Inhibition of Silencing and Accelerated Aging by Nicotinamide, a Putative Negative Regulator of Yeast Sir2 and Human SIRT1

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 B3. We show here that nicotinamide strongly inhibits yeast silencing, increases rDNA recombination, and shortens replicative life span to that of asir2 mutant. Nicotinamide abolishes silencing and leads to an eventual delocalization of Sir2 even in G1-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 (IC50< 50 μm) 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. 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 B3. We show here that nicotinamide strongly inhibits yeast silencing, increases rDNA recombination, and shortens replicative life span to that of asir2 mutant. Nicotinamide abolishes silencing and leads to an eventual delocalization of Sir2 even in G1-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 (IC50< 50 μm) 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. Transcriptional silencing involves the heritable modification of chromatin at distinct sites in the genome. Silencing is referred to as long range repression as it is promoter nonspecific and often encompasses an entire genomic locus (1Courey A.J. Jia S. Genes Dev. 2001; 15: 2786-2796Google Scholar, 2Moazed D. Mol. Cell. 2001; 8: 489-498Google Scholar). In yeast these silent regions, which are similar to the heterochromatin of higher eukaryotes, are subject to a wide variety of modifications (3Gasser M. Cockell M.M. Gene (Amst.). 2001; 279: 1-16Google Scholar). Among the best studied of these modifications is the reversible acetylation of histones (reviewed by Refs. 4Eberharter A. Becker P.B. EMBO Repts. 2002; 3: 224-229Google Scholar and 5Kuo M.H. Allis C.D. Bioessays. 1998; 20: 615-626Google Scholar). There are two types of enzymes that affect the acetylation state of histones: histone acetyltransferases and the opposing histone deacetylases (HDACs). 1The abbreviations used are: HDAC, histone deacetylase; TSA, trichostatin A; SC, synthetic medium; 5-FOA, 5-fluoroorotic acid; FACS, fluorescence-activated cell sorter; HA, hemagglutinin; GFP, green fluorescent protein.Compared with more transcriptionally active areas of the genome, histones within silent regions of chromatin are known to be hypoacetylated, specifically on the NH2-terminal tails of core histones H3 and H4 (6Bernstein B.E. Tong J.K. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13708-13713Google Scholar). Three classes of histone deacetylases have been described and classified based on homology to yeast proteins. Proteins in class I (Rpd3-like) and class II (Hda1-like) are characterized by their sensitivity to the inhibitor trichostatin A (TSA) (7Fischle W. Kiermer V. Dequiedt F. Verdin E. Biochem. Cell Biol. 2001; 79 (K.): 337-348WGoogle Scholar, 8Marks P. Rifkind R.A. Breslow V.M. Miller R. Kelly T. Kelly W.K. Nat. Rev. Cancer. 2001; 1: 194-202Google Scholar). Studies using this inhibitor have provided a wealth of information regarding the biochemistry and cellular function of these proteins (reviewed by Ref. 9Yoshida M. Furumai R. Nishiyama M. Komatsu Y. Nishino N. Horinouchi S. Cancer Chemother. Pharmacol. 2001; 48 (suppl.): S20-S26Google Scholar). Yeast Sir2 is the founding member of Class III HDACs. Sir2-like deacetylases are not inhibited by TSA and have the unique characteristic of being NAD+-dependent (10Smith J.S. Brachmann C.B. Celic I. Kenna M.A. Muhammad S. Starai V.J. Avalos J.L. Escalante-Semerena J.C. Grubmeyer C. Wolberger C. Boeke J.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6658-6663Google Scholar, 11Tanner K.G. Landry J. Sternglanz R. Denu J.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14178-14182Google Scholar, 12Landry J. Sutton A. Tafrov S.T. Heller R.C. Stebbins J. Pillus L. Sternglanz R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5807-5811Google Scholar, 13Imai S. Armstrong C.M. Kaeberlein M. Guarente L. Nature. 2000; 403: 795-800Google Scholar). Proteins of this class are found in a wide array of organisms, ranging from bacteria to humans. At least two Sir2 homologues, yeast Hst2 and human SIRT2, are localized to the cytoplasm and human SIRT1, a nuclear protein, has recently been shown to target p53 for deacetylation (11Tanner K.G. Landry J. Sternglanz R. Denu J.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14178-14182Google Scholar, 13Imai S. Armstrong C.M. Kaeberlein M. Guarente L. Nature. 2000; 403: 795-800Google Scholar, 14Brachmann C.B. Sherman J.M. Devine S.E. Cameron E.E. Pillus L. Boeke J.D. Genes Dev. 1995; 9: 2888-2902Google Scholar, 15Tanny J.C. Moazed D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 415-420Google Scholar). These results indicate that only a subset of the Sir2 family are likely to be histone deacetylases. Although insensitive to TSA, several synthetic small molecule inhibitors of Sir2 have been isolated and have provided novel insights into the biology of these proteins (16Grozinger C.M. Chao E.D. Blackwell H.E. Moazed D. Schreiber S.L. J. Biol. Chem. 2001; 276: 38837-38843Google Scholar, 17Bedalov A. Gatbonton T. Irvine W.P. Gottschling D.E. Simon J.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 15113-15118Google Scholar). The term silent informationregulator (SIR) was first coined to describe a set of nonessential genes required for repression of yeast mating-type loci (HML and HMR) (18Rine J. Herskowitz I. Genetics. 1987; 116: 9-22Google Scholar). Transcriptional silencing in yeast is also observed at telomeres and the ribosomal DNA (rDNA) locus (2Moazed D. Mol. Cell. 2001; 8: 489-498Google Scholar, 19Wood J.G. Sinclair D.A. Trends Pharmacol. Sci. 2002; 23: 1-4Google Scholar). The formation of silent heterochromatin at mating-type loci and the poly(TG1–3) tracts of yeast telomeres is mediated by a complex of Sir2, Sir3, and Sir4 (20Strahl-Bolsinger S. Hecht A. Luo K. Grunstein M. Genes Dev. 1997; 11: 83-93Google Scholar, 21Hecht A. Strahl-Bolsinger S. Grunstein M. Nature. 1996; 383: 92-96Google Scholar). At the rDNA locus, Sir2 is part of the RENT (regulator of nucleolar silencing and telophase exit) complex, which includes Net1 and Cdc14 (22Ghidelli S. Donze D. Dhillon N. Kamakaka R.T. EMBO. 2001; 20: 4522-4535Google Scholar, 23Shou W. Sakamoto K.M. Keener J. Morimoto K.W. Traverso E.E. Azzam R. Hoppe G.J. Feldman R.M.R. DeModena J. Moazed D. Charbonneaux H. Nomura M. Deshaies R.J. Mol. Cell. 2001; 8: 45-55Google Scholar). Of these proteins, Sir2 is the only factor that is indispensable for silencing at all three silent regions (24Gottschling D.E. Aparicio O.M. Billington B.L. Zakian V.A. Cell. 1990; 63: 751-762Google Scholar, 25Smith J.S. Boeke J.D. Genes Dev. 1997; 11: 241-254Google Scholar, 26Bryk M. Banerjee M. Murphy M. Knudsen K.E. Garfinkel D.J. Curcio M.J. Genes Dev. 1997; 11: 255-269Google Scholar). The yeast rDNA locus (RDN1) consists of 100–200 tandemly repeated 9-kb units encoding ribosomal RNAs. A major cause of yeast aging has been shown to stem from recombination between these repeats (27Sinclair D.A. Guarente L. Cell. 1997; 91: 1033-1042Google Scholar, 28Kaeberlein M. McVey M. Guarente L. Genes Dev. 1999; 13: 2570-2580Google Scholar, 29Park P.U. Defossez P.A. Guarente L. Mol. Cell. Biol. 1999; 19: 3848-3856Google Scholar), which can lead to the excision of an extrachromosomal rDNA circle. Extrachromosomal rDNA circles can accumulate to a DNA content greater than that of the entire yeast genome in old cells and are thought to kill cells by titrating essential transcription and/or replication factors (30Sinclair D.A. Mills K. Guarente L. Trends Biochem. Sci. 1998; 23: 131-134Google Scholar). Although Sir2 silences polymerase II-transcribed genes integrated at the rDNA, there is evidence that its primary function at this locus is to suppress rDNA recombination. Deletion of SIR2 eliminates rDNA silencing and increases the frequency that a marker gene is recombined of the rDNA by 10-fold (31Gottlieb S. Esposito R.E. Cell. 1989; 56: 771-776Google Scholar). Sir2 is a limiting component of yeast longevity. A single extra copy of the SIR2 gene suppresses recombination and extends life span by 40% (28Kaeberlein M. McVey M. Guarente L. Genes Dev. 1999; 13: 2570-2580Google Scholar, 32Lin S.J. Defossez P.A. Guarente L. Science. 2000; 289: 2126-2128Google Scholar, 33Anderson R.M. Bitterman K.J. Wood J.G. Medvedik O. Cohen H. Lin S.S. Manchester J.K. Gordon J.I. Sinclair D.A. J. Biol. Chem. 2002; 277: 18881-18890Google Scholar), whereas deletion of SIR2 increases extrachromosomal rDNA circle formation and dramatically shortens life span (31Gottlieb S. Esposito R.E. Cell. 1989; 56: 771-776Google Scholar, 34Kennedy B.K. Austriaco Jr., N.R. Zhang J. Guarente L. Cell. 1995; 80: 485-496Google Scholar). Recently, it has been shown that SIR2 is essential for the increased longevity provided by calorie restriction (32Lin S.J. Defossez P.A. Guarente L. Science. 2000; 289: 2126-2128Google Scholar), a regimen that extends the life span of every organism it has been tested on. Moreover, increased dosage of the Sir2 homologuesir-2.1 has been shown to extend the life span of the nematode Caenorhabditis elegans (35Tissenbaum H.A. Guarente L. Nature. 2001; 410: 227-230Google Scholar), and the nearest human homologue SIRT1 has been shown to inhibit apoptosis through deacetylation of p53 (36Vaziri H. Dessain S.K. Eaton E.N. Imai S.I. Frye R.A. Pandita T.K. Guarente L. Weinberg R.A. Cell. 2001; 107: 149-159Google Scholar, 37Luo J. Nikolaev A.Y. Imai S. Chen D. Su F. Shiloh A. Guarente L. Gu W. Cell. 2001; 107: 137-148Google Scholar). These findings suggest that Sir2 and its homologues have a conserved role in the regulation of survival at both the cellular and organismal levels. Recently, a great deal of insight has been gained into the biochemistry of Sir2-like deacetylases (reviewed by Ref. 38Moazed D. Curr. Opin. Cell Biol. 2001; 13: 232-238Google Scholar). In vitro, Sir2 has specificity for lysine 16 of histone H4 and lysines 9 and 14 of histone H3 (10Smith J.S. Brachmann C.B. Celic I. Kenna M.A. Muhammad S. Starai V.J. Avalos J.L. Escalante-Semerena J.C. Grubmeyer C. Wolberger C. Boeke J.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6658-6663Google Scholar, 12Landry J. Sutton A. Tafrov S.T. Heller R.C. Stebbins J. Pillus L. Sternglanz R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5807-5811Google Scholar, 13Imai S. Armstrong C.M. Kaeberlein M. Guarente L. Nature. 2000; 403: 795-800Google Scholar). Although TSA-sensitive HDACs catalyze deacetylation without the need of a cofactor, Sir2 requires NAD+, perhaps allowing for regulation of Sir2 activity through changes in the availability of this co-substrate (10Smith J.S. Brachmann C.B. Celic I. Kenna M.A. Muhammad S. Starai V.J. Avalos J.L. Escalante-Semerena J.C. Grubmeyer C. Wolberger C. Boeke J.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6658-6663Google Scholar, 11Tanner K.G. Landry J. Sternglanz R. Denu J.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14178-14182Google Scholar, 12Landry J. Sutton A. Tafrov S.T. Heller R.C. Stebbins J. Pillus L. Sternglanz R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5807-5811Google Scholar, 13Imai S. Armstrong C.M. Kaeberlein M. Guarente L. Nature. 2000; 403: 795-800Google Scholar). The first step in Sir2-catalyzed deacetylation is the cleavage of the high energy glycosidic bond that joins the ADP-ribose moiety of NAD+ to nicotinamide. Upon cleavage, Sir2 then catalyzes the transfer of an acetyl group to ADP-ribose (10Smith J.S. Brachmann C.B. Celic I. Kenna M.A. Muhammad S. Starai V.J. Avalos J.L. Escalante-Semerena J.C. Grubmeyer C. Wolberger C. Boeke J.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6658-6663Google Scholar, 11Tanner K.G. Landry J. Sternglanz R. Denu J.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14178-14182Google Scholar, 15Tanny J.C. Moazed D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 415-420Google Scholar, 39Sauve A.A. Celic I. Avalos J. Deng H. Boeke J.D. Schramm V.L. Biochemistry. 2001; 40: 15456-15463Google Scholar). The product of this transfer reaction is O-acetyl-ADP-ribose, a novel metabolite that has recently been shown to cause a delay/block in the cell cycle and oocyte maturation of embryos (40Borra M.T. O'Neill F.J. Jackson M.D. Marshall B. Verdin E. Foltz K.R. Denu J.M. J. Biol. Chem. 2002; 277: 12632-12641Google Scholar). The other product of deacetylation is nicotinamide, a form of vitamin B3 (41Dietrich L.S. Am. J. Clin. Nutr. 1971; 24: 800-804Google Scholar). High doses of nicotinamide and its acid derivative, nicotinic acid, are often used interchangeably to self-treat a number of conditions including anxiety, osteoarthritis, and psychosis. Furthermore, nicotinamide is currently in clinical trials as a therapy for cancer and type I diabetes (42Kaanders J.H. Pop L.A. Marres H.A. Bruaset I. van den Hoogen F.J. Merkx M.A. van der Kogel A.J. Int. J. Radiat. Oncol. Biol. Phys. 2002; 52: 769-778Google Scholar). The long term safety of the high doses used in these treatments has been questioned (43Knip M. Dovek I.F. Moore W.P. Gillmor H.A. McLean A.E. Bingley P.J. Gale E.A. Diabetologia. 2000; 43: 1337-1345Google Scholar), and the effects of these compounds at the molecular level are even less clear. Interestingly, nicotinamide has recently been shown to inhibit yeast Hst2 in vitro (44Landry J. Slama J.T. Sternglanz R. Biochem. Biophys. Res. Commun. 2000; 278: 685-690Google Scholar), although the effects of this compound at the organismal level or on other yeast Sir2 family members have not been investigated. In most organisms, there are two pathways of NAD+biosynthesis. NAD+ may be synthesized de novofrom tryptophan or recycled in four steps from nicotinamide via the NAD+ salvage pathway (45Foster J.W. Park Y.K. Penfound T. Fenger T. Spector M.P. J. Bacteriol. 1990; 172: 4187-4196Google Scholar) (Fig.1). In the salvage pathway, nicotinamide produced from NAD+ cleavage is converted to nicotinic acid by Pnc1, a nicotinamidase (46Ghislain M. Talla E. Francois J.M. Yeast. 2002; 19: 215-224Google Scholar). Nicotinic acid is subsequently converted into nicotinic acid mononucleotide by a phosphoribosyltransferase encoded by NPT1. We recently demonstrated that increased dosage of NAD+ salvage pathway genes increases silencing at the rDNA locus, telomeres, and mating-type loci. We also showed that a single extra copy of the NPT1gene extends life span by 60% without increasing total steady-state NAD+ levels or NAD+/NADH ratios (33Anderson R.M. Bitterman K.J. Wood J.G. Medvedik O. Cohen H. Lin S.S. Manchester J.K. Gordon J.I. Sinclair D.A. J. Biol. Chem. 2002; 277: 18881-18890Google Scholar). This suggests that Sir2 may be regulated either by nuclear specific changes in NAD+ availability, flux through the salvage pathway, or by levels of an inhibitory molecule (33Anderson R.M. Bitterman K.J. Wood J.G. Medvedik O. Cohen H. Lin S.S. Manchester J.K. Gordon J.I. Sinclair D.A. J. Biol. Chem. 2002; 277: 18881-18890Google Scholar). With regards to the later hypothesis, we wished to examine whether Sir2 enzymes might be negatively regulated by nicotinamide, a product of the deacetylation reaction. Here, we show that nicotinamide strongly inhibits silencing at yeast telomeres, rDNA, and mating-type loci, whereas the related nicotinic acid has no effect. Nicotinamide also increases recombination at the rDNA locus and shortens yeast life span to that of a sir2 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 Sir2, even in non-dividing cells. Physiological concentrations of nicotinamide inhibit Sir2 and human SIRT1 noncompetitively in vitro, raising the possibility that nuclear nicotinamide negatively regulates Sir2 activity in vivo. Our findings also suggest that the medicinal use of nicotinamide should be given careful consideration. All yeast strains used in this study are listed in Table I. Cells were grown at 30 °C on YPD medium (1% yeast extract, 2% bactopeptone, 2% glucose, w/v) unless otherwise stated. The extent of silencing at the ribosomal DNA locus was determined by growingRDN1::MET15 strains on Pb2+-containing medium (0.3% peptone, 0.5% yeast extract, 4% glucose, 0.02% (w/v) ammonium acetate, 0.07% Pb(NO3)2, and 2% agar). ADE2-based telomeric and HM locus silencing assays were performed as described previously (32Lin S.J. Defossez P.A. Guarente L. Science. 2000; 289: 2126-2128Google Scholar). To monitor silencing of theRDN1::URA3 marker, log phase cultures were preincubated in YPD with or without 5 mm nicotinamide for 2 h. Following this, cells were spread onto either synthetic complete (SC) medium (1.67% yeast nitrogen base, 2% glucose, 40 mg/liter each of histidine, uridine, tryptophan, adenine, and leucine) or SC medium containing 0.4 mg/ml 5-fluoroorotic acid (5-FOA) each with or without 5 mm nicotinamide. Colonies were counted after 48 h using Bio-Rad Quantity One software.Table IYeast strains used in this studyStrainGenotypeW303AR5W303MAT a, ade2–1, leu2–3, 112, can1–100, trp1–1, ura3–52, his3–11, 15, RDN1∷ADE2, RAD5YDS878W303MAT a, ade2–1, leu2–3, 112, can1–100, trp1–1, ura3–52, his3–11, 15, RDN1∷ADE2, RAD5, sir2:TRP1YDS1572W303 MAT a,ade2–1, leu2–3, 112, can1–100, trp1–1, ura3–52, his3–11, 15, RDN1∷ADE2, RAD5, LEU2/SIR2YDS1595W303MAT a, ade2–1, leu2–3, 112, can1–100, trp1–1, ura3–52, his3–11, 15, RAD5YDS1596W303 MAT a,ADE2, leu2–3, 112, can1–100, trp1–1, ura3–52, his3–11, 15, RAD5YDS1097W303 MAT a, ade2–1, leu2–3, 112, can1–100, trp1–1, ura3–52, his3–11, 15, RDN, RAD5, GFP-Sir4∷URA3YDS1099W303 MAT a,ade2–1, leu2–3, 112, can1–100, trp1–1, ura3–52, his3–11, 15, RDN, RAD5, GFP-Sir3∷LEU2YDS1109W303MAT a, ade2–1, leu2–3, 112, can1–100, trp1–1, ura3–52, his3–11, 15, RDN, RAD5, GFP-Sir3∷LEU2, sir2:TRP1YSB0163W303 MATα,ade2–1, leu2–3, 112, can1–100, trp1–1, ura3–1, his3–11, 15, MATα hmrWT∷TRP1, HMR∷URA3∷ADE2YDS1183W303 MAT a, ade2–1, leu2–3, 112, can1–100, trp1–1, ura3–52, his3–11, 15, RDN1∷ADE2, RAD5, SIR2-HA∷URA3YDS1782W303MAT a, ade2–1, leu2–3, 112, can1–100, trp1–1, ura3–52, his3–11, 15, RDN1∷URA3, sir2∷TRPYDS1078W303 MAT a, ade2–1, leu2–3, 112, can1–100, trp1–1, ura3–52, his3–11, 15, RDN1∷ADE2, RAD5, Sir2-GFP∷LEU2YDS1784W303MAT a, ade2–1, leu2–3, 112, can1–100, trp1–1, ura3–52, his3–11, 15, RDN1∷ADE2, RAD5, Sir2-GFP, leu2∷URA3, Δhml∷LEU2PSY316ATMATα, ura3–53 leu2–3, 112 his3-Δ200 ade2–1, 01 can1–100 ADE2-TEL V-RYDS1594PSY316 MATα, ura3–53 leu2–3, 112 his3-Δ200 ade2–1,01 can1–100 ADE2-TEL V-R, sir2:TRP1YDS970PSY316 MATα, ura3–53 leu2–3, 112 his3-Δ200 ade2–1,01 can1–100 ADE2-TEL V-R, HMR∷GFPYDS1005PSY316 MATα, ura3–53 leu2–3, 112 his3-Δ200 ade2–1,01 can1–100 ADE2-TEL V-R, HMR∷GFPYDS1499PSY316 MATα, ura3–53 leu2–3, 112 his3-Δ200 ade2–1,01 can1–100 ADE2-TEL V-R, HMR∷GFP , sir4:HIS3YDS1690PSY316MATα, ura3–53 leu2–3, 112 his3-Δ200 ade2–1,01 can1–100 ADE2-TEL V-R, HMR∷GFP, Δhml∷LEU2YDS1652PSY316AT, MATα, ura3–53 leu2–3, 112 his3-Δ200 ade2–1,01 can1–100 ADE2-TEL V-R RDN1∷URA3YDS1795PSY316AT,MATα, ura3–53 leu2–3, 112 his3-Δ200 ade2–1,01 can1–100 ADE2-TEL V-R, pSP400-URA3JS209MATα, his3Δ200, leu2Δ1, met15Δ200, trp1Δ63, ura3–167JS241JS209 MATα,his3Δ200, leu2Δ1,met15Δ200, trp1Δ63, ura3–167, Ty1-MET15JS237JS209 MATα, his3Δ200, leu2Δ1, met15Δ200, trp1Δ63, ura3–167, RDN1∷Ty1-MET15JS218JS237 MATα,his3Δ200, leu2Δ1,met15Δ200, trp1Δ63, ura3–167, RDN1∷Tyl-MET15, sir2∷HIS3YDS1583JS237 MATα, his3Δ200, leu2Δ1, met15Δ200, trp1Δ63, ura3–167, RDN1∷Ty1-MET15, LEU2/SIR2 Open table in a new tab Ribosomal DNA recombination frequencies were determined as described (44Landry J. Slama J.T. Sternglanz R. Biochem. Biophys. Res. Commun. 2000; 278: 685-690Google Scholar). Replicative life span determination was performed by micromanipulation as described (25Smith J.S. Boeke J.D. Genes Dev. 1997; 11: 241-254Google Scholar). A minimum of 40 cells were examined per experiment and each experiment was performed at least twice independently. Statistical significance of life span differences was determined using the Wilcoxon rank sum test. Differences are stated to be significant when the confidence is higher than 95%. GFP fluorescence in strain YDS1005 was quantified by fluorescence-activated cell sorting (FACS) using a FACSCalibur flow cytometer (BD Biosciences) as described (45Foster J.W. Park Y.K. Penfound T. Fenger T. Spector M.P. J. Bacteriol. 1990; 172: 4187-4196Google Scholar). For G1-arrest experiments, cells were treated with 10 μg/ml α-factor mating pheromone (Sigma) for 2 h. DNA content was determined by FACS analysis of fixed cells stained with propidium iodide (Sigma) as described (47Mills K.D. Sinclair D.A. Guarente L. Cell. 1999; 97: 609-620Google Scholar). Typically 20,000 cells were analyzed per sample. Data acquisition and analysis were performed using CELLQuest software (BD Biosciences). Western blots were performed using standard techniques. The HA epitope tag was detected using monoclonal antibody HA.11 (CRP, Richmond, CA). Actin was detected with monoclonal antibody MAB1501R (Chemicon, Temecula, CA). GFP fluorescence was visualized in live cells grown to log phase in SC medium. For arrest experiments, strain YDS1078 was first deleted forLEU2 using a URA3-based disruption plasmid. The resulting strain was deleted for HML using aLEU2-based plasmid and the disruption was confirmed by Southern blot. Disrupted cells were grown to early log phase and treated with 10 μg/ml α-factor for 2 h after which time the culture was split and treated with either 0 or 5 mmnicotinamide. Images were captured using a Nikon Eclipse E600 microscope and analyzed with Scion Image software. Recombinant glutathioneS-transferase-tagged yeast Sir2p (gift of D. Moazed) and recombinant human SIRT1 (48Langley E. Pearson M. Faretta M. Bauer U.M. Frye R.A. Minucci S. Pelicci P.G. Kouzarides T. EMBO J. 2002; 21: 2383-2396Google Scholar) were assayed for deacetylase activity using the HDAC fluorescent activity assay/drug discovery kit (AK-500, BIOMOL Research Laboratories). This assay system allows detection of a fluorescent signal upon deacetylation of a histone substrate when treated with developer. Fluorescence was measured on a fluorometric reader (Cytofluor II 400 series PerSeptive Biosystems) with excitation set at 360 nm and emission detection set at 460 nm. Reactions consisted of either 5 μg of glutathione S-transferase-Sir2 or 2.5 μg of SIRT1, incubated with 250 μm acetylated histone substrate, 1 mm dithiothreitol, and a range of NAD+ concentrations as described. Reactions with the yeast and human proteins were carried out at 30 and 37 °C, respectively, for 30 min. For inhibitor assays, reactions were performed in the presence of 200 μm NAD+ and either nicotinamide (0, 50, 150, or 300 μm) (Sigma) or 50 μm of the following compounds: nicotinic acid (Sigma), sirtinol, M15 (Chembridge), splitomicin (47Mills K.D. Sinclair D.A. Guarente L. Cell. 1999; 97: 609-620Google Scholar), or TSA (BIOMOL). To examine whether nicotinamide could affect Sir2 activity in vivo, we examined strains with eitherADE2 or MET15 integrated at the rDNA locus (RDN1). Silencing of ADE2 results in the accumulation of a red pigment on plates with limiting adenine, whereas silencing of MET15 leads to production of a brown pigment on Pb2+-containing medium. We used two marker genes to ensure that the effects we observed were not simply because of changes in adenine or methionine biosynthesis. Strains with a single extra copy ofSIR2 (2xSIR2) or lacking SIR2(sir2::TRP1) were included as controls for increased silencing and lack of silencing, respectively. As shown in Fig. 2 A, we observed a dramatic reduction in silencing when cells were grown in the presence of 5 mm nicotinamide. Silencing of an ADE2marker at this locus was similarly attenuated by addition of nicotinamide (data not shown). To test whether this effect was specific to the rDNA or influenced all heterochromatic regions, we also examined silencing at telomeres and the mating-type locus. To monitor telomeric silencing, we used a strain in which the ADE2 gene was integrated at the subtelomeric (Y′) region of the right arm of chromosome V (24Gottschling D.E. Aparicio O.M. Billington B.L. Zakian V.A. Cell. 1990; 63: 751-762Google Scholar). On plates with limiting adenine, colonies have red/white sectors because of variegated expression of the ADE2 marker. In the presence of 5 mm nicotinamide colonies were white indicating a loss of silencing at this locus (Fig. 2 B). To monitor silencing of mating-type genes, a strain with an ADE2 marker integrated in the HMR locus was treated with 0 or 5 mmnicotinamide. Similar to the effect at the rDNA and telomeres, treatment with nicotinamide led to a dramatic loss of repression at this locus (Fig. 2 C). We wished to obtain a more quantitative measure of the extent of desilencing induced by nicotinamide. To do this, we utilized a strain containing a URA3 marker integrated at the RDN1locus and a wild-type strain with an endogenous nonsilencedURA3 gene. An equivalent number of cells of each strain were plated for single colonies onto SC medium or SC medium containing 5-FOA, in the presence or absence of nicotinamide. As shown in Fig.2 D, in the presence of 5 mm nicotinamide the number of colonies able to grow on 5-FOA decreased ∼8-fold to a level similar to that of Ura+ cells. This increase in 5-FOA sensitivity is indicative of increased URA3 expression resulting from a severe abrogation of silencing. Nicotinic acid, another intermediate in the NAD+ salvage pathway, is structurally similar to nicotinamide (see Fig.3 C). Nicotinic acid is taken up efficiently by yeast cells and a specific transporter for this compound, Tna1, was recently identified (49Llorente B. Dujon B. FEBS Lett. 2000; 475: 237-241Google Scholar, 50Sandmeier J.J. Celic I. Boeke J.D. Smith J.S. Genetics. 2002; 160: 877-889Google Scholar). In each of the above assays, we examined the effect of 5 mm nicotinic acid on Sir2-dependent silencing and in each case found that nicotinic acid had no effect (data not shown). Although the most likely explanation for the above observations was that Sir2 is catalytically inactivated by nicotinamide, it was plausible that Sir2 was down-regulated in the presence of this compound. To test this, we compared Sir2 protein levels in the presence and absence of nicotinamide after 2 h, a time at which silencing is almost entirely lost (see below). As shown in Fig. 3 A, we observed no significant difference in Sir2 levels in the presence of 0, 1, and 5 mm nicotinamide. Next, we wished to address whether the loss of silencing was because of inhibition of Sir2 activity, in which case nicotinamide-treated cells should mimic a sir2Δ strain. One of the best characterized phenotypes of a sir2 mutant is an increased frequency of rDNA recombination. Therefore, the loss of an ADE2 marker integrated at the rDNA locus was scored for wild-type, 2xSIR2, and sir2 strains, in the presence and absence of nicotinamide. As shown in Fig. 3 B, treatment of wild-type and 2xSIR2 cells with nicotinamide increased the frequency of marker loss 7-fold, similar to that of a sir2mutant. Importantly, treatment of the sir2 strain did not further increase recombination, arguing that the observed marker loss was because of inhibition of Sir2. Recombination of the rDNA locus has been shown to be a major cause of yeast replicative aging (27Sinclair D.A. Guarente L. Cell. 1997; 91: 1033-1042Google Scholar, 28Kaeberlein M. McVey M. Guarente L. Genes Dev. 1999; 13: 2570-2580Google Scholar). We therefore examined the effect of nicotinamide on yeast life span. Cells were grown for 2 days on fresh YPD medium to ensure that they were not calorie restricted prior to the assay. Daughter cells that emerged from previously unbudded (virgin) mother cells were micro-manipulated away and scored. Fig. 3 Dshows representative life span curves of both wild-type (triangles) and the short-lived sir2 mutant (circles). Cells grown on medium containing 5 mmnicotinamide (closed diamonds) exhibited an average life span ∼45% that of wild-type, equivalent to that of thesir2 mutant. Treatment of the sir2 strain with nicotinamide did not further shorten life span (squares). Consistent with the silencing data, we observed no detrimental effect on replicative