Single-cell 3D genome imaging shows super-enhancer hubs are rare, nonspecific, and neither necessary nor sufficient for transcriptional bursting.

Super-enhancer (SE) hubs have been proposed to coordinate gene expression through 3D genome organization and transcriptional condensates. Using multiplexed imaging, we mapped hundreds of SEs in thousands of mouse embryonic stem cells and also paired SE position with nascent transcription measurements. We found that most SEs are spatially isolated, with multiway SE hubs occurring in only a small fraction of cells. Rare hubs were largely promiscuous, cooperative aggregates shaped by genomic proximity, nuclear speckle association, and general transcription machinery occupancy. Perturbing cohesin, CTCF, BET proteins, or RNA polymerase II showed that normal genome organization generally suppresses SE clustering. Combined RNA and DNA imaging demonstrated that SE hubs were neither necessary nor sufficient for transcriptional bursting, although larger hubs weakly increased burst probability. These results challenge models in which SE hubs are a dominant mechanism of enhancer function and instead suggest rare transcriptional crosstalk.