Transcription condensates are promoter hubs that enhance transcriptional bursts.

Transcription of eukaryotic genes by RNA Polymerase II occurs in temporal bursts and spatial clusters. It is regulated by dozens of transcription factor and coactivator proteins and guided by epigenetic histone marks. Colocalization of transcription machinery in dense foci suggests that cooperative effects orchestrate the process. Factory or condensate models provide a framework for the spatial assembly of the transcription machinery at highly active chromatin loci. But conventional methods lack the resolution to determine how chromatin regulatory elements interact with spatial clusters of the transcription machinery, and whether chromatin structural features modulate functional output. Here, we use super-resolution microscopy to elucidate nanoscale organization of regulatory chromatin at Pol II clusters across scales. We find that Pol II clusters exist on a continuous spectrum of sizes and represent promoter chromatin hubs. We uncover a layered organization of regulatory chromatin, where Pol II clusters form at H3K27ac and H3K4me3-rich domains while H3K4me1 positions peripherally at the surface of large Pol II clusters. Perturbation experiments are consistent with a model in which cohesin loop extrusion forms the active chromatin scaffold underlying transcription assemblies while condensate-driven interactions play only a minor role in genome organization at these sites. Importantly, the number and size of transcriptional burst size increases with Pol II cluster size, revealing directly the cooperative benefits of transcription organization in promoter hubs and a functional consequence of local chromatin structure.