Quantitative Dissection of Steroid Receptor Function

描述（由申请人提供）：本研究的长期目标是确定高级真核生物基因调控的机制。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、雄激素受体和矿物皮质激素受体——在结构上是相似的。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，特别是当其其余家族成员存在时。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同作用的能量；b)类固醇受体在不同的启动子上表现出的协同作用的能量学是不同的。因此，我们假设基因被特定类固醇受体激活的程度与该基因启动子组装时受体协同性的能量学相关。因此，合作结合能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。在目的1中，我们将确定小鼠乳腺肿瘤病毒（MMTV）启动子上类固醇受体组装的微观结合能量值。我们将使用分析性超离心来分析每个受体的DNA独立自结合特性，我们将使用定量DNA酶足迹滴定法来确定启动子上受体组装的内在和合作结合自由能。在Aim 2中，我们将描述前列腺癌中发现的雄激素受体转录激活突变体。我们将使用分析性超离心检查它们的自结合特性，我们将使用足迹滴定分析这些突变体与MMTV启动子的微态结合能量。在Aim 3中，我们将把受体的结合能与它们在细胞内的启动子占用率和转录激活能力联系起来。启动子上的细胞占用将通过染色质免疫沉淀（ChIP）测定，mRNA产生的程度将通过定量RT-PCR测定。

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.