Structure of the yeast Hst2 protein deacetylase in ternary complex with 2'-O-acetyl ADP ribose and histone peptide

Sir2 proteins are NAD(+)-dependant protein deactylases that have been implicated in playing roles in gene silencing, DNA repair, genome stability, longevity, metabolism, and cell physiology. To define the mechanism of Sir2 activity, we report the 1.5 A crystal structure of the yeast Hst2 (yHst2) Sir2 protein in ternary complex with 2'-O-acetyl ADP ribose and an acetylated histone H4 peptide. The structure captures both ligands meeting within an enclosed tunnel between the small and large domains of the catalytic protein core and permits the assignment of a detailed catalytic mechanism for the Sir2 proteins that is consistent with solution and enzymatic studies. Comparison of the ternary complex with the yHst2/NAD(+) complex, also reported here, and nascent yHst2 structure also reveals that NAD(+) binding accompanies intramolecular loop rearrangement for more stable NAD(+) and acetyl-lysine binding, and that acetyl-lysine peptide binding induces a trimer-monomer protein transition involving nonconserved Sir2 residues.     

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

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(+).     

Coenzyme specificity of Sir2 protein deacetylases: implications for physiological regulation

Sir2 (silent information regulator 2) enzymes catalyze a unique protein deacetylation reaction that requires the coenzyme NAD(+) and produces nicotinamide and a newly discovered metabolite, O-acetyl-ADP-ribose (OAADPr). Conserved from bacteria to humans, these proteins are implicated in the control of gene silencing, metabolism, apoptosis, and aging. Here we examine the role of NAD(+) metabolites/derivatives and salvage pathway intermediates as activators, inhibitors, or coenzyme substrates of Sir2 enzymes in vitro. Also, we probe the coenzyme binding site using inhibitor binding studies and alternative coenzyme derivatives as substrates. Sir2 enzymes showed an exquisite selectivity for the nicotinamide base coenzyme, with the most dramatic losses in binding affinity/reactivity resulting from relatively minor changes in the nicotinamide ring, either by reduction, as in NADH, or by converting the amide to its acid analogue. Both ends of the dinucleotide NAD(+) are shown to be critical for high selectivity and high affinity. Among the NAD(+) metabolites tested none were able to allosterically activate, although all led to various extents of inhibition, consistent with competition at the coenzyme binding site. Nicotinamide was the most potent inhibitor examined, suggesting that cellular nicotinamide levels would provide an effective small molecule regulator of protein deacetylation and generation of OAADPr. The presented findings also suggest that changes in the physiological NAD(+):NADH ratio, without a change in NAD(+), would yield little alteration in Sir2 activity. That is, NADH is an extremely ineffective inhibitor of Sir2 enzymes (average IC(50) of 17 mm). We propose that changes in both free nicotinamide and free NAD(+) afford the greatest contribution to cellular activity of Sir2 enzymes but with nicotinamide having a more dramatic effect during smaller fluctuations in concentration.     

A role for the Saccharomyces cerevisiae RENT complex protein Net1 in HMR silencing

Silencing in the yeast Saccharomyces cerevisiae is known in three classes of loci: in the silent mating-type loci HML and HMR, in subtelomeric regions, and in the highly repetitive rDNA locus, which resides in the nucleolus. rDNA silencing differs markedly from the other two classes of silencing in that it requires a DNA-associated protein complex termed RENT. The Net1 protein, a central component of RENT, is required for nucleolar integrity and the control of exit from mitosis. Another RENT component is the NAD(+)-dependent histone deacetylase Sir2, which is the only silencing factor known to be shared among the three classes of silencing. Here, we investigated the role of Net1 in HMR silencing. The mutation net1-1, as well as NET1 expression from a 2micro-plasmid, restored repression at silencing-defective HMR loci. Both effects were strictly dependent on the Sir proteins. We found overexpressed Net1 protein to be directly associated with the HMR-E silencer, suggesting that Net1 could interact with silencer binding proteins and recruit other silencing factors to the silencer. In agreement with this, Net1 provided ORC-dependent, Sir1-independent silencing when artificially tethered to the silencer. In contrast, our data suggested that net1-1 acted indirectly in HMR silencing by releasing Sir2 from the nucleolus, thus shifting the internal competition for Sir2 from the silenced loci toward HMR.     

Identification of a novel allele of SIR3 defective in the maintenance, but not the establishment, of silencing in Saccharomyces cerevisiae

Using a screen for genes that affect telomere function, we isolated sir3-P898R, an allele of SIR3 that reduces telomeric silencing yet does not affect mating. While sir3-P898R mutations cause no detectable mating defect in quantitative assays, they result in synergistic mating defects in combination with mutations such as sir1 that affect the establishment of silencing. In contrast, sir3-P898R in combination with a cac1 mutation, which affects the maintenance of silencing, does not result in synergistic mating defects. MATa sir3-P898R mutants form shmoo clusters in response to alpha-factor, and sir3-P898R strains are capable of establishing silencing at a previously derepressed HML locus with kinetics like that of wild-type SIR3 strains. These results imply that Sir3-P898Rp is defective in the maintenance, but not the establishment of silencing. In addition, overexpression of a C-terminal fragment of Sir3-P898R results in a dominant nonmating phenotype: HM silencing is completely lost at both HML and HMR. Furthermore, HM silencing is most vulnerable to disruption by the Sir3-P898R C terminus immediately after S-phase, the time when new silent chromatin is assembled onto newly replicated DNA.     

Sir2 deacetylates histone H3 lysine 56 to regulate telomeric heterochromatin structure in yeast

At telomeric heterochromatin in yeast, the Sir protein complex spreads from Rap1 sites to silence adjacent genes. This cascade is believed to occur when Sir2, an NAD(+)-dependent enzyme, deacetylates histone H3 and H4 N termini, in particular histone H4 K16, enabling more Sir protein binding. Lysine 56 of histone H3 is located at the entry-exit points of the DNA superhelix surrounding the nucleosome, where it may control DNA compaction. We have found that K56 substitutions disrupt silencing severely without decreasing Sir protein binding at the telomere. Our in vitro and in vivo data indicate that Sir2 deacetylates K56 directly in telomeric heterochromatin to compact chromatin and prevent access to RNA polymerase and ectopic bacterial dam methylase. Since the spread of Sir proteins is necessary but not sufficient for silencing, we propose that silencing occurs when Sir2 deacetylates H3 K56 to close the nucleosomal entry-exit gates, enabling compaction of heterochromatin.     

The artificial evolution of an enzyme by random mutagenesis: the development of formaldehyde dehydrogenase

A unique variant of glutathione independent formaldehyde dehydrogenase of Pseudomonas putida was obtained by random mutagenesis using the PCR-reaction. This YM042 mutant, S318G, was a cold-adapted formaldehyde dehyrogenase. The activity at 29 degrees C of the variant was 1.7-fold higher than that of the wild type. The K(m) values of the mutant at 37 degrees C were 0.40 mM for NAD(+) and 2.5 mM for formaldehyde, while those of the wild-type were 0.18 mM for NAD(+) and 2.1 mM for formaldehyde. The catalytic efficiency for formaldehyde was about 1.5-fold greater in the mutant than in the wild-type enzyme. The optimum pHs and temperatures of the mutant and the wild-type enzyme were 7.5, and 8.0 and 37 degrees C, and 47 degrees C, respectively. The thermal stability of the mutant was lower than that of the wild type.     

Manipulation of a nuclear NAD+ salvage pathway delays aging without altering steady-state NAD+ levels

Yeast deprived of nutrients exhibit a marked life span extension that requires the activity of the NAD(+)-dependent histone deacetylase, Sir2p. Here we show that increased dosage of NPT1, encoding a nicotinate phosphoribosyltransferase critical for the NAD(+) salvage pathway, increases Sir2-dependent silencing, stabilizes the rDNA locus, and extends yeast replicative life span by up to 60%. Both NPT1 and SIR2 provide resistance against heat shock, demonstrating that these genes act in a more general manner to promote cell survival. We show that Npt1 and a previously uncharacterized salvage pathway enzyme, Nma2, are both concentrated in the nucleus, indicating that a significant amount of NAD(+) is regenerated in this organelle. Additional copies of the salvage pathway genes, PNC1, NMA1, and NMA2, increase telomeric and rDNA silencing, implying that multiple steps affect the rate of the pathway. Although SIR2-dependent processes are enhanced by additional NPT1, steady-state NAD(+) levels and NAD(+)/NADH ratios remain unaltered. This finding suggests that yeast life span extension may be facilitated by an increase in the availability of NAD(+) to Sir2, although not through a simple increase in steady-state levels. We propose a model in which increased flux through the NAD(+) salvage pathway is responsible for the Sir2-dependent extension of life span.     

Sir2 regulates histone H3 lysine 9 methylation and heterochromatin assembly in fission yeast

Hypoacetylated histones are a hallmark of heterochromatin in organisms ranging from yeast to humans. Histone deacetylation is carried out by both NAD(+)-dependent and NAD(+)-independent enzymes. In the budding yeast Saccharomyces cerevisiae, deacetylation of histones in heterochromatic chromosomal domains requires Sir2, a phylogenetically conserved NAD(+)-dependent deacetylase. In the fission yeast Schizosaccharomyces pombe, NAD(+)-independent histone deacetylases are required for the formation of heterochromatin, but the role of Sir2-like deacetylases in this process has not been evaluated. Here, we show that spSir2, the S. pombe Sir2-like protein that is the most closely related to the S. cerevisiae Sir2, is an NAD(+)-dependent deacetylase that efficiently deacetylates histone H3 lysine 9 (K9) and histone H4 lysine 16 (K16) in vitro. In sir2 Delta cells, silencing at the donor mating-type loci, telomeres, and the inner centromeric repeats (imr) is abolished, while silencing at the outer centromeric repeats (otr) and rDNA is weakly reduced. Furthermore, Sir2 is required for hypoacetylation and methylation of H3-K9 and for the association of Swi6 with the above loci in vivo. Our findings suggest that the NAD(+)-dependent deacetylase Sir2 plays an important and conserved role in heterochromatin assembly in eukaryotes.     

γH2A is a component of yeast heterochromatin required for telomere elongation

Histones of heterochromatin are deacetylated in yeast and methylated in more complex eukaryotes to regulate heterochromatin structure and gene silencing. Here, we report that histone H2A phosphorylated at serine 129 (γH2A) in Saccharomyces cerevisiae is a conceptually new type of heterochromatin modification that functions downstream of silent chromatin assembly. We show that γH2A is enriched throughout yeast telomeric and silent mating locus (HM) heterochromatin where γH2A results from the action of kinases Tel1 and Mec1. Interestingly, mutation of γH2A has no apparent effect on the binding of Sir (silent information regulator) complex or on gene silencing. In contrast, deletion of SIR3 abolishes the formation of γH2A at heterochromatin. To address the function of γH2A, we used a Δrif1 mutant strain in which telomeres are excessively elongated to show that γH2A is required for the optimal recruitment of Cdc13, a regulator of telomere elongation, and for telomere elongation itself. Thus, a histone modification that parallels Sir3 protein binding is shown here to be dispensable for the formation of a silent structure but is important for a crucial heterochromatin-specific downstream function in telomere homeostasis.