RESUMO
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.
Assuntos
Mapeamento Cromossômico/métodos , Proteínas Fúngicas/genética , Genes Fúngicos , Saccharomyces cerevisiae/genética , Telômero/ultraestrutura , Cromossomos Fúngicos , Deleção de Genes , Genoma Fúngico , Polimorfismo Genético , Locos de Características Quantitativas , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/genética , Troca de Cromátide IrmãRESUMO
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.
Assuntos
Inibidores Enzimáticos/farmacologia , Inibidores de Histona Desacetilases , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/antagonistas & inibidores , Sirtuínas/antagonistas & inibidores , Sequência de Aminoácidos , Northern Blotting , Relação Dose-Resposta a Droga , Farmacorresistência Fúngica , Genes Fúngicos , Genes Reporter , Genoma Fúngico , Histona Desacetilases/química , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , NAD/metabolismo , Naftalenos/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Plasmídeos/metabolismo , Pironas/farmacologia , Proteínas de Saccharomyces cerevisiae/química , Homologia de Sequência de Aminoácidos , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/química , Sirtuína 2 , Sirtuínas/química , Telômero/ultraestrutura , beta-Galactosidase/metabolismoRESUMO
Nicotine adenine dinucleotide (NAD(+)) performs key roles in electron transport reactions, as a substrate for poly(ADP-ribose) polymerase and NAD(+)-dependent protein deacetylases. In the latter two processes, NAD(+) is consumed and converted to ADP-ribose and nicotinamide. NAD(+) levels can be maintained by regeneration of NAD(+) from nicotinamide via a salvage pathway or by de novo synthesis of NAD(+) from tryptophan. Both pathways are conserved from yeast to humans. We describe a critical role of the NAD(+)-dependent deacetylase Hst1p as a sensor of NAD(+) levels and regulator of NAD(+) biosynthesis. Using transcript arrays, we show that low NAD(+) states specifically induce the de novo NAD(+) biosynthesis genes while the genes in the salvage pathway remain unaffected. The NAD(+)-dependent deacetylase activity of Hst1p represses de novo NAD(+) biosynthesis genes in the absence of new protein synthesis, suggesting a direct effect. The known Hst1p binding partner, Sum1p, is present at promoters of highly inducible NAD(+) biosynthesis genes. The removal of HST1-mediated repression of the NAD(+) de novo biosynthesis pathway leads to increased cellular NAD(+) levels. Transcript array analysis shows that reduction in cellular NAD(+) levels preferentially affects Hst1p-regulated genes in comparison to genes regulated with other NAD(+)-dependent deacetylases (Sir2p, Hst2p, Hst3p, and Hst4p). In vitro experiments demonstrate that Hst1p has relatively low affinity toward NAD(+) in comparison to other NAD(+)-dependent enzymes. These findings suggest that Hst1p serves as a cellular NAD(+) sensor that monitors and regulates cellular NAD(+) levels.
