Role of the conserved Sir3-BAH domain in nucleosome binding and silent chromatin assembly

Silent chromatin domains in Saccharomyces cerevisiae represent examples of epigenetically heritable chromatin. The formation of these domains involves the recruitment of the SIR complex, composed of Sir2, Sir3, and Sir4, followed by iterative cycles of NAD-dependent histone deacetylation and spreading of SIR complexes over adjacent chromatin domains. We show here that the conserved bromo-adjacent homology (BAH) domain of Sir3 is a nucleosome- and histone-tail-binding domain and that its binding to nucleosomes is regulated by residues in the N terminus of histone H4 and the globular domain of histone H3 on the exposed surface of the nucleosome. Furthermore, using a partially purified system containing nucleosomes, the three Sir proteins, and NAD, we observe the formation of SIR-nucleosome filaments with a diameter of less than 20 nm. Together, these observations suggest that the SIR complex associates with an extended chromatin fiber through interactions with two different regions in the nucleosome.     

Ordered nucleation and spreading of silenced chromatin in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, silencing at the HM loci depends on Sir proteins, which are structural components of silenced chromatin. To explore the structure and assembly of silenced chromatin, the associations of Sir proteins with sequences across the HMR locus were examined by chromatin immunoprecipitation. In wild-type cells, Sir2p, Sir3p, and Sir4p were spread throughout and coincident with the silenced region at HMR. Sir1p, in contrast, associated only with the HMR-E silencer, consistent with its role in establishment but not maintenance of silencing. Sir4p was required for the association of other Sir proteins with silencers. In contrast, in the absence of Sir2p or Sir3p, partial assemblies of Sir proteins could form at silencers, where Sir protein assembly began. Spreading across HMR required Sir2p and Sir3p, as well as the deacetylase activity of Sir2p. These data support a model for the spreading of silenced chromatin involving cycles of nucleosome deacetylation by Sir2p followed by recruitment of additional Sir2p, Sir3p, and Sir4p to the newly deacetylated nucleosome. This model suggests mechanisms for boundary formation, and for maintenance and inheritance of silenced chromatin. The principles are generalizable to other types of heritable chromatin states.     

Dependence of ORC silencing function on NatA-mediated Nalpha acetylation in Saccharomyces cerevisiae

N(alpha) acetylation is one of the most abundant protein modifications in eukaryotes and is catalyzed by N-terminal acetyltransferases (NATs). NatA, the major NAT in Saccharomyces cerevisiae, consists of the subunits Nat1p, Ard1p, and Nat5p and is necessary for the assembly of repressive chromatin structures. Here, we found that Orc1p, the large subunit of the origin recognition complex (ORC), required NatA acetylation for its role in telomeric silencing. NatA functioned genetically through the ORC binding site of the HMR-E silencer. Furthermore, tethering Orc1p directly to the silencer circumvented the requirement for NatA in silencing. Orc1p was N(alpha) acetylated in vivo by NatA. Mutations that abrogated its ability to be acetylated caused strong telomeric derepression. Thus, N(alpha) acetylation of Orc1p represents a protein modification that modulates chromatin function in S. cerevisiae. Genetic evidence further supported a functional link between NatA and ORC: (i) nat1Delta was synthetically lethal with orc2-1 and (ii) the synthetic lethality between nat1Delta and SUM1-1 required the Orc1 N terminus. We also found Sir3p to be acetylated by NatA. In summary, we propose a model by which N(alpha) acetylation is required for the binding of silencing factors to the N terminus of Orc1p and Sir3p to recruit heterochromatic factors and establish repression.     

Targeting of the yeast Ty5 retrotransposon to silent chromatin is mediated by interactions between integrase and Sir4p

The Ty5 retrotransposons of Saccharomyces cerevisiae integrate preferentially into regions of silent chromatin at the telomeres and silent mating loci (HMR and HML). We define a Ty5-encoded targeting domain that spans 6 amino acid residues near the C terminus of integrase (LXSSXP). The targeting domain establishes silent chromatin when it is tethered to a weakened HMR-E silencer, and it disrupts telomeric silencing when it is overexpressed. As determined by both yeast two-hybrid and in vitro binding assays, the targeting domain interacts with the C terminus of Sir4p, a structural component of silent chromatin. This interaction is abrogated by mutations in the targeting domain that disrupt integration into silent chromatin, suggesting that recognition of Sir4p by the targeting domain is the primary determinant in Ty5 target specificity.     

Tolerance of Sir1p/origin recognition complex-dependent silencing for enhanced origin firing at HMRa

The HMR-E silencer is a DNA element that directs the formation of silent chromatin at the HMRa locus in Saccharomyces cerevisiae. Sir1p is one of four Sir proteins required for silent chromatin formation at HMRa. Sir1p functions by binding the origin recognition complex (ORC), which binds to HMR-E, and recruiting the other Sir proteins (Sir2p to -4p). ORCs also bind to hundreds of nonsilencer positions distributed throughout the genome, marking them as replication origins, the sites for replication initiation. HMR-E also acts as a replication origin, but compared to many origins in the genome, it fires extremely inefficiently and late during S phase. One postulate to explain this observation is that ORC's role in origin firing is incompatible with its role in binding Sir1p and/or the formation of silent chromatin. Here we examined a mutant HMR-E silencer and fusions between robust replication origins and HMR-E for HMRa silencing, origin firing, and replication timing. Origin firing within HMRa and from the HMR-E silencer itself could be significantly enhanced, and the timing of HMRa replication during an otherwise normal S phase advanced, without a substantial reduction in SIR1-dependent silencing. However, although the robust origin/silencer fusions silenced HMRa quite well, they were measurably less effective than a comparable silencer containing HMR-E's native ORC binding site.     

Sir3-nucleosome interactions in spreading of silent chromatin in Saccharomyces cerevisiae

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.     

The origin recognition complex and Sir4 protein recruit Sir1p to yeast silent chromatin through independent interactions requiring a common Sir1p domain

Sir1p is one of four SIR (silent information regulator) proteins required for silencing the cryptic mating-type locus HMRa in the budding yeast Saccharomyces cerevisiae. A Sir1p interaction with Orc1p, the largest subunit of the origin recognition complex (ORC), is critical for Sir1p's ability to bind HMRa and function in the formation of silent chromatin. Here we show that a discrete domain within Sir1p, the ORC interaction region (OIR), was necessary and sufficient for a Sir1p-ORC interaction. The OIR contains the originally defined silencer recognition-defective region as well as additional amino acids. In addition, a Sir1p-Sir4p interaction required a larger region of Sir1p that included the OIR. Amino acid substitutions causing defects in either a Sir1p-Orc1p or a Sir1p-Sir4p interaction reduced HMRa silencing and Sir1p binding to HMRa in chromatin. These data support a model in which Sir1p's association with HMRa is mediated by separable Sir1p-ORC and Sir1p-Sir4p interactions requiring a common Sir1p domain, and they indicate that a Sir1p-ORC interaction is restricted to silencers, at least in part, through interactions with Sir4p.