The Role of PARP-1 in Hormone-Regulated Transcription

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DESCRIPTION (provided by applicant): The coordinated regulation of gene expression in response to hormonal signals is a fundamental process in biology. Lipophilic hormones (e.g., estrogens in vertebrates, and ecdysteroids in insects) comprise a structurally diverse array of compounds that play important roles in many physiological processes, including growth, development, reproduction, and metabolism. Aberrations in the signaling pathways controlled by these hormones lead to disease states, including developmental abnormalities and cancers. The lipophilic hormones exert their effects through nuclear receptor (NR) proteins that function as DNA-binding transcription factors in the chromatin environment of the nucleus. The long term objective of these studies is to test the broad hypothesis that transcriptional outcomes in NR signaling pathways are determined by physical and functional interactions among the receptors, their associated coregulatory proteins, and chromatin. In particular, we will investigate a possible link between metabolic cofactors and signal-regulated transcription, focusing on the role of poly (ADP-ribose) polymerase- 1 (PARP-1) in NR-dependent gene regulation. PARP-1 is an intriguing coregulatory protein whose activity is regulated by the metabolic cofactor nicotinamide adenine dinucleotide (NAD¿). We will use a variety of complementary experimental systems (e.g., human cells and Drosophila larvae) and approaches (e.g., biochemical, cell-based, and organismal) to examine the mechanisms underlying PARP-1 coregulatory activity with NRs. The approaches include a unique biochemical ("cell-free") assay that accurately recreates hormone-dependent transcription with chromatin templates assembled in vitro, as well as microarray-based chromatin immunoprecipitation assays ("CHIP on chips") and immunofluorescent staining of Drosophila polytene chromosomes to generate a global view of factor distribution at hormone-regulated genes. These studies should lead to significant advances in the way we analyze hormone-regulated gene expression and increase our understanding of how these processes occur in normal and disease states.