Together with their co-activators and RNA polymerase II (Pol II), transcription factors (TFs) form biomolecular condensates at their target sites within super-enhancers. Although this ‘phase separation’ has been shown to be a pre-requisite for assembly of the transcription apparatus on enhancers, evidence of a functional role on enhancer activity was lacking. A recent report now reveals the physicochemical properties of enhancer assembly and cooperation in response to ligand-induced activation.
Using human breast cancer cells, Nair et al. investigated the response to 17β-oestradiol (E2), which activates ~7,000–8,000 enhancers across the genome. Of these, ~16.5% are activated robustly (hereafter referred to as MegaTrans enhancers, because they indirectly recruit megadalton-sized TF complexes). MegaTrans enhancers are characterized by higher levels of chromatin accessibility, oestrogen receptor-α (ERα), Pol II, co-activators and MegaTrans complex upon induction than weakly activated ERα-bound enhancers.
Ligand-induced activation led to the recruitment of ERα and co-activators to target enhancers, transcription of enhancer RNA (eRNA) and recruitment of condensin complexes. Recruited proteins, including ERα itself, formed condensates at MegaTrans enhancers but not at basally activated enhancers. This process was dependent on the assembly of an eRNA–ribonucleoprotein complex, as evidenced by chemical inhibition of phase separation and subsequent global run-on sequencing (GRO-seq), which showed a genome-wide decrease in eRNA transcription specifically at MegaTrans enhancers.
Focusing on the MegaTrans enhancer TFF1e, the team applied circular chromosome conformation capture sequencing (4C-seq) to assess chromatin conformation and found that E2 induced chromatin interactions with a second enhancer region located at a genomic distance of 1.9 Mb in a different topologically associating domain. This finding was supported by detailed microscopic analyses. Spatial proximity and activation of enhancer target gene expression correlated inversely, suggesting that ligand-induced proximity of MegaTrans enhancers leads to cooperative transcriptional activation of targets. Moreover, spatial proximity could be abrogated by chemical disruption of phase separation.
Under chronic E2 stimulation, condensates at MegaTrans enhancers underwent changes in their physical properties to a more “gel-like state”. These changes were associated with decreased ligand-induced proximity of MegaTrans enhancers and reduced target gene expression. While this study focused on distinguishing acute from chronic E2-induced effects on chromosomal conformation and transcriptional activation, the broader concepts of condensate formation at enhancers and subsequent cooperative gene regulation may be transferable to other TFs.
Nair, S. J. et al. Phase separation of ligand-activated enhancers licenses cooperative chromosomal enhancer assembly. Nat. Struct. Mol. Biol. 26, 193–203 (2019)