Identification and Dynamic Remodeling of Bivalent Chromatin Regions in Sheep Early Embryos and ESCs

Bivalent chromatin, marked by H3K4me3 and H3K27me3, contributes to maintaining developmental genes in a poised state. However, it remains unclear whether sheep embryonic stem cells (ESCs) establish bivalent domains. In this study, we identified bivalent chromatin regions and characterized the dynamics of H3K4me3, chromatin accessibility, and expression of bivalent chromatin-associated genes in sheep early embryos and ESCs. Using an overlap threshold of >200 bp between H3K4me3 and H3K27me3 peaks, we identified 40,543 bivalent regions in sheep ESCs. These regions preferentially occurred at promoters and were enriched in RNA processing, translation, and ribonucleoprotein complex biogenesis. By integrating Cleavage Under Targets and Tagmentation (CUT&Tag), Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), and RNA-seq data across preimplantation development from MII oocytes to hatching blastocysts, we found that ESC-defined bivalent regions underwent dynamic changes in H3K4me3 enrichment and chromatin accessibility, with distinct patterns across developmental stages. Bivalent-associated genes also exhibited stage-specific expression patterns, and key pluripotency- and lineage-associated transcription factors, including SOX2, SOX17, POU5F1, and ZFP42, showed distinct developmental expression profiles. Moreover, formative ESCs displayed higher transcriptional activity and chromatin accessibility at bivalent regions than that of primed ESCs. Together, these results characterize the genomic distribution of bivalent chromatin in sheep ESCs and its stage-specific remodeling during preimplantation development, providing a resource for further investigation of epigenetic regulation during early embryonic development in sheep.