Epigenetic regulation and three-dimensional chromatin structural changes during myofiber formation in chicken embryos

Skeletal muscle development is a major determinant of poultry meat production, yet the stage-specific regulatory mechanisms remain poorly understood. In this study, pectoral muscle samples from eight developmental stages of chickens (E9-E18, D1, D7) were subjected to transcriptome sequencing, with two representative stages (E9 and D1) further analyzed through integrative whole-genome bisulfite sequencing (WGBS), chromatin immunoprecipitation sequencing (ChIP-seq), and high-throughput chromosome conformation capture (Hi-C) to investigate epigenetic and chromatin structural regulation. Transcriptome analysis revealed extensive gene expression remodeling after hatching, with stage-specific gene sets associated with cell cycle, differentiation, and neural development downregulated, while protein translation and cell morphology genes were upregulated. WGBS identified 265,022 differentially methylated loci, with 15,745 E9-specific and 7,391 D1-specific hypermethylated differentially methylated regions (DMRs) enriched in gene promoters and first introns, containing abundant bHLH transcription factor motifs. ChIP-seq identified 240 and 672 H3K27ac-marked super-enhancers at E9 and D1, respectively, demonstrating stage-specific histone modifications. Hi-C analysis detected stage-associated changes in topologically associating domains (TADs) and chromatin loops. Multi-omics integration demonstrated that epigenetic modifications and three-dimensional chromatin architecture are closely associated with the transcriptional dynamics of key genes including MyoG, MyoD1, and Grb10 through interactions among super-enhancers, TADs, and promoters, potentially contributing to the developmental transition of skeletal muscle. This study confirms the pivotal roles of epigenetic regulation and chromatin spatial organization in controlling chicken skeletal muscle development and provides a theoretical foundation for the molecular breeding of poultry meat production traits.