hSIR2(SIRT1) functions as an NAD-dependent p53 deacetylase. (41/361)

DNA damage-induced acetylation of p53 protein leads to its activation and either growth arrest or apoptosis. We show here that the protein product of the gene hSIR2(SIRT1), the human homolog of the S. cerevisiae Sir2 protein known to be involved in cell aging and in the response to DNA damage, binds and deacetylates the p53 protein with a specificity for its C-terminal Lys382 residue, modification of which has been implicated in the activation of p53 as a transcription factor. Expression of wild-type hSir2 in human cells reduces the transcriptional activity of p53. In contrast, expression of a catalytically inactive hSir2 protein potentiates p53-dependent apoptosis and radiosensitivity. We propose that hSir2 is involved in the regulation of p53 function via deacetylation.  (+info)

The S. cerevisiae SET3 complex includes two histone deacetylases, Hos2 and Hst1, and is a meiotic-specific repressor of the sporulation gene program. (42/361)

Set3 is one of two proteins in the yeast Saccharomyces cerevisiae that, like Drosophila Trithorax, contains both SET and PHD domains. We found that Set3 forms a single complex, Set3C, with Snt1, YIL112w, Sif2, Cpr1, and two putative histone deacetylases, Hos2 and NAD-dependent Hst1. Set3C includes NAD-dependent and independent deacetylase activities when assayed in vitro. Homology searches suggest that Set3C is the yeast analog of the mammalian HDAC3/SMRT complex. Set3C represses genes in early/middle of the yeast sporulation program, including the key meiotic regulators ime2 and ndt80. Whereas Hos2 is only found in Set3C, Hst1 is also present in a complex with Sum1, supporting previous characterizations of Hst1 and Sum1 as repressors of middle sporulation genes during vegetative growth. However, Hst1 is not required for meiotic repression by Set3C, thus implying that Set3C (-Hst1) and not Hst1-Sum1, is the meiotic-specific repressor of early/middle sporulation genes.  (+info)

Identification of a small molecule inhibitor of Sir2p. (43/361)

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. In vitro, splitomicin inhibits NAD(+)-dependent histone deacetylase activity (HDA) of the Sir2 protein. Mutations in SIR2 that confer resistance to the drug map to the likely acetylated histone tail binding domain of the protein. By using splitomicin as a chemical genetic probe, we demonstrate that continuous HDA of Sir2p is required for maintaining a silenced state in nondividing cells.  (+info)

hpr1Delta affects ribosomal DNA recombination and cell life span in Saccharomyces cerevisiae. (44/361)

Multiple genetic pathways have been shown to regulate life span and aging in the yeast Saccharomyces cerevisiae. Here we show that loss of a component of the RNA polymerase II complex, Hpr1p, results in a decreased life span. Although hpr1Delta mutants have an increased rate of recombination within the ribosomal DNA (rDNA) array, this is not accompanied by an increase in extrachromosomal rDNA circles (ERCs). Analyses of mutants that affect replication of the rDNA array and suppressors that reverse the phenotypes of the hpr1Delta mutant show that the reduced life span is associated with increased genomic instability but not with increased ERC formation. The hpr1Delta mutant acts in a pathway distinct from previously described mutants that reduce life span.  (+info)

Conserved enzymatic production and biological effect of O-acetyl-ADP-ribose by silent information regulator 2-like NAD+-dependent deacetylases. (45/361)

Silent information regulator 2 (Sir2) family of enzymes has been implicated in many cellular processes that include histone deacetylation, gene silencing, chromosomal stability, and aging. Yeast Sir2 and several homologues have been shown to be NAD(+)-dependent histone/protein deacetylases. Previously, it was demonstrated that the yeast enzymes catalyze a unique reaction mechanism in which the cleavage of NAD(+) and the deacetylation of substrate are coupled with the formation of O-acetyl-ADP-ribose, a novel metabolite. We demonstrate that the production of O-acetyl-ADP-ribose is evolutionarily conserved among Sir2-like enzymes from yeast, Drosophila, and human. Also, endogenous yeast Sir2 complex from telomeres was shown to generate O-acetyl-ADP-ribose. By using a quantitative microinjection assay to examine the possible biological function(s) of this newly discovered metabolite, we demonstrate that O-acetyl-ADP-ribose causes a delay/block in oocyte maturation and results in a delay/block in embryo cell division in blastomeres. This effect was mimicked by injection of low nanomolar levels of active enzyme but not with a catalytically impaired mutant, indicating that the enzymatic activity is essential for the observed effects. In cell-free oocyte extracts, we demonstrate the existence of cellular enzymes that can efficiently utilize O-acetyl-ADP-ribose.  (+info)

Evidence that Set1, a factor required for methylation of histone H3, regulates rDNA silencing in S. cerevisiae by a Sir2-independent mechanism. (46/361)

Several types of histone modifications have been shown to control transcription. Recent evidence suggests that specific combinations of these modifications determine particular transcription patterns. The histone modifications most recently shown to play critical roles in transcription are arginine-specific and lysine-specific methylation. Lysine-specific histone methyltransferases all contain a SET domain, a conserved 130 amino acid motif originally identified in polycomb- and trithorax-group proteins from Drosophila. Members of the SU(VAR)3-9 family of SET-domain proteins methylate K9 of histone H3. Methylation of H3 has also been shown to occur at K4. Several studies have suggested a correlation between K4-methylated H3 and active transcription. In this paper, we provide evidence that K4-methylated H3 is required in a negative role, rDNA silencing in Saccharomyces cerevisiae. In a screen for rDNA silencing mutants, we identified a mutation in SET1, previously shown to regulate silencing at telomeres and HML. Recent work has shown that Set1 is a member of a complex and is required for methylation of K4 of H3 at several genomic locations. In addition, we demonstrate that a K4R change in H3, which prevents K4 methylation, impairs rDNA silencing, indicating that Set1 regulates rDNA silencing, directly or indirectly, via H3 methylation. Furthermore, we present several lines of evidence that the role of Set1 in rDNA silencing is distinct from that of the histone deacetylase Sir2. Together, these results suggest that Set1-dependent H3 methylation is required for rDNA silencing in a Sir2-independent fashion.  (+info)

Structural identification of 2'- and 3'-O-acetyl-ADP-ribose as novel metabolites derived from the Sir2 family of beta -NAD+-dependent histone/protein deacetylases. (47/361)

The Sir2 (silent information regulator 2) family of histone/protein deacetylases has been implicated in a wide range of biological activities, including gene silencing, life-span extension, and chromosomal stability. Their dependence on beta-NAD(+) for activity is unique among the known classes of histone/protein deacetylase. Sir2 enzymes have been shown to couple substrate deacetylation and beta-NAD(+) cleavage to the formation of O-acetyl-ADP-ribose, a newly described metabolite. To gain a better understanding of the catalytic mechanism and of the biological implications of producing this molecule, we have performed a detailed enzymatic and structural characterization of O-acetyl-ADP-ribose. Through the use of mass spectrometry, rapid quenching techniques, and NMR structural analyses, 2'-O-acetyl-ADP-ribose and 3'-O-acetyl-ADP-ribose were found to be the solution products produced by the Sir2 family of enzymes. Rapid quenching approaches under single-turnover conditions identified 2'-O-acetyl-ADP-ribose as the enzymatic product, whereas 3'-O-acetyl-ADP-ribose was formed by intramolecular transesterification after enzymatic release into bulk solvent, where 2'- and 3'-O-acetyl-ADP-ribose exist in equilibrium (48:52). In addition to (1)H and (13)C chemical shift assignments for each regioisomer, heteronuclear multiple-bond correlation spectroscopy was used to assign unambiguously the position of the acetyl group. These findings are highly significant, because they differ from the previous conclusion, which suggested that 1'-O-acetyl-ADP-ribose was the solution product of the reaction. Possible mechanisms for the generation of 2'-O-acetyl-ADP-ribose are discussed.  (+info)

Telomeric and rDNA silencing in Saccharomyces cerevisiae are dependent on a nuclear NAD(+) salvage pathway. (48/361)

The Sir2 protein is an NAD(+)-dependent protein deacetylase that is required for silencing at the silent mating-type loci, telomeres, and the ribosomal DNA (rDNA). Mutations in the NAD(+) salvage gene NPT1 weaken all three forms of silencing and also cause a reduction in the intracellular NAD(+) level. We now show that mutation of a highly conserved histidine residue in Npt1p results in a silencing defect, indicating that Npt1p enzymatic activity is required for silencing. Deletion of another NAD(+) salvage pathway gene called PNC1 caused a less severe silencing defect and did not significantly reduce the intracellular NAD(+) concentration. However, silencing in the absence of PNC1 was completely dependent on the import of nicotinic acid from the growth medium. Deletion of a gene in the de novo NAD(+) synthesis pathway BNA1 resulted in a significant rDNA silencing defect only on medium deficient in nicotinic acid, an NAD(+) precursor. By immunofluorescence microscopy, Myc-tagged Bna1p was localized throughout the whole cell in an asynchronously growing population. In contrast, Myc-tagged Npt1p was highly concentrated in the nucleus in approximately 40% of the cells, indicating that NAD(+) salvage occurs in the nucleus in a significant fraction of cells. We propose a model in which two components of the NAD(+) salvage pathway, Pnc1p and Npt1p, function together in recycling the nuclear nicotinamide generated by Sir2p deacetylase activity back into NAD(+).  (+info)