NIPBL-mediated 3D genome folding translates enhancer priming into gene activation and safeguards lineage fidelity during embryonic transitions

Precise gene control by complex regulatory landscapes is fundamental to embryo development, yet the instructive role of 3D genome architecture remains controversial. While acute cohesin depletion completely disrupts genome folding, it yields modest transcriptional impacts, but these findings are often confounded by cohesin’s essential roles in cell division and proliferation. Here, we resolve this discrepancy by decoupling architectural functions from cell-cycle roles using an acute NIPBL degron system. By integrating single-gene imaging with single-cell and bulk multi-omics during mouse pluripotency transitions and germ-layer specification, we show that NIPBL-mediated cohesin function is required for proper de novo activation of lineage-specifying genes. Mechanistically, NIPBL translates epigenetic priming into transcriptional outputs by physically bringing distal enhancers and target promoters into proximity. We further uncover a dual regulatory role: an acute requirement for establishing new enhancer–promoter interactions during cell state transitions and a long-term role in safeguarding transcriptional fidelity by preventing ectopic gene de-repression. Our findings demonstrate that NIPBL/cohesin-orchestrated genome folding facilitates the faithful execution of developmental gene expression programs. Highlights Acute NIPBL depletion decouples the architectural functions of cohesin from its essential roles in chromosome segregation and cell cycle progression. NIPBL-mediated loop extrusion is required to translate the epigenetic priming of distal enhancers into de novo gene activation during embryonic state transitions. NIPBL is a “rate-limiting physical relay” required to bring distal enhancers and target promoters into proximity to initiate transcription. 3D genome architecture serves a dual role: enabling acute enhancer–promoter communication and safeguarding long-term lineage fidelity by preventing ectopic gene de-repression.