Determining the mechanism of how GATA4 directs ERalpha binding in osteoblasts

说明(申请人提供)：雌激素对维持骨密度和预防骨质疏松症(或低骨量)非常重要，骨质疏松症是一种影响美国2950多万人的疾病。雌激素受体α(ER�)在雌激素的作用下，通过促进成骨细胞的增殖和分化来增加骨密度。GATA4作为一种先锋因子(一种蛋白质，可以在沉默的染色质内与靶基因结合，启动染色质重塑，并帮助将其他转录因子招募到同一位点)，帮助确定成骨细胞中ER�基因调控的这些组织特异性效应。然而，GATA4何时何地与脱氧核糖核酸结合，以及GATA4如何引导ER�与增强剂结合以调节成骨细胞分化目前尚不清楚。这一建议的中心假设是，GATA4通过开放染色质使ER�与增强剂结合来促进间充质干细胞向成骨细胞的分化。这一假设将通过以下三个具体目标进行检验。目的1通过体内和体外时间调控基因的表达，确定在分化过程中，ER、�和GATA4是调控成骨细胞分化所必需的。目标2将利用最先进的高通量全基因组测序技术确定ER、�和GATA4与DNA结合的位置，以调节成骨细胞的分化。目的3阐明GATA4调节ER�与成骨细胞增强剂结合的机制。这些实验将首次描述新的成骨细胞转录因子GATA4如何调节骨骼中的雌激素生物学。这些结果将通过表征ER�在骨骼中的组织特异性而产生有价值的影响，从而进一步了解雌激素如何预防骨质疏松症。

DESCRIPTION (provided by applicant): Estrogens are important for the maintenance of bone mineral density and the prevention of osteoporosis (or low bone mass), a disease that affects more than 29.5 million people in the United States. Estrogen receptor alpha (ER�), in response to estrogens, increases bone mineral density by increasing osteoblast proliferation and differentiation. GATA4, as a pioneer factor (a protein that can bind to target DNA within silent chromatin and initiate chromatin remodeling and helps recruit other transcription factors to the same locus), helps determine these tissue-specific effects of ER� gene regulation in osteoblasts. However, when and where GATA4 binds to DNA, and how GATA4 directs ER� binding to enhancers to regulate osteoblast differentiation is currently unknown. The central hypothesis of this proposal is that GATA4 increases differentiation of mesenchymal stem cells (MSCs) to osteoblasts by opening chromatin for ER� binding to enhancers. This hypothesis will be tested with the following three specific aims. Aim 1 will determine when during differentiation ER� and GATA4 are necessary to regulate osteoblast differentiation using temporally regulated gene expression in vitro and in vivo. Aim 2 will identify where ER� and GATA4 bind to DNA to regulate osteoblast differentiation using state-of-the-art high throughput, whole-genome sequencing technology. Aim 3 will elucidate the mechanism by which GATA4 regulates ER� binding to osteoblast enhancers. Together these experiments will for the first time describe how the novel osteoblast transcription factor GATA4 regulates estrogen biology in bone. These results will have valuable impact through characterization of ER� tissue-specificity in bone and hence will further understanding of how estrogens prevent osteoporosis.