Gene expression is controlled by transcription factors (TFs) that consist of DNA-binding domains (DBDs) and activation domains (ADs). The DBDs have been well-characterized, but little is known about the mechanisms by which ADs effect gene activation. Here we report that diverse ADs form phase-separated condensates with the Mediator coactivator. For the OCT4 and GCN4 TFs, we show that the ability to form phase-separated droplets with Mediator in vitro and the ability to activate genes in vivo are dependent on the same amino acid residues. For the estrogen receptor (ER), a ligand-dependent activator, we show that estrogen enhances phase separation with Mediator, again linking phase separation with gene activation. These results suggest that diverse TFs can interact with Mediator through the phase-separating capacity of their ADs and that formation of condensates with Mediator is involved in gene activation.

The synthesis of pre-mRNA by RNA polymerase II (Pol II) involves the formation of a transcription initiation complex and a transition to an elongation complex 1 – 4 . The large subunit of Pol II contains an intrinsically disordered C-terminal domain (CTD), which is phosphorylated by cyclin-dependent kinases (CDKs) during the initiation-to-elongation transition, thus influencing the CTD’s interaction with different components of the initiation or the RNA splicing apparatus ( Fig. 1a ) 5 , 6 . Recent observations suggest that this model provides only a partial picture of the effects of CTD phosphorylation. Both the transcription initiation machinery and the splicing machinery can form phase-separated condensates containing large numbers of component molecules; hundreds of Pol II and Mediator molecules are concentrated in condensates at super-enhancers 7 , 8 and large numbers of splicing factors are concentrated in nuclear speckles, some of which occur at highly active transcription sites 9 – 12 . Here we investigate whether phosphorylation of the CTD regulates its incorporation into phase-separated condensates associated with transcription initiation and splicing. We find that the hypophosphorylated Pol II CTD is incorporated into Mediator condensates and that phosphorylation by regulatory CDKs reduces this incorporation. We also find that the hyperphosphorylated CTD is preferentially incorporated into condensates formed by splicing factors. These results suggest that Pol II CTD phosphorylation drives an exchange from condensates involved in transcription initiation to those involved in RNA processing and implicates phosphorylation as a mechanism to regulate condensate preference.

The nucleus contains diverse phase-separated condensates that compartmentalize and concentrate biomolecules with distinct physicochemical properties. Here, we investigated whether condensates concentrate small-molecule cancer therapeutics such that their pharmacodynamic properties are altered. We found that antineoplastic drugs become concentrated in specific protein condensates in vitro and that this occurs through physicochemical properties independent of the drug target. This behavior was also observed in tumor cells, where drug partitioning influenced drug activity. Altering the properties of the condensate was found to affect the concentration and activity of drugs. These results suggest that selective partitioning and concentration of small molecules within condensates contributes to drug pharmacodynamics and that further understanding of this phenomenon may facilitate advances in disease therapy.