Transcriptional silencing in Saccharomyces cerevisiae is processed by binding and spreading of silent information regulator (Sir) proteins to form a compacted chromatin structure, or heterochromatin. My research is mainly focused on two of these Sir proteins, Sir2 and Sir3 respectively.

A screen for yeast temperature-sensitive silencing mutants identified a strain with a point mutation in the SIR2 gene. Haploid strains of either mating type carrying the iv mutation were severely defective at mating at 37° but almost normal at 25°. A rapid loss of silencing at HMR was observed upon shifting the mutant from the low to the high temperature, but it took >8 hours to reestablish silencing after a shift back to 25°. Silencing at the rDNA locus was also temperature sensitive, while telomeric silencing was totally defective at both temperatures. In enzymatic assays, the mutant exhibited defective deacetylase activity at both 37° and 25°. Interestingly, the mutant had much more NAD ⁺-nicotinamide exchange activity than wild type, as did a mutation in the same region of the protein in the Sir2 homolog, Hst2. Thus, mutations in this region of the NAD+ binding pocket of the protein are able to carry out cleavage of NAD+ to nicotinamide but are defective at the subsequent deacetylation step of the reaction.

In the studies on Sir3, it was found that Sir3 is mono-sumolyated on K247 and Siz1 is the primary SUMO E3 ligase responsible. In a ris1 Δ mutant, the level of sumoylated Sir3 increases, while the total Sir3 level is not affected. Mutation of the RING finger or of the ATPase motif of Ris1 also caused the accumulation of Sir3-SUMO. Overexpression of Sir3 causes it to spread from the silent chromatin into euchromatin and is somewhat toxic to yeast. This toxicity depends on Sir2 and Sir4. Abolishment of Sir3 sumoylation, deletion of ris1, or both, causes further spreading of Sir3 from the silent chromatin and leads to a more severe growth defect. We propose that down-regulation of sumoylated Sir3 by Ris1 provides a protection mechanism for the cell from inappropriate spreading of Sir3 and improper silencing of transcriptionally active regions.