Synergy between regulatory elements can render cohesin dispensable for distal enhancer function

Enhancers are critical genetic elements controlling transcription from promoters, yet how they convey regulatory information across large genomic distances remains unclear. Here, we engineer pluripotent stem cells in which cohesin loop extrusion can be inducibly disrupted without confounding cell cycle defects. Transcriptional dysregulation is cell type-specific, and not all loci with distal enhancers depend equally on cohesin extrusion. Using comparative genome editing, we demonstrate that enhancer-promoter communication over just 20 kilobases can require cohesin. However, promoter-proximal elements can support long-range, cohesin-independent enhancer action – even across strong CTCF insulators. Finally, transcriptional dynamics and the emergence of embryonic cell types remain largely robust despite disrupted extrusion. Beyond establishing novel strategies to study cohesin in enhancer biology, our work provides mechanistic insight into cell type- and genomic context-specificity.