Mutational Analysis of the Sir3 BAH Domain Reveals Multiple Points of Interaction with Nucleosomes

Abstract

Sir3, a component of the transcriptional silencing complex in the yeast Saccharomyces cerevisiae, has an N-terminal BAH domain that is crucial for the protein's silencing function. Previous work has shown that the N-terminal alanine residue of Sir3 (Ala2) and its acetylation play an important role in silencing. Here we show that the silencing defects of Sir3 Ala2 mutants can be suppressed by mutations in histones H3 and H4, specifically, by H3 D77N and H4 H75Y mutations. Additionally, a mutational analysis demonstrates that three separate regions of the Sir3 BAH domain are important for its role in silencing. Many of these BAH mutations also can be suppressed by the H3 D77N and H4 H75Y mutations. In agreement with the results of others, in vitro experiments show that the Sir3 BAH domain can interact with partially purified nucleosomes. The silencing-defective BAH mutants are defective for this interaction. These results, together with the previously characterized interaction between the C-terminal region of Sir3 and the histone H3/H4 tails, suggest that Sir3 utilizes multiple domains to interact with nucleosomes.Transcriptional silencing in the yeast Saccharomyces cerevisiae occurs at the silent mating type loci, HML and HMR, at genes near telomeres, and at the ribosomal DNA. The silencer elements flanking the HM loci recruit the DNA binding proteins Rap1, Abf1, and Orc1, while telomeric sequences bind Rap1. These DNA-bound proteins in turn recruit the silent information regulator (SIR) proteins. Multiple protein-protein interactions lead to spreading of a Sir2, Sir3, and Sir4 complex to nearby nucleosomes (reviewed in references 11 and 35). Sir2 plays a crucial role in this spreading by deacetylating histone H4 K16, thus allowing Sir3 and Sir4 to bind to nucleosomes and allowing further spreading of the Sir complex (reviewed in references 11, 26, and 35). Sir3 protein levels seem to control the extent of silencing. Silencing normally spreads up to 4 kb from the telomeres but can go as far as 20 kb away from the telomeric ends when Sir3 is overexpressed (16,34). While the exact mechanism by which transcription is silenced at these loci is not yet clear, it seems to involve a specialized chromatin structure that prevents transcription at either initiation or elongation (6,36).The N-terminal tails of histones H4 and H3 are important for silencing. Mutations in these tail regions show loss of silencing because they cause reduced binding of the Sir2, -3, and -4 complex (17,19,41). In addition to the N-terminal tails, a group of residues in the core domain of H3 and H4 are important for silencing (31, 42). These residues cluster around H3 K79, a site of methylation by Dot1, forming a patch on the surface of the nucleosome that could be a potential site for interaction with the silencing complex (23,44,49). Silencing is restricted to silent loci by many redundant mechanisms that prevent the spread of the silencing complex from the silent chromatin to active chromatin. Acetylation at H4 K16, methylation at H3 K4 and K7...

Promoter Strength Influences the S Phase Requirement for Establishment of Silencing at the Saccharomyces cerevisiae Silent Mating Type Loci

“…In dot1D and set1D mutants, establishment of silencing was more rapid than in wild-type cells, probably because active transcription was compromised by the hypomethylated chromatin and hence was less resistant to silencing (Osborne et al 2009). However, it may also have been caused by the better binding of Sir proteins to hypomethylated histones (Onishi et al 2007;Sampath et al 2009). …”

The Nuts and Bolts of Transcriptionally Silent Chromatin in Saccharomyces cerevisiae

“…Genetic suppressor studies over the course of many years suggested that the domain mediates binding of nucleosomes. Mutations that mapped to the BAH domain suppressed mutations of either the H4 tail or a patch on the nucleosome surface known as the LRS domain (Johnson et al 1990;Thompson et al 2003;Norris et al 2008;Sampath et al 2009). The BAH domain was sufficient for partial silencing when forced to dimerize, strengthening the notion that the BAH domain was sufficient for nucleosome recognition (Connelly et al 2006).…”

“…Mutations that block N a acetylation of Sir3 abolish silencing and impede assembly of extended silent chromatin domains (Whiteway et al 1987;Mullen et al 1989;Ruault et al 2011). Genetic studies suggested and biochemical studies proved that Sir3 N a acetylation increases the affinity of Sir3 for nucleosomes (Connelly et al 2006;Onishi et al 2007;van Welsem et al 2008;Sampath et al 2009). Crystallographic studies showed that the Sir3 modification stabilizes the surface of the Sir3 BAH domain at the nucleosome-binding interface (Arnaudo et al 2013;Yang et al 2013).…”

Symmetry, asymmetry, and kinetics of silencing establishment in Saccharomyces cerevisiae revealed by single-cell optical assays

“…First, Dot1 catalyzes the mono-, di-, and trimethylation states of H3 K79 (27,28,30,31). This particular lysine is located on the loss of rDNA silencing (LRS) face of H3, a surface whose electrostatic properties are important for association between the nucleosome and the BAH domain of Sir3 (32)(33)(34)(35)(36). H3 K79 methylation, therefore, interferes with the nucleosome's ability to adequately bind Sir3 (9).…”