The Role of PARP-1 in Hormone-Regulated Transcription

描述(申请人提供)：对荷尔蒙信号作出反应的基因表达的协调调节是生物学中的一个基本过程。亲脂激素(如脊椎动物中的雌激素和昆虫中的蜕皮类固醇)由一系列结构多样的化合物组成，这些化合物在许多生理过程中发挥重要作用，包括生长、发育、生殖和新陈代谢。由这些激素控制的信号通路的异常会导致疾病状态，包括发育异常和癌症。亲脂激素通过核受体蛋白发挥作用，核受体蛋白在核染色质环境中作为DNA结合转录因子发挥作用。 这些研究的长期目标是检验一个广泛的假设，即NR信号通路的转录结果由受体、其相关的协同调节蛋白和染色质之间的物理和功能相互作用决定。特别是，我们将研究代谢辅助因子和信号调控转录之间的可能联系，重点是聚(ADP-核糖)聚合酶-1(PARP-1)在NR依赖的基因调控中的作用。PARP-1是一种耐人寻味的共调节蛋白，其活性受代谢辅因子烟酰胺腺嘌呤二核苷酸(NAD？)调节。我们将使用各种互补的实验系统(例如，人类细胞和果蝇幼虫)和方法(例如，生化、细胞和有机体)来研究PARP-1与NRs协同调节活性的机制。这些方法包括一种独特的生化(“无细胞”)分析，它通过在体外组装的染色质模板准确地重建激素依赖的转录，以及基于微阵列的染色质免疫沉淀分析(“芯片上的芯片”)和果蝇多线染色体的免疫荧光染色，以生成激素调节基因中因子分布的全球视图。这些研究应该会在我们分析激素调节的基因表达的方式上取得重大进展，并增加我们对这些过程在正常和疾病状态下如何发生的理解。

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.