The Role of PARP-1 in Hormone-Regulated Transcription

描述（由申请人提供）：基因表达的转录控制需要在dna结合激活剂、转录共调节剂、RNA聚合酶II转录机制和染色质模板之间精心编排一套物理和功能相互作用。在这篇论文中，我们探讨了聚（ADP-核糖）聚合酶-1 (PARP-1)，一种核小体结合蛋白，在染色质依赖的基础和雌激素调节的转录控制中的作用。PARP-1具有内在的酶活性，可催化靶蛋白上adp核糖链与供体烟酰胺腺嘌呤二核苷酸（NAD+）分子的聚合。最近的研究表明PARP-1作为雌激素受体依赖性转录的调节剂具有重要作用。然而，在许多具有酶活性的基因调控因子中，PARP-1是最不被充分表征的一个。特别是，PARP-1在基因组中指向特定目标启动子的机制，PARP-1对启动子染色质组成和结构的影响，以及核NAD+代谢在调节PARP-1活性中的作用尚不清楚。这些研究的长期目标是更好地理解PARP-1及其相关因子控制基础和信号调节转录的染色质依赖分子机制。我们的假设是，PARP-1的基因调控活性取决于(1)局部染色质环境（如染色质组成、组蛋白修饰），(2)PARP-1、信号调节的dna结合激活因子（如雌激素受体1、ER1）和其他协同调节因子之间的物理和功能相互作用，以及(3)细胞核内NAD+的可用性。在本提案中，我们概述了一系列实验，使用综合方法与一套互补的生化，生物物理和基于细胞的分析，将测试上述广泛的假设，并解决三个具体目标，将确定：(1)细胞内PARP-1及其相关因子调控基础基因表达的分子机制；(2)细胞内PARP-1及其相关因子调控雌激素依赖性基因表达的分子机制；(3)体外PARP-1及其相关因子调控染色质依赖性基因表达的生化机制。总的来说，这些研究将为染色质背景下PARP-1基因调控活性的分子机制提供新的见解，包括那些与er1依赖性转录相关的机制。此外，这些研究将为细胞核NAD+信号在激素调节转录中的作用提供新的见解，这是一个令人兴奋的新领域，现在才刚刚开始被理解。鉴于PARP-1和ER1在人类疾病中的作用，我们的研究也可能导致利用这些因子作为治疗靶点的新方法。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.