Mechanistic Studies of Progesterone 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分子机制。所采用的模型系统是人孕激素受体（PR），它是配体激活转录因子核受体超家族的成员。PR以两种同工型共存：83 kD a受体（PR- a）和99 kD b受体（PR- b）。PR-B的同工异构体是相同的，只是在其N端多了164个B-unique残基序列（BUS）。尽管它们的序列相近，但这两种受体产生的分子、生理和临床结果却截然不同。这些差异的分子起源尚不清楚。本实验室的研究已经确定，每个异构体的合作DNA结合程度是由BUS调节的。还确定了异构体特异性协同性可以通过启动子结构进行调节。因此，协同结合能的分布预测PR-A和PR-B启动子占用率与它们的转录激活谱密切相关。由于只有协同受体结合才能与有效的协同激活子募集相结合（从而导致转录激活），因此假设协同性的程度和类型可能是亚型特异性基因控制的关键生理调节因子。为了支持这一点，本实验室发现Na+和K+分别是同型特异性协同性的正调节因子和负调节因子。由于PR-A和PR-B对编码Na+， K+- atp酶β 1基因的启动子有不同的调控，离子结合和bus调节的协同性之间的联系可能定义了一种异构体特异性β 1基因调控的分子机制。该启动子的协同异构体结合的热力学和动力学机制将被确定为所述假设的测试。目的1：利用分析式超离心确定阳离子依赖性PR-A和PR-B自组装的能量学和驱动力。阳离子结合亲和力和化学计量学也将被确定。目的2：利用定量足迹和统计热力学模型确定Na+， K+- atp酶启动子上的同工异构体特异性组装的热力学和动力学机制。这些研究将作为阳离子类型的函数进行，以评估这些离子在调节组装中的作用。目标3 - sr3共激活因子对Na+， K+- atp酶启动子的同型特异性募集的能量学，化学计量学和驱动力将通过定量足迹确定，并作为阳离子型的功能。

DESCRIPTION (provided by applicant): The long-term goal of this research is to determine the molecular mechanisms responsible for higher eukaryotic gene regulation. The model system employed is the human progesterone receptor (PR), a member of the nuclear receptor superfamily of ligand-activated transcription factors. PR co-exist naturally as two isoforms: an 83 kD A-receptor (PR-A) and a 99 kD B-receptor (PR-B). The isoforms are identical except that PR-B has an additional 164 residue B-unique sequence (BUS) at its N- terminus. Despite their near sequence identity, the two receptors generate distinctly different molecular, physiological and clinical outcomes. The molecular origins of these differences are unknown. Studies by this laboratory have determined that the extent of cooperative DNA binding seen for each isoform is modulated by BUS. It has also been determined that isoform-specific cooperativity can be regulated by promoter architecture. As a consequence, the distributions of cooperative binding energetics predict PR-A and PR-B promoter occupancies well correlated with their transcriptional activation profiles. Since only cooperative receptor binding is coupled to efficient coactivator recruitment (thus leading to transcriptional activation), it is hypothesized that the extent and type of cooperativity may be the key physiological regulator of isoform-specific gene control. In support of this, this laboratory has discovered that Na+ and K+ are positive and negative regulators of isoform-specific cooperativity, respectively. Since PR-A and PR-B differentially regulate the promoter encoding the Na+, K+-ATPase beta1 gene, the linkage between ion binding and BUS-modulated cooperativity may define a molecular mechanism for isoform-specific beta1 gene regulation. The thermodynamic and kinetic mechanisms of cooperative isoform binding at this promoter will be determined as a test of the stated hypothesis. Aim 1 The energetics and driving forces responsible for cation-dependent PR-A and PR-B self-assembly will be determined using analytical ultracentrifugation. The cation binding affinities and stoichiometries will additionally be determined. Aim 2 The thermodynamic and kinetic mechanisms of isoform-specific assembly at the Na+, K+- ATPase promoter will be determined using quantitative footprinting and statistical thermodynamic modeling. These studies will be carried out as a function of cation type in order to assess the role of these ions in regulating assembly. Aim 3 - The energetics, stoichiometry and driving forces responsible for isoform-specific recruitment of the SRC3 coactivator to the Na+, K+-ATPase promoter will be determined using quantitative footprinting, and as a function of cation-type.