Estrogenic hormones, such as 17(3-estradiol (E2), play important roles in normal physiologic processes, as well as disease states. The long-term objective of these studies is to gain a better understanding of the molecular mechanisms underlying the control of gene expression by E2 acting through estrogen receptor alpha (ERa), a ligand-regulated, DMA-binding transcription factor. In particular, we are interested how the activity of ERa is modulated by its associated coregulatory proteins in the chromatin environment of the nucleus. In this regard, we are focusing our studies on poly(ADP-ribose) polymerase-1 (PARP-1), a chromatin-dependent transcriptional coregulator, and macroH2A, an ADP-ribose-binding histone variant that binds to PARP-1. PARP-1, the most abundant member of a family of poly(ADP-ribose) polymerases, is a chromatin-binding nuclear enzyme that catalyzes the polymerization of ADP-ribose chains on target proteins from donor nicotinamide adenine dinucleotide (NAD+) molecules. Recent studies have revealed important roles for PARP-1 as a coregulator of ERa. Our broad hypothesis is that the coregulatory activity of PARP- 1 at E2-regulated promoters is determined by (1) the local chromatin environment (e.g., chromatin composition, histone modifications) and (2) physical and functional interactions among PARP-1, ERa, other coregulators, and components of chromatin. We have planned a series of experiments using a multidisciplinary approach with a complementary set of biochemical, biophysical, cell-based, and genomic approaches that will test our broad hypothesis and address three specific aims: (1) Define the set of E2-regulated genes that are direct targets for coregulation by PARP-1 and macroH2A by using genomic approaches, (2) Determine the molecular mechanisms underlying the chromatin-dependent regulation of E2 target genes by PARP-1 and macroH2A by using cell-based assays, and (3) Determine the biochemical mechanisms underlying the chromatin-dependent regulation of ERa transcriptional activity by PARP-1 and macroH2A by using biochemical assays. An underlying theme in this work is the connection between nuclear NAD+ metabolism and the regulation of PARP-1 and macroH2A activity. Collectively, these studies will provide new insights into the molecular mechanisms of PARP-1's coregulatory activity, especially those relevant to ERa-dependent transcription in the context of macroH2A-containing chromatin. In addition, these studies will provide new insights into the role of nuclear NAD+ signaling in hormone-regulated transcription, an exciting new area that is only now beginning to be understood. Given the roles of PARP-1 and ERa in human disease, our studies could also lead to new ways to exploit these factors as therapeutic targets.
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