Transcriptional control of gene expression requires a carefully orchestrated set of physical and functional interactions among DNA-binding activators, transcriptional coregulators, the RNA polymerase II transcriptional machinery, and the chromatin template. In this proposal, we explore the role of poly(ADP- ribose) polymerase-1 (PARP-1), a nucleosome-binding protein, in the chromatin-dependent control of both basal and estrogen-regulated transcription. PARP-1 has an intrinsic enzymatic activity that catalyzes the polymerization of ADP-ribose chains on target proteins from donor nicotinamide adenine dinucleotide (NAD+) molecules. Recent studies have revealed an important role for PARP-1 as a modulator of estrogen receptor-dependent transcription. Yet, among the many gene regulatory factors with enzymatic activities, PARP-1 is one of the least well characterized. In particular, the mechanisms directing PARP-1 to specific target promoters in the genome, the effects of PARP-1 on the composition and structure of promoter chromatin, and the role of nuclear NAD+ metabolism in regulating PARP-1 activity are not well understood. The long-term objective of these studies is to achieve a better understanding of the chromatin- dependent molecular mechanisms underlying the control of basal and signal-regulated transcription by PARP-1 and its associated factors. Our broad hypothesis is that the gene regulatory activity of PARP-1 is determined by (1) the local chromatin environment (e.g., chromatin composition, histone modifications), (2) physical and functional interactions among PARP-1, signal-regulated DNA-binding activators (e.g., estrogen receptor 1;ER1), and other coregulators, and (3) the availability of NAD+ in the nucleus. In this proposal, we outline a series of experiments using an integrated approach with a complementary set of biochemical, biophysical, and cell-based assays that will test the broad hypothesis noted above and address three specific aims which will determine: (1) the molecular mechanisms underlying the modulation of basal gene expression by PARP-1 and its associated factors in cells, (2) the molecular mechanisms underlying the regulation of estrogen-dependent gene expression by PARP-1 and its associated factors in cells, and (3) the biochemical mechanisms underlying the chromatin- dependent regulation of gene expression by PARP-1 and its associated factors in vitro. Collectively, the studies outlined in these aims will provide new insights into the molecular mechanisms of PARP-1's gene regulatory activity in the context of chromatin, including those relevant to ER1-dependent transcription. 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 ER1 in human disease, our studies could also lead to new ways to exploit these factors as therapeutic targets.
Statement Poly(ADP-ribose) polymerase-1 (PARP-1) and estrogen receptor alpha (ER1) are two nuclear factors that play key roles in human diseases, such as breast cancers. Understanding the molecular actions of PARP- 1 and ER1, as well as the functional interplay between these two proteins, may suggest new ways to target these factors for the treatment of breast cancers and other hormone-regulated diseases.
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