The Role of Nuclear PARPs in Signal-Regulated Transcription

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Modified Project Summary/Abstract Section ADPRylation (ADPRylation) is a reversible of post-translational modification (PTM) of proteins resulting in the covalent attachment of ADP-ribose (ADPR) units derived from β-NAD+ on a variety of substrates. It plays key roles in the control of cellular processes, such as transcriptional regulation, that drive physiological outcomes, such as adipogenesis. ADPRylation is catalyzed by the poly(ADP-ribose) polymerase (PARP) family of enzymes, including the nuclear enzyme PARP-1. Although PARP-1 and ADPRylation have historically been linked to DNA repair, growing evidence now supports their role in the regulation of gene expression. However, key gaps in knowledge remain. For example, the molecular mechanisms underlying PARP-1-mediated gene regulation have not been fully elucidated in signal-regulated biological systems. Moreover, the role of site-specific ADPRylation as a mediator of tissue-specific physiological processes is poorly understood. Our recent studies have begun to address these questions. We have shown that PARP-1 modulates transcriptional responses in preadipocytes and macrophages that are (1) linked to site-specific modification of core histones and (2) controlled by signal-regulated transcription factors (TFs) (e.g., C/EBPβ). The long-term objective of these studies is to achieve a better understanding of the molecular, biochemical, genomic mechanisms underlying the control of signal-regulated transcription by PARP-1-mediated site-specific ADPRylation of key regulatory proteins, as well as the downstream physiological consequences of these regulatory events in adipose tissue. Our broad hypothesis is that the gene regulatory activities of PARP-1 are mediated through site-specific ADPRylation of histones and TFs. We will test this hypothesis using an integrated approach with a complementary of set tools from biochemistry, molecular biology, cell biology, chemical biology, proteomics, genomics, and mouse genetics. In addition, we will test specific mechanistic hypotheses related to the role of PARP-1 in signal-regulated gene expression driven by ADPRylation of histones and C/EBPβ in preadipocytes and macrophages in fat tissue. Our specific aims are to: (1) Explore the role of site-specific histone ADPRylation in signal-regulated gene expression in preadipocytes, as well as macrophages (Aim 1); (2) Determine how dynamic ADPRylation of C/EBPβ regulates enhancer function and target gene expression in macrophages (Aim 2); and (3) Determine the effects of site-specific ADPRylation of C/EBPβ on adipogenesis (Aim 3). Collectively, these studies will provide new insights into the molecular mechanisms of gene regulation by PARP-1-mediated site-specific ADPRylation in adipogenesis. Given the important role of PARP-1 in human disease, as well its potential “drugability,” our studies could lead to new ways to exploit these factors as therapeutic targets. Furthermore, our studies have the potential to reveal new information that will shape the future of the field and may shed light more generally on the functions of ADPRylation.