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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 independently 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 poly(ADP-ribosyl)ation 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.

Poly(ADP-ribose) polymerase-1 (PARP-1) and poly(ADP-ribose) glycohydrolase (PARG) are enzymes that modify target proteins by the addition and removal, respectively, of ADP-ribose polymers. Although a role for PARP-1 in gene regulation has been well established, the role of PARG is less clear. To investigate how PARP-1 and PARG coordinately regulate global patterns of gene expression, we used short hairpin RNAs to stably knock down PARP-1 or PARG in MCF-7 cells followed by expression microarray analyses. Correlation analyses showed that the majority of genes affected by the knockdown of one factor were similarly affected by the knockdown of the other factor. The most robustly regulated common genes were enriched for stress-response and metabolic functions. In chromatin immunoprecipitation assays, PARP-1 and PARG localized to the promoters of positively and negatively regulated target genes. The levels of chromatin-bound PARG at a given promoter generally correlated with the levels of PARP-1 across the subset of promoters tested. For about half of the genes tested, the binding of PARP-1 at the promoter was dependent on the binding of PARG. Experiments using stable re-expression of short hairpin RNA-resistant catalytic mutants showed that PARP-1 and PARG enzymatic activities are required for some, but not all, target genes. Collectively, our results indicate that PARP-1 and PARG, nuclear enzymes with opposing enzymatic activities, localize to target promoters and act in a similar, rather than antagonistic, manner to regulate gene expression.

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(+).

Nucleosome-binding proteins act to modulate the promoter chromatin architecture and transcription of target genes. We used genomic and gene-specific approaches to show that two such factors, histone H1 and poly(ADP-ribose) polymerase-1 (PARP-1), exhibit a reciprocal pattern of chromatin binding at many RNA polymerase II-transcribed promoters. PARP-1 was enriched and H1 was depleted at these promoters. This pattern of binding was associated with actively transcribed genes. Furthermore, we showed that PARP-1 acts to exclude H1 from a subset of PARP-1-stimulated promoters, suggesting a functional interplay between PARP-1 and H1 at the level of nucleosome binding. Thus, although H1 and PARP-1 have similar nucleosome-binding properties and effects on chromatin structure in vitro, they have distinct roles in determining gene expression outcomes in vivo.