Roles of the BRD4 short isoform in phase separation and active gene transcription

Abstract

BRD4, a major tandem-bromodomain-containing transcription regulator, has two isoforms. The long isoform (BRD4L) has an extended C terminus that binds transcription cofactors, while the short isoform (BRD4S) lacks this C-terminal extension. Unlike BRD4L, the role of BRD4S in gene transcription remains unclear. Here, we report that, in human cancer cells, BRD4S forms nuclear puncta that possess liquid-like properties and that colocalize with BRD4L, MED1 and sites of histone H3 lysine 27 acetylation. BRD4 puncta are correlated with BRD4S but not BRD4L expression levels. BRD4S knockdown reduces BRD4S condensation, and ectopic expression promotes puncta formation and target gene transcription. BRD4S nuclear condensation is mediated by its intrinsically disordered regions and binding of its bromodomains to DNA and acetylated chromatin, respectively, and BRD4S phosphorylation diminishes BRD4 condensation. Our study illuminates a previously unappreciated role of BRD4S in organizing chromatin and transcription factors through phase separation to sustain gene transcription in chromatin for cancer cell proliferation.

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Fig. 1: Discrete BRD4 condensates are clustered on chromatin.
Fig. 2: BRD4 condensates exhibit LLPS properties.
Fig. 3: BRD4S constitutes BRD4 condensates on chromatin for active gene transcription.
Fig. 4: BRD4S LLPS is modulated by phosphorylation.
Fig. 5: Characterization of DNA binding activity of BRD4S.
Fig. 6: BRD4S phase-separated condensates promote in vitro gene transcription.

Data availability

Source data for Figs. 3a,c and 6c,e and Supplementary Figs. 2b,c and 9b are available with the paper online. All other data are available from the authors upon reasonable request.

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Acknowledgements

We thank C.-M. Chiang of UT Southwestern Medical Center for providing valuable BRD4S-specific antibody and BRD4 mutant plasmids, S. Fu of the First Hospital at Jilin University for cancer cell lines, R.G. Roeder of Rockefeller University for helpful discussion and F. Wang and J. Zhang for assistance with microscopy. We thank the State Key Laboratory of Supramolecular Structure and Materials at Jilin University for the use of their research facilities. This work was supported in part by the research fund from the First Hospital of Jilin University (Changchun, China); the Open Project of the State Key Laboratory for Supramolecular Structure and Materials, JLU (grant no. SKLSSM201602); the JLU Science and Technology Innovative Research Team (grant no. JLUSTIRT, grant no. 2017TD-25); the International Center of Future Science, JLU; and the National Natural Science Foundation of China (grant no. 31770780; L.Z.).

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Contributions

L.Z. and M.-M.Z. conceived the project. X.H. and Q.W. performed BRD4 LLPS experiments. D.Y. and M.Y. performed BRD4 knockdown, transfection and microscopic imaging experiments in cells. R.G. performed EMSA and thermal shift assays. Y.J., X.Y., C.Z., H.Y., A.J. and Q.Z. performed protein expression and purification. N.B. contributed to florescence anisotropy and mass spectrometry studies. L.Z. and M.-M.Z. wrote the manuscript with input from all of the coauthors.

Corresponding authors

Correspondence to Ming-Ming Zhou or Lei Zeng.

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Competing interests

M.-M.Z. is a founder, director and shareholder of Parkside Scientific Inc.

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Peer review information Beth Moorefield was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Supplementary information

41594_2020_394_MOESM3_ESM.mov

A movie showing BRD4 GFP–BD2–CPS liquid droplets. Droplets were formed by GFP–BD2–CPS (21 μM) in 65 mM NaCl and 2% PEG-6000 and were observed at room temperature with an upright Zeiss (Germany) confocal microscope using a ×63 oil immersion lens. The movie is represented as 8.8-fold speed-up. Scale bars, 10 μm.

Supplementary Information

Supplementary Figs. 1–10 and Tables 1–6.

Reporting Summary

Supplementary Video 1

A movie showing BRD4 GFP–BD2–CPS liquid droplets. Droplets were formed by GFP–BD2–CPS (21 μM) in 65 mM NaCl and 2% PEG-6000 and were observed at room temperature with an upright Zeiss (Germany) confocal microscope using a ×63 oil immersion lens. The movie is represented as 8.8-fold speed-up. Scale bars, 10 μm.

Supplementary Data 1

Uncropped gel (or blot) images of Figs. 3a,c and 6c,e, and Supplementary Figs. 2b,c and 9b are shown in Supplementary Data Set 1.

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Han, X., Yu, D., Gu, R. et al. Roles of the BRD4 short isoform in phase separation and active gene transcription. Nat Struct Mol Biol 27, 333–341 (2020). https://doi.org/10.1038/s41594-020-0394-8

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