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

Abstract

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.

Transcription Factors Activate Genes through the Phase-Separation Capacity of Their Activation Domains

“…Fluorescent microscopy of the droplet mixture revealed that OCT4 alone did not form droplets throughout the range of concentrations tested (Figure 3B). In contrast, purified recombinant MED1-IDR-GFP fusion protein exhibited concentration-dependent liquid-liquid phase separation (Figure 3B), as described previously (Sabari et al, 2018). …”

“…This enrichment was not seen using a randomly selected nuclear position (Figure S1). These results confirm that OCT4 occurs in puncta at the same SEs where Mediator forms condensates (Sabari et al, 2018) and where ChIP-seq shows co-occupancy of OCT4 and MED1 (Figure 1). …”

“…For example, serine residues are required for MED1 phase separation (Sabari et al, 2018). We asked whether amino acid enrichments in the OCT4 ADs might point to a mechanism for interaction.…”

“…ChIP-Seq tracks shown in Figure 1 are derived from GSM1082340 (OCT4) and GSM560348 (MED1) from Whyte et al, 2013. Super-enhancers and typical enhancers and their associated genes in cells grown in 2i conditions were downloaded from Sabari et al, 2018. Distributions of occupancy fold-changes were calculated using bamToGFF (https://github.com/BradnerLab/pipeline) to quantify coverage in super-enhancers and typical enhancers from cells grown in 2i conditions.…”

“…We recently proposed that transcriptional control may be driven by the formation of phase-separated condensates (Hnisz et al, 2017), and demonstrated that the coactivator proteins MED1 and BRD4 form phase-separated condensates at superenhancers (SEs) (Sabari et al, 2018). Here, we report that diverse TF ADs phase separate with the Mediator coactivator.…”