The Role of PARP-1 in Hormone-Regulated Transcription

描述(申请人提供)：基因表达的转录控制需要DNA结合激活物、转录共调节因子、RNA聚合酶II转录机制和染色质模板之间一套精心安排的物理和功能相互作用。在这个建议中，我们探索了聚(ADP-核糖)聚合酶-1(PARP-1)，一种核小体结合蛋白，在基础和雌激素调节的转录的染色质依赖的控制中的作用。PARP-1具有催化供体烟酰胺腺嘌呤二核苷酸(NAD+)分子在靶蛋白上聚合ADP-核糖链的内在酶活性。最近的研究表明，PARP-1作为雌激素受体依赖的转录调节因子具有重要作用。然而，在众多具有酶活性的基因调控因子中，PARP-1是特征最差的因子之一。特别是，PARP-1与基因组中特定的靶启动子结合的机制、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活性中的作用还不是很清楚。这些研究的长期目标是更好地理解PARP-1及其相关因素控制基础转录和信号调节转录的染色质依赖的分子机制。我们的广泛假设是，PARP-1的基因调控活性取决于(1)局部染色质环境(例如，染色质组成、组蛋白修饰)，(2)PARP-1、信号调节的DNA结合激活剂(例如，雌激素受体1；ER1)和其他辅助调节因子之间的物理和功能相互作用，以及(3)核内NAD+的可用性。在这项建议中，我们概述了一系列实验，利用一套互补的生化、生物物理和基于细胞的分析方法来测试上述广泛的假设，并解决三个特定目标，这些实验将确定：(1)PARP-1及其相关因子调控基础基因表达的分子机制；(2)PARP-1及其相关因子调控细胞内雌激素依赖基因表达的分子机制；以及(3)PARP-1及其相关因子体外对基因表达依赖染色质调控的生化机制。总之，这些目标中概述的研究将为在染色质背景下PARP-1‘S基因调控活性的分子机制提供新的见解，包括那些与ER1依赖的转录相关的机制。此外，这些研究将为核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，我们的研究也可能带来利用这些因素作为治疗靶点的新方法。 公共卫生相关性：声明多聚(ADP-核糖)聚合酶-1(PARP-1)和雌激素受体α(ER1)是两个在人类疾病(如乳腺癌)中发挥关键作用的核因子。了解PARP-1和ER1的分子作用，以及这两种蛋白之间的功能相互作用，可能会为乳腺癌和其他激素调节疾病的治疗提供新的靶向。

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.