Regulating repression: roles for the sir4 N-terminus in linker DNA protection and stabilization of epigenetic states

PLoS Genet. 2012;8(5):e1002727. doi: 10.1371/journal.pgen.1002727. Epub 2012 May 24.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chromatin / genetics
  • Cyclin-Dependent Kinases
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Epigenesis, Genetic / genetics
  • G2 Phase Cell Cycle Checkpoints / genetics
  • Gene Expression Regulation, Fungal
  • Gene Silencing
  • Genes, Mating Type, Fungal* / genetics
  • Mitosis
  • Phosphorylation
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism
  • Silent Information Regulator Proteins, Saccharomyces cerevisiae / genetics*
  • Silent Information Regulator Proteins, Saccharomyces cerevisiae / metabolism
  • Telomere / genetics*
  • Transcription, Genetic*
  • Transcriptional Activation

Substances

  • Chromatin
  • DNA-Binding Proteins
  • SIR3 protein, S cerevisiae
  • SIR4 protein, S cerevisiae
  • Saccharomyces cerevisiae Proteins
  • Silent Information Regulator Proteins, Saccharomyces cerevisiae
  • YKU70 protein, S cerevisiae
  • Cyclin-Dependent Kinases