Single molecule studies of chromatin structure in transcription condensates

Transcription condensates concentrate regulatory factors at super-enhancers. We use single molecule microscopy to characterize structure and dynamics in endogenous transcription condensates. Dual-color dSTORM reveals that active promoter and enhancer marks are differentially distributed at condensates. While promoters concentrate inside condensates, enhancers localize preferentially to the condensate surface. Single condensates harbor multiple active chromatin clusters, suggesting that active chromatin elements coalesce in condensates to form regulatory hubs. The distribution of Pol II isoforms in condensates corroborates this model. Initiating Pol II is enriched in the condensate core, while elongating Pol II forms multiple distinct foci per condensate. Pol II single particle tracking data supports the structural model. Inside condensates, the Pol II is mostly in a bound state. Inhibiting transcription initiation increases Pol II mobility globally and inside condensates; and purges Pol II from condensates over time. Thus, DNA-binding contributes to Pol II partitioning into condensates. Our single molecule characterizations shed new light on the structural underpinnings of chromatin structure at transcription condensates and molecular mechanisms that drive condensate organization at the molecular scale.