Super-enhancers (SEs) are clusters of enhancers that cooperatively assemble a high density of 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 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 control of key cell identity genes.

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 RNA-guided endonuclease Cpf1 is a promising tool for genome editing in eukaryotic cells1–7. However, the utility of the commonly used Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1) and Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1) is limited by their requirement of a TTTV protospacer adjacent motif (PAM) in the DNA substrate. To address this limitation, we performed a structure-guided mutagenesis screen to increase the targeting range of Cpf1. We engineered two AsCpf1 variants carrying the mutations S542R/K607R and S542R/K548V/N552R, which recognize TYCV and TATV PAMs, respectively, with enhanced activities in vitro and in human cells. Genome-wide assessment of off-target activity using BLISS7 assay indicated that these variants retain high DNA targeting specificity, which we further improved by introducing an additional non-PAM-interacting mutation. Introducing the identified mutations at their corresponding positions in LbCpf1 similarly altered its PAM specificity. Together, these variants increase the targeting range of Cpf1 by approximately three-fold in human coding sequences to one cleavage site per ~11 bp.

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.

Highlights d Signaling factors incorporate into Mediator condensates at super-enhancers d b-Catenin IDRs are required for both phase separation and target gene activation d Both condensate interactions and TF interactions contribute to b-catenin localization

SUMMARY Transcriptional dysregulation of the MYC oncogene is among the most frequent events in aggressive tumor cells, and this is generally accomplished by acquisition of a super-enhancer somewhere within the 2.8 Mb TAD where MYC resides. We find that these diverse cancer-specific super-enhancers, differing in size and location, interact with the MYC gene through a common and conserved CTCF binding site located 2 kb upstream of the MYC promoter. Genetic perturbation of this enhancer-docking site in tumor cells reduces CTCF binding, super-enhancer interaction, MYC gene expression, and cell proliferation. CTCF binding is highly sensitive to DNA methylation, and this enhancer-docking site, which is hypomethylated in diverse cancers, can be inactivated through epigenetic editing with dCas9-DNMT. Similar enhancer-docking sites occur at other genes, including genes with prominent roles in multiple cancers, suggesting a mechanism by which tumor cell oncogenes can generally hijack enhancers. These results provide insights into mechanisms that allow a single target gene to be regulated by diverse enhancer elements in different cell types.

SummaryThe RNA-guided endonuclease Cpf1 is a promising tool for genome editing in eukaryotic cells1-5. Compared to other genome editing platforms, Cpf1 offers distinct advantages, such as the ability to easily target multiple genes simultaneously3, as well as low rates of off-target activity4, 5. However, the Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1), which has been successfully harnessed for genome editing, can only robustly cleave target sites preceded by a TTTV protospacer adjacent motif (PAM), which may limit its practical utility. To address this limitation, we used a structure- guided saturation mutagenesis screen to increase the targeting range of Cpf1. We engineered two variants of AsCpf1 with the mutations S542R/K607R and S542R/K548V/N552R that can cleave target sites with TYCV/CCCC and TATV PAMs, respectively, with enhanced activities in vitro and in human cells. Genome-wide assessment of off-target activity indicated that these variants retain a high level of DNA targeting specificity, which can be further improved by introducing mutations in non-PAM-interacting domains. Together, these variants increase the targeting range of AsCpf1 to one cleavage site for every ~8.7 bp in non-repetitive regions of the human genome, providing a useful addition to the CRISPR/Cas genome engineering toolbox.