Chromosome-wide histone deacetylation by sirtuins prevents hyperactivation of DNA damage-induced signaling upon replicative stress

2706–2726 Nucleic Acids Research, 2016, Vol. 44, No. 6 doi: 10.1093/nar/gkv1537 Published online 8 January 2016 Chromosome-wide histone deacetylation by sirtuins prevents hyperactivation of DNA damage-induced signaling upon replicative stress Antoine Simoneau1,2 , Étienne Ricard1,2 , Sandra Weber3 , Ian Hammond-Martel1,2 , Lai Hong Wong4 , Adnane Sellam5,6 , Guri Giaever4 , Corey Nislow4 , Martine Raymond3,7,* and Hugo Wurtele1,8,* 1 Maisonneuve-Rosemont Hospital Research Center, 5415 Assomption boulevard, Montreal, H1T 2M4, Canada, Molecular biology program, Université de Montréal, P.O. Box 6128, Succursale Centre-ville, Montreal, H3C 3J7, Canada, 3 Institute for Research in Immunology and Cancer, Université de Montréal, P.O. Box 6128, Succursale Centre-Ville, Montreal, H3C 3J7, Canada, 4 Department of Pharmaceutical Sciences, University of British Columbia, Vancouver, V6T 1Z3, Canada, 5 Infectious Diseases Research Centre-CRI, CHU de Québec Research Center (CHUQ), Université Laval, Québec, G1V 4G2, Canada, 6 Department of Microbiology-Infectious Disease and Immunology, Faculty of Medicine, Université Laval, Québec, G1V 0A6, Canada, 7 Department of Biochemistry and Molecular Medicine, Université de Montréal, C.P. 6128, Succursale Centre-ville, Montréal, H3C 3J7, Canada and 8 Department of Medicine, Université de Montréal, Montreal, H3T 1J4, Canada 2 ABSTRACT The Saccharomyces cerevisiae genome encodes five sirtuins (Sir2 and Hst1–4), which constitute a conserved family of NAD-dependent histone deacetylases. Cells lacking any individual sirtuin display mild growth and gene silencing defects. However, hst3Δ hst4Δ double mutants are exquisitely sensitive to genotoxins, and hst3Δ hst4Δ sir2Δ mutants are inviable. Our published data also indicate that pharmacological inhibition of sirtuins prevents growth of several fungal pathogens, although the biological basis is unclear. Here, we present genome-wide fitness assays conducted with nicotinamide (NAM), a pan-sirtuin inhibitor. Our data indicate that NAM treatment causes yeast to solicit specific DNA damage response pathways for survival, and that NAMinduced growth defects are mainly attributable to inhibition of Hst3 and Hst4 and consequent elevation of histone H3 lysine 56 acetylation (H3K56ac). Our results further reveal that in the presence of constitutive H3K56ac, the Slx4 scaffolding protein and PP4 phosphatase complex play essential roles in preventing hyperactivation of the DNA damage-response kinase Rad53 in response to spontaneous DNA damage caused by reactive oxygen species. Overall, our data support the concept that chromosome-wide hi- stone deacetylation by sirtuins is critical to mitigate growth defects caused by endogenous genotoxins. INTRODUCTION Post-translational modification of histones can directly influence chromatin structure, or serve as platforms for the recruitment of regulatory factors, thereby modulating DNA-associated processes (1). Acetylation of histone lysine residues is catalyzed by histone acetyltransferases (HATs), and reversed by histone deacetylases (HDACs). Sirtuins are an evolutionarily conserved family of HDACs that deacetylate lysines in a reaction that consumes nicotinamide adenine dinucleotide (NAD+ ) and releases nicotinamide and O-acetyl ADP ribose (2,3). These enzymes are found in archaea, eubacteria and eukaryotes (2) where they regulate key cellular pathways, e.g. metabolic processes, DNA replication and repair, telomere structure and function, gene expression and replicative lifespan (4). The Saccharomyces cerevisiae genome contains five sirtuin genes: HST1–4 and SIR2 (5,6). Yeast Sir2 is the founding member of this family of enzymes, and was identified on the basis of its role in regulating gene silencing at the yeast mating loci (6), rDNA (7) and telomeres (8). These functions of Sir2 can be attributed in part to reversal of histone H4 lysine 16 acetylation (H4K16ac), an abundant and conserved modification of transcriptionally active chromatin (9,10). Sir2 activity influences replicative life-span by limiting recombination in rDNA and consequent forma- * To whom correspondence should be addressed. Tel: +514-252-3400 ext.: 4302; Fax: +514-252-3430; Email: Correspondence may also be addressed to Martine Raymond. Tel: +514-343-6746; Fax: +514-343-6843; Email:  C The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact Received August 14, 2015; Revised December 01, 2015; Accepted December 24, 2015 Nucleic Acids Research, 2016, Vol. 44, No. 6 2707 vents growth of the pathogenic fungus Candida albicans by causing constitutive H3K56ac (24). To further understand this phenomenon, we performed genome-wide fitness assays to identify genes that influence growth of Saccharomyces cerevisiae in the presence of NAM. The data reveal that sirtuin-mediated deacetylation of H3K56ac promotes cell growth by preventing persistent activation of DNA damage-induced kinases in response to endogenous genotoxins. MATERIALS AND METHODS Yeast strains and growth conditions Strains used in this study are listed in Table 1 and were generated and propagated using standard yeast genetics methods. Nicotinamide and methyl methanesulfonate (MMS) were purchased from Sigma-Aldrich. Growth assays in 96 well plates Cells were grown overnight in YPD in a humid chamber at 30◦ C. Cells were then diluted to OD600 0.0005 in 100 ␮l YPD containing nicotinamide in flat-bottomed 96 well plates. Plates were incubated for 48 h at 30◦ C in a humid chamber and OD630 was measured using a Biotek EL800 plate reader equipped with Gen5 version 1.05 software (Biotek instruments). OD630 from blank wells (YPD) was subtracted from OD630 readings and growth was normalized to untreated controls for each strain. Experiments were performed at least in triplicate and error bars represent the standard error of the mean of normalized growth. To calculate population doubling time, cells were grown overnight in YPD at 30◦ C. Cells were then diluted to OD600 0.01 in 100 ␮l YPD with or without 20 mM NAM in flat-bottomed 96 well plates. Cells were incubated for 48 h at room temperature with shaking in a Biotek ELX808 and OD630 readings taken every 30 min. OD630 readings were plotted on a graph, and exponential regression was used to calculate doubling times. Cell synchronization and treatment with MMS Cells were grown overnight in YPD medium at 25◦ C and arrested in G1 at 30◦ C in YPD containing 5 ␮g/ml ␣-factor for 90 min, followed by addition of a second dose of 5 ␮g/ml ␣-factor for 75 min. Cells were released into the cell cycle by washing them once with YPD a (...truncated)