Examination of functional interplay between NMNAT-1 (a nuclear NAD+ synthase) and PARP-1 (a nuclear poly(ADP-ribose) polymerase)

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