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Manipulation of a Nuclear NAD+ Salvage Pathway Delays Aging without Altering Steady-state NAD+ Levels*

Open AccessPublished:May 24, 2002DOI:https://doi.org/10.1074/jbc.M111773200
      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.
      Physiological studies and, more recently, DNA array analysis of gene expression patterns have confirmed that aging is a complex biological process. In contrast, genetic studies in model organisms have demonstrated that relatively minor changes to an organism's environment or genetic makeup can dramatically slow the aging process. For example, the life span of many diverse organisms can be greatly extended simply by limiting calorie intake, in a dietary regime known as caloric restriction (
      • Masoro E.J.
      ,
      • Vanfleteren J.R.
      • Braeckman B.P.
      ,
      • Zainal T.A.
      • Oberley T.D.
      • Allison D.B.
      • Szweda L.I.
      • Weindruch R.
      ).
      How can simple changes have such profound effects on a complex process such as aging? A picture is emerging in which all eukaryotes possess a surprisingly conserved regulatory system that governs the pace of aging (,
      • Guarente L.
      • Kenyon C.
      ). Such a regulatory system may have arisen in evolution to allow organisms to survive in adverse conditions by redirecting resources from growth and reproduction to pathways that provide stress resistance (,
      • Kirkwood T.B.
      • Rose M.R.
      ).
      One model that has proven particularly useful in the identification of regulatory factors of aging is the budding yeast Saccharomyces cerevisiae. Replicative life span in S. cerevisiae is typically defined as the number of buds or “daughter cells” produced by an individual “mother cell” (
      • Barton A.
      ). Mother cells undergo age-dependent changes, including an increase in size, a slowing of the cell cycle, enlargement of the nucleolus, an increase in steady-state NAD+ levels, increased gluconeogenesis and energy storage, and sterility resulting from the loss of silencing at telomeres and mating-type loci (
      • Sinclair D.A.
      • Mills K.
      • Guarente L.
      ,
      • Mortimer R.K.
      • Johnston J.R.
      ,
      • Kennedy B.K.
      • Austriaco N.R., Jr.
      • Guarente L.
      ,
      • Kim S.
      • Villeponteau B.
      • Jazwinski S.M.
      ,
      • Ashrafi K.
      • Lin S.S.
      • Manchester J.K.
      • Gordon J.I.
      ,
      • Lin S.S.
      • Manchester J.K.
      • Gordon J.I.
      ). An alternative measure of yeast life span, known as chronological aging, is the length of time a population of non-dividing cells remains viable when deprived of nutrients (
      • Longo V.D.
      ). Increased chronological life span correlates with increased resistance to heat shock and oxidative stress, suggesting that cumulative damage to cellular components is a major cause of this type of aging (
      • Longo V.D.
      ,
      • Jazwinski S.M.
      ). The extent of overlap between replicative and chronological aging is currently unclear.
      One cause of yeast replicative aging has been shown to stem from the instability of the repeated ribosomal DNA (rDNA)
      The abbreviations used are: rDNA
      ribosomal DNA
      NaMN
      nicotinic acid mononucleotide
      NaAD
      desamido-NAD+
      NaM
      nicotinamide
      NaMNAT
      nicotinate mononucleotide adenylyltransferase
      ORF
      open reading frame
      GFP
      green fluorescence protein
      HA
      hemagglutinin
      SC
      synthetic complete
      MMS
      methylmethane sulfonate
      3xHA
      triple hemagglutinin epitope
      1The abbreviations used are: rDNA
      ribosomal DNA
      NaMN
      nicotinic acid mononucleotide
      NaAD
      desamido-NAD+
      NaM
      nicotinamide
      NaMNAT
      nicotinate mononucleotide adenylyltransferase
      ORF
      open reading frame
      GFP
      green fluorescence protein
      HA
      hemagglutinin
      SC
      synthetic complete
      MMS
      methylmethane sulfonate
      3xHA
      triple hemagglutinin epitope
      locus (
      • Sinclair D.A.
      • Guarente L.
      ). This instability gives rise to circular forms of rDNA called extrachromosomal rDNA circles that replicate but fail to segregate to daughter cells. Eventually, extrachromosomal rDNA circles accumulate to over 1000 copies, which are thought to kill cells by titrating essential transcription and/or replication factors (
      • Sinclair D.A.
      • Guarente L.
      ,
      • Kaeberlein M.
      • McVey M.
      • Guarente L.
      ,
      • Park P.U.
      • Defossez P.A.
      • Guarente L.
      ). Regimens that reduce rDNA recombination such as caloric restriction or a fob1 deletion extend replicative life span (
      • Kaeberlein M.
      • McVey M.
      • Guarente L.
      ,
      • Lin S.J.
      • Defossez P.A.
      • Guarente L.
      ,
      • Defossez P.A.
      • Prusty R.
      • Kaeberlein M.
      • Lin S.J.
      • Ferrigno P.
      • Silver P.A.
      • Keil R.L.
      • Guarente L.
      ).
      A key regulator of aging in yeast is the Sir2 silencing protein (
      • Kaeberlein M.
      • McVey M.
      • Guarente L.
      ), a nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase (
      • Tanner K.G.
      • Landry J.
      • Sternglanz R.
      • Denu J.M.
      ,
      • Imai S.
      • Armstrong C.M.
      • Kaeberlein M.
      • Guarente L.
      ,
      • Smith 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.
      ,
      • Landry J.
      • Sutton A.
      • Tafrov S.T.
      • Heller R.C.
      • Stebbins J.
      • Pillus L.
      • Sternglanz R.
      ). Sir2 is a component of the heterotrimeric Sir2/3/4 complex that catalyzes the formation of silent heterochromatin at telomeres and the two silent mating-type loci (
      • Laurenson P.
      • Rine J.
      ). Sir2 is also a component of the regulator of nucleolar silencing and telophase exit complex that is required for silencing at the rDNA locus and exit from telophase (
      • Straight A.F.
      • Shou W.
      • Dowd G.J.
      • Turck C.W.
      • Deshaies R.J.
      • Johnson A.D.
      • Moazed D.
      ,
      • Shou W.
      • Seol J.H.
      • Shevchenko A.
      • Baskerville C.
      • Moazed D.
      • Chen Z.W.
      • Jang J.
      • Charbonneau H.
      • Deshaies R.J.
      ). This complex has also recently been shown to directly stimulate transcription of rRNA by polymerase I and to be involved in the regulation of nucleolar structure (
      • Shou 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.
      ).
      Biochemical studies have shown that Sir2 can readily deacetylate the amino-terminal tails of histones H3 and H4, resulting in the formation of O-acetyl-ADP-ribose and nicotinamide (
      • Tanner K.G.
      • Landry J.
      • Sternglanz R.
      • Denu J.M.
      ,
      • Imai S.
      • Armstrong C.M.
      • Kaeberlein M.
      • Guarente L.
      ,
      • Smith 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.
      ,
      • Tanny J.C.
      • Moazed D.
      ). Strains with additional copies of SIR2 display increased rDNA silencing (
      • Smith J.S.
      • Caputo E.
      • Boeke J.D.
      ) and a 30% longer life span (
      • Kaeberlein M.
      • McVey M.
      • Guarente L.
      ). It has recently been shown that additional copies of the Caenorhabditis elegans SIR2 homolog, sir-2.1, greatly extend life span in that organism (
      • Tissenbaum H.A.
      • Guarente L.
      ). This implies that the SIR2-dependent regulatory pathway for aging arose early in evolution and has been well conserved (). Yeast life span, like that of metazoans, is also extended by interventions that resemble caloric restriction (
      • Lin S.J.
      • Defossez P.A.
      • Guarente L.
      ,
      • Jiang J.C.
      • Jaruga E.
      • Repnevskaya M.V.
      • Jazwinski S.M.
      ). Mutations that reduce the activity of the glucose-responsive cAMP (adenosine 3′,5′-monophosphate)-dependent (protein kinase A) pathway extend life span in wild type cells but not in mutant sir2 strains, demonstrating that SIR2 is a key downstream component of the caloric restriction pathway (
      • Lin S.J.
      • Defossez P.A.
      • Guarente L.
      ).
      In bacteria, NAD+ is synthesized de novo from tryptophan and recycled in four steps from nicotinamide via the NAD+ salvage pathway (see Fig. 5 below). The first step in the bacterial NAD+ salvage pathway, the hydrolysis of nicotinamide to nicotinic acid and ammonia, is catalyzed by the pncA gene product (
      • Foster J.W.
      • Kinney D.M.
      • Moat A.G.
      ). An S. cerevisiae gene with homology to pncA, YGL037, was recently assigned the name PNC1 (
      • Ghislain M.
      • Talla E.
      • Francois J.M.
      ). A nicotinate phosphoribosyltransferase, encoded by the NPT1 gene in S. cerevisiae, converts the nicotinic acid from this reaction to nicotinic acid mononucleotide (NaMN) (
      • Wubbolts M.G.
      • Terpstra P.
      • van Beilen J.B.
      • Kingma J.
      • Meesters H.A.
      • Witholt B.
      ,
      • Vinitsky A.
      • Teng H.
      • Grubmeyer C.T.
      ,
      • Imsande J.
      ,
      • Grubmeyer C.T.
      • Gross J.W.
      • Rajavel M.
      ). At this point, the NAD+ salvage pathway and the de novoNAD+ pathway converge and NaMN is converted to desamido-NAD+ (NaAD) by a nicotinate mononucleotide adenylyltransferase (NaMNAT). In S. cerevisiae, there are two putative ORFs with homology to bacterial NaMNAT genes,YLR328 (
      • Emanuelli M.
      • Carnevali F.
      • Lorenzi M.
      • Raffaelli N.
      • Amici A.
      • Ruggieri S.
      • Magni G.
      ) and an uncharacterized ORF, YGR010(
      • Smith 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.
      ,
      • Emanuelli M.
      • Carnevali F.
      • Lorenzi M.
      • Raffaelli N.
      • Amici A.
      • Ruggieri S.
      • Magni G.
      ). We refer to these two ORFs as NMA1 and NMA2, respectively. In Salmonella, the final step in the regeneration of NAD+ is catalyzed by an NAD synthetase (
      • Hughes K.T.
      • Olivera B.M.
      • Roth J.R.
      ). An as yet uncharacterized ORF, QNS1, is predicted to encode an NAD synthetase (
      • Smith 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.
      ).
      Figure thumbnail gr5
      Figure 5Multiple limiting components in the NAD+ salvage pathway. A, the putative steps in NAD+ biosynthesis in S. cerevisiae based on the known steps in Salmonella. The yeast genes that are thought to mediate each step are shown in italics. NaMN, nicotinic acid mononucleotide; NaAD, desamido-NAD+; NaM, nicotinamide; Na, nicotinic acid. Adapted from Smith et al. (
      • Smith 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.
      ).B, silencing of ADE2 at the rDNA locus in strains ADE2 (YDS1596), wild type (W303AR5), 2x NPT1(YDS1503), 2x PNC1 (YDS1588), 2x NMA2 (YDS1589), 2x NMA1 (YDS1590), and 2x QNS1 (YDS1614). Increased silencing is indicated by growth retardation on media lacking adenine.C, strains with an ADE2 marker at the telomere were streaked onto SC medium containing limiting amounts of adenine. Silencing is indicated by the accumulation of a red pigment. Strains tested: wild type (PSY316AT), 2x NPT1 (YDS1544), 5x NPT1 (YDS1548), sir2::TRP1(YDS1594), 2x PNC1 (YDS1591), 2x NMA2 (YDS1592), and 2x NMA1 (YDS1593).
      In yeast, null mutations in NPT1 reduce steady-state NAD+ levels by ∼2-fold (
      • Smith 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.
      ) and abolish the longevity provided by limiting calories (
      • Lin S.J.
      • Defossez P.A.
      • Guarente L.
      ). One current hypothesis explaining how caloric restriction extends replicative life span is that decreased metabolic activity causes an increase in NAD+ levels, which then stimulate Sir2 activity (reviewed in Campisi (
      • Campisi J.
      ) and Guarente (
      • Guarente L.
      )). In this study, we tested this theory by examining whether additional copies of NPT1 can promote Sir2-dependent life span extension and whether this correlates with increased NAD+ levels. We show that additional NPT1 extends replicative life span in a SIR2-dependent manner via the caloric restriction pathway. We find that these long-lived strains do not have increased NAD+ levels or altered NAD+/NADH ratios, despite the fact that every SIR2-dependent process we examined was enhanced. Interestingly, increased dosage of SIR2 or NPT1 provides resistance to heat shock, indicating that these genes act in a general manner to promote cell survival.
      We find that additional copies of all the salvage pathway genes increase rDNA and telomeric silencing with exception of QNS1. We show that Npt1 and Nma2 are concentrated in the nucleus, raising the possibility that a substantial fraction of NAD+ is recycled within this organelle. We discuss the potential for extending life span in higher organisms by stimulation of the conserved NAD+ salvage pathway.

      DISCUSSION

      NPT1 encodes a key component of the yeast salvage pathway that recycles NAD+, a cofactor of Sir2. We have shown that additional copies of NPT1 increase life span by up to 60% in a SIR2-dependent manner. It has been proposed that longevity in yeast may be associated with increased NAD+ levels. However, we have shown that in strains with additional copies of NPT1, steady-state NAD+levels are unaltered. Furthermore, the NAD+/NADH ratios are also similar to wild type cells, indicating that total cellular redox state is not dramatically altered either.
      We have also shown that sir2 mutants have wild type NAD+ levels, implying that Sir2 is not a major consumer of NAD+. Nevertheless, by virtue of its ability to convert NAD+ to nicotinamide, Sir2 should be responsive to increased flux through the salvage pathway (Fig. 6). Thus, although steady-state levels of NAD+ remain constant, the turnover of this molecule may be elevated. Localization of GFP-tagged enzymes indicated that at least two of the enzymes in the NAD+ salvage pathway are concentrated in the nucleus. Consistent with this, Nma1 and Nma2 have been shown by high throughput two-hybrid screening to interact with Srp1, a protein that acts as a receptor for nuclear localization sequences (
      • Uetz P.
      • Giot L.
      • Cagney G.
      • Mansfield T.A.
      • Judson R.S.
      • Knight J.R.
      • Lockshon D.
      • Narayan V.
      • Srinivasan M.
      • Pochart P.
      • Qureshi-Emili A., Li, Y.
      • Godwin B.
      • Conover D.
      • Kalbfleisch T.
      • Vijayadamodar G.
      • Yang M.
      • Johnston M.
      • Fields S.
      • Rothberg J.M.
      ). The same two-hybrid screen also found that Nma1 and Nma2 can interact with themselves and with each other. Perhaps Nma proteins exist as dimers, as is the case for the Bacillus subtilis NaMNAT (
      • Olland A.M.
      • Underwood K.W.
      • Czerwinski R.M.
      • Lo M.C.
      • Aulabaugh A.
      • Bard J.
      • Stahl M.L.
      • Somers W.S.
      • Sullivan F.X.
      • Chopra R.
      ), or as hexamers, as is the case for Methanococcus jannaschii (
      • D'Angelo I.
      • Raffaelli N.
      • Dabusti V.
      • Lorenzi T.
      • Magni G.
      • Rizzi M.
      ) and Methanobacterium thermoautotrophicum NaMNAT s (
      • Saridakis V.
      • Christendat D.
      • Kimber M.S.
      • Dharamsi A.
      • Edwards A.M.
      • Pai E.F.
      ). It is worth nothing that strains disrupted for either NMA1 or NMA2 are viable, arguing that they may be functionally redundant, given that the conversion of NaMN to NAD+ is apparently essential for viability (
      • Winzeler E.A.
      • Shoemaker D.D.
      • Astromoff A.
      • Liang H.
      • Anderson K.
      • Andre B.
      • Bangham R.
      • Benito R.
      • Boeke J.D.
      • Bussey H.
      • Chu A.M.
      • Connelly C.
      • Davis K.
      • Dietrich F.
      • Dow S.W., El
      • Bakkoury M.
      • Foury F.
      • Friend S.H.
      • Gentalen E.
      • Giaever G.
      • Hegemann J.H.
      • Jones T.
      • Laub M.
      • Liao H.
      • Davis R.W.
      • et al.
      ).
      Figure thumbnail gr6
      Figure 6Model for life span extension via increased flux through the NAD+ salvage pathway. Type III histone deacetylases such as Sir2 and Hst1–4 catalyze a key step in the salvage pathway by converting NAD+ to nicotinamide. Additional copies of PNC1, NPT1, NMA1, and NMA2 increase flux through the NAD+ salvage pathway, which stimulates Sir2 activity and increases life span. Additional copies of QNS1 fail to increase silencing. Unlike other steps in the pathway, its substrate cannot be supplied from a source outside the salvage pathway and is therefore limiting for the reaction. Abbreviations: NAD +, nicotinamide adenine dinucleotide; NaMN, nicotinic acid mononucleotide; NaAD, desamido-NAD+.
      In vertebrates, NaMNAT/NMNAT activity is primarily observed in the nuclear fraction of liver cell extracts (
      • Hogeboom G.
      • Schneider W.
      ), suggesting that nuclear compartmentalization of the pathway may be a universal property of eukaryotic cells. Having the salvage pathway in proximity to chromatin may allow NAD+ to be rapidly regenerated for silencing proteins. Alternatively, it may permit the coordination of a variety of nuclear activities via the alteration of nuclear NAD+ pools. Testing of these hypotheses will not be a simple task but one that will be greatly assisted by the development of a molecular probe for intracellular NAD+.
      In yeast and many metazoans, a number of long-lived mutants display increased stress resistance. However, there are many examples of mutations that extend life span but provide little protection against stress, indicating that this relationship is not straightforward (). For example, in yeast the life span extension provided by a cdc25-10 mutation is not accompanied by heat-shock resistance (
      • Lin S.J.
      • Defossez P.A.
      • Guarente L.
      ). We have shown that additional copies of NPT1 or SIR2 extend life span but do not provide protection against MMS, paraquat, or starvation. Thus, in S. cerevisiae, longevity is not linked to a general increase in stress resistance. The only stress-related phenotype that correlated with longevity was heat-shock resistance. Based on genome-wide analyses of gene expression in sir2Δ strains, it has been proposed that Sir2 regulates genes other than those at the three silent loci (
      • Bernstein B.E.
      • Tong J.K.
      • Schreiber S.L.
      ), although this interpretation is debated (
      • Bedalov A.
      • Gatbonton T.
      • Irvine W.P.
      • Gottschling D.E.
      • Simon J.A.
      ). If the interpretation is correct, then it is plausible that the heat-shock resistance we observed in 2x NPT1 and 2x SIR2 strains results from Sir2-mediated silencing of genes that suppress heat-shock resistance.
      In bacteria, the Npt1 homolog PncB catalyzes a rate-limiting step in the NAD+ salvage pathway (
      • Wubbolts M.G.
      • Terpstra P.
      • van Beilen J.B.
      • Kingma J.
      • Meesters H.A.
      • Witholt B.
      ,
      • Imsande J.
      ,
      • Grubmeyer C.T.
      • Gross J.W.
      • Rajavel M.
      ). In this study we show that additional copies of PNC1, NPT1, NMA1, or NMA2 all increase rDNA and telomeric silencing. The implication is that, in yeast, multiple steps can affect the rate of the pathway. Such a proposal is consistent with Metabolic Control Analysis, a theory based on the observation that flux through most metabolic pathways is controlled by multiple enzymes, rather than by a single rate-liming step (
      • Fell D.
      ). Of all the genes in the salvage pathway, only QNS1 had no effect on silencing, suggesting that it is the only enzyme in the pathway limited by substrate availability. This is likely due to the fact that the predicted substrate for Qns1, desamido-NAD+, is the only intermediate that cannot be supplied from a source outside the salvage pathway (see Fig. 6).
      In yeast and metazoans there are multiple members of the Sir2 family, many of which have been shown (or are predicted) to be NAD+-dependent deacetylases (
      • Landry J.
      • Sutton A.
      • Tafrov S.T.
      • Heller R.C.
      • Stebbins J.
      • Pillus L.
      • Sternglanz R.
      ,
      • Landry J.
      • Slama J.T.
      • Sternglanz R.
      ). This finding, combined with the fact that some Sir2 family members are cytoplasmic (
      • Perrod S.
      • Cockell M.M.
      • Laroche T.
      • Renauld H.
      • Ducrest A.L.
      • Bonnard C.
      • Gasser S.M.
      ,
      • Afshar G.
      • Murnane J.P.
      ), suggests that reversible acetylation may be a much more prevalent regulatory mechanism than previously thought (
      • Shore D.
      ). This would place the NAD+ salvage pathway in a pivotal position, coordinating the activity of this group of effector proteins in response to cellular energy status.
      It is now widely accepted that there are conserved pathways for the regulation of longevity (,
      • Guarente L.
      • Kenyon C.
      ). The extent of this conservation is exemplified by the discovery that additional copies of C. elegans sir-2.1 also extend life span in that organism (
      • Tissenbaum H.A.
      • Guarente L.
      ). Our findings show that several SIR2-dependent processes can be enhanced by manipulation of the NAD+ salvage pathway in yeast, and this may hold true for higher organisms. We have identified NPT1 homologs in every genome we have examined, and all possess a highly conserved region around a histidine residue that, in Salmonella, greatly stimulates catalysis when phosphorylated (
      • Rajavel M.
      • Lalo D.
      • Gross J.W.
      • Grubmeyer C.
      ). This mode of regulation may permit the design of mutations or small molecules that increase Npt1 activity. Together, our findings show that Npt1 and other members of the salvage pathway are attractive targets for small molecules that may mimic the beneficial effects of caloric restriction.

      ACKNOWLEDGEMENTS

      We thank D. Moazed, J. Smith, C. Grubmeyer, M. Bryk, F. Winston, A. Andalis, and G. Fink for reagents and advice. We also thank S. Luikenhuis for help with manuscript preparation.

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