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负责高等真核基因调控的分子机制。所采用的模型系统是人孕酮受体（PR），配体激活的转录因子的核受体超家族的成员。PR作为两种同种型天然共存：83 kD A-受体（PR-A）和99 kD B-受体（PR-B）。除了PR-B在其N-末端具有额外的164个残基B-独特序列（BUS）之外，同种型是相同的。尽管它们的序列接近同一性，但这两种受体产生明显不同的分子、生理和临床结果。这些差异的分子起源是未知的。该实验室的研究已经确定，BUS调节了每种亚型的协同DNA结合程度。还已经确定，异构体特异性协同性可以通过启动子结构来调节。因此，协同结合能的分布预测PR-A和PR-B启动子的位置与它们的转录激活谱有很好的相关性。由于只有合作受体结合耦合到有效的辅激活剂招聘（从而导致转录激活），它是假设的程度和类型的协同性可能是异构体特异性基因控制的关键生理调节。为了支持这一点，本实验室已经发现，Na+和K+是异构体特异性协同性的正和负调节剂，分别。由于PR-A和PR-B差异调节编码Na+，K+-ATP酶β 1基因的启动子，离子结合和BUS调节的协同性之间的联系可能定义异构体特异性β 1基因调控的分子机制。热力学和动力学机制的合作异构体结合在这个启动子将被确定为所述假设的测试。要求1 负责阳离子依赖性PR-A和PR-B自组装的能量学和驱动力将使用分析超离心来确定。将另外测定阳离子结合亲和力和化学计量。目的2 将使用定量足迹法和统计热力学建模来确定在Na+，K+-ATP酶启动子处的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。目的3 -将使用定量足迹法并作为阳离子类型的函数来确定负责SRC 3辅激活剂向Na+，K+-ATP酶启动子的同种型特异性募集的能量学、化学计量学和驱动力。

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.