Structure and Function of the Saccharomyces cerevisiae Sir3 BAH Domain

Abstract

Previous work has shown that the N terminus of the Saccharomyces cerevisiae Sir3 protein is crucial for the function of Sir3 in transcriptional silencing. Here, we show that overexpression of N-terminal fragments of Sir3 in strains lacking the full-length protein can lead to some silencing of HML and HMR. Sir3 contains a BAH (bromo-adjacent homology) domain at its N terminus. Overexpression of this domain alone can lead to silencing as long as Sir1 is overexpressed and Sir2 and Sir4 are present. Overexpression of the closely related Orc1 BAH domain can also silence in the absence of any Sir3 protein. A previously characterized hypermorphic sir3 mutation, D205N, greatly improves silencing by the Sir3 BAH domain and allows it to bind to DNA and oligonucleosomes in vitro. A previously uncharacterized region in the Sir1 N terminus is required for silencing by both the Sir3 and Orc1 BAH domains. The structure of the Sir3 BAH domain has been determined. In the crystal, the molecule multimerizes in the form of a left-handed superhelix. This superhelix may be relevant to the function of the BAH domain of Sir3 in silencing.Epigenetic silencing is a term used to describe the heritable transmission of a transcriptionally inactive state. The silent mating type loci HML and HMR and telomeres of the budding yeast Saccharomyces cerevisiae are examples of loci that undergo this type of transcriptional silencing and have served as a paradigm for studying this process.HML and HMR harbor copies of the mating type information genes, ␣ and a, respectively. They are involved in mating type interconversion with the actively transcribed MAT locus. Transcriptional silencing at these loci relies on the existence of cis-acting DNA regulatory elements, termed silencers (E and I), which flank both loci. These elements recruit the DNA binding proteins Rap1, Abf1, and ORC, which then serve to recruit the silent information regulators (Sir) 1, 2, 3, and 4 (11, 13, 33). The widely accepted view of silencing at these loci (and at telomeres) is that histone tails are deacetylated through the action of Sir2, a NAD-dependent histone deacetylase, creating a binding surface on nucleosomes for the binding of Sir3 and Sir4. Multiple rounds of deacetylation lead to the formation of a Sir2/3/4 polymer that spreads on the nucleosomes of the silent region, altering the chromatin and making it unavailable for transcription. The detailed structure of silent chromatin is not known, and exactly how transcription is prevented is a matter of dispute (6, 34).Sir3 is essential for the establishment and maintenance of the silent state at the HM loci and telomeres. Genetic, twohybrid, and biochemical studies have identified interactions of Sir3 with histones H3 and H4, Sir4, Rap1, Abf1, and Sir3 itself (reviewed in references 11, 13, and 33). Interestingly, all these interactions are within the C-terminal two-thirds of the Sir3 protein. Nevertheless, expression of a Sir3 construct lacking the N-terminal region (hereafter referred to as the N terminus) is not suffici...

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

“…The sequences of Sir3 and Orc1 have diverged considerably, and these proteins cannot complement each other (31). However one domain, the nucleosomebinding BAH domain, is 50% identical and 65% similar between ScOrc1 and ScSir3 and has a highly conserved tertiary structure (31)(32)(33). Nonduplicated orthologs of Orc1 and Sir3 display more sequence similarity to ScOrc1 than to ScSir3, and this accelerated sequence divergence in Sir3 has led to the hypothesis that the silencing function of Sir3 arose through neofunctionalization (29).…”

Elaboration, Diversification and Regulation of the Sir1 Family of Silencing Proteins in Saccharomyces

“…We found SIR1 orthologs in S. paradoxus, S. mikatae, S. kudriavzevii, S. bayanus, and S. castellii ( Figure 1A). The SIR1 genes from these yeast species all contained the Cterminal OIR (Gardner et al 1999) which binds the BAH domain of Orc1 (Bose et al 2004) and the Nterminal duplication of the OIR, called OIR9 (or SIR1N) (Connelly et al 2006). The Sir1's from the different species were between 77 and 58% identical to S. cerevisiae Sir1 across the entire protein sequence (Table 2).…”

“…The predicted Kos3 protein lacked the first 145 amino acids corresponding to the OIR9 domain of Sir1 ( Figure 1C and supplemental Figure 1) (Connelly et al 2006). However, the N termini of the Kos3 proteins were strongly conserved relative to each other (supplemental Figure 1).…”

“…A revision of the Sir1 protein primary structure: Comparison of all the Sir1 and Kos proteins across the Saccharomyces species suggested that the sequence of Sir1 of S. cerevisiae as represented in the Saccharomyces Genome Database (SGD) and earlier publications (Stone et al 1991;Gardner et al 1999;Gardner and Fox 2001;Hou et al 2005;Hsu et al 2005;Connelly et al 2006) contains a 24 amino acid N-terminal extension compared to most other species. The S. cerevisiae SIR1 ORF contains three in-frame methionine codons, 1 (Met1), 5 (Met5), and 25 (Met25) residues into the ORF, the latter of which corresponded to the first methionine codon in the Sir1 ORFs of other species ( Figure 3A).…”

“…Sir1 is recruited to the silencers by interaction with Orc1 (Triolo and Sternglanz 1996;Fox et al 1997) through the Orc1 interaction region (OIR) of Sir1, located in the C-terminal half of the protein, and the bromo adjacent homology (BAH) domain of Orc1 (Gardner et al 1999;Zhang et al 2002;Hou et al 2005;Hsu et al 2005). A duplication of the OIR of Sir1, called OIR9, has been proposed to bind to the bromo-associated homology (BAH) domain in the N-terminal region of Sir3, although no direct binding of these two domains has yet been detected (Connelly et al 2006). Thus, these two similar domains within Sir1 may act as a scaffold to bring Orc1 and Sir3 into juxtaposition.…”