Dissecting the Functional Energetics of the Human Glucocorticoid Receptor

描述（由申请人提供）：高等真核生物基因调控是一个复杂的过程，在机制水平上知之甚少。类固醇反应调节不当可导致多种疾病并影响肿瘤的发展和进展。在类固醇受体领域，一个悬而未决的问题是不同的类固醇受体如何在识别几乎相同的DNA序列的同时调节基因的差异。类固醇控制基因的启动子通常包含多个反应元件结合位点，从而使相邻结合的受体之间能够协同相互作用。本研究以糖皮质激素和孕激素受体为模型系统，探讨了协同性和差异基因调控之间的功能联系。该提案的假设是，GR和PR激活基因的程度与GR和PR特异性合作相互作用的程度相关，当它们聚集在该基因的启动子上时。目的1将集中在微观结合能量的热力学解剖，定义糖皮质激素受体和HRE2启动子之间的相互作用。定量足迹和统计力学分析将用于实现这些目标。这些受体特异性能量差异在体外测量的细胞相关性将在Aim 2中使用ChIP测定和定量RT-PCR分析。目的3将通过测量体外复合结合能将本研究扩展到GR和PR天然启动子。

DESCRIPTION (provided by applicant): Higher eukaryotic gene regulation is a complex process that is poorly understood at the mechanistic level. Improper regulation of steroid responses can result in a wide range of diseases and affect tumor development and progression. An unanswered question in the steroid receptor field is how the different steroid receptors differentially regulate genes while recognizing nearly identical DNA sequences. The promoters of steroid-controlled genes often contain multiple response element binding sites, thereby enabling cooperative interactions between adjacently bound receptors. This proposal addresses the functional connection between cooperativity and differential gene regulation using the glucocorticoid and progesterone receptors as a model system. The hypothesis of this proposal is that the extent to which GR and PR activate a gene correlates with the extent of GR- and PR- specific cooperative interactions when assembling on the promoter of that gene. Aim 1 will focus on a thermodynamic dissection of the microscopic binding energetics that define the interaction between the glucocorticoid receptor and HRE2 promoter. Quantitative footprints and statistical mechanical analyses will be used to carry out these Aims. The cellular relevance of these receptor- specific energetic differences measured in vitro will be analyzed in Aim 2 using ChIP assays and quantitative RT-PCR. Aim 3 will extend this study to GR and PR natural promoters by measuring in vitro composite binding energetics cellular measurements of promoter occupancies.