Quantitative Dissection of Steroid Receptor Function

描述(申请人提供)：这项研究的长期目标是确定负责更高真核基因调控的机制。作为一个模型系统，我们正在研究配体激活的转录因子的类固醇受体家族的五个成员。这些蛋白质--雌激素受体、孕激素受体、糖皮质激素受体、雄激素受体和盐皮质激素受体--结构相似。然而，每个受体执行其独特的基因调控功能的基础尚不清楚，特别是在其剩余家庭成员在场的情况下。我们的研究表明，a)不同的类固醇受体在与相同的启动子结合时表现出不同的位点间协作性能量学；以及b)类固醇受体在不同的启动子上表现出不同的协作性能量学。因此，我们假设，基因被特定类固醇受体激活的程度与当在该基因的启动子上组装时受体协同作用的能量相关。因此，合作结合能量学的差异将决定每个类固醇受体成功占据启动子的程度，从而决定受体特异性基因调控的程度。在目标1中，我们将确定类固醇受体在小鼠乳腺肿瘤病毒(MMTV)启动子上组装的微观结合能。我们将使用分析超速离心法解析每个受体的DNA独立自结合特性，并将使用定量DNA酶足迹滴定来确定受体在启动子上组装的固有结合自由能和协同结合自由能。在目标2中，我们将鉴定在前列腺癌中发现的雄激素受体转录激活突变。我们将使用分析超速离心法检测它们的自结合特性，并使用足迹滴定分析这些突变体与MMTV启动子的微观结合能。在目标3中，我们将把受体的结合能与它们在细胞内的启动子占有率和转录激活能力联系起来。启动子的细胞占有率将通过染色质免疫沉淀(ChIP)分析来确定，mRNA的产生程度将通过定量RT-PCR来确定。

DESCRIPTION (provided by applicant): The long-term goal of this research is to determine the mechanisms responsible for higher eukaryotic gene regulation. As a model system we are studying the five members of the steroid receptor family of ligand-activated transcription factors. These proteins - the estrogen receptor, progesterone receptor, glucocorticoid receptor, androgen receptor and mineralocorticoid receptor - are structurally similar. However, the basis by which each receptor executes its unique gene regulatory functions is unclear, particularly so when in the presence of its remaining family members. Our research indicates that a) different steroid receptors exhibit variable energetics of inter-site cooperativity when binding to an identical promoter; and b) the energetics of cooperativity exhibited by a steroid receptor can vary at different promoters. We therefore hypothesize that the extent to which a gene is activated by a particular steroid receptor correlates with the energetics of receptor cooperativity when assembling at the promoter of that gene. Differences in cooperative binding energetics will thus dictate the degree of successful promoter occupancy by each steroid receptor, and thus the extent of receptor-specific gene regulation. In Aim 1, we will determine the microscopic binding energetics for steroid receptor assembly at the mouse mammary tumor virus (MMTV) promoter. We will resolve the DNA- independent self-association properties of each receptor using analytical ultracentrifugation, and we will determine the intrinsic and cooperative binding free energies for receptor assembly at the promoter using quantitative DNase footprint titration. In Aim 2, we will characterize androgen receptor transcriptional activation mutants identified in prostate carcinomas. We will examine their self-association properties using analytical ultracentrifugation, and we will dissect the microstate binding energetics of these mutants to the MMTV promoter using footprint titrations. In Aim 3, we will correlate the binding energetics of the receptors with their promoter occupancy and transcriptional activation ability within the cell. Cellular occupancy at the promoter will be determined by chromatin immunoprecipitation (ChIP) assays, and the extent of mRNA production will be determined by quantitative RT-PCR.