Dual H3K27me3 and H3K9 methylation establish epigenetic barriers to extraembryonic plasticity

During preimplantation development, embryonic cells progressively lose extraembryonic lineage plasticity as they commit to the epiblast, but the epigenetic mechanisms underlying this restriction remain unclear. Here, we identify H3K27me3 and H3K9 methylation as complementary epigenetic barriers progressively established at extraembryonic lineage genes. Integrative chromatin analyses revealed acquisition of these repressive chromatin states during transition from the inner cell mass to embryonic stem cells. Transient relief of both histone marks restored extraembryonic competence, generating chemically-induced extraembryonic-like cells (iExLCs) exhibiting both trophectoderm (TE)- and primitive endoderm (PrE)-associated features. Single-cell transcriptomics, blastoid formation, morula aggregation and embryo transfer demonstrated that iExLCs contribute to both TE and PrE lineages. CUT&Tag profiling showed that loss of H3K9me3 and H3K27me3 is associated with selective activation of extraembryonic lineage genes during iExLC induction. Together, our findings establish dual H3K27me3 and H3K9me3 as epigenetic barriers that progressively stabilize embryonic identity by restricting extraembryonic lineage plasticity.