Title: The role of Sir3 in spreading of silent chromatin in Saccharomyces cerevisiae
Description: Silent chromatin in Saccharomyces cerevisiae is a heterochromatin-like structure with important roles in genome stability and gene repression. S. cerevisiae silent chromatin is established in a step-wise process at the silent mating type cassettes and telomeres. The SIR complex, comprised of Sir2, Sir3 and Sir4, is recruited to specific silencing elements and subsequently spreads along the chromatin fiber through multiple cycles of Sir2-mediated histone deacetylation and recruitment of additional SIR components. In this study, we analyzed the role of the structural component Sir3 in spreading of the silencing complex. In order to identify mutations that disrupt the spreading process, we performed a targeted screen for alleles of SIR3 that dominantly disrupt gene silencing. 21 of the 22 recovered mutations map to a single surface in the N-terminal BAH domain, while one, L738P, lies in the AAA+ domain within the C-terminal half of Sir3. Using a series of chromatin immunoprecipitation experiments, we determined that the mutants are recruited to silent domains in the presence of wild-type SIR3, indicating that they act directly at the level of chromatin. All of the mutants whose behavior we analyzed further (D17G, E84K, K202E and L738P) are recruited to the end of chromosomes in absence of wild-type SIR3 but are unable to spread, confirming that the defect is not due to a failure in the initial recruitment step but occurs during downstream spreading. None of the mutants tested disrupt SIR complex assembly or Sir3 oligomerization.

Recently, a study from our laboratory has demonstrated that the BAH domain binds to nucleosomes. The three BAH point mutants, but not L738P, disrupt this interaction. In contrast, in an in vitro binding assay, L738P binds to the N-terminal tail of histone H4 more strongly than wild-type Sir3 or the BAH mutants, indicating that the C-terminal histone binding activity of Sir3 is misregulated in L738P. This study, therefore, underscores the importance of the proper interaction between the multiple histone-binding domains of Sir3 and the nucleosome, and demonstrates that misregulation in either domain can disrupt spreading of the silencing complex.