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 NADdependent deacetylases may constitute novel anticancer agents. (Cancer Res 2006; 66(8): 4368-77)
Sir2p is an NAD ؉ -dependent histone deacetylase required for chromatin-dependent silencing in yeast. In a cell-based screen for inhibitors of Sir2p, we identified a compound, splitomicin, that creates a conditional phenocopy of a sir2 deletion mutant in Saccharomyces cerevisiae. Cells grown in the presence of the drug have silencing defects at telomeres, silent mating-type loci, and the ribosomal DNA. In addition, whole genome microarray experiments show that splitomicin selectively inhibits Sir2p. Portions of the eukaryotic genome can be maintained in a transcriptionally inactive, or silenced, state as the result of the local chromatin structure. Silent chromatin may encompass regions ranging from a few thousand base pairs, as in the silent mating-type genes of the yeast Saccharomyces cerevisiae (1), to whole chromosomes such as the inactive X chromosome in mammals (2). The formation of silent chromatin, which is best understood at the S. cerevisiae silent mating-type loci HMR and HML, and telomeres depends on DNA elements, or silencers. The HM silencers are located in proximity to the genes that they regulate and contain a combination of binding sites for Rap1p, Abf1p, and the origin recognition complex (1). These proteins recruit the SIR (silent information regulator) protein complex (Sir2p-4p) through protein-protein interactions. Once recruited to silencers, the SIR complex is thought to spread along the chromatin through binding of Sir3p and Sir4p to the NH 2 -terminal tails of histone H3 and H4 (reviewed in ref. 3). Among the many requirements for silent chromatin (reviewed in ref. 4), posttranslational modification (i.e., acetylation, phosphorylation, methylation, and ubiquitination) of histones seems to be critical. For example, the NH 2 -terminal tails of histones H3 and H4 are hypoacetylated in silent chromatin compared with other regions of the genome (5). Of the SIR proteins, Sir2p has been shown to be an NAD ϩ -dependent histone deacetylase and is responsible for the hypoacetylated state of histones in silent chromatin (6-9). Sir2p also acts at the rRNA-encoding DNA (rDNA) in the RENT protein complex, which does not include Sir3p or Sir4p (10).The yeast SIR2 gene is the defining member of a broadly conserved family of NAD ϩ -dependent deacetylases found in organisms ranging from bacteria to humans (11). In S. cerevisiae alone four additional homologues have been identified (see below). However, it shares the greatest similarity with genes found in other eukaryotes, where it is believed that these closely related homologues serve a comparable role in silencing. Interestingly, SIR2 and its homologues have been implicated in the genetic regulation of aging in both yeast and Caenorhabditis elegans (12, 13) and in metazoan development (M. I. Rosenberg and S. M. Parkhurst, personal communication), although the details of how it affects these fundamental processes are still mysterious.To provide a new tool to dissect the functional role of Sir2p in vivo further, we undertook a phenotypic screen for sma...
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.
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