Mechanistic Studies of Progesterone Receptor Function

描述(申请人提供)：这项研究的长期目标是确定负责更高真核基因调控的分子机制。所使用的模型系统是人孕酮受体(PR)，它是配体激活的转录因子核受体超家族的成员。PR以两种亚型共存：83kD的A受体(PR-A)和99kD的B受体(PR-B)。异构体是相同的，除了PR-B在其N端有另外164个残基B-唯一序列(BUS)。尽管这两个受体的序列相近，但它们产生了截然不同的分子、生理和临床结果。这些差异的分子起源尚不清楚。该实验室的研究已经确定了每个异构体的协同DNA结合的程度是由BUS调节的。已经确定，启动子的结构可以调节异构体特异性的协同作用。因此，协同结合能量学的分布预测PR-A和PR-B启动子的占有率与它们的转录激活谱很好地相关。由于只有协同受体结合才能与有效的辅助激活因子结合(从而导致转录激活)，因此推测协同作用的程度和类型可能是异构体特异性基因控制的关键生理调节因素。为了支持这一点，该实验室发现，钠和钾分别是异构体特异性协作性的正和负调节因子。由于PR-A和PR-B对编码Na，K-ATPase Beta1基因的启动子起着不同的调控作用，离子结合和Bus调节的协同作用之间的联系可能定义了一种同型特异性的Beta1基因调控的分子机制。该启动子上的协同异构体结合的热力学和动力学机制将被确定为对所述假说的检验。目的1利用分析超速离心法确定阳离子依赖的PR-A和PR-B自组装的能量和驱动力。另外还将测定阳离子结合亲和力和化学计量比。目的2利用定量足迹和统计热力学模型研究Na，K-ATPase启动子上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-利用定量足迹和阳离子类型的函数，将确定SRC3辅活化子与Na，K-ATPase启动子的异构体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.