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负责高等真核基因调控的机制。作为一个模型系统，我们正在研究类固醇受体家族的配体激活的转录因子的五个成员。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-在结构上相似。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，特别是在其剩余家族成员存在的情况下。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可变的位点间协同性能量;和B）类固醇受体表现出的协同性能量在不同的启动子处可以变化。因此，我们假设基因被特定类固醇受体激活的程度与在该基因启动子处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，我们将确定类固醇受体组装在小鼠乳腺肿瘤病毒（MMTV）启动子的微观结合能。我们将使用分析性超离心来解析每个受体的DNA独立自缔合性质，并且我们将使用定量DNA酶足迹滴定来确定启动子处受体组装的内在和合作结合自由能。在目标2中，我们将描述在前列腺癌中发现的雄激素受体转录激活突变体。我们将研究他们的自缔合特性，使用分析超离心，我们将解剖这些突变体的微观结合力的MMTV启动子使用足迹滴定。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用和转录激活能力相关联。通过染色质免疫沉淀（ChIP）试验确定启动子处的细胞占有率，并通过定量RT-PCR确定mRNA产生的程度。 公共卫生相关性：这项研究的长期目标是确定负责人类基因调控的机制。作为一个模型系统，我们正在研究类固醇受体及其与复杂的启动子序列的相互作用。这项工作的结果应该揭示一个新的和更定量的方式来了解受体介导的基因调控。因此，这些研究应该为药物设计开辟一些新的途径。

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. PUBLIC HEALTH RELEVANCE: The long-term goal of this research is to determine the mechanisms responsible for human gene regulation. As a model system we are studying steroid receptors and their interactions with complex promoter sequences. The results of this work should reveal a new and more quantitative way to understand receptor-mediated gene regulation. As a result, these studies should open up a number of new avenues for drug design.