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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

Structured Abstract

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.

Abstract

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.
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Supplementary Material

Summary

Materials and Methods
Figs. S1 to S10
Tables S1 to S3
References (6982)
Data S1

Resources

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File (aar3958_sabari_sm_table_s2.xlsx)
File (aar3958_sabari_sm_table_s3.xlsx)

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Science
Volume 361 | Issue 6400
27 July 2018

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Received: 4 November 2017
Accepted: 6 June 2018
Published in print: 27 July 2018

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Acknowledgments

We thank W. Salmon of the W. M. Keck Microscopy Facility; D. Richardson and S. Terclavers of the Harvard Center for Biological Imaging; and T. Volkert, D. Reynolds, S. Mraz, and S. Gupta of the Whitehead Genome Technologies Core for technical assistance. We thank the Imaging Platform at the Broad Institute for assistance with CellProfiler. Funding: The work was supported by NIH grants GM123511 (R.A.Y.) and P01-CA042063 (P.A.S.), NSF grant PHY-1743900 (A.K.C., R.A.Y., and P.A.S.), Koch Institute Support (core) grant P30-CA14051 from the NCI (P.A.S.), Damon Runyon Cancer Research Foundation Fellowship 2309-17 (B.R.S.), Swedish Research Council Postdoctoral Fellowship VR 2017-00372 (A.B.), a Hope Funds for Cancer Research fellowship (B.J.A.), an NSF Graduate Research Fellowship (A.V.Z.), a Cancer Research Institute Irvington Fellowship (Y.E.G.), American Cancer Society New England Division Postdoctoral Fellowship PF-16-146-01-DMC (D.S.D.), and a NWO Rubicon Fellowship (J.S.). Author contributions: B.R.S., A.D., and R.A.Y. conceptualized and organized the project and wrote the manuscript. A.D., A.B., J.C.M., and Y.E.G. performed cell-imaging experiments and image analysis. I.A.K. and A.V.Z. generated endogenously tagged cell lines. B.R.S. and A.B. performed ChIP-seq. B.R.S. and E.L.C. performed in vitro droplet assays and optoIDR experiments. K.S. and B.J.A. developed and performed image analysis and produced visualizations. B.J.A. performed ChIP-seq analysis and produced visualizations. N.M.H. produced and purified recombinant proteins. A.V.Z. helped with biochemical experiments. C.H.L. performed protein amino acid analysis. D.S.D. performed ChIA-PET analysis and visualization. B.R.S., I.A.K., E.L.C., J.S., and A.V.Z. generated constructs. S.M. performed in vitro transcription assays. D.H., E.V., T.I.L., I.I.C., R.G.R., P.A.S., A.K.C., and R.A.Y. provided input into experimental design and interpretation. P.A.S., A.K.C., and R.A.Y. acquired funding for this study. R.A.Y. supervised the project with help from T.I.L. and A.K.C. All authors contributed to editing the manuscript. Competing interests: The Whitehead Institute filed a patent application based on this paper. R.A.Y. is a founder and shareholder of Syros Pharmaceuticals, Camp4 Therapeutics, and Omega Therapeutics. B.J.A. and T.I.L. are shareholders of Syros Pharmaceuticals, and T.I.L. is a consultant to Camp4 Therapeutics. All other authors declare no competing interests. Data and materials availability: Datasets generated in this study have been deposited in the Gene Expression Omnibus under accession number GSE112808.

Authors

Affiliations

Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA 02142, USA.
Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA 02142, USA.
Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA 02142, USA.
Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA 02142, USA.
Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02215, USA.
Eliot L. Coffey
Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA 02142, USA.
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Institute for Medical Engineering & Science, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA 02142, USA.
Nancy M. Hannett
Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA 02142, USA.
Alicia V. Zamudio
Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA 02142, USA.
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA 02142, USA.
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA 02142, USA.
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Yang E. Guo
Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA 02142, USA.
Daniel S. Day
Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA 02142, USA.
Jurian Schuijers
Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA 02142, USA.
Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York, NY 10065, USA.
Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA 02142, USA.
Tong Ihn Lee
Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA 02142, USA.
Department of Physics, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York, NY 10065, USA.
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Institute for Medical Engineering & Science, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Department of Physics, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard, Cambridge, MA 02139, USA.
Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA 02142, USA.
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

Funding Information

Hope Funds for Cancer Research:

Notes

*
These authors contributed equally to this work.
†Corresponding author. Email: [email protected]wi.mit.edu

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