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.
Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.
Stephanie
S
Kueng
Monika
M
Tsai-Pflugfelder
Mariano
M
Oppikofer
Helder C
HC
Ferreira
Emma
E
Roberts
Chinyen
C
Tsai
Tim-Christoph
TC
Roloff
Ragna
R
Sack
Susan M
SM
Gasser
D002843:Chromatin / Q000235:genetics; D018844:Cyclin-Dependent Kinases; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D044127:Epigenesis, Genetic / Q000235:genetics; D059565:G2 Phase Cell Cycle Checkpoints / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D020868:Gene Silencing; D049770:Genes, Mating Type, Fungal / Q000235:genetics; D008938:Mitosis; D010766:Phosphorylation; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D016615:Telomere / Q000235:genetics*; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D002843:Chromatin; D004268:DNA-Binding Proteins; C084122:SIR3 protein, S cerevisiae; C061573:SIR4 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae; C520769:YKU70 protein, S cerevisiae; D018844:Cyclin-Dependent Kinases