Abstract
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.
Highlights
NADϩ is required for nuclear enzymes that regulate chromatin and gene expression
Using a similar in vitro transcription assay with chromatin templates, we examined whether the nuclear NADϩ synthase nicotinamide mononucleotide adenylyltransferase (NMNAT)-1 could produce NADϩ to support the activity of Poly(ADP-ribose) polymerase-1 (PARP-1) and relieve the PARP-1-mediated repression in these assays
Using cell-free biochemical assays, we show that NMNAT-1 can produce NADϩ to support the catalytic activity of PARP-1 (Fig. 1, A–E), but can enhance the PAR synthesis and PAR chain elongation activities of PARP-1 independently of NADϩ production (Fig. 1, F and G)
Summary
NADϩ is required for nuclear enzymes that regulate chromatin and gene expression. Results: The nuclear NADϩ synthase NMNAT-1 is required for PARP-1-dependent gene regulation. Conclusion: The enzymatic activities of NMNAT-1 and PARP-1 are linked to the regulation of common target genes through functional interactions at gene promoters. 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. Several mechanisms for transcriptional regulation by chromatin-associated PARP-1 have emerged from gene-specific studies (24 –26, 28 –34) In many cases, these actions of PARP-1 require its enzymatic activity [22,23,24,25, 27,28,29,30,31,32]. Our results indicate a functional link between the enzymatic activities of NMNAT-1 and PARP-1 at gene promoters that regulates the expression of a set of common target genes
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