Immunofluorescence against BRD4 and MED1, coupled with DNA-FISH or RNA-FISH against SEs or SE-driven nascent transcripts, was performed in mESCs to visualize the colocalization between BRD4 or MED1 puncta and SEs. BRD4 and MED1 were endogenously tagged with mEGFP in mESCs to visualize the organization of BRD4 and MED1 and to study their dynamics by FRAP and drug treatments in live cells. ChIP-seq was performed to investigate the effect of 1,6-hexanediol treatment on the chromatin occupancy of BRD4, MED1, and RNA Pol II. Recombinant BRD4-IDR and MED1-IDR were purified to test their capacity to phase-separate in vitro. The optoIDR assay (45) was implemented to test the capacity of a section of MED1-IDR to phase-separate in live cells. Mutations were introduced into MED1-IDR to study the sequence determinants of MED1-IDR phase separation. BRD4-IDR and MED1-IDR fused to different fluorescent tags were used to demonstrate the capacity of MED1-IDR droplets to compartmentalize and concentrate BRD4-IDR. Formation of MED1-IDR droplets in a transcriptionally competent nuclear extract was used to study the ability of MED1-IDR droplets to compartmentalize and concentrate BRD4 and RNA Pol II from a complex extract. In vitro transcription assays were used to measure the effect of synthetic droplet formation on transcription. All procedures are described in detail in the supplementary materials.
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