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.

Nicotine adenine dinucleotide (NADNAD ϩ and its pyridine or dihydropyridine congeners NADP ϩ , NADH, and NADPH play crucial roles in electron transport processes (46). NAD ϩ also serves as an essential cofactor in enzymatic reactions that do not involve oxidative metabolism. For example, cleavage of the glycosidic bond between nicotinamide and ADP-ribose and concomitant transfer of ADP-ribose to arginine or histidine residues of G-proteins is identified as the mechanism of action of bacterial virulence factors such as the cholera and pertussis toxins (28). A related reaction carried out by poly(ADP-ribose) polymerase couples glycosidic bond hydrolysis with formation of ADP-ribose polymers in response to DNA damage in metazoans (12). More recently, the cleavage of the glycosidic bond of NAD ϩ was shown to be a requisite step in the deacetylation reaction carried out by class III deacetylases of the Saccharomyces cerevisiae Sir2p family (16,20,39). Deacetylation is accomplished through the transfer of the acetyl group from the substrate to ADP-ribose, the NAD ϩ breakdown product, to generate Oacetyl ADP-ribose and the release of free nicotinamide (33,41,42). Yeast Sir2p, the founding member of this family of enzymes, is required for silencing at the silent mating loci, telomeres, and rRNA genes (rDNA) and is responsible for the hypoacetylated state of histones at these locations (reviewed in reference 25). In addition to silencing (38), SIR2 suppresses recombination between tandem copies of rRNA genes (10) and thus reduces the formation of extrachromosomal ribosomal DNA (rDNA) circles and their accumulation in mother cells, a function critical for maintenance of mother-cell life span (36). SIR2 homologues in higher eukaryotes have been implicated in a wide range of biological processes involving promotion of longevity in Caenorhabditis elegans (43), development and epigenetic regulation in Drosophila melanogaster

Sir2 and Hst1 are NAD؉ -dependent deacetylases involved in transcriptional repression in yeast. The two enzymes are highly homologous yet have different sensitivity to the small-molecule inhibitor splitomicin (compound 1) (Bedalov, A., Gatbonton, T., Irvine, W. P., Gottschling, D. E., and Simon, J. A. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 15113-15118). We have now defined a critical amino acid residue within a small helical module of Hst1 that confers relative resistance to splitomicin. Parallel cell-based screens of 100 splitomicin analogues led to the identification of compounds that exhibit a higher degree of selectivity toward Sir2 or Hst1. A series of compounds based on a splitomicin derivative, dehydrosplitomicin (compound 2), effectively phenocopied a yeast strain that lacked Hst1 deacetylase while having no effect on the silencing activities of Sir2. In addition, we identified a compound with improved selectivity for Sir2. Selectivity was affirmed using whole-genome DNA microarray analysis. This study underscores the power of phenotypic screens in the development and characterization of selective inhibitors of enzyme functions.Sir2-like enzymes constitute a family of NAD ϩ -dependent deacetylases found in diverse organisms ranging from bacteria to humans (1, 2). These highly conserved enzymes catalyze a reaction that requires the consumption of NAD ϩ for the removal of the acetyl group from substrate lysine residues to generate nicotinamide, O-acetyl-ADP-ribose, and lysine (3-10). Initially characterized as histone deacetylases, this family of enzymes was subsequently shown to have a broad range of substrates including p53, BCL6, and ␣-tubulin in mammalian cells and acetyl-CoA synthetase in bacteria (11-17). The yeast Saccharomyces cerevisiae has five Sir2-like proteins: Sir2p, Hst1p (homologue of Sir two), Hst2p, Hst3p, and Hst4p (homologues of Sir two) (1, 18). Sir2p and its closest homologue, Hst1p, act as transcriptional repressors by promoting targeted histone deacetylation (11, 19 -21). These two enzymes, however, play distinct cellular roles, since they are directed to different chromatin regions by specificity factors. Sir2p, which is found in two separate multiprotein complexes, is critical for transcriptional silencing of large chromosomal domains at three loci: telomeres, the silent mating-type loci (HMR and HML), and the ribosomal RNA-encoding DNA (reviewed in Ref. 22). Hst1p-mediated repression, in contrast, is restricted to specific genes through the DNA-binding protein Sum1p and the tethering factor Rfm1p (23, 24). Hst1p has previously been known to participate in repression of middle sporulation genes during mitotic growth (23) but has recently been shown to serve as a sensor and a regulator of cellular NAD ϩ levels through controlling the expression of genes involved in de novo NAD ϩ biosynthesis and the import of nicotinic acid (25). Little is known about cellular functions of other NAD ϩ -dependent deacetylases in yeast. Hst2p is a cytoplasmic enzyme that accounts for the majorit...