Academic journal article Genetics

Proliferating Cell Nuclear Antigen and ASF1 Modulate Silent Chromatin in Saccharomyces Cerevisiae Via Lysine 56 on Histone H3

Academic journal article Genetics

Proliferating Cell Nuclear Antigen and ASF1 Modulate Silent Chromatin in Saccharomyces Cerevisiae Via Lysine 56 on Histone H3

Article excerpt

ABSTRACT

The formation and stability of epigenetically regulated chromatin is influenced by DNA replication and factors that modulate post-translational modifications on histones. Here we describe evidence that PCNA can affect silencing in Saccharomyces cerevisiae by facilitating deposition of H3 K56ac onto chromosomes. We propose that PCNA participates in this process through a pathway that includes replication factor C, the chromatin assembly factor Asf1p, and the K56-specific acetyltransferase Rtt109p. We show that mutation of POL30 or loss of K56-acetylation in rtt109 and histone H3 mutants enhances silencing at the crippled HMR locus HMRae** via restoring Sir binding and that pol30 mutants with silencing phenotypes have reduced levels of H3 K56ac. Although loss of acetylation on H3 K56 was generally compatible with silencing, mutations at this residue also led to defects in silencing an ADE2 reporter at HMR and abolished silencing when combined with cac1 or pol30-8. These silencing phenotypes are analogous to those in asf1 mutants or pol30-6 and pol30-79 mutants with defects in ASF1-dependent pathways. On the basis of these findings, we propose that mutations in DNA replication factors alter acetylation of H3 K56. We show that this defect, in turn, contributes to misregulation of epigenetic processes as well as of cellular responses to DNA damage.

THE foundation supporting epigenetically regulated chromatin is nucleosomal DNA. In Saccharomyces cerevisiae, the strength and stability of silent chromatin depends on the characteristics of histones present not only at silenced loci but also elsewhere throughout the genome. In yeast, transcribed loci are enriched in both acetylated and methylated histones whereas histones in silenced loci are hypoacetylated and hypomethylated (BRAUNSTEIN et al. 1996; SUKA et al. 2001; BERNSTEIN et al. 2002; BRYK et al. 2002; HOPPE et al. 2002; RUSCHÉ et al. 2002; NG et al. 2003; SANTOS- ROSA et al. 2004; KATAN-KHAYKOVICH and STRUHL 2005; RUDNER et al. 2005; LI et al. 2006). Loss of acetylation in silent chromatin is mediated by the NAD1- dependent histone deacetylase Sir2p (RUSCHÉ et al. 2003 and references therein). Sir2p enables the major structural components of silent chromatin, Sir2, -3, and -4 proteins, to bind preferentially to hypoacetylated histones and spread across the chromosome (HOPPE et al. 2002; LUO et al. 2002; RUSCHÉ et al. 2002). The resulting silent chromatin then blocks transcription and prevents histone-modifying enzymes from reaching their targets. Silencing defects at the HM loci and telomeres in yeast can occur upon overexpression or loss of histone-modifying enzymes. In these instances, inappropriately modified histones may prevent Sir proteins from interacting stably with silent loci. In addition, global loss of histone modifications is thought to result in the redistribution of Sir protein binding throughout the genome, thereby limiting the pool of Sir proteins available for forming silent chromatin at appropriate sites (SINGER et al. 1998; VAN LEEUWEN et al. 2002; RUSCHÉ et al. 2003).

Silencing defects are also observed in cells with altered expression of proteins that load histones onto DNA. Cells lacking the chromatin assembly factors CAF-I (composed of Cac1p, Cac2p, and Cac3p) and Asf1p exhibit silencing defects (KAUFMAN et al. 1997; MONSON et al. 1997; ENOMOTO and BERMAN 1998; TYLER et al. 1999). These defects are more severe in asf1 cac1 and asf1 cac2 mutants relative to single mutants (TYLER et al. 1999; SUTTON et al. 2001). Overexpression of Asf1p also leads to defects in silencing at both theHMloci and telomeres (LE et al. 1997; SINGER et al. 1998). The silencing defects associated with cac1 and asf1 mutants may reflect both improper chromatin assembly and altered post-translational modifications on histones. Cells lacking CAC1 have reduced levels of histone H3 as well as Sir proteins at HMR and telomere VIR (TAMBURINI et al. 2006; HUANG et al. …

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