Chromatin structure and protein dynamics in transcription condensates at single molecule resolution

Transcription condensates concentrate key regulatory proteins at specific chromatin elements. But their small size near the diffraction limit of optical microscopy makes it difficult to investigate condensate biophysical properties in situ. Single molecule microscopy is uniquely suited to overcome this hurdle. Here, we use 3D super-resolution microscopy to uncover chromatin organization at condensates. We find a layered structure of chromatin associated with condensates. Gene promoters concentrate in the core and enhancers decorate the condensate surface. Single particle tracking reveals that RNA Polymerase II is enriched in these condensates because it binds promoter chromatin rather than through phase separation mediated by its C-terminal domain. Acute Pol II degradation does not affect condensate composition but results in nanoscale changes to chromatin structure at condensates. Our findings suggest a model in which Pol II freely enters a chromatin-supported condensate environment rich in BRD4 and Mediator. Inside the condensate, Pol II is rapidly loaded onto promoter DNA and initiates transcription, leading to the observed enrichment in condensate foci. Other condensate constituents but not Pol II anchor active chromatin at condensates.