Super-resolution imaging of transcription in live mammalian cells

Gene activation is thought to involve a multistep process whereby transcription factors bind to distal enhancer sites, recruit the Mediator complex which contacts the pre‐initiating RNA Polymerase II (Pol II) complex assembled at the start site of the gene. The interaction of Mediator and Pol II has yet to be observed in the nucleus of living cells and the dynamics of this interaction are not yet elucidated. Here we use quantitative live cell super‐resolution and light sheet imaging to study the organization and dynamics of endogenous Mediator and Pol II directly in living mouse embryonic stem cells. In addition to forming transient clusters with average lifetimes of 11.1 (± 0.9) s, and 12.1 (± 1.4) s respectively, Mediator and Pol II also form previously uncharacterized large and stable clusters in stem cells (~15 stable clusters per cell). The large and stable Mediator and Pol II clusters gradually disappear within hours after induction of stem cell differentiation. Stable Mediator and Pol II clusters co‐localize with each other, as well as with pluripotency factors. Inhibition of Brd4 bromodomains necessary for enhancer association eliminates both Mediator and Pol II stable clusters, and inhibition of transcription elongation selectively eliminates stable Pol II but not stable Mediator clusters. Tracking of Mediator and Pol II stable clusters suggests they are chromatin associated and they coalesce upon contact, a property associated with phase separated droplets. We conclude that Mediator and Pol II associate in diffraction‐sized condensates with a define lifetime dependent on active transcription in living stem cells