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. I...