Unliganded nuclear receptors have been implicated in ligand-dependent gene regulation. However, the underlying mechanisms are not fully understood. Here we demonstrate that unliganded ERα binds to specific sites in the genome thereby pre-marking them as future functional enhancers. Upon ligand exposure, ERα binds to several EREs relatively proximal to the pre-marked, or persistent, ERα-bound sites. Interestingly, the persistent sites interact extensively, via chromatin looping, with the proximal transiently bound sites forming ERα 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 ERα 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 ERα. Our results establish the role of persistent unliganded ERα binding in priming enhancer clusters in 3D that drive transient, but robust, gene expression in a ligand-dependent fashion.

Ligand dependent gene regulation by transient ERα clustered enhancers