Title: The role of nucleosomes in heterochromatin assembly in Saccharomyces cerevisiae
Description: Silent chromatin, or heterochromatin, refers to regions of the genome from which gene expression is constitutively repressed. These domains are important for maintaining genome stability, as well as the silenced state of developmentally regulated genes. In Saccharomyces cerevisiae , the telomeres and silent mating type cassettes are assembled into silent chromatin domains by the Silent Information Regulator (SIR) complex (Sir2/Sir3/Sir4), which binds, deacetylates, and condenses chromatin, forming a repressed chromatin structure. Many questions remain regarding the interactions between Sir proteins and nucleosomes that are required for silent chromatin assembly. To address these questions, we developed a method of affinity purifying native yeast nucleosome arrays containing canonical or variant histones. Mono-nucleosomes were isolated from these purifications and analyzed by mass spectrometry, revealing differences in the post-translational modifications between canonical and variant nucleosomes.

The NAD-dependent histone deacetylase, Sir2, is unable to deacetylate histones in vitro once they have been assembled into nucleosomes, suggesting that the presence of additional chromatin proteins that alter chromatin structure may be necessary. We have developed a partially purified system in which deacetylation of nucleosomes by Sir2 can be observed. This assay has allowed us to identify several chromatin proteins that may be involved in facilitating the Sir2 deacetylation reaction. Analysis of Sir3-histone interactions also led us to the identification of the conserved bromo-adjacent-homology (BAH) domain of Sir3 as a nucleosome binding domain that is sensitive to the modification states of histone H4K16 and histone H3K79. We also observed the formation of filaments with a diameter of 10-20nm by electron microscopy, in a reaction containing nucleosomes, the SIR complex, and NAD. The necessary components for filament formation mirror the in vivo requirements for silencing, and they are proposed to represent a step in the formation of silent chromatin in vivo . Our investigations have led to further insight into how silent chromatin is assembled in vivo . We propose that the fundamental mechanisms underlying heterochromatin formation in higher eukaryotes is likely to be analogous to the mechanism of silent chromatin assembly in S. cerevisiae that we have described here.