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.
Highlights
The eukaryotic genome is organized into euchromatic and heterochromatic domains that generally reflect their potential for gene expression
Three Silent Information Regulator (SIR) proteins Sir2, Sir3, and Sir4 are involved in the epigenetic gene silencing of the homothallic mating (HM) loci and of telomere-proximal genes in budding yeast
We report here that Sir4 N- and Cterminal domains have distinct functions: The Sir4 Cterminus binds all proteins essential for Silent information regulator (SIR)–mediated silencing and is sufficient to repress homothallic mating type (HM) loci, but surprisingly it is not sufficient to efficiently repress at telomeres
Summary
The eukaryotic genome is organized into euchromatic and heterochromatic domains that generally reflect their potential for gene expression. Chromatin repressed by the Silent information regulator (SIR) complex in the budding yeast Saccharomyces cerevisiae shares many key features with heterochromatin in higher eukaryotes. Unlike the situation in other eukaryotes, where histone H3 lysine 9 methylation and its specific ligands mediate repression, heritable transcriptional silencing in S. cerevisiae relies on the association of a trimeric SIR complex with unmodified histones (reviewed in [9,10,11,12]). This heterotrimeric complex contains equimolar amounts of Sir, Sir and Sir4 [13], each of which is essential for the repression of promoters at the homothallic mating type loci, HMR and HML [14] and in subtelomeric domains [15]. In analogy to centromeric position effect variegation in flies, repression at telomeres has been called telomere position effect, or TPE
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