Insights into the impact of histone acetylation and methylation on Sir protein recruitment, spreading and silencing in Saccharomyces cerevisiae

Bo Yang, Purdue University

Abstract

Sir protein spreading along chromosomes and silencing in Saccharomyces cerevisiae requires the NAD+-dependent histone deacetylase activity of Sir2p. We tested whether this requirement could be bypassed in cells containing a catalytically inactive mutant of Sir2p, sir2-345p, plus histone mutants that mimic the hypoacetylated state normally created by Sir2p. Sir protein spreading was rescued in sir2-345 mutants expressing histones in which key lysine residues in their N-termini had been mutated to arginine. Silencing at the HM loci in these mutants was also partially restored upon overexpression of Sir3p. Together, these results indicate that histone hypoacetylation is sufficient for Sir protein spreading in the absence of production of 2’-O-acetyl-ADP-ribose by Sir2p, and Sir2p’s enzymatic function for silencing can be bypassed in a subset of cells. Sir spreading across hypoacetylated chromatin could disrupt SET1 and DOT1-dependent histone methylation without silencing underlying genes. Loss of acetylation but not methylation facilitated both Sir recruitment and spreading. Our data indicate loss of methylation of K4 and K79 on histone H3 reflects intermediate events during the formation of silent chromatin. Another histone modification that potentially affects silencing is the recently discovered, evolutionary conserved H3 K56ac. H3 K56ac is essential for replisome stability during DNA replication and chromosome integrity in S. cerevisiae. H3 K56 acetylation is catalyzed by Rtt109p during S phase, and is removed by Hst3p and Hst4p during G2/M phase. Here we investigated the influence of H3 K56ac on epigenetic gene regulation. Our results indicated H3 K56 was hypoacetylated in silent chromatin. Deletion of HST3 and HST4 resulted in hyperacetylation of H3 K56 and in the loss of telomeric silencing without significantly disrupting Sir protein binding. This result also indicates H3 K56 deacetylation works downstream of Sir protein binding during the establishment of silencing. Silencing defects in hst3Δ hst4Δ cells could be suppressed by deletion of RTT109. In contrast, overexpression of Sir2p did not rescue H3 K56 hypoacetylation or silencing at subtelomeric regions in hst3Δ hst4Δ mutants. Together, our findings argue that HST3 and HST4 play critical roles in maintaining the hypoacetylated state of K56 on histone H3 within silent chromatin.

Degree

Ph.D.

Advisors

Kirchmaier, Purdue University.

Subject Area

Genetics

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