Ligand Dependent Gene Regulation By Transient Er Alpha Clustered Enhancers

Unliganded Estrogen receptor alpha (ER alpha) has been implicated in ligand-dependent gene regulation. Upon ligand exposure, ER alpha binds to several EREs relatively proximal to the pre-marked, unliganded ER alpha-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 ER alpha-bound sites, forming Ligand Dependent ER alpha 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 ER alpha 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 ER alpha. Our results establish, at TFF1 and NRIP1 locus, a direct link between ER alpha condensates, ER alpha enhancer clusters, and transient, but robust, gene expression in a ligand-dependent fashion.Author summaryER alpha occupies its cognate binding sites on the genome, upon estrogen stimulation, to bring about the signaling mediated response. However, ER alpha has been implicated in gene regulation even in the absence of hormone and its underlying mechanisms are not known. Here we show that, unliganded ER alpha-bound genomic sites are necessary for the emergence of active enhancers upon estrogen stimulation. Several ER alpha sites emerge in proximity to these unliganded ER alpha sites which loop with each other in three-dimensional nuclear space forming the enhancer:ER alpha clusters that we call Ligand dependent enhancer clusters (LDEC). These clusters are essential for expression of genes that are associated with the LDEC's as deletion of unliganded ER alpha-bound site completely perturbs the formation of clusters and expression of genes within the same TAD while clusters in neighboring TAD remain unaffected. At the mesoscale, these LDEC's appear as ER alpha condensates that emerge dynamically only upon addition of hormone. Further, the disappearance of clusters coincide with the loss of gene expression and condensate disappearance, marking the end of signaling response.