Chaperones, chromatin, and transcriptional control by PR

描述（由申请人提供）：了解基因表达的调控对于理解发育和分化以及这些过程出错时可能出现的癌症和其他疾病至关重要。生物调节系统具有确保信号及时发生和消散的机制。在类固醇受体的情况下，激素配体的结合启动了一个精心设计的序列，最终调节靶基因的表达。这种调节通常在转录起始步骤进行，但机制基础仍然知之甚少。更不为人所知的是介导激活信号停止和生物反应系统重置的生物机制。这些研究提出了一种体外方法来阐明孕激素受体的活性周期的机制。我们已经开发了一种孕酮受体依赖的体外转录系统，使用染色质模板，并已纳入分子伴侣介导的受体回收系统的一个组成部分。伴侣活性不仅是纯化受体恢复激素结合所必需的，而且也是额外步骤所必需的。第一个目的是阐明伴侣在体外孕酮受体活性周期中的作用。有了这个激素和受体依赖的无细胞转录系统，我们还将探索染色质修饰和重塑在打开和关闭靶基因受体作用中的作用。由于不同的类固醇受体辅激活剂已被证明具有组蛋白甲基转移酶和组蛋白乙酰转移酶活性，我们将评估这些辅激活剂的受体的激素激活的贡献。我们以前的研究已经证明了从染色质模板转录绝对需要乙酰辅酶A。乙酰辅酶A是所需的preinitiation复杂的装配在所有的启动子，包括类固醇受体依赖性启动子，表明假定的乙酰化步骤是一个基本的。我们将使用一个纯化的转录系统来鉴定被乙酰化的染色质蛋白，并研究哪些蛋白在转录过程中起核心作用。总之，这些研究将提供基础知识，促进我们对类固醇受体在人类健康和疾病中作用的理解。

DESCRIPTION (provided by applicant): Understanding the regulation of gene expression is essential to a comprehension of development and differentiation, and thus, cancer and other disorders that may arise when these processes go awry. Biological regulatory systems have mechanisms to ensure the timely onset and dissipation of a signal. In the case of steroid receptors, binding of the hormonal ligand initiates an elaborate sequence that culminates in the regulation of target gene expression. This regulation is often imposed at the transcription initiation step but the mechanistic basis remains poorly understood. Even less well understood are the biological mechanisms that mediate the cessation of the activation signal and the resetting of the biological response system. These studies propose an in vitro approach to elucidate the mechanisms that govern the activity cycle of the progesterone receptor. We have developed a progesterone receptor-dependent in vitro transcription system that uses chromatin templates and have incorporated chaperone-mediated receptor recycling as an integral part of the system. Not only is chaperone activity required for restoration of hormone binding by purified receptors, it is required for an additional step as well. A first aim is to elucidate the roles of chaperones in the progesterone receptor activity cycle in vitro. With this hormone- and receptor dependent cell-free transcription system, we will also explore the roles of chromatin modification and remodeling in turning on and turning off receptor action at target genes. Since different steroid receptor coactivators have been shown to possess histone methyltransferase and histone acetyltransferase activity, we will evaluate the contribution of these coactivators to hormonal activation of receptor. Our previous studies have demonstrated an absolute requirement for acetyl CoA for transcription from chromatin templates. The acetyl CoA is required for preinitiation complex assembly at all promoters, including steroid receptor-dependent promoters, indicating that the presumptive acetylation step is a fundamental one. We will use a purified transcription system to identify the chromatin proteins that are being acetylated and investigate which of these are playing a central role in the process of transcription. Together, the studies proposed will provide fundamental knowledge that will advance our understanding of steroid receptor action in human health and disease.