Genetic variation at the 8q24 locus is linked with the greater susceptibility to prostate cancer in men of African ancestry. One such African ancestry specific rare variant, rs72725854 (A>G/T) (~6% allele frequency) has been associated with a~2-fold increase in prostate cancer risk. However, the functional relevance of this variant is unknown. Here we show that the variant rs72725854 is present in a prostate cancer-specific enhancer at 8q24 locus. Chromatin-conformation capture and dCas9 mediated enhancer blocking establish a direct regulatory link between this enhancer and lncRNAs PCAT1, PRNCR1 and PVT1. The risk allele ('T') is associated with higher expression of PCAT1, PVT1 and c-myc in prostate tumors. Further, enhancer with the risk allele gains response to androgen stimulation by recruiting the transcription factor SPDEF whereas, non-risk alleles remain non-responsive. Elevated expression of these lncRNAs and c-myc in risk allele carriers may explain their greater susceptibility to prostate cancer.

Unliganded Estrogen receptor alpha (ERα) has been implicated in ligand-dependent gene regulation. Upon ligand exposure, ERα binds to several EREs relatively proximal to the premarked, unliganded ERα-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 ERα-bound sites, forming Ligand Dependent ERα 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 ERα 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 ERα. Our results establish, at TFF1 and NRIP1 locus, a direct link between ERα condensates, ERα enhancer clusters, and transient, but robust, gene expression in a ligand-dependent fashion. Author summaryERα occupies its cognate binding sites on the genome, upon estrogen stimulation, to bring about the signaling mediated response. However, ERα has been implicated in gene regulation even in the absence of hormone and its underlying mechanisms are not known. Here we show that, unliganded ERα-bound genomic sites are necessary for the emergence of active enhancers upon estrogen stimulation. Several ERα sites emerge in proximity to these unliganded ERα sites which loop with each other in three-dimensional nuclear space forming the enhancer:ERα clusters that we call Ligand dependent enhancer PLOS Genetics | https://doi.

Unliganded nuclear receptors have been implicated in ligand-dependent gene regulation. However, the underlying mechanisms are not fully understood. Here we demonstrate that unliganded ERa binds to specific sites in the genome thereby pre-marking them as future functional enhancers. Upon ligand exposure, ERa binds to several EREs relatively proximal to the pre-marked, or persistent, ERa-bound sites. Interestingly, the persistent sites interact extensively, via chromatin looping, with the proximal transiently bound sites forming ERa clustered enhancers in 3D. CRISPR-based deletion of TFF1 persistent site disrupts the formation of its clustered enhancer resulting in the loss of E2-dependent induced expression of TFF1 and its neighboring genes within the same cluster. The clustered enhancers overlap with nuclear ERa puncta that coalesce in a ligand-dependent manner. Furthermore, formation of clustered enhancers, as well as puncta, 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 the role of persistent unliganded ERa binding in priming enhancer clusters in 3D that drive transient, but robust, gene expression in a ligand-dependent fashion.