Abstract
The Saccharomyces cerevisiae Sir2 protein is an NAD(+)-dependent histone deacetylase that plays a critical role in transcriptional silencing, genome stability, and longevity. A human homologue of Sir2, SIRT1, regulates the activity of the p53 tumor suppressor and inhibits apoptosis. The Sir2 deacetylation reaction generates two products: O-acetyl-ADP-ribose and nicotinamide, a precursor of nicotinic acid and a form of niacin/vitamin B(3). We show here that nicotinamide strongly inhibits yeast silencing, increases rDNA recombination, and shortens replicative life span to that of a sir2 mutant. Nicotinamide abolishes silencing and leads to an eventual delocalization of Sir2 even in G(1)-arrested cells, demonstrating that silent heterochromatin requires continual Sir2 activity. We show that physiological concentrations of nicotinamide noncompetitively inhibit both Sir2 and SIRT1 in vitro. The degree of inhibition by nicotinamide (IC(50) < 50 microm) is equal to or better than the most effective known synthetic inhibitors of this class of proteins. We propose a model whereby nicotinamide inhibits deacetylation by binding to a conserved pocket adjacent to NAD(+), thereby blocking NAD(+) hydrolysis. We discuss the possibility that nicotinamide is a physiologically relevant regulator of Sir2 enzymes.
Highlights
Transcriptional silencing involves the heritable modification of chromatin at distinct sites in the genome
Nicotinamide Abolishes Silencing at the ribosomal DNA (rDNA), Telomeres, and Mating-type Loci—To examine whether nicotinamide could affect Sir2 activity in vivo, we examined strains with either ADE2 or MET15 integrated at the rDNA locus (RDN1)
Measurement of DNA content by fluorescenceactivated cell sorting (FACS) confirmed that the cells remained in G1 for the duration of the experiment (Fig. 4C, right column). These results demonstrate that exogenous nicotinamide derepresses silent chromatin even in nondividing cells and suggests that heterochromatin is an unstable structure in the absence of Sir2 deacetylation
Summary
W303 MATa, ade, leu 112, can100, trp, ura, his , RDN1ϻADE2, RAD5 W303 MATa, ade, leu 112, can100, trp, ura, his , RDN1ϻADE2, RAD5, sir2:TRP1 W303 MATa, ade, leu 112, can100, trp, ura, his , RDN1ϻADE2, RAD5, LEU2/SIR2 W303 MATa, ade, leu 112, can100, trp, ura, his , RAD5 W303 MATa, ADE2, leu 112, can100, trp, ura, his , RAD5 W303 MATa, ade, leu 112, can100, trp, ura, his , RDN, RAD5, GFP-Sir4ϻURA3 W303 MATa, ade, leu 112, can100, trp, ura, his , RDN, RAD5, GFP-Sir3ϻLEU2 W303 MATa, ade, leu 112, can100, trp, ura, his , RDN, RAD5, GFP-Sir3ϻLEU2, sir2:TRP1 W303 MAT␣, ade, leu 112, can100, trp, ura, his , MAT␣ hmrWTϻTRP1, HMRϻURA3ϻADE2 W303 MATa, ade, leu 112, can100, trp, ura, his , RDN1ϻADE2, RAD5, SIR2-HAϻURA3 W303 MATa, ade, leu 112, can100, trp, ura, his , RDN1ϻURA3, sir2ϻTRP W303 MATa, ade, leu 112, can100, trp, ura, his , RDN1ϻADE2, RAD5, Sir2-GFPϻLEU2 W303 MATa, ade, leu 112, can100, trp, ura, his , RDN1ϻADE2, RAD5, Sir2-GFP, leu2ϻURA3, ⌬hmlϻLEU2 MAT␣, ura leu 112 his3-⌬200 ade can100 ADE2-TEL V-R PSY316 MAT␣, ura leu 112 his3-⌬200 ade can100 ADE2-TEL V-R, sir2:TRP1 PSY316 MAT␣, ura leu 112 his3-⌬200 ade can100 ADE2-TEL V-R, HMRϻGFP PSY316 MAT␣, ura leu 112 his3-⌬200 ade can100 ADE2-TEL V-R, HMRϻGFP PSY316 MAT␣, ura leu 112 his3-⌬200 ade can100 ADE2-TEL V-R, HMRϻGFP, sir4:HIS3 PSY316 MAT␣, ura leu 112 his3-⌬200 ade can100 ADE2-TEL V-R, HMRϻGFP, ⌬hmlϻLEU2 PSY316AT, MAT␣, ura leu 112 his3-⌬200 ade can100 ADE2-TEL V-R RDN1ϻURA3 PSY316AT, MAT␣, ura leu 112 his3-⌬200 ade can100 ADE2-TEL V-R, pSP400-URA3 MAT␣, his3⌬200, leu2⌬1, met15⌬200, trp1⌬63, ura167 JS209 MAT␣, his3⌬200, leu2⌬1, met15⌬200, trp1⌬63, ura167, Ty1-MET15 JS209 MAT␣, his3⌬200, leu2⌬1, met15⌬200, trp1⌬63, ura167, RDN1ϻTy1-MET15 JS237 MAT␣, his3⌬200, leu2⌬1, met15⌬200, trp1⌬63, ura167, RDN1ϻTyl-MET15, sir2ϻHIS3 JS237 MAT␣, his3⌬200, leu2⌬1, met15⌬200, trp1⌬63, ura167, RDN1ϻTy1-MET15, LEU2/SIR2. Nicotinamide increases recombination at the rDNA locus and shortens yeast life span to that of a sir mutant. We use this inhibitor to show that maintenance of silenced chromatin and the localization of Sir2/3/4 to telomeres require the continual activity of Sir, even in nondividing cells. Physiological concentrations of nicotinamide inhibit Sir and human SIRT1 noncompetitively in vitro, raising the possibility that nuclear nicotinamide negatively regulates Sir activity in vivo. Our findings suggest that the medicinal use of nicotinamide should be given careful consideration
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