Regulating repression: Roles for the Sir4 N-terminus in linker DNA protection and stabilization of epigenetic states

Silent information regulator proteins sir2,sir3,and sir4 form a heterotrimeric complex that represses transcription at subtelomeric regions and homothallic mating type (hm) loci in budding yeast. we have performed a detailed biochemical and genetic analysis of the largest sir protein,sir4. the n-terminal half of sir4 is dispensable for sir-mediated repression of hm loci in vivo,except in strains that lack yku70 or have weak silencer elements. for hm silencing in these cells,the c-terminal domain (sir4c,residues 747-1,358) must be complemented with an n-terminal domain (sir4n; residues 1-270),expressed either independently or as a fusion with sir4c. nonetheless,recombinant sir4c can form a complex with sir2 and sir3 in vitro,is catalytically active,and has sedimentation properties similar to a full-length sir4-containing sir complex. sir4c-containing sir complexes bind nucleosomal arrays and protect linker dna from nucleolytic digestion,but less effectively than wild-type sir complexes. consistently,full-length sir4 is required for the complete repression of subtelomeric genes. supporting the notion that the sir4 n-terminus is a regulatory domain,we find it extensively phosphorylated on cyclin-dependent kinase consensus sites,some being hyperphosphorylated during mitosis. mutation of two major phosphoacceptor sites (s63 and s84) derepresses natural subtelomeric genes when combined with a serendipitous mutation (p2a),which alone can enhance the stability of either the repressed or active state. the triple mutation confers resistance to rapamycin-induced stress and a loss of subtelomeric repression. we conclude that the sir4 n-terminus plays two roles in sir-mediated silencing: it contributes to epigenetic repression by stabilizing the sir-mediated protection of linker dna; and,as a target of phosphorylation,it can destabilize silencing in a regulated manner. © 2012 kueng et al.