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.     

Synthetic lethal screens identify gene silencing processes in yeast and implicate the acetylated amino terminus of Sir3 in recognition of the nucleosome core

Dot1 methylates histone H3 lysine 79 (H3K79) on the nucleosome core and is involved in Sir protein-mediated silencing. Previous studies suggested that H3K79 methylation within euchromatin prevents nonspecific binding of the Sir proteins, which in turn facilitates binding of the Sir proteins in unmethylated silent chromatin. However, the mechanism by which the Sir protein binding is influenced by this modification is unclear. We performed genome-wide synthetic genetic array (SGA) analysis and identified interactions of DOT1 with SIR1 and POL32. The synthetic growth defects found by SGA analysis were attributed to the loss of mating type identity caused by a synthetic silencing defect. By using epistasis analysis, DOT1, SIR1, and POL32 could be placed in different pathways of silencing. Dot1 shared its silencing phenotypes with the NatA N-terminal acetyltransferase complex and the conserved N-terminal bromo adjacent homology (BAH) domain of Sir3 (a substrate of NatA). We classified all of these as affecting a common silencing process, and we show that mutations in this process lead to nonspecific binding of Sir3 to chromatin. Our results suggest that the BAH domain of Sir3 binds to histone H3K79 and that acetylation of the BAH domain is required for the binding specificity of Sir3 for nucleosomes unmethylated at H3K79.     

Phylogenetic Conservation and Homology Modeling Help Reveal a Novel Domain within the Budding Yeast Heterochromatin Protein Sir1

The yeast Sir1 protein's ability to bind and silence the cryptic mating-type locus HMRa requires a protein-protein interaction between Sir1 and the origin recognition complex (ORC). A domain within the C-terminal half of Sir1, the Sir1 ORC interaction region (Sir1OIR), and the conserved bromo-adjacent homology (BAH) domain within Orc1, the largest subunit of ORC, mediate this interaction. The structure of the Sir1OIR-Orc1BAH complex is known. Sir1OIR and Orc1BAH interacted with a high affinity in vitro, but the Sir1OIR did not inhibit Sir1-dependent silencing when overproduced in vivo, suggesting that other regions of Sir1 helped it bind HMRa. Comparisons of diverged Sir1 proteins revealed two highly conserved regions, N1 and N2, within Sir1's poorly characterized N-terminal half. An N-terminal portion of Sir1 (residues 27 to 149 [Sir1^27-149]) is similar in sequence to the Sir1OIR; homology modeling predicted a structure for Sir1^27-149 in which N1 formed a submodule similar to the known Orc1BAH-interacting surface on Sir1. Consistent with these findings, two-hybrid assays indicated that the Sir1 N terminus could interact with BAH domains. Amino acid substitutions within or near N1 or N2 reduced full-length Sir1's ability to bind and silence HMRa and to interact with Orc1BAH in a two-hybrid assay. Purified recombinant Sir1 formed a large protease-resistant structure within which the Sir1OIR domain was protected, and Orc1BAH bound Sir1OIR more efficiently than full-length Sir1 in vitro. Thus, the Sir1 N terminus exhibited both positive and negative roles in the formation of a Sir1-ORC silencing complex. This functional duality might contribute to Sir1's selectivity for silencer-bound ORCs in vivo.     

A histone variant, Htz1p, and a Sir1p-like protein, Esc2p, mediate silencing at HMR

Silencing at HMR requires silencers, and one of the roles of the silencer is to recruit Sir proteins. This work focuses on the function of Sir1p once it is recruited to the silencer. We have generated mutants of Sir1p that are recruited to the silencer but are unable to silence, and we have utilized these mutants to identify four proteins, Sir3p, Sir4p, Esc2p, and Htz1p, that when overexpressed, restored silencing. The isolation of Sir3p and Sir4p validated this screen. Molecular analysis suggested that Esc2p contributed to silencing in a manner similar to Sir1p and probably helped recruit or stabilize the other Sir proteins, while Htz1p present at HMR assembled a specialized chromatin structure necessary for silencing.     

Yeast two-hybrid analysis of the origin recognition complex of Saccharomyces cerevisiae: interaction between subunits and identification of binding proteins

Origin recognition complex (ORC), a six-protein complex (Orc1p-6p), is the most likely initiator of chromosomal DNA replication in eukaryotes. Although ORC of Saccharomyces cerevisiae has been studied extensively from biochemical and genetic perspectives, its quaternary structure remains unknown. Previous studies suggested that ORC has functions other than DNA replication, such as gene silencing, but the molecular mechanisms of these functions have not been determined. In this study, we used yeast two-hybrid analysis to examine the interaction between ORC subunits and to search for ORC-binding proteins. As well as the known Orc4p-Orc5p interaction, we revealed strong interactions between Orc2p and Ord3p (2p-3p), Orc2p and Ord5p (2p-5p), Orc2p and Ord6p (2p-6p) and Orc3p and Ord6p (3p-6p) and weaker interactions between Orc1p and Ord4p (1p-4p), Orc3p and Ord4p (3p-4p), Orc2p and Ord3p (3p-5p) and Orc5p and Ord3p (5p-6p). These results suggest that 2p-3p-6p may form a core complex. Orc2p and Orc6p are phosphorylated in vivo, regulating initiation of DNA replication. However, replacing the phosphorylated amino acid residues with others that cannot be phosphorylated, or that mimic phosphorylation, did not affect subunit interactions. We also identified several proteins that interact with ORC subunits; Sir4p and Mad1p interact with Orc2p; Cac1p and Ykr077wp with Orc3p; Rrm3p and Swi6p with Orc5p; and Mih1p with Orc6p. We discuss roles of these interactions in functions of ORC.     

Dimerization of Sir3 via its C‐terminal winged helix domain is essential for yeast heterochromatin formation

Gene silencing in budding yeast relies on the binding of the Silent Information Regulator (Sir) complex to chromatin, which is mediated by extensive interactions between the Sir proteins and nucleosomes. Sir3, a divergent member of the AAA+ ATPase‐like family, contacts both the histone H4 tail and the nucleosome core. Here, we present the structure and function of the conserved C‐terminal domain of Sir3, comprising 138 amino acids. This module adopts a variant winged helix‐turn‐helix (wH) architecture that exists as a stable homodimer in solution. Mutagenesis shows that the self‐association mediated by this domain is essential for holo‐Sir3 dimerization. Its loss impairs Sir3 loading onto nucleosomes in vitro and eliminates silencing at telomeres and HM loci in vivo. Replacing the Sir3 wH domain with an unrelated bacterial dimerization motif restores both HM and telomeric repression in sir3Δ cells. In contrast, related wH domains of archaeal and human members of the Orc1/Sir3 family are monomeric and have DNA binding activity. We speculate that a dimerization function for the wH evolved with Sir3's ability to facilitate heterochromatin formation.     

Locus specificity determinants in the multifunctional yeast silencing protein Sir2

Yeast SIR2, the founding member of a conserved gene family, acts to modulate chromatin structure in three different contexts: silent (HM) mating-type loci, telomeres and rDNA. At HM loci and telomeres, Sir2p forms a complex with Sir3p and Sir4p. However, Sir2p's role in rDNA silencing is Sir3/4 independent, requiring instead an essential nucleolar protein, Net1p. We describe two novel classes of SIR2 mutations specific to either HM/telomere or rDNA silencing. Despite their opposite effects, both classes of mutations cluster in the same two regions of Sir2p, each of which borders on a conserved core domain. A surprising number of these mutations are dominant. Several rDNA silencing mutants display a Sir2p nucleolar localization defect that correlates with reduced Net1p binding. Although the molecular defect in HM/telomere-specific mutants is unclear, they mimic an age-related phenotype where Sir3p and Sir4p relocalize to the nucleolus. Artificial targeting can circumvent the silencing defect in a subset of mutants from both classes. These results define distinct functional domains of Sir2p and provide evidence for additional Sir2p-interacting factors with locus-specific silencing functions.     

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.     

Bypassing the catalytic activity of SIR2 for SIR protein spreading in Saccharomyces cerevisiae

Sir protein spreading along chromosomes and silencing in Saccharomyces cerevisiae requires the NAD+-dependent histone deacetylase activity of Sir2p. We tested whether this requirement could be bypassed at the HM loci and telomeres in cells containing a stably expressed, but catalytically inactive mutant of Sir2p, sir2-345p, plus histone mutants that mimic the hypoacetylated state normally created by Sir2p. Sir protein spreading was rescued in sir2-345 mutants expressing histones in which key lysine residues in their N-termini had been mutated to arginine. Mating in these mutants was also partially restored upon overexpression of Sir3p. Together, these results indicate that histone hypoacetylation is sufficient for Sir protein spreading in the absence of production of 2'-O-acetyl-ADP ribose by sir2p and Sir2p's enzymatic function for silencing can be bypassed in a subset of cells in a given population. These results also provide genetic evidence for the existence of additional critical substrates of Sir2p for silencing in vivo.