Quantitative changes in 3D contact predict changes in enhancer-promoter regulation upon cohesin depletion

Enhancers are drivers of gene regulation thought to act via 3D interactions with promoters. However, genome-wide cohesin depletion causes only limited changes in gene expression, despite widespread loss of contact domains, raising questions about how 3D contacts and cohesin influence enhancer function. Here, we introduce CRISPRi of regulatory elements upon degron operation (CRUDO), to quantitatively assess how changes in contact frequency affect enhancer-driven gene regulation. We analyzed 1039 candidate enhancers near five cohesin-dependent genes and identified 30 significant enhancer-gene pairs, 16 with cohesin-dependent effects. While all cohesin-dependent enhancers were distal (>50 Kb), not all distal enhancers were sensitive to cohesin. Instead, changes in enhancer effects correlated with changes in enhancer-promoter 3D contact frequency, which appeared to depend on both genomic distance and other locus-specific factors. Across the genome, most predicted enhancer-gene regulatory interactions do not show substantial contact changes upon cohesin depletion, explaining why only a small subset of genes are cohesin-sensitive. Together, our results illuminate how 3D contacts tune enhancer effects on endogenous gene expression.