Super-enhancer interactomes from single cells link clustering and transcription.

Regulation of gene expression hinges on the interplay between enhancers and promoters, traditionally explored through pairwise analyses. Recent advancements in mapping genome folding, like GAM, SPRITE, and multi-contact Hi-C, have uncovered multi-way interactions among super-enhancers (SEs), spanning megabases, yet have not measured their frequency in single cells or the relationship between clustering and transcription. To close this gap, here we used multiplexed imaging to map the 3D positions of 376 SEs across thousands of mammalian nuclei. Notably, our single-cell images reveal that while SE-SE contacts are rare, SEs often form looser associations we termed "communities". These communities, averaging 4-5 SEs, assemble cooperatively under the combined effects of genomic tethers, Pol2 clustering, and nuclear compartmentalization. Larger communities are associated with more frequent and larger transcriptional bursts. Our work provides insights about the SE interactome in single cells that challenge existing hypotheses on SE clustering in the context of transcriptional regulation.