Mediator and RNA polymerase II clusters associate in transcription-dependent condensates
Abstract
Models of gene control have emerged from genetic and biochemical studies, with limited consideration of the spatial organization and dynamics of key components in living cells. We used live-cell superresolution and light-sheet imaging to study the organization and dynamics of the Mediator coactivator and RNA polymerase II (Pol II) directly. Mediator and Pol II each form small transient and large stable clusters in living embryonic stem cells. Mediator and Pol II are colocalized in the stable clusters, which associate with chromatin, have properties of phase-separated condensates, and are sensitive to transcriptional inhibitors. We suggest that large clusters of Mediator, recruited by transcription factors at large or clustered enhancer elements, interact with large Pol II clusters in transcriptional condensates in vivo.
Additional Information
© 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works http://www.sciencemag.org/about/science-licenses-journal-article-reuse This is an article distributed under the terms of the Science Journals Default License. Received for publication November 6, 2017. Resubmitted April 17, 2018. Accepted for publication June 11, 2018. We thank L. D. Lavis (HHMI, Janelia) and J. Grimm (HHMI, Janelia) for the gift of the JF646-Halo dyes and E. Calo (MIT) for the wild-type R1 cells and differentiation protocol. We thank J. Wysocka (Stanford) for the CARGO material. We thank R. Young (MIT) and members of the Young, Sharp, and Chakraborty groups (MIT) for helpful discussions and R. Young and J. Gore (MIT) for helpful comments on the manuscript. We acknowledge the students of the Cissé lab rotation in the 2017 Marine Biology Laboratory physiology course for participation in early aspects of Dendra2–Pol II characterization in mESCs and J. O. Andrews for assistance with the quantitative superresolution analysis software. The lattice light-sheet microscope was home built in the Cissé lab at MIT Physics under license from HHMI, Janelia Research Campus, and we thank E. Betzig (HHMI, Janelia) and W. Legant (HHMI, Janelia) for their critical support in the process. FRAP experiments were performed at the W. M. Keck Microscopy Facility at the Whitehead Institute. We thank L. Boyer for help with stem cell culture. This work was supported primarily by the NIH director's New Innovator award (DP2CA195769 to I.I.C.) and also by the Pew Charitable Trusts through the Pew Biomedical Scholars Program grant (to I.I.C.). I.I.C. is also supported by the NIH 4D Nucleome through NOFIC. J.-H.S. is supported by a postdoctoral fellowship from the German Research Foundation (DFG, SP1680/1-1). Author contributions: W.-K.C., J.-H.S., and I.I.C. conceived of and designed the study; W.-K.C. and J.-H.S. performed experiments and analyzed data with help from M.H., C.Le., and V.G.; M.H. cloned CRISPR repair templates and single-guide RNA plasmids and genotyped cell lines; C.Li conducted and analyzed the Western blot and chromatin immunoprecipitation sequencing (ChIP-seq) assays; W.-K.C., J.-H.S., and I.I.C. wrote the manuscript with input from all coauthors; and I.I.C. supervised all aspects of the project. The authors declare that they have no competing interests. Data and materials availability: All data and materials will be provided upon reasonable request to the corresponding author. ChIP-seq datasets generated in this study have been deposited in the Gene Expression Omnibus under accession number GSE115436. Other data described in the text are presented in the supplementary materials.Errata
Correction (9 July 2018): Since First Release, the y-axis label in fig. S5B has been corrected to refer to Pol II rather than Mediator, and minor editorial changes have been made in the legends to figs. S6 and S9.Attached Files
Accepted Version - nihms-1030474.pdf
Supplemental Material - aar4199-Cho-SM_revision1.pdf
Supplemental Material - aar4199_S3.mov
Supplemental Material - aar4199_s1.mov
Supplemental Material - aar4199_s2.mov
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Additional details
- PMCID
- PMC6543815
- Eprint ID
- 106659
- Resolver ID
- CaltechAUTHORS:20201112-143949440
- NIH
- DP2CA195769
- Pew Charitable Trust
- Deutsche Forschungsgemeinschaft (DFG)
- SP1680/1-1
- Created
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2020-11-16Created from EPrint's datestamp field
- Updated
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2021-11-16Created from EPrint's last_modified field