Project description:Unliganded Estrogen receptor alpha (ERa) has been implicated in ligand-dependent gene regulation. Upon ligand exposure, ERa binds to several EREs relatively proximal to the pre-marked, or persistent, ERa-bound sites and affects transient but robust gene expression. However, the underlying mechanisms are not fully understood. Here we demonstrate that upon ligand stimulation, persistent sites interact extensively, via chromatin looping, with the proximal transiently ERa-bound sites, forming Ligand Dependent ERa Enhancer Cluster in 3D (LDEC). The E2-target genes are regulated by these clustered enhancers but not by the H3K27Ac super-enhancers. Further, CRISPR-based deletion of TFF1 persistent site disrupts the formation of its LDEC resulting in the loss of E2-dependent expression of TFF1 and its neighboring genes within the same TAD. The LDEC overlap with nuclear ERa condensates that coalesce in a ligand and persistent site dependent manner. Furthermore, formation of clustered enhancers, as well as condensates, coincide with the active phase of signaling and their later disappearance results in the loss of gene expression even though persistent sites remain bound by ERa. Our results establish a direct link between ERa condensates, ERa enhancer clusters, and transient, but robust, gene expression in a ligand-dependent fashion.
Project description:Estrogen clearly prevents osteoporotic bone loss by attenuating bone resorption. The molecular basis of how this is accomplished, however, remains elusive. Here we report a critical role of osteoclastic ERa in mediating estrogen action on bone in females. We selectively ablated ERa in differentiated osteoclasts (ERa dOc/dOc). ERa dOc/dOc females, but not males, exhibited clear trabecular bone loss, similar to the osteoporotic bone phenotype in post-menopausal women. Recovery of bone loss by estrogen treatment of the ovariectomized ERa dOc/dOc females was ineffective in the trabecular areas of the long bones and lumbar vertebral bodies. Osteoclastic apoptosis, induced by estrogen, occurred simultaneously with up-regulation of Fas ligand (FasL) expression in intact trabecular bones of ERa +/+mice, but not in ERa dOc/dOc mice. ERa was also required for similar effects of estrogen and tamoxifen in cultured osteoclasts. These findings suggest that the osteoprotective actions of estrogen and SERMS are mediated at least in part through osteoclastic ERa in trabecular bone; and the life span of mature osteoclasts is regulated through activation of the Fas/FasL system. Experiment Overall Design: Wild type and osteoclast-specific Estrogen Receptor alpha knock-out mice were ovariectomized. The number of both genotypes of mice was eight. The mice of each genotypes were divided to vehicle control and estrogen treated group. Four hours after chemical treatment, the distal 5 mm of the left femurs were harvested after sacrificing by cervical dislocation and total RNAs were purified for Affymetix GeneChip microarray analysis without pooling. Therefore, this experiment consists of four groups with four replicates per group.
Project description:Estrogen Receptor a (ERa) bindning to DNA was profiled by ChIP-seq in MCF-7 and T47D cells transduced with either control sgRNA, or sgRNA targeting a specific enhancer region (enhancer588). ERa in MCF-7 and T47D control or enhancer588-targeted cells
Project description:In this study, we investigated the functions of the most common GATA3 mutation (X308_Splice) that we called “neoGATA3”. Analysis of available molecular data from >3000 breast cancer patients revealed a dysregulation of the ERa-dependent transcriptional response in tumors carrying this mutations. ChIP-Seq analysis indicated that ERa binding is reduced in neoGATA3-expressing cells, especially at distal regions, suggesting that neoGATA3 interferes with the fine-tuning of ERa-dependent gene expression.
Project description:To examine the miRNA changes that occur during osteoclast differentiation we used the miRCURY LNATM microRNA Array (7th Gen) to profile monocyte-derived osteoclasts and monocyte-derived macrophages. Of the 2046 probes present on the assay, we detected expression of ~400 (20%) miRNAs across all samples.