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

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Author(s)

Stephanie Kueng, Monika Tsai-Pflugfelder, Mariano Oppikofer, Helder C. Ferreira, Emma Roberts, Chinyen Tsai, Tim-Christoph Roloff, Ragna Sack, Susan M. Gasser

School/Research organisations

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.

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Details

Original languageEnglish
Article numbere1002727
Number of pages19
JournalPLoS Genetics
Volume8
Issue number5
DOIs
Publication statusPublished - May 2012

    Research areas

  • Histone deacetylation, Phosphorylation sites, In-vitro, Yeast silent chromatin, Transcriptional activation, Saccharomyces-cerevisiae telomeres, Cell-cycle, Mating-type loci, Mediated repression, Drosophila embryos

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