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Enzymes in the NAD+ Salvage Pathway Regulate SIRT1 Activity at Target Gene Promoters*

  • Tong Zhang
    Affiliations
    Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853
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  • Jhoanna G. Berrocal
    Affiliations
    Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853

    Graduate Field of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853
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  • Kristine M. Frizzell
    Affiliations
    Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853

    Graduate Field of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853
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  • Matthew J. Gamble
    Affiliations
    Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853
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  • Michelle E. DuMond
    Affiliations
    Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853
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  • Raga Krishnakumar
    Affiliations
    Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853

    Graduate Field of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853
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  • Tianle Yang
    Affiliations
    Department of Pharmacology, Weill Medical College of Cornell University, New York, New York 10021
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  • Anthony A. Sauve
    Affiliations
    Department of Pharmacology, Weill Medical College of Cornell University, New York, New York 10021
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  • W. Lee Kraus
    Correspondence
    To whom correspondence should be addressed: Dept. of Molecular Biology and Genetics, Cornell University, 465 Biotechnology Bldg., Ithaca, NY 14853. Tel.: 607-255-6087; Fax: 607-255-6249
    Affiliations
    Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853

    Graduate Field of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853

    Department of Pharmacology, Weill Medical College of Cornell University, New York, New York 10021
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grants R01 DK069710 (NIDDK, to W. L. K.) and R01 DK73466 (NIDDK, to A. A. S.). This work was also supported by grants from the Endocrine Society (to W. L. K.) and the Ellison Medical Foundation (to A. A. S.), postdoctoral fellowships from the New York State Health Research Science Board (to T. Z.) and the American Heart Association (AHA; to M. J. G.), and predoctoral fellowships from the Alfred P. Sloan Foundation (to J. G. B.) and the AHA (to K. M. F.).
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1 and Figs. S1–S6.
Open AccessPublished:May 28, 2009DOI:https://doi.org/10.1074/jbc.M109.016469
      In mammals, nic o tin a mide phosphoribosyltransferase (NAMPT) and nic o tin a mide mononucleotide ad en y lyltransferase 1 (NMNAT-1) constitute a nuclear NAD+ salvage pathway which regulates the functions of NAD+-de pend ent enzymes such as the protein deacetylase SIRT1. One of the major functions of SIRT1 is to regulate target gene transcription through modification of chromatin-associated proteins. However, little is known about the molecular mechanisms by which NAD+ biosynthetic enzymes regulate SIRT1 activity to control gene transcription in the nucleus. In this study we show that stable short hairpin RNA-mediated knockdown of NAMPT or NMNAT-1 in MCF-7 breast cancer cells reduces total cellular NAD+ levels and alters global patterns of gene expression. Furthermore, we show that SIRT1 plays a key role in mediating the gene regulatory effects of NAMPT and NMNAT-1. Specifically, we found that SIRT1 binds to the promoters of genes commonly regulated by NAMPT, NMNAT-1, and SIRT1 and that SIRT1 histone deacetylase activity is regulated by NAMPT and NMNAT-1 at these promoters. Most significantly, NMNAT-1 interacts with, and is recruited to target gene promoters by SIRT1. Collectively, our results reveal a mechanism for the direct control of SIRT1 deacetylase activity at a set of target gene promoters by NMNAT-1. This mechanism, in collaboration with NAMPT-de pend ent regulation of nuclear NAD+ production, establishes an important pathway for transcription regulation by NAD+.
      Nicotinamide adenine dinucleotide (NAD+), a coenzyme in metabolic processes and redox reactions, is an important signaling molecule. NAD+ is (i) a substrate for mono- and poly-ADP-ribosylation of proteins, (ii) required for NAD+-dependent protein deacetylation, and (iii) a precursor for calcium mobilizing agents (
      • Berger F.
      • Ramírez-Hernández M.H.
      • Ziegler M.
      ). As a signaling molecule, NAD+ is consumed as a donor of ADP-ribose, releasing nicotinamide (NAM)
      The abbreviations used are: NAM
      nicotinamide
      ChIP
      chromatin immunoprecipitation
      NAMPT
      nicotinamide phosphoribosyltransferase
      NMN
      nicotinamide mononucleotide
      NMNAT-1
      nicotinamide mononucleotide adenylyltransferase 1
      qPCR
      quantitative real-time PCR
      RT
      reverse transcription
      PARP
      poly(ADP-ribose) polymerase
      GO
      gene ontology
      GST
      glutathione S-transferase
      shRNA
      short hairpin RNA
      HPLC
      high performance liquid chromatography
      Luc
      luciferase.
      2The abbreviations used are: NAM
      nicotinamide
      ChIP
      chromatin immunoprecipitation
      NAMPT
      nicotinamide phosphoribosyltransferase
      NMN
      nicotinamide mononucleotide
      NMNAT-1
      nicotinamide mononucleotide adenylyltransferase 1
      qPCR
      quantitative real-time PCR
      RT
      reverse transcription
      PARP
      poly(ADP-ribose) polymerase
      GO
      gene ontology
      GST
      glutathione S-transferase
      shRNA
      short hairpin RNA
      HPLC
      high performance liquid chromatography
      Luc
      luciferase.
      as a byproduct. Consequently, resynthesis of NAD+ is crucial for maintaining the functions of a wide variety of NAD+-dependent enzymes in the cytoplasm and nucleus.
      In mammalian cells the enzymes nicotinamide phosphoribosyltransferase (NAMPT) and nicotinamide mononucleotide adenylyltransferase (NMNAT) constitute an NAD+ salvage/recycling pathway using NAM as the precursor (see Fig. 1A) (
      • Rongvaux A.
      • Andris F.
      • Van Gool F.
      • Leo O.
      ). NAMPT, a unique enzyme encoded by a single gene, catalyzes the conversion of NAM to nicotinamide mononucleotide (NMN). NAMPT localizes to both the cytosol and nucleus (
      • Rongvaux A.
      • Shea R.J.
      • Mulks M.H.
      • Gigot D.
      • Urbain J.
      • Leo O.
      • Andris F.
      ,
      • Kitani T.
      • Okuno S.
      • Fujisawa H.
      ).
      K. M. Frizzell and W. L. Kraus, unpublished information.
      3K. M. Frizzell and W. L. Kraus, unpublished information.
      Interestingly, an extracellular form of NAMPT has also been described, although controversy exists regarding its function (
      • Imai S.I.
      ,
      • Garten A.
      • Petzold S.
      • Körner A.
      • Imai S.
      • Kiess W.
      ). NMN produced by NAMPT is further converted into NAD+ by NMNAT. Three NMNAT enzymes encoded by distinct genes are found in mammals (
      • Emanuelli M.
      • Carnevali F.
      • Saccucci F.
      • Pierella F.
      • Amici A.
      • Raffaelli N.
      • Magni G.
      ,
      • Raffaelli N.
      • Sorci L.
      • Amici A.
      • Emanuelli M.
      • Mazzola F.
      • Magni G.
      ,
      • Schweiger M.
      • Hennig K.
      • Lerner F.
      • Niere M.
      • Hirsch-Kauffmann M.
      • Specht T.
      • Weise C.
      • Oei S.L.
      • Ziegler M.
      ,
      • Yalowitz J.A.
      • Xiao S.
      • Biju M.P.
      • Antony A.C.
      • Cummings O.W.
      • Deeg M.A.
      • Jayaram H.N.P.
      ). Among them, NMNAT-1 is localized exclusively in the nucleus, whereas NMNAT-2 and NMNAT-3 are found in the Golgi and mitochondria, respectively (
      • Berger F.
      • Lau C.
      • Dahlmann M.
      • Ziegler M.
      ). In the nucleus, NAMPT and NMNAT-1 form a nuclear NAD+ salvage pathway that supplies NAD+ as a substrate for a variety of NAD+-dependent enzymes, including the protein deacetylase SIRT1 and the poly(ADP-ribose) polymerase PARP-1 (Fig. 1A).
      Figure thumbnail gr1
      FIGURE 1Enzymes in the nuclear NAD+ salvage pathway regulate cellular NAD+ levels in MCF-7 cells. A, the nuclear NAD+ salvage pathway produces NAD+ for protein deacetylation by SIRT1. PRPP, phosphoribosylpyrophosphate; OAADPR, O-acetyl-ADP-ribose. B, stable shRNA-mediated knockdown of NMNAT-1 and NAMPT in MCF-7 cells. The Luc shRNA sequence was used as a control. NMNAT-1 and NAMPT protein levels were determined by Western blot analysis. C, total cellular NAD+ levels in control and NAMPT or NMNAT-1 knockdown cells. The concentrations of NAD+ in whole cell extracts were measured using a quantitative HPLC/mass spectrometry method with 18O standards. Error bars, S.E.; n = 8 independent biological replicates. *, significantly different from NAD+ levels in Luc control cells, p < 0.02 (Student's t test).
      Many recent studies have examined the biological functions of NAMPT (
      • Revollo J.R.
      • Grimm A.A.
      • Imai S.
      ,
      • Yang H.
      • Yang T.
      • Baur J.A.
      • Perez E.
      • Matsui T.
      • Carmona J.J.
      • Lamming D.W.
      • Souza-Pinto N.C.
      • Bohr V.A.
      • Rosenzweig A.
      • de Cabo R.
      • Sauve A.A.
      • Sinclair D.A.
      ,
      • van der Veer E.
      • Ho C.
      • O'Neil C.
      • Barbosa N.
      • Scott R.
      • Cregan S.P.
      • Pickering J.G.
      ,
      • van der Veer E.
      • Nong Z.
      • O'Neil C.
      • Urquhart B.
      • Freeman D.
      • Pickering J.G.
      ,
      • Revollo J.R.
      • Körner A.
      • Mills K.F.
      • Satoh A.
      • Wang T.
      • Garten A.
      • Dasgupta B.
      • Sasaki Y.
      • Wolberger C.
      • Townsend R.R.
      • Milbrandt J.
      • Kiess W.
      • Imai S.
      ,
      • Fulco M.
      • Cen Y.
      • Zhao P.
      • Hoffman E.P.
      • McBurney M.W.
      • Sauve A.A.
      • Sartorelli V.
      ,
      • Dvir-Ginzberg M.
      • Gagarina V.
      • Lee E.J.
      • Hall D.J.
      ). These studies found that NAMPT expression is regulated by nutrients and stress in a number of human cell lines and primary rat tissues (
      • Yang H.
      • Yang T.
      • Baur J.A.
      • Perez E.
      • Matsui T.
      • Carmona J.J.
      • Lamming D.W.
      • Souza-Pinto N.C.
      • Bohr V.A.
      • Rosenzweig A.
      • de Cabo R.
      • Sauve A.A.
      • Sinclair D.A.
      ). Increased NAMPT levels protect cells against genotoxic stress through regulation of NAD+ levels and SIRT3 and SIRT4 functions in mitochondria (
      • Yang H.
      • Yang T.
      • Baur J.A.
      • Perez E.
      • Matsui T.
      • Carmona J.J.
      • Lamming D.W.
      • Souza-Pinto N.C.
      • Bohr V.A.
      • Rosenzweig A.
      • de Cabo R.
      • Sauve A.A.
      • Sinclair D.A.
      ). Nutrient restriction also stimulates NAMPT expression in skeletal myoblasts (
      • Fulco M.
      • Cen Y.
      • Zhao P.
      • Hoffman E.P.
      • McBurney M.W.
      • Sauve A.A.
      • Sartorelli V.
      ), leading to modulation of cellular NAD+/NADH ratio as well as NAM levels and to SIRT1-dependent impairment of myoblast differentiation (
      • Fulco M.
      • Cen Y.
      • Zhao P.
      • Hoffman E.P.
      • McBurney M.W.
      • Sauve A.A.
      • Sartorelli V.
      ). Similarly, NAMPT is up-regulated during human vascular smooth muscle cell maturation (
      • van der Veer E.
      • Nong Z.
      • O'Neil C.
      • Urquhart B.
      • Freeman D.
      • Pickering J.G.
      ). This process is accompanied by increased cellular NAD+ levels and requires NAD+-dependent deacetylase activity (
      • van der Veer E.
      • Nong Z.
      • O'Neil C.
      • Urquhart B.
      • Freeman D.
      • Pickering J.G.
      ). Intriguingly, NAMPT levels decline in aging smooth muscle cells, and ectopic expression of NAMPT delays smooth muscle cell senescence in a SIRT1-dependent pathway (
      • van der Veer E.
      • Ho C.
      • O'Neil C.
      • Barbosa N.
      • Scott R.
      • Cregan S.P.
      • Pickering J.G.
      ). Together, these studies illustrate a critical role of NAMPT in muscle cell differentiation, maturation, and senescence. Consistent with these observations, knock-out mouse studies indicate that NAMPT is essential for early embryo development (
      • Revollo J.R.
      • Körner A.
      • Mills K.F.
      • Satoh A.
      • Wang T.
      • Garten A.
      • Dasgupta B.
      • Sasaki Y.
      • Wolberger C.
      • Townsend R.R.
      • Milbrandt J.
      • Kiess W.
      • Imai S.
      ) as well as lymphocyte differentiation (
      • Rongvaux A.
      • Galli M.
      • Denanglaire S.
      • Van Gool F.
      • Drèze P.L.
      • Szpirer C.
      • Bureau F.
      • Andris F.
      • Leo O.
      ). Recent studies have also shown that NAMPT is required to modulate circadian gene expression (
      • Nakahata Y.
      • Sahar S.
      • Astarita G.
      • Kaluzova M.
      • Sassone-Corsi P.
      ,
      • Ramsey K.M.
      • Yoshino J.
      • Brace C.S.
      • Abrassart D.
      • Kobayashi Y.
      • Marcheva B.
      • Hong H.K.
      • Chong J.L.
      • Buhr E.D.
      • Lee C.
      • Takahashi J.S.
      • Imai S.
      • Bass J.
      ).
      In comparison to NAMPT, our knowledge of the physiological functions of NMNAT-1 is limited to observations made using the Wallerian degeneration slow (WldS) mouse model. The axonal protective phenotype of these mice results from overexpression of a chimeric nuclear protein WldS with NMNAT-1 activity (
      • Conforti L.
      • Tarlton A.
      • Mack T.G.
      • Mi W.
      • Buckmaster E.A.
      • Wagner D.
      • Perry V.H.
      • Coleman M.P.
      ,
      • Mack T.G.
      • Reiner M.
      • Beirowski B.
      • Mi W.
      • Emanuelli M.
      • Wagner D.
      • Thomson D.
      • Gillingwater T.
      • Court F.
      • Conforti L.
      • Fernando F.S.
      • Tarlton A.
      • Andressen C.
      • Addicks K.
      • Magni G.
      • Ribchester R.R.
      • Perry V.H.
      • Coleman M.P.
      ). Both mammalian NMNAT-1 and Drosophila NMNAT exhibit neuronal protective activity, although in some studies with reduced efficacy compared to WldS protein (
      • Araki T.
      • Sasaki Y.
      • Milbrandt J.
      ,
      • Wang J.
      • Zhai Q.
      • Chen Y.
      • Lin E.
      • Gu W.
      • McBurney M.W.
      • He Z.
      ,
      • Conforti L.
      • Fang G.
      • Beirowski B.
      • Wang M.S.
      • Sorci L.
      • Asress S.
      • Adalbert R.
      • Silva A.
      • Bridge K.
      • Huang X.P.
      • Magni G.
      • Glass J.D.
      • Coleman M.P.
      ,
      • Zhai R.G.
      • Zhang F.
      • Hiesinger P.R.
      • Cao Y.
      • Haueter C.M.
      • Bellen H.J.
      ,
      • Zhai R.G.
      • Cao Y.
      • Hiesinger P.R.
      • Zhou Y.
      • Mehta S.Q.
      • Schulze K.L.
      • Verstreken P.
      • Bellen H.J.
      ,
      • MacDonald J.M.
      • Beach M.G.
      • Porpiglia E.
      • Sheehan A.E.
      • Watts R.J.
      • Freeman M.R.
      ,
      • Hoopfer E.D.
      • McLaughlin T.
      • Watts R.J.
      • Schuldiner O.
      • O'Leary D.D.
      • Luo L.
      ). Controversy exists over the mechanism of WldS action; in some experimental systems NMNAT enzymatic activity is critical for the neuronal protection phenotype (
      • Araki T.
      • Sasaki Y.
      • Milbrandt J.
      ,
      • Wang J.
      • Zhai Q.
      • Chen Y.
      • Lin E.
      • Gu W.
      • McBurney M.W.
      • He Z.
      ), whereas in other systems mechanisms independent of the enzymatic activity have been proposed (
      • Conforti L.
      • Fang G.
      • Beirowski B.
      • Wang M.S.
      • Sorci L.
      • Asress S.
      • Adalbert R.
      • Silva A.
      • Bridge K.
      • Huang X.P.
      • Magni G.
      • Glass J.D.
      • Coleman M.P.
      ,
      • Zhai R.G.
      • Zhang F.
      • Hiesinger P.R.
      • Cao Y.
      • Haueter C.M.
      • Bellen H.J.
      ,
      • Zhai R.G.
      • Cao Y.
      • Hiesinger P.R.
      • Zhou Y.
      • Mehta S.Q.
      • Schulze K.L.
      • Verstreken P.
      • Bellen H.J.
      ,
      • Fainzilber M.
      • Twiss J.L.
      ).
      The most important mediators of NAMPT and NMNAT-1 actions identified so far are the sirtuin family of NAD+-dependent enzymes, especially SIRT1 (
      • Revollo J.R.
      • Grimm A.A.
      • Imai S.
      ,
      • Yang H.
      • Yang T.
      • Baur J.A.
      • Perez E.
      • Matsui T.
      • Carmona J.J.
      • Lamming D.W.
      • Souza-Pinto N.C.
      • Bohr V.A.
      • Rosenzweig A.
      • de Cabo R.
      • Sauve A.A.
      • Sinclair D.A.
      ,
      • van der Veer E.
      • Ho C.
      • O'Neil C.
      • Barbosa N.
      • Scott R.
      • Cregan S.P.
      • Pickering J.G.
      ,
      • van der Veer E.
      • Nong Z.
      • O'Neil C.
      • Urquhart B.
      • Freeman D.
      • Pickering J.G.
      ,
      • Fulco M.
      • Cen Y.
      • Zhao P.
      • Hoffman E.P.
      • McBurney M.W.
      • Sauve A.A.
      • Sartorelli V.
      ,
      • Dvir-Ginzberg M.
      • Gagarina V.
      • Lee E.J.
      • Hall D.J.
      ,
      • Araki T.
      • Sasaki Y.
      • Milbrandt J.
      ). SIRT1 is a nuclear NAD+-dependent deacetylase that connects cell metabolism to transcriptional regulation (
      • Guarente L.
      ). SIRT1 regulates chromatin structure and gene transcription through modification of chromatin-associated proteins, including histones, transcription factors, and coregulators as well as components of the basal transcriptional machinery (
      • Michan S.
      • Sinclair D.
      ,
      • Feige J.N.
      • Auwerx J.
      ,
      • Yang T.
      • Fu M.
      • Pestell R.
      • Sauve A.A.
      ). SIRT1 directly interacts with DNA binding transcription factors and coregulators and is recruited to gene promoters through these interactions. Many of these same factors are direct targets of deacetylation by SIRT1, including p53, Ku70, NF-κB, the FOXO family of transcription factors, liver X receptor, estrogen receptor α, SOX9, PGC-1α, and p300 (
      • Dvir-Ginzberg M.
      • Gagarina V.
      • Lee E.J.
      • Hall D.J.
      ,
      • Michan S.
      • Sinclair D.
      ,
      • Feige J.N.
      • Auwerx J.
      ,
      • Kim M.Y.
      • Woo E.M.
      • Chong Y.T.
      • Homenko D.R.
      • Kraus W.L.
      ). Once at gene promoters, SIRT1 can modify additional chromatin-associated proteins, including histones and other coregulators, to control the transcriptional outcome (
      • Dvir-Ginzberg M.
      • Gagarina V.
      • Lee E.J.
      • Hall D.J.
      ,
      • Feige J.N.
      • Auwerx J.
      ). Transcription regulation by SIRT1 can be either activation or repression, depending upon the specific factors involved. Interestingly, a recent study has demonstrated that DNA damage induces global redistribution of SIRT1 on chromatin, leading to transcriptional deregulation of SIRT1-associated genes (
      • Oberdoerffer P.
      • Michan S.
      • McVay M.
      • Mostoslavsky R.
      • Vann J.
      • Park S.K.
      • Hartlerode A.
      • Stegmuller J.
      • Hafner A.
      • Loerch P.
      • Wright S.M.
      • Mills K.D.
      • Bonni A.
      • Yankner B.A.
      • Scully R.
      • Prolla T.A.
      • Alt F.W.
      • Sinclair D.A.
      ). Overall, SIRT1-dependent transcriptional regulation plays a key role in cell defense, survival, metabolism, and cellular signaling responses.
      Mounting evidence suggests that NAD+ biosynthesis has a broad impact on cellular functions through transcription regulation. Of particular focus is the role of NAMPT in controlling SIRT1 activity through intracellular as well as systemic NAD+ production (
      • Imai S.I.
      ,
      • Garten A.
      • Petzold S.
      • Körner A.
      • Imai S.
      • Kiess W.
      ). Many of the functions of NAMPT are mediated by SIRT1, presumably through changes in NAD+ and possibly NAM levels (
      • Revollo J.R.
      • Grimm A.A.
      • Imai S.
      ,
      • van der Veer E.
      • Ho C.
      • O'Neil C.
      • Barbosa N.
      • Scott R.
      • Cregan S.P.
      • Pickering J.G.
      ,
      • van der Veer E.
      • Nong Z.
      • O'Neil C.
      • Urquhart B.
      • Freeman D.
      • Pickering J.G.
      ,
      • Fulco M.
      • Cen Y.
      • Zhao P.
      • Hoffman E.P.
      • McBurney M.W.
      • Sauve A.A.
      • Sartorelli V.
      ). However, details on the regulatory events that occur at gene promoters in response to NAD+ production are not clear. In this study we focused on the target genes regulated by the mammalian NAD+ salvage pathway and explored the molecular mechanism that connects the NAD+ biosynthetic enzymes to SIRT1-dependent transcriptional regulation. Our results reveal a mechanism for the direct control of SIRT1 deacetylase activity at a set of target gene promoters by NMNAT-1, in collaboration with NAMPT.

      DISCUSSION

      In this study we examined the gene regulatory function of two enzymes in the nuclear NAD+ biosynthetic pathway, NAMPT and NMNAT-1, as well as the client nuclear NAD+-dependent protein deacetylase, SIRT1. Using shRNA-mediated knockdown and expression microarray analyses, we identified a set of genes commonly regulated by NAMPT, NMNAT-1, and SIRT1 in MCF-7 cells. Using ChIP assays, we showed that SIRT1 binds specifically to the promoter regions of these genes. Although we have not examined the mode of SIRT1 recruitment in our studies, previous studies have shown that SIRT1 can be recruited to gene promoters through direct interaction with sequence-specific transcription factors (e.g. FOXO family members, NF-κB, p53, SOX9) as well as transcription coregulators (e.g. p300, NCoR, SMRT) (
      • Dvir-Ginzberg M.
      • Gagarina V.
      • Lee E.J.
      • Hall D.J.
      ,
      • Feige J.N.
      • Auwerx J.
      ). In our study we showed that the deacetylase activity of SIRT1 at target gene promoters is controlled by NAMPT and NMNAT-1, establishing one possible mechanism for NAMPT and NMNAT-1-dependent transcription regulation. Interestingly, we found that NMNAT-1 is recruited to target gene promoters through interaction with SIRT1. This colocalization of NMNAT-1 and SIRT1 on chromatin may provide a unique mechanism for NAD+-dependent transcriptional regulation (Fig. 7).
      Figure thumbnail gr7
      FIGURE 7A model for the regulation of SIRT1 activity at target gene promoters by NAMPT and NMNAT-1. SIRT1 binds to target gene promoters and regulates the acetylation status of transcription factors, histones, and other chromatin-associated proteins in an NAD+-dependent manner. Both NAMPT and NMNAT-1 localize to the nucleus and constitute an NAD+ recycling pathway. Nuclear NAD+ production by NAMPT and NMNAT-1 supports SIRT1 deacetylase activity. In addition, NMNAT-1 interacts with SIRT1 and is recruited to SIRT1 target gene promoters. Interactions between NMNAT-1 and SIRT1 on chromatin may underlie novel mechanisms for transcriptional regulation by SIRT1. TF, transcription factor. CoReg, coregulator.
      A somewhat surprising observation from our expression microarray analysis is that the gene sets regulated by NAMPT or NMNAT-1 show only a moderate overlap (15–20%, Fig. 2, A and B). The enzymes are in a linear NAD+ biosynthetic pathway, and their knockdown have similar effects on cellular NAD+ levels (Fig. 1C). Therefore, genes that are regulated by nuclear NAD+ levels are likely to respond to both enzymes in a similar way. However, both NAMPT and NMNAT-1 may have gene regulatory activities independent of NAD+ production in the nucleus. NAMPT is found in cytoplasm, including mitochondria (
      • Rongvaux A.
      • Shea R.J.
      • Mulks M.H.
      • Gigot D.
      • Urbain J.
      • Leo O.
      • Andris F.
      ,
      • Kitani T.
      • Okuno S.
      • Fujisawa H.
      ,
      • Yang H.
      • Yang T.
      • Baur J.A.
      • Perez E.
      • Matsui T.
      • Carmona J.J.
      • Lamming D.W.
      • Souza-Pinto N.C.
      • Bohr V.A.
      • Rosenzweig A.
      • de Cabo R.
      • Sauve A.A.
      • Sinclair D.A.
      ), where it can modulate NAD+ levels and may impact gene expression independent of the nuclear NAD+ pathway. Importantly, one consequence of NAMPT enzymatic activity is to remove NAM (
      • Fulco M.
      • Cen Y.
      • Zhao P.
      • Hoffman E.P.
      • McBurney M.W.
      • Sauve A.A.
      • Sartorelli V.
      ), an inhibitor of sirtuin and PARP activities. Direct measurement of NAM failed to show any changes in total cellular NAM levels in our NAMPT or NMNAT-1 knockdown cells (data not shown). However, we cannot rule out the possibility that some effects of NAMPT on gene expression are mediated through NAM removal in a microenvironment. NMNAT-1 is an exclusively nuclear protein and is recruited to gene promoters (Fig. 6A). It interacts with nuclear proteins such as SIRT1 (Fig. 6, C and D) and PARP-1 (
      • Berger F.
      • Lau C.
      • Ziegler M.
      ).
      T. Zhang and W. L. Kraus unpublished data.
      The unique localization and interaction partners of NMNAT-1 may contribute to gene regulation independent of nuclear NAD+ production. Based on these considerations, for the mechanistic studies we focused on a subset of genes that are commonly regulated by NAMPT, NMNAT-1, and SIRT1 and are, therefore, most likely targets of transcription regulation by both the NAD+ salvage pathway and SIRT1.
      Previous studies have shown that the cellular functions of SIRT1 are regulated by NAMPT and NMNAT-1 (
      • Revollo J.R.
      • Grimm A.A.
      • Imai S.
      ,
      • van der Veer E.
      • Ho C.
      • O'Neil C.
      • Barbosa N.
      • Scott R.
      • Cregan S.P.
      • Pickering J.G.
      ,
      • van der Veer E.
      • Nong Z.
      • O'Neil C.
      • Urquhart B.
      • Freeman D.
      • Pickering J.G.
      ,
      • Fulco M.
      • Cen Y.
      • Zhao P.
      • Hoffman E.P.
      • McBurney M.W.
      • Sauve A.A.
      • Sartorelli V.
      ,
      • Dvir-Ginzberg M.
      • Gagarina V.
      • Lee E.J.
      • Hall D.J.
      ,
      • Araki T.
      • Sasaki Y.
      • Milbrandt J.
      ). In the case of NAMPT, its actions have been linked to increases in cellular NAD+ production (
      • Revollo J.R.
      • Grimm A.A.
      • Imai S.
      ,
      • Yang H.
      • Yang T.
      • Baur J.A.
      • Perez E.
      • Matsui T.
      • Carmona J.J.
      • Lamming D.W.
      • Souza-Pinto N.C.
      • Bohr V.A.
      • Rosenzweig A.
      • de Cabo R.
      • Sauve A.A.
      • Sinclair D.A.
      ,
      • van der Veer E.
      • Ho C.
      • O'Neil C.
      • Barbosa N.
      • Scott R.
      • Cregan S.P.
      • Pickering J.G.
      ,
      • Fulco M.
      • Cen Y.
      • Zhao P.
      • Hoffman E.P.
      • McBurney M.W.
      • Sauve A.A.
      • Sartorelli V.
      ,
      • Rongvaux A.
      • Galli M.
      • Denanglaire S.
      • Van Gool F.
      • Drèze P.L.
      • Szpirer C.
      • Bureau F.
      • Andris F.
      • Leo O.
      ). Additionally, NAMPT can reduce cellular NAM levels (
      • Fulco M.
      • Cen Y.
      • Zhao P.
      • Hoffman E.P.
      • McBurney M.W.
      • Sauve A.A.
      • Sartorelli V.
      ). Both of these actions of NAMPT can positively stimulate SIRT1 activity. Relatively little was known, however, about the molecular mechanisms by which NAMPT and NMNAT-1 affects SIRT1-dependent regulation of target gene promoters. NAMPT can enhance the repressive actions of SIRT1 in a reporter gene assay (
      • Revollo J.R.
      • Grimm A.A.
      • Imai S.
      ). In human chondrocytes, SIRT1 is recruited to the promoter of a cartilage-specific gene through interactions with SOX9 (
      • Dvir-Ginzberg M.
      • Gagarina V.
      • Lee E.J.
      • Hall D.J.
      ). SIRT1 recruitment is accompanied by cofactor binding and histone modification, leading to transcription activation. NAMPT stimulates SIRT1 activity and target gene expression through NAD+ production (
      • Dvir-Ginzberg M.
      • Gagarina V.
      • Lee E.J.
      • Hall D.J.
      ). Our results build upon these previous studies to present a detailed molecular mechanism for the regulation of SIRT1 activity at endogenous target gene promoters by nuclear NAD+ biosynthetic enzymes. Both NAMPT and NMNAT-1 regulate cellular NAD+ production as well as SIRT1 activity at the promoter of common target genes, highlighting the central role of NAD+ production in SIRT1-dependent transcriptional regulation. More importantly, colocalization of NMNAT-1 and SIRT1 at target gene promoters suggests novel regulatory mechanisms dependent upon the interaction between NMNAT-1 and SIRT1.
      Why might SIRT1 recruit an NAD+-producing enzyme such as NMNAT-1 to target gene promoters for localized actions? Given the very rapid diffusion rate of small molecules in cells, the need for localized NAD+ production seems unnecessary, and the ability to accumulate promoter-localized pools of elevated NAD+ seems unlikely. Colocalization of NMNAT-1 and SIRT1 at target gene promoters may, however, regulate SIRT1 activity in several ways. For example, close proximity of NMNAT-1 and SIRT1 may facilitate more efficient NAD+ utilization by SIRT1, perhaps through a substrate channeling mechanism (
      • Srere P.A.
      ). In this regard, Grubisha et al. (
      • Grubisha O.
      • Smith B.C.
      • Denu J.M.
      ) have hypothesized that NAD+ biosynthetic enzymes may form a complex with SIRT1 and channel NAD+ directly to SIRT1, creating a microdomain of high NAD+ concentration for regulation of SIRT1 activity. Our results provide compelling evidence in support of this hypothesis. Interestingly, our results demonstrate that a 30–45% decrease in total cellular NAD+ levels upon NAMPT or NMNAT-1 knockdown (Fig. 1C) can lead to as much as a 10-fold increase in H4K16 acetylation levels at SIRT1 target gene promoters (Fig. 5D). One explanation for this observation is that the changes in nuclear NAD+ levels upon NAMPT or NMNAT-1 knockdown may well exceed those observed in the cell as a whole. Grubisha et al. (
      • Grubisha O.
      • Smith B.C.
      • Denu J.M.
      ) have previously proposed that localized NAD+ production at the site of SIRT1 function, rather than total cellular NAD+ levels, may play a more significant role in controlling SIRT1 activity.
      Another way in which the colocalization of NMNAT-1 and SIRT1 at target gene promoters may regulate SIRT1 activity is to promote allosteric interactions that enhance the enzymatic activity of either or both enzymes, as shown for NMNAT-1 and PARP-1 (
      • Berger F.
      • Lau C.
      • Ziegler M.
      ). Finally, interactions between NMNAT-1 and SIRT1 may allow for regulation by cellular signaling inputs, providing an additional level of regulatory control.
      With respect to the latter point, both NAD+ production and SIRT1 activity are regulated by a wide array of extracellular signals (
      • Guarente L.
      ,
      • Sauve A.A.
      ,
      • Sauve A.A.
      • Wolberger C.
      • Schramm V.L.
      • Boeke J.D.
      ). Stress, nutrient availability, and cellular differentiation regulate the expression of NAMPT (
      • Yang H.
      • Yang T.
      • Baur J.A.
      • Perez E.
      • Matsui T.
      • Carmona J.J.
      • Lamming D.W.
      • Souza-Pinto N.C.
      • Bohr V.A.
      • Rosenzweig A.
      • de Cabo R.
      • Sauve A.A.
      • Sinclair D.A.
      ,
      • van der Veer E.
      • Ho C.
      • O'Neil C.
      • Barbosa N.
      • Scott R.
      • Cregan S.P.
      • Pickering J.G.
      ,
      • van der Veer E.
      • Nong Z.
      • O'Neil C.
      • Urquhart B.
      • Freeman D.
      • Pickering J.G.
      ,
      • Fulco M.
      • Cen Y.
      • Zhao P.
      • Hoffman E.P.
      • McBurney M.W.
      • Sauve A.A.
      • Sartorelli V.
      ), a rate-limiting enzyme in the NAD+ recycling pathway (
      • Revollo J.R.
      • Grimm A.A.
      • Imai S.
      ). Consequently, these signals can control NMNAT-1-dependent NAD+ synthesis through regulation of NMN production. Additionally, signal inputs from protein kinases may control the interaction between NMNAT-1 and NAD+-utilizing enzymes (
      • Berger F.
      • Lau C.
      • Ziegler M.
      ), leading to dynamic regulation of NMNAT-1 recruitment to specific sites on chromatin. Our results suggest that the integrated input from these signaling pathways is likely to be an important factor in determining NAD+ production and SIRT1-dependent transcriptional regulation at target gene promoters.
      The production of small molecule substrates by nuclear metabolic enzymes for use by chromatin-modifying or transcription-regulating enzymes is an emerging theme. For example, acetyl-CoA production by a nuclear acetyl-CoA synthetase in the yeast Saccharomyces cerevisiae has been shown to regulate the activity of histone acetyltransferases (
      • Takahashi H.
      • McCaffery J.M.
      • Irizarry R.A.
      • Boeke J.D.
      ). The extent to which acetyl-CoA synthetase and other substrate-producing metabolic enzymes are, like NMNAT-1, recruited to target gene promoters has yet to be determined, but this mode of action may be a general and relatively unexplored mechanism for transcriptional control.

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