Transcription Factor Interactions in Inhibin Regulation

这项研究的总体目标是在分子水平上了解转录因子及其共调节蛋白之间的相互作用如何介导生殖组织中的依赖性基因表达。 这些研究将使用编码α亚基的基因进行，α亚基是一种调节垂体促卵泡激素（FSH）合成和分泌的性腺蛋白。抑制素基因表达受FSH调节，通过涉及cAMP依赖性蛋白激酶（PKA）激活和转录因子CREB（cAMP反应元件（CRE）结合蛋白）磷酸化的途径，CREB结合至FSH α亚基启动子中的非典型CRE。 FSH的作用与孤儿核受体类固醇生成因子-1（SF-1）共同依赖并增强，SF-1结合至与α-受体结合素α亚基基因中CRE相邻的非典型靶位点。 目前还不清楚这种相互依赖的性质是什么，也不知道这些功能性相互作用是如何作为正常生殖周期中发生的激素和细胞变化的结果进行调节的。 目标1中的实验将通过研究激素刺激后CREB和SF-1的启动子占用的时间过程以及通过测试两种蛋白质之间在Eclobin α亚基启动子上的直接相互作用来解决这些问题。 目的2中的实验将研究转录共激活因子CBP和SRC-1可能通过促进包括这些共调节蛋白的高阶蛋白质复合物的形成而部分介导CREB和SF-1之间观察到的功能相互依赖性的假设。 最后，目标3中的实验将使用基于NMR的方法研究功能重要的蛋白质-DNA和蛋白质-蛋白质相互作用的结构基础。 这些研究将提供机制的见解，将增加我们的知识，卵巢基因调控在生殖周期，并将适用于了解缺陷的基因表达和调节，可能发生在生殖疾病的重要性，以人类健康。

The overall objective of this research proposal is to understand at a molecular level how interactions between transcription factors and their co-regulatory proteins mediate hormone-dependent gene expression in reproductive tissues. These studies will be performed using the gene encoding the alpha subunit of inhibin, a gonadal protein that modulates pituitary follicle-stimulating hormone (FSH) synthesis and secretion. Inhibin gene expression is regulated by FSH through a pathway involving the activation of the cAMP-dependent protein kinase (PKA) and the phosphorylation of the transcription factor CREB (cAMP-response element (CRE) binding protein), which binds to an atypical CRE in the inhibin alpha subunit promoter. FSH action is co-dependent on and augmented by the orphan nuclear receptor steroidogenic factor-1 (SF-1), which binds to an atypical target site adjacent to the CRE in the inhibin alpha subunit gene. It remains unknown what the nature of this interdependence is or how these functional interactions are regulated as a consequence of the hormonal and cellular changes that occur during a normal reproductive cycle. Experiments in aim 1 will address these issues by investigating the time course of promoter occupancy by CREB and SF-1 following hormone stimulation and by testing for direct interactions between the two proteins on the inhibin alpha subunit promoter. Experiments in aim 2 will investigate the hypothesis that the transcriptional coactivators CBP and SRC-1 may mediate in part the functional interdependence observed between CREB and SF-1 by facilitating formation of a higher-order protein complex including these co-regulatory proteins. Finally, experiments in aim 3 will investigate the structural basis for functionally important protein-DNA and protein-protein interactions using NMR-based approaches. These studies will provide mechanistic insights that will increase our knowledge of ovarian gene regulation during the reproductive cycle and will be applicable to understanding defects in gene expression and regulation that may occur in reproductive disease of importance to human health.