Klf5-adjacent super-enhancer functions as a 3D genome structure-dependent transcriptional driver to safeguard ESC identity.

Cell-specific super-enhancers (SEs) and master transcription factors (TFs) dynamically remodel embryonic stem cell (ESC) fate, yet their regulatory interplay remains unclear. By integrating multi-omics data (H3K27ac, Hi-C, scRNA-seq) across ESC states, we identified SEs interacting with master TFs, exemplified by the Klf5-adjacent SE (K5aSE). K5aSE deletion impaired proliferation, differentiation, and Klf5 expression, partially rescued by KLF5 reintroduction. Despite phenotypic similarities between Klf5-KO and K5aSE-KO ESCs, scRNA-seq of embryoid bodies revealed distinct differentiation trajectories, suggesting K5aSE targets beyond Klf5. High-throughput 3D genome screening demonstrated K5aSE activates four distal genes via chromatin looping. CRISPRa-mediated activation of these targets rescued K5aSE-KO phenotypes and uncovered their regulatory roles. Furthermore, CTCF depletion disrupted topologically associated domains (TADs) near K5aSE, suppressing Klf5 and target gene expression, indicating CTCF-mediated TADs sustain K5aSE activity. Our study unveils a 3D genome-dependent mechanism by which SEs govern ESC identity through coordinated TF interaction and multi-gene regulation.