Identification of a small molecule inhibitor of Sir2p

Abstract

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...

In Vivo and in Vitro Examination of Stability of Primary Hyperoxaluria-associated Human Alanine:Glyoxylate Aminotransferase

“…A major advantage of this assay is that it not only allows identification of compounds that directly bind AGT but also allows for the identification of compounds that act indirectly, such as those that may stimulate protein chaperones. Previous high throughput compound library screens with yeast growth assays have successfully targeted a variety of proteins, such as polyglutamine-containing proteins (46,47), Sir2 (48), and HSP90 ATPase (49).…”

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“…1c). To investigate whether bax and mdm2 transcriptions were still regulated by Sirtuins in this cell lines, pharmacological modulators of Sirtuins activity were used (Landry et al, 2000;Bedalov et al, 2001;Howitz et al, 2003). Cells pretreated with Resveratrol (Res) showed a remarkable decrease in bax and mdm2 luciferase activity, as measured by transient luciferase reporter assays (Fig.…”

Chronological and replicative life-span extension in Saccharomyces cerevisiae by increased dosage of alcohol dehydrogenase 1

“…5(c) shows that the duplication times of WT-UT and 26ADH1-UT grown on SC medium were 196 and 238 min, respectively. To demonstrate that this difference in duplication time was Sir2p-dependent, the addition of two well-known Sir2p inhibitors, splitomicin (Bedalov et al, 2001) and NAM (Bitterman et al, 2002;Jackson et al, 2003) was used.…”

“…5(c) shows that the duplication times of WT-UT and 26ADH1-UT grown on SC medium were 196 and 238 min, respectively. To demonstrate that this difference in duplication time was Sir2p-dependent, the addition of two well-known Sir2p inhibitors, splitomicin (Bedalov et al, 2001) and NAM (Bitterman et al, 2002;Jackson et al, 2003) was used.Derepression of the URA3 reporter should result in growth enhancement on medium lacking uracil. Addition of 5 mM splitomicin or 1 mM NAM to the medium abrogated the differences in duplication time observed between WT-UT and 26ADH1-UT.…”