Coactivator condensation at super-enhancers links phase separation and gene control

Phase separation and gene control Many components of eukaryotic transcription machinery—such as transcription factors and cofactors including BRD4, subunits of the Mediator complex, and RNA polymerase II—contain intrinsically disordered low-complexity domains. Now a conceptual framework connecting the nature and behavior of their interactions to their functions in transcription regulation is emerging (see the Perspective by Plys and Kingston). Chong et al. found that low-complexity domains of transcription factors form concentrated hubs via functionally relevant dynamic, multivalent, and sequence-specific protein-protein interaction. These hubs have the potential to phase-separate at higher concentrations. Indeed, Sabari et al. showed that at super-enhancers, BRD4 and Mediator form liquid-like condensates that compartmentalize and concentrate the transcription apparatus to maintain expression of key cell-identity genes. Cho et al. further revealed the differential sensitivity of Mediator and RNA polymerase II condensates to selective transcription inhibitors and how their dynamic interactions might initiate transcription elongation. Science, this issue p. eaar2555, p. eaar3958, p. 412; see also p. 329 Phase-separated condensates compartmentalize the transcription apparatus at super-enhancers of key cell-identity genes. INTRODUCTION 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. RATIONALE 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. RESULTS 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. CONCLUSION 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 why SEs that facilitate transcriptional dysregulation in disease can be especially sensitive to transcriptional inhibitors. Phase separation of coactivators compartmentalizes and concentrates the transcription apparatus. Enhancers are gene regulatory elements bound by transcription factors that recruit coactivators and the transcription apparatus (not shown) to regulate gene expression. Super-enhancers are clusters of enhancers bound by master transcription factors that concentrate high densities of coactivators and the transcription apparatus to drive robust expression of genes that play prominent roles in cell identity. This is achieved by the phase separation of coactivators, which is driven in part by high-valency and low-affinity interactions of intrinsically disordered regions. Super-enhancers (SEs) are clusters of enhancers that cooperatively assemble a high density of the transcriptional apparatus to drive robust expression of genes with prominent roles in cell identity. Here we demonstrate that the SE-enriched transcriptional coactivators BRD4 and MED1 form nuclear puncta at SEs that exhibit properties of liquid-like condensates and are disrupted by chemicals that perturb condensates. The intrinsically disordered regions (IDRs) of BRD4 and MED1 can form phase-separated droplets, and MED1-IDR droplets can compartmentalize and concentrate the transcription apparatus from nuclear extracts. These results support the idea that coactivators form phase-separated condensates at SEs that compartmentalize and concentrate the transcription apparatus, suggest a role for coactivator IDRs in this process, and offer insights into mechanisms involved in the control of key cell-identity genes.