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Genome-wide Analysis of Functional Sirtuin Chromatin Targets in Yeast

ABSTRACT: Sir2 and the homologous proteins, Hst1, Hst2, Hst3, and Hst4 from Saccharomyces cerevisiae are NAD+-dependent histone deacetylases of the sirtuin protein family. Sir2 functions in transcriptional silencing at the silent mating-type loci, telomeres, and rDNA locus, but also promotes replicative lifespan. To gain a better understanding of the chromatin-regulatory roles carried out by Sir2 and the Hst proteins, we performed ChIP-sequencing analysis on all five sirtuins and Sum1, the DNA binding partner for Hst1. Sir2, Hst1, and Sum1 were abundantly, and functionally co-enriched at several major targets, including the telomeric repeats, where they were required for maintaining proper telomere repeat length. At tRNA target genes they were required for efficient cohesin and condensin deposition. Across the open reading frames of glycolytic and ribosomal protein genes, Sir2 and Hst1 functioned in NAD+-dependent transcriptional repression at the diauxic shift, directly linking Sir2 to glucose metabolism, which could have implications for longevity.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE41415 | GEO | 2013/05/10

SECONDARY ACCESSION(S): PRJNA177035

REPOSITORIES: GEO

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Genome-wide Analysis of Functional Sirtuin Chromatin Targets in Yeast

Project description:Sir2 and the homologous proteins, Hst1, Hst2, Hst3, and Hst4 from Saccharomyces cerevisiae are NAD+-dependent histone deacetylases of the sirtuin protein family. Sir2 functions in transcriptional silencing at the silent mating-type loci, telomeres, and rDNA locus, but also promotes replicative lifespan. To gain a better understanding of the chromatin-regulatory roles carried out by Sir2 and the Hst proteins, we performed ChIP-sequencing analysis on all five sirtuins and Sum1, the DNA binding partner for Hst1. Sir2, Hst1, and Sum1 were abundantly, and functionally co-enriched at several major targets, including the telomeric repeats, where they were required for maintaining proper telomere repeat length. At tRNA target genes they were required for efficient cohesin and condensin deposition. Across the open reading frames of glycolytic and ribosomal protein genes, Sir2 and Hst1 functioned in NAD+-dependent transcriptional repression at the diauxic shift, directly linking Sir2 to glucose metabolism, which could have implications for longevity. Six factors and Input ChIP-seq samples were analyzed in Saccharomyces eerevisiae.

2013-05-10 | E-GEOD-41415 | biostudies-arrayexpress

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Yeast expression data from conditions that inhibit sirtuins

Project description:Sir2 is an NAD+-dependent histone deacetylase, and is the founding member of a large, phylogentically conserved, family of such deacetylases called the Sirtuins. The budding yeast, Saccharomyces cerevisiae, harbors 4 paralogs of Sir2, known as Hst1, Hst2, Hst3, and Hst4. Reducing the intracellular NAD+ concentration is inhibitory for the Sirtuins, and raising the intracellular nicotinamide (NAM) concentration is inhibitory. Microarray gene expression analysis was used to identify novel classes of yeast genes whose expression is altered when either NAD+ concentration is reduced or NAM is elevated. A subset of genes involved in thiamine biosynthesis was identified as being upregulated when Sir2 or Hst1 was inactivated. Several mutants in the NAD+ biosynthesis and salvage pathways were tested, along with WT and WT grown in the presence of 5 mM nicotinamide, which inhibits the Sirtuins. Duplicate cultures in rich YPD media were grown into log phase and then harvested. The deletion mutants npt1M-bM-^HM-^F, bna1M-bM-^HM-^F, tna1M-bM-^HM-^F, and pnc1M-bM-^HM-^F were compared to a wild-type (WT) strain. Additionally, a WT strain grown in the presence of 5 mM NAM was compared to WT without NAM. Total RNA was isolated and used for microarray hybridizations onto Affymetrix Yeast 2.0 Gene arrrays.

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Project description:The hst3hst4 strain (FY background) has the HST3 and HST4 genes, encoding putative NAD-dependent deacetylases that regulate histone 3 K56 acetylation, deleted. Expression profiling using Affymetrix microarrays was used to assess the change in the gene expression in this strain in comparison to wild-type under normal growth conditions. Experiment Overall Design: Total RNA was extracted from two independent cultures of the hst3hst4 strain and the wt strain, and was hybridized to microarrays. Gene expression levels of each mutant culture was compared to each wt culture.

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