Mammalian genes that play prominent roles in healthy and diseased cellular states are often controlled by special DNA elements called super-enhancers (SEs). SEs are clusters of enhancers that are occupied by an unusually high density of interacting factors and drive higher levels of transcription than most typical enhancers. This high-density assembly at SEs has been shown to exhibit sharp transitions of formation and dissolution, forming in a single nucleation event and collapsing when chromatin factors or nucleation sites are deleted. These features led us to postulate that SEs are phase-separated multimolecular assemblies, also known as biomolecular condensates. Phase-separated condensates, such as the nucleolus and other membraneless cellular bodies, provide a means to compartmentalize and concentrate biochemical reactions within cells.

SEs are formed by the binding of master transcription factors (TFs) at each component enhancer, and these recruit unusually high densities of coactivators, including Mediator and BRD4. Mediator is a large (~1.2 MDa) multisubunit complex that has multiple roles in transcription, including bridging interactions between TFs and RNA polymerase II (RNA Pol II). BRD4 facilitates the release of RNA Pol II molecules from the site of transcription initiation. The presence of MED1, a subunit of Mediator, and BRD4 can be used to define SEs. We reasoned that if transcriptional condensates are formed at SEs, then MED1 and BRD4 should be visualized as discrete bodies at SE elements in cell nuclei. These bodies should exhibit behaviors described for liquid-like condensates. We investigated these possibilities by using murine embryonic stem cells (mESCs), in which SEs were originally described. Because intrinsically disordered regions (IDRs) of proteins have been implicated in condensate formation, we postulated that the large IDRs present in MED1 and BRD4 might be involved.

We found that MED1 and BRD4 occupy discrete nuclear bodies that occur at SEs in mESCs. These bodies exhibit properties of other well-studied biomolecular condensates, including rapid recovery of fluorescence after photobleaching and sensitivity to 1,6-hexanediol, which disrupts liquid-like condensates. Disruption of MED1 and BRD4 bodies by 1,6-hexanediol was accompanied by a loss of chromatin-bound MED1 and BRD4 at SEs, as well as a loss of RNA Pol II at SEs and SE-driven genes. The IDRs of both MED1 and BRD4 formed phase-separated liquid droplets in vitro, and these droplets exhibited features characteristic of condensates formed by networks of weak protein-protein interactions. The MED1-IDR droplets were found to concentrate BRD4 and RNA Pol II from transcriptionally competent nuclear extracts, which may reflect their contribution to compartmentalizing and concentrating biochemical reactions associated with transcription at SEs in cells.

Our results show that coactivators form phase-separated condensates at SEs and that SE condensates compartmentalize and concentrate the transcription apparatus at key cell-identity genes. These results have implications for the mechanisms involved in the control of genes in healthy and diseased cell states. We suggest that SE condensates facilitate the compartmentalization and concentration of transcriptional components at specific genes through the phase-separating properties of IDRs in TFs and cofactors. SE condensates may thus ensure robust transcription of genes essential to cell-identity maintenance. These properties may also explain why cancer cells acquire large SEs at driver oncogenes and …