Activity of Estrogen Receptor Enhancers with Chromatin

雌激素的细胞信号传导在多种组织的发育、生理学和病理学中起关键作用，所述组织包括生殖器官、乳腺、骨、心脏、脉管系统、脂肪、肝脏和中枢神经系统，以及相同组织的常见疾病。雌激素的分子作用是通过雌激素受体蛋白（例如，ER β），其作为转录因子起作用，通过促进转录增强子在ER β结合位点的协调组装来驱动细胞类型特异性的基因表达模式。过去十年的研究发现了短的非编码RNA，它们是由活性增强子转录的。这些增强子RNA（eRNA）的功能还不清楚，但新出现的证据表明，eRNA可能有助于增强子组装和功能，以及靶基因表达。这些观察结果导致人们对信号调节eRNA的机制和功能越来越感兴趣，但仍存在许多问题。我们仍然不知道eRNA的哪一部分是有功能的。此外，我们不知道eRNA如何（1）与信号调节转录因子（如ER β）和辅助调节因子合作以调节增强子活性，（2）调节增强子组装和拆卸的动力学，（3）驱动增强子-启动子环和靶基因的激活，以及（4）调节哺乳动物增强子和体内基因表达。 这些研究的长期目标是更好地了解雌激素信号传导和基因调控的分子机制。具体而言，该提案侧重于从雌激素调节增强子转录的eRNA与配体ER β合作调节基因表达的全球模式并控制生物学结果的机制。我们广泛的假设是，eRNA与配体和DNA结合的ER β合作，为多蛋白增强子复合物的组装构建支架，该复合物驱动环到雌激素调节的靶基因，其表达决定雌激素依赖的生物反应。我们建议探索eRNA介导的ER β增强子组装和活性的机制，以及eRNA在体内的生物学作用，使用一套完整的实验方法。我们的具体目标是：（1）探索雌激素调节的eRNA与靶基因表达之间的功能联系（目标1）;（2）确定功能性雌激素调节的eRNA促进增强子活性的分子机制（目标2）;（3）在体内小鼠子宫中鉴定雌激素调节的eRNA，并对其在增强子组装和功能中的作用进行初步分析（目标3）。 我们对eRNA作用于ER β增强子的分子机制和功能进行了综合研究， 将详细阐述雌激素信号通路的一个方面，这在很大程度上仍然是未知的。雌激素分子作用的知识将为预防、诊断和治疗雌激素相关疾病提供新的方法。此外，这些研究将揭示相关转录因子的基因调控。 （二）

Cellular signaling by estrogens plays a critical role in the development, physiology, and pathology of a wide variety of tissues, including the reproductive organs, mammary glands, bone, heart, vasculature, adipose, liver, and central nervous system, as well as common diseases of the same tissues. The molecular actions of estrogens are mediated through estrogen receptor proteins (e.g., ER), which function as transcription factors that drive cell type-specific patterns of gene expression by promoting the coordinated assembly of transcriptional enhancers at ER binding sites. Studies over the past decade have led to the discovery of short noncoding RNAs that are transcribed by from active enhancers. The functions of these enhancer RNAs (eRNAs) are not well understood, but emerging evidence suggests that eRNAs may contribute to enhancer assembly and function, and target gene expression. These observations have led to a growing interest in the mechanisms and functions of signal-regulated eRNAs, but many questions remain. We still do not know what fraction of eRNAs is functional. In addition, we do not know how eRNAs (1) cooperate with signal-regulated transcription factors, like ER, and coregulators to regulate enhancer activity, (2) regulate the kinetics of enhancer assembly and disassembly, (3) drive enhancer-promoter looping and the activation of target genes, and (4) regulate mammalian enhancers and gene expression in vivo. The long-term objective of these studies is to better understand the molecular mechanisms of estrogen signaling and gene regulation. Specifically, this proposal focuses on the mechanisms by which eRNAs transcribed from estrogen-regulated enhancers cooperate with liganded ER to regulate global patterns of gene expression and control biological outcomes. Our broad hypothesis is that eRNAs cooperate with ligand- and DNA-bound ER to build a scaffold for the assembly of a multi-protein enhancer complex that drives looping to estrogen-regulated target genes, whose expression determines estrogen-dependent biological responses. We propose to explore the mechanisms of eRNA-mediated ER enhancer assembly and activity, as well as the biological roles of eRNAs in vivo, using an integrated set of experimental approaches. Our specific aims are to: (1) Explore the functional links between estrogen-regulated eRNAs and target gene expression (Aim 1); (2) Determine the molecular mechanisms by which functional estrogen- regulated eRNAs promote enhancer activity (Aim 2); and (3) Identify estrogen-regulated eRNAs in mouse uterus in vivo and perform an initial analysis of their roles in enhancer assembly and function (Aim 3). Our integrative studies on the molecular mechanisms and functions of eRNAs acting at ER enhancers will elaborate a facet of the estrogen signaling pathway that remains largely unexplored. This knowledge of the molecular actions of estrogens will suggest new ways to prevent, diagnose, and treat estrogen-related diseases. In addition, these studies will shed light on gene regulation by related transcription factors. 2)