ABSTRACT
The SIR2 homologues HST3 and HST4 have been implicated in maintenance of genome integrity in the yeast Saccharomyces cerevisiae. We find that Hst3 has NAD-dependent histone deacetylase activity in vitro and that it functions during S phase to deacetylate the core domain of histone H3 at lysine 56 (H3K56). In response to genotoxic stress, Hst3 undergoes rapid Mec1-dependent phosphorylation and is targeted for ubiquitin-mediated proteolysis, thus providing a mechanism for the previously observed checkpoint-dependent accumulation of Ac-H3K56 at sites of DNA damage. Loss of Hst3-mediated regulation of H3K56 acetylation results in a defect in the S phase DNA damage checkpoint. The pathway that regulates H3K56 acetylation acts in parallel with the Rad9 pathway to transmit a DNA damage signal from Mec1 to Rad53. We also observe that loss of Hst3 function impairs sister chromatid cohesion (SCC). Both S phase checkpoint and SCC defects are phenocopied by H3K56 point mutants. Our findings demonstrate that Hst3-regulated H3K56 acetylation safeguards genome stability by controlling the S phase DNA damage response and promoting SCC.
Subject(s)
Chromatids/genetics , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Fungal , Histone Deacetylases/physiology , Histones/metabolism , Lysine/chemistry , Saccharomyces cerevisiae Proteins/physiology , Cell Cycle , Cell Cycle Proteins/metabolism , DNA Damage , Histone Deacetylases/biosynthesis , Histone Deacetylases/metabolism , Intracellular Signaling Peptides and Proteins , Models, Biological , Phenotype , Phosphorylation , Protein Serine-Threonine Kinases , Saccharomyces cerevisiae Proteins/biosynthesis , Saccharomyces cerevisiae Proteins/metabolism , Ubiquitin/metabolismABSTRACT
SIRT1 and other NAD-dependent deacetylases have been implicated in control of cellular responses to stress and in tumorigenesis through deacetylation of important regulatory proteins, including p53 and the BCL6 oncoprotein. Hereby, we describe the identification of a compound we named cambinol that inhibits NAD-dependent deacetylase activity of human SIRT1 and SIRT2. Consistent with the role of SIRT1 in promoting cell survival during stress, inhibition of SIRT1 activity with cambinol during genotoxic stress leads to hyperacetylation of key stress response proteins and promotes cell cycle arrest. Treatment of BCL6-expressing Burkitt lymphoma cells with cambinol as a single agent induced apoptosis, which was accompanied by hyperacetylation of BCL6 and p53. Because acetylation inactivates BCL6 and has the opposite effect on the function of p53 and other checkpoint pathways, the antitumor activity of cambinol in Burkitt lymphoma cells may be accomplished through a combined effect of BCL6 inactivation and checkpoint activation. Cambinol was well tolerated in mice and inhibited growth of Burkitt lymphoma xenografts. Inhibitors of NAD-dependent deacetylases may constitute novel anticancer agents.
Subject(s)
Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Histone Deacetylase Inhibitors , Naphthalenes/pharmacology , Pyrimidinones/pharmacology , Sirtuins/antagonists & inhibitors , Acetylation/drug effects , Animals , Burkitt Lymphoma/drug therapy , Burkitt Lymphoma/enzymology , Burkitt Lymphoma/metabolism , Cell Line, Tumor , DNA-Binding Proteins/metabolism , Humans , Mice , Proto-Oncogene Proteins c-bcl-6 , Sirtuin 1 , Sirtuin 2 , Tubulin/metabolism , Tumor Suppressor Protein p53/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Telomere length-variation in deletion strains of Saccharomyces cerevisiae was used to identify genes and pathways that regulate telomere length. We found 72 genes that when deleted confer short telomeres, and 80 genes that confer long telomeres relative to those of wild-type yeast. Among identified genes, 88 have not been previously implicated in telomere length control. Genes that regulate telomere length span a variety of functions that can be broadly separated into telomerase-dependent and telomerase-independent pathways. We also found 39 genes that have an important role in telomere maintenance or cell proliferation in the absence of telomerase, including genes that participate in deoxyribonucleotide biosynthesis, sister chromatid cohesion, and vacuolar protein sorting. Given the large number of loci identified, we investigated telomere lengths in 13 wild yeast strains and found substantial natural variation in telomere length among the isolates. Furthermore, we crossed a wild isolate to a laboratory strain and analyzed telomere length in 122 progeny. Genome-wide linkage analysis among these segregants revealed two loci that account for 30%-35% of telomere length-variation between the strains. These findings support a general model of telomere length-variation in outbred populations that results from polymorphisms at a large number of loci. Furthermore, our results laid the foundation for studying genetic determinants of telomere length-variation and their roles in human disease.
