Dissecting Oct4 enhancer function in pluripotent stem cells and mouse embryogenesis

OCT4 is a master regulator of pluripotency, with expression restricted to pluripotent and germ cells. Its expression is controlled by two cis-regulatory elements: the distal (DE) and proximal (PE) enhancers. Although widely used as markers for pluripotent stem cells (PSCs), their biological roles have remained incompletely defined. Here, we generated PSC lines and mouse models with targeted deletions of the Oct4 DE and PE. Our findings reveal that the DE is dispensable for sustaining the primed pluripotent state but required for the naive state, whereas the PE is necessary for the primed state but not for the naive state. Notably, PE-deficient naive mouse PSCs retained the capacity to differentiate into somatic lineages in vitro and to contribute chimeras. In contrast, deletion of either enhancer in vivo resulted in early embryonic lethality. These models offer powerful genetic tools to dissect the regulation of Oct4 expression during pluripotency and early development.