ABSTRACT Proper expressions of Homeobox A cluster genes ( HoxA ) are essential for embryonic stem cells (ESC) differentiation and individual development. However, the mechanisms underlying controlling precise spatiotemporal expressions of HoxA during early ESC differentiation remain poorly understood. Herein, we identified a functional CTCF-binding element (CBE +47 ) closest to the 3’-end of HoxA within the same topologically associated domain (TAD) in ESC. CRISPR-Cas9 mediated the CBE +47 deletion significantly promotes retinoic acid (RA)-induced HoxA expression and early ESC abnormal differentiation. In delineating its underlying mechanisms, we find that CBE +47 can precisely organize chromatin interactions between its adjacent enhancers and HoxA by using chromosome conformation capture assay (Capture-C). Furthermore, we also find that its adjacent enhancers as enhancer-enhancer interaction complex (EEIC) are required for RA-induced HoxA activation. Collectively, these results provide a new insight for RA-induced HoxA expression, also highlight the unique and precise regulatory roles of CBE in ESC differentiation.

CTCF-binding element regulates ESC differentiation via orchestrating long-range chromatin interaction between enhancers and HoxA