Publications
Mediator and RNA polymerase II clusters associate in transcription-dependent condensates. WK Cho*, JH Spille*, M Hecht, C Lee, C Li, V Grube, II Cisse. Science, eaar4199 (2018).
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.
While hunting transient Pol II clusters using the lattice light sheet microscope that I had built at MIT I found that mouse embryonic stem cells actually have very bright and stable Pol II foci that we had not seen before. (They do also have the transient ones.) We found that the Mediator complex coactivator showed the same behavior, and Mediator and Pol II foci associate in transcription-dependent condensates. Both components are rapidly exchanged between condensates and the nuclear environment and dissolve upon hexanediol treatment. Most striking are the prominent merger events that we can see under the lattice light sheet microscope. We used pulse-chase labelling of nascent RNA to show that what we now call transcription condensates are indeed hotspots of transcription, and used FISH to show that condensates occur at super-enhancer controlled gene loci. However, questions remained because it was always just a fraction of the cells that had a condensate at the gene locus. When we inserted a live cell RNA label we discovered that the condensates dynamically overlapped with the gene locus in every single cell, but only for a fraction of the time.
Fun fact: I observed the first merger of two (actually three) Pol II condensates while teaching at the MBL Physiology course in Woodshole. At first I was very hesitant to believe that what I saw was real because famously in Woodshole everything you look at turns out to be a condensate (and everybody was completely sleep-deprived) - but our follow-up experiments confirmed the findings (leading to more sleep deprivation and this publication).
Our work was covered in the following articles:
