Regulating Repression: Roles for the Sir4 N-Terminus in Linker DNA Protection and Stabilization of Epigenetic States

et al. (2012) Regulating Repression: Roles for the Sir4 N-Terminus in Linker DNA Protection and Stabilization of Epigenetic States. PLoS Genet 8(5): e1002727. doi:10.1371/journal.pgen.1002727 Regulating Repression: Roles for the Sir4 N-Terminus in Linker DNA Protection and Stabilization of Epigenetic States Stephanie Kueng 0 Monika Tsai-Pflugfelder 0 Mariano Oppikofer 0 Helder C. Ferreira 0 Emma Roberts 0 Chinyen Tsai 0 Tim-Christoph Roloff 0 Ragna Sack 0 Susan M. Gasser 0 Hiten D. Madhani, University of California San Francisco, United States of America 0 1 Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland, 2 Faculty of Natural Sciences, University of Basel , Basel , Switzerland 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 Cterminal 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 Nterminus 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. - Funding: The Gasser laboratory is supported by the Novartis Research Foundation and the EU training network Nucleosome 4D. SK was supported by an EMBO long-term fellowship, a Schro dinger fellowship from the FWF, and the Swiss SystemsX.ch initiative/C-CINA; HCF by an EMBO long-term fellowship. All these funding agencies did not influence the scientific content of the study or contribute to its content or the manuscript in any other way than financial support. They had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. 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. Notably, it has hypoacetylated nucleosomes [1,2], is less accessible to DNA-binding enzymes than is euchromatin [3 5], it replicates late in S phase [6] and is spatially sequestered at the nuclear envelope or near the nucleolus [7]. The genes found within heterochromatin are generally silent, and in complex organisms this gene repression is crucial for the proper development of differentiated tissues and organs [8]. 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 [912]). This heterotrimeric complex contains equimolar amounts of Sir2, Sir3 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. The SIR complex is targeted to the genes it represses by interacting with sequence-specific DNA-binding proteins that bind silencers or telomeric TG repeats. This binding initiates or nucleates the formation of silent chromatin on adjacent genes. Repressor activator protein 1 (Rap1; [16]) is a key factor for SIRmediated repression, because it has high affinity sites both at telomeres and in silencer elements [16,17]. Furthermore, Rap1 interacts with both Sir3 and Sir4 [18]. HM silencer elements contain sites for two further sequence-specific factors, namely Abf1 (ARSbinding factor 1) and ORC (Origin recognition complex) [19,20]. Abf1 recruits the SIR complex by binding to Sir3 [10], and the largest subunit of ORC, Orc1, enhances SIR recruitment by binding Sir1, an intermediary protein that in turn binds Sir4 [21]. 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. They bind as a heterotrimeric complex to chromatin, repressing the underlying genes. Sir2 has an essential histone deacetylase activity, and Sir3 binds nucleosomes, with a high specificity for unmodified histones. We explored Sir4, whose role had largely remained a mystery. We report here that Sir4 N- and Cterminal domains have distinct functions: The Sir4 Cterminus binds all proteins essential for SIRmediated silencing and is sufficient to repress HM loci, but surprisingly it is not sufficient to efficiently repress at telomeres. The Sir4 N-terminus binds DNA, which strengthens the SIRchromatin interaction and helps target Sir4 to telomeric loci. In addition the Sir4 N-terminus binds sequence-specific factors that recruit Sir4 to sites of repression. We find that the Sir4 N-terminus is a target of mitotic phosphorylation. Mutation of the phosphoacceptor sites indicates that they help fine-tune subtelomeric repression. We propose therefore that phosphorylation of the Sir4 N-terminal domain modulates epigenetic repression at telomeres in response to cell cycle and/or stress situations. The initial recruitment of Sir4 or Sir3 to telomeric TG-repeats or to silencers, brings in Sir2, a histone deacetylase [2224], which generates high-affinity binding sites for Sir3 by r (...truncated)