Distal enhancers regulate mammalian early embryonic lineage differentiation through long-range interactions.

Histone modifications are key regulators of cell lineage differentiation; however, the roles of histone H3 lysine 27 acetylation (H3K27ac) and high-order chromatin structure in the trophectoderm (TE) and inner cell mass (ICM) remain unclear. Using ultra-low-input multi-omics, we profiled H3K27ac, chromatin accessibility, and the transcriptome in both the TE and ICM. Lineage-specific H3K27ac was predominantly enriched at distal enhancers rather than promoters, indicating that first-lineage differentiation relies on distal enhancer activity. Integration with chromatin accessibility data identified key transcription factors cooperating with H3K27ac during peri-implantation. Analysis of high-order chromatin structure revealed lineage-specific enhancer-promoter loops. Further, we identified and functionally validated BRD4, p300, and YY1 at these enhancers, demonstrating their essential roles in morula-to-blastocyst transition. Notably, CBP/p300 inhibition selectively disrupted distal H3K27ac-marked enhancers with minimal effects on promoters, leading to down-regulation of placenta-related genes. Despite similar accessibility and transcription factor binding between the TE and ICM, enhancer activity requires H3K27ac acquisition rather than accessibility alone. These results indicate that H3K27ac-marked distal enhancers regulate early lineage differentiation by promoting lineage-specific gene expression through long-range chromatin interactions. Cross-species comparisons between mice and humans further revealed stronger H3K27ac conservation in the TE than in the ICM of mammalian embryos, highlighting the conserved regulatory role of TE-specific enhancers during early development.