Abstract #42

On establishment, containment and evolution of silencing in Saccharomyces. Jasper Rine, Joshua Babiarz, Jennifer Gallagher, Jeff Halley, Erin Osborne, Bilge Ozydin, Leonid Teytelman, Oliver Zill. Dept Molecular & Cell Biol, Univ California, Berkeley, CA.
   Silencing at HM loci requires binding of the Sir proteins with proteins at the silencer, and subsequent spreading of Sir proteins to barriers that require histone H2A.Z. We have re-evaluated cell-cycle requirements for silencing using mating type itself as the benchmark for silencing. We inject a single-cell dose of wild-type Sir proteins into sir- mutants using yeast mating, and then monitor the establishment of silencing at the single cell level by the acquisition of sensitivity to mating pheromone. Single-cell studies reveal a lag between phenotype and genotype, reflecting an increasing sub-population of cells that have switched from On to Off at HML and HMR. The probability of switching from On to Off increases with successive cell divisions, with evidence of patterns to the switches, and increased switches in mutants lacking distinct histone modifications, suggesting that the removal of some histone marks increases the probability of switching to the silenced state. Histone H2A.Z is conserved from yeast to man. We showed that S. cerevisiae H2A.Z is acetylated on four N-terminal lysines by a combination of NuA4 and SAGA. H2A.Z deposition by SWR-Com is a pre-requisite for acetylation, which is critical in H2A.Z’s function as a boundary for telomeric heterochromatin. Because bromo domains can bind acetylated lysines, we tested the role of each bromodomain protein to heterochromatin boundary function. Two bromo domain proteins specifically bind the acetylated H2A.Z amino terminal tail in vitro and in vivo. Synthetic lethal interactions with bromo domain mutants are leading to deeper understanding of the post-translational marks. The Rap1 binding site at silencers is a variant of the consensus found at most locations, and the variant is conserved among silencer is sensu stricto strains. Substituting the Rap1 consensus sequence in the silencer creates a conditional silencer, underscoring the importance of the variant binding site.

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