Mediator and Pol II clusters co-associate in transcription-dependent dynamic condensates in living stem cells

At the onset of transcription, a mediator multiprotein complex assembled at a distal enhancer site contacts the pre-initiating RNA Polymerase II (Pol II) complex to help effect transcription activation. How mediator and Pol II co-organize and interact in the 3D nucleus of living cells remains elusive. Here we use quantitative live cell super-resolution and light sheet imaging to study the organization and dynamics of endogenous mediator and Pol II with unprecedented quantitative detail, 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 +/-1.4s respectively, mediator and Pol II also form previously uncharacterized large stable clusters in stem cells (~15 stable clusters per cell). The large 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 reminiscent of phase separated droplets. Our study demonstrates mediator and Pol II association in diffraction-sized condensates depending on active transcription in living stem cells.