A phylogenetically conserved NAD ⁺ -dependent protein deacetylase activity in the Sir2 protein family

Abstract

The yeast Sir2 protein, required for transcriptional silencing, has an NAD ؉ -dependent histone deacetylase (HDA) activity. Yeast extracts contain a NAD ؉ -dependent HDA activity that is eliminated in a yeast strain from which SIR2 and its four homologs have been deleted. This HDA activity is also displayed by purified yeast Sir2p and homologous Archaeal, eubacterial, and human proteins, and depends completely on NAD ؉ in all species tested. The yeast NPT1 gene, encoding an important NAD ؉ synthesis enzyme, is required for rDNA and telomeric silencing and contributes to silencing of the HM loci. Null mutants in this gene have significantly reduced intracellular NAD ؉ concentrations and have phenotypes similar to sir2 null mutants. Surprisingly, yeast from which all five SIR2 homologs have been deleted have relatively normal bulk histone acetylation levels. The evolutionary conservation of this regulated activity suggests that the Sir2 protein family represents a set of effector proteins in an evolutionarily conserved signal transduction pathway that monitors cellular energy and redox states. T ranscriptional silencing is a regulatory mechanism that results in the inactivation of large blocks of chromosomes via an altered chromatin structure. In Saccharomyces cerevisiae, silencing is observed at the HM silent mating type loci (reviewed in ref. 1), telomeres (2), and at the rDNA locus (3, 4). Although a different subset of proteins is required for silencing at each of the three loci, all types of silencing require Sir2p (3, 5). The Sir2 family of proteins is highly conserved and found in Archaea, eubacteria, and metazoa (6-9). A recent study showed that yeast and mouse Sir2p have NAD ϩ -dependent HDA activity on histone peptides specific for Lys-16 of histone H4 (10), an important residue for silencing (11-13). Earlier work had suggested that Sir2p might have HDA activity. Acetylated histones were inefficiently immunoprecipitated from the silent mating type (HM) loci relative to the expressed mating type (MAT) locus, and overexpression of Sir2p led to changes in levels of bulk histone acetylation (14,15). Other recent papers demonstrated a phosphotransferase activity for Sir2p, with NAD ϩ as the source of phosphate and a variety of proteins implicated as targets of ADP ribosylation (9, 16). A sir2 missense mutation that destroys this in vitro activity also destroys silencing in vivo. These results suggest that the Sir2p family is a group of ADP-ribosyl transferases (ARTs).We show here that Archaeal, eubacterial, and human Sir2 proteins, like Sir2p, have potent NAD ϩ -dependent HDA activity in vitro. The importance of NAD ϩ to the in vivo activity of Sir2p is underscored by our finding that mutations in the S. cerevisiae NPT1 gene lead to severe silencing defects. NPT1 encodes a nicotinate phosphoribosyltransferase, required for NAD ϩ synthesis through a salvage pathway. Intracellular NAD ϩ levels are significantly lower in npt1 null mutants than in the wild type, providing independent evidence that NAD ϩ is critic...

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