SIRT1-Dependent Gene Regulation Through Promoter-Directed Recruitment of a Nuclear NAD+ Synthase
Ontology highlight
ABSTRACT: In mammals, nicotinamide phosphoribosyltransferase (NAMPT) and nicotinamide mononucleotide adenylyltransferase 1 (NMNAT-1) constitute a nuclear NAD+ salvage pathway, regulating cellular functions of the NAD+-dependent deacetylase SIRT1. However, little is known about the molecular mechanisms by which NAD+ biosynthesis controls gene transcription in the nucleus. In this study, we show that stable knockdown of NAMPT or NMNAT-1 in MCF-7 breast cancer cells significantly reduced total cellular NAD+ levels. Expression microarray analyses demonstrate that both enzymes have broad and overlapping functions in gene regulation. SIRT1 is a key mediator of NAMPT- and NMNAT-1-dependent gene regulation, and is found at promoters of many of the target genes. Furthermore, SIRT1 deacetylase activity at these promoters is regulated by NAMPT and NMNAT-1. Most significantly, NMNAT-1 interacts with SIRT1 and is recruited to target gene promoters by SIRT1. Our results reveal an unexpected mechanism for the direct control of SIRT1 deacetylase activity at target gene promoters by NMNAT-1. Interactions between NMNAT-1 and SIRT1 at gene promoters may provide a platform for integration of multiple signaling pathways that regulate transcription. This SuperSeries is composed of the SubSeries listed below.
Project description:Type 2 diabetes (T2D) has become an epidemic in our modern lifestyle, likely due to calorie-rich diets overwhelming our adaptive metabolic pathways. One such pathway is mediated by nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in mammalian NAD+ biosynthesis, and the NAD+-dependent protein deacetylase SIRT1. Here we show that NAMPT-mediated NAD+ biosynthesis is severely compromised in metabolic organs by high-fat diet (HFD). Strikingly, nicotinamide mononucleotide (NMN), a product of the NAMPT reaction and a key NAD+ intermediate, ameliorates glucose intolerance by restoring NAD+ levels in HFD-induced T2D mice. NMN also enhances hepatic insulin sensitivity and restores gene expression related to oxidative stress, inflammatory response, and circadian rhythm, partly through SIRT1 activation. Furthermore, NAD+ and NAMPT levels show significant decreases in multiple organs during aging, and NMN improves glucose intolerance and lipid profiles in age-induced T2D mice. These findings provide critical insights into a novel intervention against diet- and age-induced T2D. 4 regular chow fed mice (RC1-4) vs 4 high-fat diet fed (HFD) (HFD1a-4a) mice were analyzed on one chip (Chip-A). 4 HFD mice (HFD1b-4b) vs 4 HFD-NMN treated mice (NMN1-4) were examined on the other chip (Chip-B).
Project description:Type 2 diabetes (T2D) has become an epidemic in our modern lifestyle, likely due to calorie-rich diets overwhelming our adaptive metabolic pathways. One such pathway is mediated by nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in mammalian NAD+ biosynthesis, and the NAD+-dependent protein deacetylase SIRT1. Here we show that NAMPT-mediated NAD+ biosynthesis is severely compromised in metabolic organs by high-fat diet (HFD). Strikingly, nicotinamide mononucleotide (NMN), a product of the NAMPT reaction and a key NAD+ intermediate, ameliorates glucose intolerance by restoring NAD+ levels in HFD-induced T2D mice. NMN also enhances hepatic insulin sensitivity and restores gene expression related to oxidative stress, inflammatory response, and circadian rhythm, partly through SIRT1 activation. Furthermore, NAD+ and NAMPT levels show significant decreases in multiple organs during aging, and NMN improves glucose intolerance and lipid profiles in age-induced T2D mice. These findings provide critical insights into a novel intervention against diet- and age-induced T2D.
Project description:Through integration of whole genome CRISPR screening and pan-cancer genetic dependency mapping, we identified nicotinamide phosphoribosyltransferase (NAMPT) and nicotinamide nucleotide adenylyltransferase 1 (NMNAT1) as acute myeloid leukemia (AML) dependencies governing NAD+ biosynthesis. While both NAMPT and NMNAT1 were required for AML, we found that the presence of NAD+ precursors bypassed the dependence of AML on NAMPT, but not NMNAT1, pointing to NMNAT1 as a gatekeeper of NAD+ biosynthesis. We provide evidence that reduced nuclear NAD+ upon deletion of NMNAT1 activated p53, which is due to attenuated deacetylation by SIRT6/7 in AML cells. Our findings reveal that NAD+ is a critical metabolic foundation for AML, and NMNAT1 is a novel therapeutic target for this disease.
Project description:Neural stem/progenitor cell (NSPC) proliferation and self-renewal, as well as insult-induced differentiation, decrease markedly with age, but the molecular mechanisms responsible for these declines remain unclear. Here we show that levels of NAD+ and nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme in mammalian NAD+ biosynthesis, decrease with age in the hippocampus. Ablation of Nampt in adult NSPCs reduced their pool and proliferation in vivo. The decrease in the NSPC pool during aging can be rescued by enhancing hippocampal NAD+ levels. Nampt is the main source of NSPC NAD+ levels and required for G1/S progression of the NSPC cell cycle. Nampt is also critical for oligodendrocytic lineage fate decisions through a mechanism mediated redundantly by Sirt1 and Sirt2. Ablation of Nampt in the adult NSPCs in vivo reduced NSPC-mediated oligodendrogenesis upon injury. These phenotypes recapitulate defects in NSPCs during aging, implicating Nampt-mediated NAD+ biosynthesis as a mediator of these age-associated functional declines. Total RNA obtained from neurospheres derived from postnatal hippocampi subjected to 48 hours in vitro of incubation with Nampt-specific inhibitor FK866 (10 nM, 4 samples) or vehicle (DMSO, 1:1000, 4 samples).
Project description:NMNAT-1 and PARP-1, two key enzymes in the NAD+ metabolic pathway, localize to the nucleus where integration of their enzymatic activities has the potential to control a variety of nuclear processes. Using a variety of biochemical, molecular, cell-based, and genomic assays, we show that NMNAT-1 and PARP-1 physically and functionally interact at target gene promoters in MCF-7 cells. Specifically, we show that PARP-1 recruits NMNAT-1 to promoters, where it produces NAD+ to support PARP-1 catalytic activity, but also enhances the enzymatic activity of PARP-1 independent of NAD+ production. Furthermore, using two-photon excitation microscopy, we show that NMNAT-1 catalyzes the production of NAD+ in a nuclear pool that may be distinct from other cellular compartments. In expression microarray experiments, depletion of NMNAT-1 or PARP-1 alters the expression of about 200 protein-coding genes each, with about 10% overlap between the two gene sets. NMNAT-1 enzymatic activity is required for PARP-1-dependent PARylation at the promoters of commonly regulated target genes, as well as the expression of those target genes. Collectively, our studies link the enzymatic activities of NMNAT-1 and PARP-1 to the regulation of a set of common target genes through functional interactions at target gene promoters. We examined the co-localization of NMNAT-1 and PARP-1 at RefSeq promoters in MCF-7 cells using ChIP-chip Five samples:(1) two FLAG-NMNAT-1 IP'd with FLAG antibody from MCF-7 cells ectopically expressig FLAG-NMNAT-1 and (2) three native PARP-1 IP'd from parental MCF-7 cells with PARP-1 antibody
Project description:In the present study, we developed a chemical method to produce dihydro nicotinamide mononucleotide (NMNH), which is the reduced-form of nicotinamide mononucleotide (NMN). We demonstrated that NMNH was a better nicotinamide adenine dinucleotide (NAD+) enhancer compared to NMN both in vitro and in vivo mediated by mononucleotide adenylyltransferase (NMNAT). Additionally, NMNH increased the reduced NAD (NADH) levels in cells and in mouse liver. Metabolomic analysis revealed that NMNH inhibited glycolysis and TCA cycle. In vitro experiments demonstrated that NMNH induced cell cycle arrest and suppressed cell growth. Nevertheless, NMNH treatment did not cause observable difference in mice. Taken together, our work demonstrates that NMNH is a potent NAD+ enhancer, and suppresses glycometabolism and cell growth.
Project description:NMNAT-1 and PARP-1, two key enzymes in the NAD+ metabolic pathway, localize to the nucleus where integration of their enzymatic activities has the potential to control a variety of nuclear processes. Using a variety of biochemical, molecular, cell-based, and genomic assays, we show that NMNAT-1 and PARP-1 physically and functionally interact at target gene promoters in MCF-7 cells. Specifically, we show that PARP-1 recruits NMNAT-1 to promoters, where it produces NAD+ to support PARP-1 catalytic activity, but also enhances the enzymatic activity of PARP-1 independent of NAD+ production. Furthermore, using two-photon excitation microscopy, we show that NMNAT-1 catalyzes the production of NAD+ in a nuclear pool that may be distinct from other cellular compartments. In expression microarray experiments, depletion of NMNAT-1 or PARP-1 alters the expression of about 200 protein-coding genes each, with about 10% overlap between the two gene sets. NMNAT-1 enzymatic activity is required for PARP-1-dependent PARylation at the promoters of commonly regulated target genes, as well as the expression of those target genes. Collectively, our studies link the enzymatic activities of NMNAT-1 and PARP-1 to the regulation of a set of common target genes through functional interactions at target gene promoters. We examined the co-localization of NMNAT-1 and PARP-1 at RefSeq promoters in MCF-7 cells using ChIP-chip
Project description:Neural stem/progenitor cell (NSPC) proliferation and self-renewal, as well as insult-induced differentiation, decrease markedly with age, but the molecular mechanisms responsible for these declines remain unclear. Here we show that levels of NAD+ and nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme in mammalian NAD+ biosynthesis, decrease with age in the hippocampus. Ablation of Nampt in adult NSPCs reduced their pool and proliferation in vivo. The decrease in the NSPC pool during aging can be rescued by enhancing hippocampal NAD+ levels. Nampt is the main source of NSPC NAD+ levels and required for G1/S progression of the NSPC cell cycle. Nampt is also critical for oligodendrocytic lineage fate decisions through a mechanism mediated redundantly by Sirt1 and Sirt2. Ablation of Nampt in the adult NSPCs in vivo reduced NSPC-mediated oligodendrogenesis upon injury. These phenotypes recapitulate defects in NSPCs during aging, implicating Nampt-mediated NAD+ biosynthesis as a mediator of these age-associated functional declines.
Project description:Pyruvate is a glycolytic metabolite used for energy production and macromolecule biosynthesis. However, little is known about its potential functions in tumorigenesis. Here,we report that exogenous pyruvate inhibits the proliferation of different types of cancer cells. This inhibitory effect of pyruvate on cell growth is attributed to its function as a signal molecule to repress histone gene expression, which leads to less compact chromatin structure and misregulation of genome-wide gene expression. Pyruvate represses histone gene expression by inducing the expression of NAD + biosynthesis enzyme, nicotinamide phosphoribosyltransferase (NAMPT), which increases NAD + levels and NAD + /NADH ratio. This increase activates the histone deacetylase activity of SIRT1. Chromatin immunoprecipitation analysis indicates that pyruvate enhances SIRT1 binding at histone gene promoters where it reduces histone acetylation. Although pyruvate delays cell entry into S phase, pyruvate represses histone gene expression independent of cell cycle progression. Moreover, we find that administration of pyruvate significantly reduces histone expression and retards tumor growth in xenograft mice without significant side effects. Using tissues from cervical cancer patients, we find intracellular pyruvate concentrations inversely correlate with histone protein levels. Together, we uncover a previously unknown function of pyruvate in histone gene expression and characterize pyruvate as a potential anti-cancer agent.
Project description:Mitochondrial biogenesis and function are controlled by anterograde regulatory pathways involving more than one thousand proteins encoded by nuclear genome. Transcriptional networks controlling the nuclear-encoded mitochondrial genes remain elucidated. Here we show that histone demethylase LSD1 knockout from adult mouse liver (LSD1-LKO) reduces one-third of all nuclear-encoded mitochondrial genes and decreases mitochondrial biogenesis and function. LSD1-modulated histone methylation epigenetically regulates nuclear-encoded mitochondrial genes. Furthermore, LSD1 targets methylation of nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1), the rate-limiting enzyme for nuclear NAD+ synthesis. Hepatic LSD1 knockout reduces NAD+-dependent Sirt1 and Sirt7 deacetylase activity, leading to hyperacetylation and hypofunctioning of GABP and PGC-1, the major transcriptional factor/cofactor for nuclear-encoded mitochondrial genes. Despite the reduced mitochondrial function, LSD1-LKO mice are protected from diet-induced hepatic steatosis and glucose intolerance, partially due to induction of hepatokine FGF21. Thus, LSD1 orchestrates a core regulatory network involving epigenetic modifications and NAD+ synthesis to control mitochondrial function and hepatokine production.