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DNA结合激活剂、转录辅助调节剂、RNA聚合酶II转录机制和染色质模板之间精心协调的一组物理和功能相互作用。在这个建议中，我们探讨的作用，聚（ADP-核糖）聚合酶-1（PARP-1），核小体结合蛋白，在染色质依赖控制的基础和雌激素调节的转录。PARP-1具有内在酶活性，可催化来自供体烟酰胺腺嘌呤二核苷酸（NAD+）分子的靶蛋白上ADP-核糖链的聚合。最近的研究揭示了PARP-1作为雌激素受体依赖性转录的调节剂的重要作用。然而，在许多具有酶活性的基因调控因子中，PARP-1是最不好表征的因子之一。特别是，将PARP-1引导至基因组中特定靶启动子的机制、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活性中的作用尚未完全了解。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性分子机制。我们的广泛假设是PARP-1的基因调节活性由（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（ER 1）和其他辅助调节因子，以及（3）细胞核中NAD+的可用性。在本提案中，我们概述了一系列实验，这些实验采用一种综合方法，并采用一组互补的生物化学、生物物理学和基于细胞的测定法，将测试上述广泛假设，并解决三个具体目标，这些目标将确定：（1）细胞中PARP-1及其相关因子调节基础基因表达的分子机制，（2）PARP-1及其相关因子在细胞中调节雌激素依赖性基因表达的分子机制，和（3）PARP-1及其相关因子在体外对基因表达的染色质依赖性调节的生化机制。总的来说，这些目标中概述的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，包括与ER 1依赖性转录相关的分子机制。此外，这些研究将为核NAD+信号在转录调控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ER 1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。 公共卫生相关性：聚腺苷二磷酸核糖聚合酶-1（PARP-1）和雌激素受体α（ER 1）是两种核因子，在乳腺癌等人类疾病中发挥关键作用。了解PARP- 1和ER 1的分子作用，以及这两种蛋白质之间的功能相互作用，可能会提出新的方法来靶向这些因素，用于治疗乳腺癌和其他乳腺癌调节疾病。

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.