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，在机制水平上了解甚少。类固醇反应的不当调节可导致广泛的疾病并影响肿瘤的发生和进展。类固醇受体领域的一个未解之谜是不同的类固醇受体如何在识别几乎相同的DNA序列的同时差异调节基因。类固醇控制的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的合作相互作用成为可能。这个建议解决了协同性和差异基因调控之间的功能连接使用糖皮质激素和孕酮受体作为模型系统。该建议的假设是，GR和PR激活基因的程度与在该基因的启动子上组装时GR和PR特异性协同相互作用的程度相关。目的1将集中在热力学解剖的微观结合能，定义糖皮质激素受体和HRE 2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。将在目标2中使用ChIP测定和定量RT-PCR分析体外测量的这些受体特异性能量差异的细胞相关性。目的3将本研究扩展到GR和PR天然启动子，通过测定体外复合结合能，细胞测量启动子占位。 公共卫生相关性：高等真核生物基因调控是一个复杂的过程，与许多疾病和癌症有关。阐明真核生物基因调控的机制，将提供一个正确的调节如何发生的理解，并将开辟新的途径，为未来的药物开发，以治疗基因调控时出现的条件是不正确的控制。

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. PUBLIC HEALTH RELEVANCE: Higher eukaryotic gene regulation is a complex process that is linked to a multitude of diseases and cancers. An elucidation of the mechanisms controlling eukaryotic gene regulation will provide an understanding of how proper regulation occurs and will open new avenues for future drug developments to treat the conditions that arise when gene regulation is improperly controlled.