded to include additional loci that it does not control during vegetative growth. These findings indicate that mitotic and postinduction EMG repressions are mediated by two separate systems that utilize different Sin3p domains.     

Stress and developmental regulation of the yeast C-type cyclin Ume3p (Srb11p/Ssn8p).

The ume3-1 allele was identified as a mutation that allowed the aberrant expression of several meiotic genes (e.g. SPO11, SPO13) during mitotic cell division in Saccharomyces cerevisiae. Here we report that UME3 is also required for the full repression of the HSP70 family member SSA1. UME3 encodes a non-essential C-type cyclin (Ume3p) whose levels do not vary through the mitotic cell cycle. However, Ume3p is destroyed during meiosis or when cultures are subjected to heat shock. Ume3p mutants resistant to degradation resulted in a 2-fold reduction in SPO13 mRNA levels during meiosis, indicating that the down-regulation of this cyclin is important for normal meiotic gene expression. Mutational analysis identified two regions (PEST-rich and RXXL) that mediate Ume3p degradation. A third destruction signal lies within the highly conserved cyclin box, a region that mediates cyclin-cyclin-dependent kinase (Cdk) interactions. However, the Cdk activated by Ume3p (Ume5p) is not required for the rapid destruction of this cyclin. Finally, Ume3p destruction was not affected in mutants defective for ubiquitin-dependent proteolysis. These results support a model in which Ume3p, when exposed to heat shock or sporulation conditions, is targeted for destruction to allow the expression of genes necessary for the cell to respond correctly to these environmental cues.     

Stable incorporation of sequence specific repressors Ash1 and Ume6 into the Rpd3L complex

Histone deacetylation by Saccharomyces cerevisiae Rpd3 represses genes regulated by the Ash1 and Ume6 DNA-binding proteins. Rpd3 exists in a small 0.6 MDa (Rpd3S) and large 1.2 MDa (Rpd3L) corepressor complex. In this report, we identify by mass spectrometry and MudPIT the subunits of the Rpd3L complex. These included Rpd3, Sds3, Pho23, Dep1, Rxt2, Sin3, Ash1, Ume1, Sap30, Cti6, Rxt3 and Ume6. Dep1 and Sds3, unique components of Rpd3L, were required for Rpd3L integrity and HDAC activity. Similar to RPD3, deletion of DEP1 enhanced telomeric silencing and derepressed INO1. Two sequence-specific repressors, Ash1 and Ume6, were stably associated with Rpd3L. While both of these proteins localized to the INO1 and HO promoters, the repression of these genes were dependent only on Ume6 and Ash1, respectively. Thus, the Rpd3L complex is directly recruited to specific promoters through multiple integral DNA-binding proteins.     

Mutations in mating-type genes of the heterothallic fungus Podospora anserina lead to self-fertility

It has been established that meiotic recombination and chromosome segregation are inhibited when meiotic DNA replication is blocked. Here we demonstrate that early meiotic gene (EMG) expression is also inhibited by a block in replication. Since early meiotic genes are required to promote meiotic recombination and DNA division, the low expression of these genes may contribute to the block in meiotic progression. We have identified three Hur- (HU reduced recombination) mutants that fail to couple meiotic recombination and gene expression with replication. One of these mutations is in RPD3, a gene required to maintain meiotic gene repression in mitotic cells. Complete deletions of RPD3 and the repression adapter SIN3 permitted recombination and early meiotic gene expression when replication was inhibited with hydroxyurea (HU). Biochemical analysis showed that the Rpd3p-Sin3p-Ume6p repression complex does exist in meiotic cells. These observations suggest that repression of early meiotic genes by SIN3 and RPD3 is critical for the normal response to inhibited replication. A second response to inhibited replication has also been discovered. HU-inhibited replication reduced the accumulation of phospho-Ume6p in meiotic cells. Phosphorylation of Ume6p normally promotes interaction with the meiotic activator Ime1p, thereby activating EMG expression. Thus, inhibited replication may also reduce the Ume6p-dependent activation of EMGs. Taken together, our data suggest that both active repression and reduced activation combine to inhibit EMG expression when replication is inhibited.     

Opi1p,Ume6p and Sin3p control expression from the promoter of the INO2 regulatory gene via a novel regulatory cascade

The INO2 gene of Saccharomyces cerevisiae is required for expression of most of the phospholipid biosynthetic genes. INO2 expression is regulated by a complex cascade that includes autoregulation, Opi1p-mediated repression and Ume6p-mediated activation. To screen for mutants with altered INO2 expression directly, we constructed an INO2-HIS3 reporter that provides a plate assay for INO2 promoter activity. This reporter was used to isolate mutants (dim1) that fail to repress expression of the INO2 gene in an otherwise wild-type strain. The dim1 mutants contain mutations in the OPI1 gene. To define further the mechanism for Ume6p regulation of INO2 expression, we isolated suppressors (rum1, 2, 3) of the ume6Delta mutation that overexpress the INO2-HIS3 gene. Two of the rum mutant groups contain mutations in the OPI1 and SIN3 genes showing that opi1 and sin3 mutations are epistatic to the ume6Delta mutation. These results are surprising given that Ume6p, Sin3p and Rpd3p are known to form a complex that represses the expression of a diverse set of yeast genes. This prompted us to examine the effect of sin3Delta and rpd3Delta mutants on INO2-cat expression. Surprisingly, the sin3Delta allele overexpressed INO2-cat, whereas the rpd3Delta mutant had no effect. We also show that the UME6 gene does not affect the expression of an OPI1-cat reporter. This suggests that Ume6p does not regulate INO2 expression indirectly by regulating OPI1 expression.     

Multiple histone deacetylases are recruited by corepressor Sin3 and contribute to gene repression mediated by Opi1 regulator of phospholipid biosynthesis in the yeast Saccharomyces cerevisiae

Yeast genes of phospholipid biosynthesis are negatively regulated by repressor protein Opi1 when precursor molecules inositol and choline (IC) are available. Opi1-triggered gene repression is mediated by recruitment of the Sin3 corepressor complex. In this study, we systematically investigated the regulatory contribution of subunits of Sin3 complexes and identified Pho23 as important for IC-dependent gene repression. Two non-overlapping regions within Pho23 mediate its direct interaction with Sin3. Previous work has shown that Sin3 recruits the histone deacetylase (HDAC) Rpd3 to execute gene repression. While deletion of SIN3 strongly alleviates gene repression by IC, an rpd3 null mutant shows almost normal regulation. We thus hypothesized that various HDACs may contribute to Sin3-mediated repression of IC-regulated genes. Indeed, a triple mutant lacking HDACs, Rpd3, Hda1 and Hos1, could phenocopy a sin3 single mutant. We show that these proteins are able to contact Sin3 in vitro and in vivo and mapped three distinct HDAC interaction domains, designated HID1, HID2 and HID3. HID3, which is identical to the previously described structural motif PAH4 (paired amphipathic helix), can bind all HDACs tested. Chromatin immunoprecipitation studies finally confirmed that Hda1 and Hos1 are recruited to promoters of phospholipid biosynthetic genes INO1 and CHO2.