Activity of Estrogen Receptor Enhancers with Chromatin

雌激素的细胞信号在多种组织的发育、生理和病理过程中起着关键作用，这些组织包括生殖器官、乳腺、骨骼、心脏、血管、脂肪、肝脏和中枢神经系统，以及这些组织的常见疾病。雌激素的分子作用是通过雌激素受体蛋白(如ER)介导的，雌激素受体蛋白作为转录因子，通过促进转录增强子在ER结合位点的协调组装来驱动特定细胞类型的基因表达模式。过去十年的研究导致了由活性增强子转录的短非编码RNA的发现。这些增强子RNA(ERNAs)的功能还不是很清楚，但新的证据表明，eRNAs可能有助于增强子的组装和功能，以及靶基因的表达。这些观察结果使人们对信号调控的eRNAs的机制和功能产生了越来越大的兴趣，但仍然存在许多问题。我们仍然不知道有多少eRNAs是有功能的。此外，我们还不知道eRNAs(1)如何与信号调控的转录因子(如ER-)合作来调节增强子的活性，(2)调节增强子的组装和拆解动力学，(3)驱动增强子-启动子环和靶基因的激活，以及(4)调节哺乳动物体内的增强子和基因表达。 这些研究的长期目标是更好地了解雌激素信号和基因调控的分子机制。具体地说，这项建议侧重于从雌激素调节增强子转录的eRNA与连接的ER合作调节基因表达的全球模式和控制生物结果的机制。我们的广泛假设是，eRNA与配体和脱氧核糖核酸结合的ER合作，为组装多蛋白增强子复合体构建支架，该多蛋白增强子复合体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。我们建议使用一套完整的实验方法来探索ERNA介导的ER增强子组装和活性的机制，以及eRNAs在体内的生物学作用。我们的具体目标是：(1)探索雌激素调节的eRNAs和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eRNAs促进增强子活性的分子机制(目标2)；以及(3)在活体小鼠子宫中识别雌激素调节的eRNAs，并初步分析它们在增强子组装和功能中的作用(目标3)。 我们对eRNA作用于ER增强子的分子机制和功能的综合研究 将详细阐述雌激素信号通路的一个方面，这在很大程度上仍未被探索。对雌激素分子作用的了解将为预防、诊断和治疗雌激素相关疾病提供新的方法。此外，这些研究还将阐明相关转录因子对基因的调控。 2)

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)