Kim, S.;Lim, S.;Lim, S.;Chu, Y.;Bu, J.;Won, Y.;Kim, K.;Kim, H.;Alev, C.;Choi, M.;Lee, C.;Cha, H.

The first fate decision in mammalian embryos segregates embryonic and extraembryonic lineages, establishing a largely irreversible transition from totipotency to pluripotency. The mechanisms that enforce this unidirectionality remain incompletely understood. Here, we identify the PRC2.1 subunit Mtf2 as a key epigenetic barrier that safeguards lineage fidelity in naïve embryonic stem cells (ESCs). Despite globally elevated H3K27me3 levels in naïve ESCs, loss of Mtf2 derepresses trophectoderm (TE) regulators, enhances cellular plasticity, generating both ICM- and TE-like populations. Mtf2-deficient cells readily acquire expanded potential with extraembryonic competence and form blastoids without trophoblast stem cell supplementation, whereas loss of the PRC2.2 subunit Jarid2 has minimal effects. Integrated CUT&Tag and transcriptomic analyses reveal that Mtf2 directly represses TE-associated genes in naïve ESCs. Thus, reduced Mtf2 occupancy permits their activation in expanded potential states. Collectively, our findings establish Mtf2 as a central epigenetic barrier that stabilizes naïve pluripotency and restricts inappropriate acquisition of extraembryonic fate.