Sir3-Nucleosome Interactions in Spreading of Silent Chromatin in Saccharomyces cerevisiae

Abstract

Silent chromatin in Saccharomyces cerevisiae is established in a stepwise process involving the SIR complex, comprised of the histone deacetylase Sir2 and the structural components Sir3 and Sir4. The Sir3 protein, which is the primary histone-binding component of the SIR complex, forms oligomers in vitro and has been proposed to mediate the spreading of the SIR complex along the chromatin fiber. In order to analyze the role of Sir3 in the spreading of the SIR complex, we performed a targeted genetic screen for alleles of SIR3 that dominantly disrupt silencing. Most mutations mapped to a single surface in the conserved N-terminal BAH domain, while one, L738P, localized to the AAA ATPase-like domain within the C-terminal half of Sir3. The BAH point mutants, but not the L738P mutant, disrupted the interaction between Sir3 and nucleosomes. In contrast, Sir3-L738P bound the N-terminal tail of histone H4 more strongly than wild-type Sir3, indicating that misregulation of the Sir3 C-terminal histone-binding activity also disrupted spreading. Our results underscore the importance of proper interactions between Sir3 and the nucleosome in silent chromatin assembly. We propose a model for the spreading of the SIR complex along the chromatin fiber through the two distinct histone-binding domains in Sir3.

Transcriptional silencing functions of the yeast protein Orc1/Sir3 subfunctionalized after gene duplication

“…The BAH domains of ScSir3 and ScOrc1 contribute to silencing in different ways. The ScSir3 BAH domain enables the spreading of the Sir complex by binding nucleosomes (45,46), whereas the ScOrc1 BAH domain recruits Sir1 to silencers. Given that there is no identifiable homolog of Sir1 in K. lactis (40), it is more likely that KlOrc1 acts at HMLα in a manner analogous to ScSir3.…”

“…To explore this hypothesis, we generated two point mutations in KlORC1. The P185L mutation is analogous to a mutation in ScSir3 that disrupts nucleosome binding and the ability of Sir proteins to spread (46), and the E124K mutation occurs in the H domain, which interacts with Sir1 in S. cerevisiae (26) (Fig. S4).…”

The Nuts and Bolts of Transcriptionally Silent Chromatin in Saccharomyces cerevisiae

“…A C-terminal fragment of the protein lacking the BAH domain binds nucleosomes, as well as peptides corresponding to just the H4 and H3 tails and the LRS domain (Hecht et al 1995;Carmen et al 2002;Santos-Rosa et al 2004;Altaf et al 2007;Ehrentraut et al 2011). Mutational analyses indicate that this C-terminal domain (actually two domains in close proximity) contributes to silencing ( Figure 5A) (Hecht et al 1995;Stone et al 2000;Buchberger et al 2008;Ehrentraut et al 2011). Importantly, acetylation or methylation of the critical lysines in histone peptides blocks binding by the Sir3 C-terminal.…”

“…Interestingly, S. cerevisiae contains a distantly-related SIR4 paralog named ASF2 with no known physiological function. Overexpression of ASF2 disrupts silencing, possibly because the gene product can compete with Sir4 for binding Sir3 (Le et al 1997;Buchberger et al 2008). …”

Telomere as an Important Player in Regulation of Microbial Pathogen Virulence

“…Either ScRap1 or yKu can recruit Sir4 to the telomere, and ScRap1 can also recruit Sir3 Martin et al 1999;Moretti & Shore 2001;Moretti et al 1994;Tsukamoto et al 1997). Together, Sir3 and Sir4 can recruit Sir2 (Bourns et al 1998;Buchberger et al 2008;Martino et al 2009;Moazed et al 1997;Strahl-Bolsinger et al 1997), which is an NAD + -dependent histone deacetylase (Landry et al 2000;Smith et al 2000;Tanny et al 1999). Sir2 activity and the interaction between Sir3/4 and histone tails are necessary for propagating of the heterochromatic structure from telomere to chromosome internal regions (Hoppe et al 2002).…”