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

Abstract

In mammals, nicotinamide phosphoribosyltransferase (NAMPT) and nicotinamide mononucleotide adenylyltransferase 1 (NMNAT-1) constitute a nuclear NAD ؉ salvage pathway which regulates the functions of NAD ؉ -dependent 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-dependent regulation of nuclear NAD ؉ production, establishes an important pathway for transcription regulation by NAD ؉ .Nicotinamide adenine dinucleotide (NAD ϩ ), a coenzyme in metabolic processes and redox reactions, is an important signaling molecule. NAD ϩ is (i) a substrate for mono-and poly-ADP-ribosylation of proteins, (ii) required for NAD ϩ -dependent protein deacetylation, and (iii) a precursor for calcium mobilizing agents (1). As a signaling molecule, NAD ϩ is consumed as a donor of ADP-ribose, releasing nicotinamide (NAM) 2 as a byproduct. Consequently, resynthesis of NAD ϩ is crucial for maintaining the functions of a wide variety of NAD ϩ -dependent enzymes in the cytoplasm and nucleus. In mammalian cells the enzymes nicotinamide phosphoribosyltransferase (NAMPT) and nicotinamide mononucleotide adenylyltransferase (NMNAT) constitute an NAD ϩ salvage/ recycling pathway using NAM as the precursor (see Fig. 1A) (2). 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 (3, 4).3 Interestingly, an extracellular form of NAMPT has also been described, although controversy exists regarding its function (5, 6). NMN produced by NAMPT is further converted into NAD ϩ by NMNAT. Three NMNAT enzymes encoded by distinct genes are found in mammals (7-10). Among them, NMNAT-1 is localized exclusively in the nucleus, whereas NMNAT-2 and NMNAT-3 are found in the Golgi and mitochondria, respectively (11). In the nucleus, NAMPT and NMNAT-1 form a nuclear NAD ϩ salvage pathway that supplies NAD ϩ as a substrate for a variety of NAD ϩ -dependent enzymes, including th...

Regulation of Poly(ADP-ribose) Polymerase-1-dependent Gene Expression through Promoter-directed Recruitment of a Nuclear NAD+ Synthase

“…pET15b and pGEX2TK for bacterial expression, pQCXIP for mammalian expression, and pSuper.Retro for shRNA-mediated knockdown) have been described previously (19,28).…”

“…Antibodies-The custom rabbit polyclonal anti-PARP-1 and anti-NMNAT-1 antisera have been described previously (19,22). Anti-FLAG M2 monoclonal antibody was from Sigma-Aldrich.…”

“…For the human NMNAT-1 and PARP-1 shRNAs, multiple target sequences were tested for each gene, and the most effective sequences were used for the studies described herein: NMNAT-1, 5Ј-AACACAAGATTCTAGTCAA-3Ј (shRNA 1); PARP-1, 5Ј-GGGCAAGCACAGTGTCAAA-3Ј (shRNA 1) and 5Ј-ACACCTCTCTACTATATAA-3Ј (shRNA 2) (19,28). Potential off-target effects of the NMNAT-1 and PARP-1 shRNAs have been assessed in detail previously and were found to be of minimal concern (19,28). An shRNA directed against firefly luciferase, 5Ј-GATATGGGCTGAATACAAA-3Ј (35), was used as a control.…”

“…The results of some studies suggest that nuclear NAD ϩ production might be a key contributing factor for these protective effects (17,18). We have recently shown that NMNAT-1 is recruited to target gene promoters through interactions with promoter-bound SIRT1, where it produces NAD ϩ that is used by SIRT1 to support its histone deacetylase activity (19). Furthermore, biochemical studies have shown that NMNAT-1 binds to automodified PARP-1 and stimulates its enzymatic activity in a phosphorylation-dependent manner (20), suggesting a signal-regulated role of NMNAT-1 in controlling PARP-1 function as well.…”

Sirtuins and inflammation: Friends or foes?

“…Mononucleotide Adenylyl Transferase (NMNAT-1), the enzyme catalyzing the last step in NAD biosynthesis from nicotinamide in mammals, has been found to bind to SIRT1 and to be recruited to selected gene promoters [59]. This close association strongly suggests a mechanism whereby neo-synthesized NAD is made directly available to SIRT1, thus providing a link between cell metabolism and the regulation of gene transcription.…”