Dissecting chromatin accessibility profiles of anatomically distinct skeletal muscles and different breeds in pigs at single-nucleus resolution

1. Constructed the chromatin accessibility landscape of pig skeletal muscle at a single nucleus resolution; 2. Dissected regulatory divergences between oxidative and glycolytic myofibers, identifying DARs and TF networks; 3. Revealed breed-specific DARs in myofibers and displayed the dynamic chromatin remodeling during myogenesis. To dissect chromatin accessibility profiles in pig skeletal muscle at single-nucleus resolution, we performed single-nucleus ATAC-seq on longissimus dorsi and psoas major muscles of Large White pigs. After quality control, 26,225 nuclei were classified into seven major cell types, including myofibers, muscle stem cells, and immune cells, using snRNA-seq-based label transfer. We identified 158,438 accessible chromatin regions, with cell-type-specific differentially accessible regions (DAR) enriching for specific functions. Myonuclei subtypes (type I, IIA, IIB) showed distinct accessibility patterns, with SIX1 and MAF transcription factor motifs enriched in fast myofibers (type II). Comparative analysis between muscles revealed that myofiber composition drove chromatin differences, with psoas major featuring more type I myofibers. Cross-breed analysis (Rongchang vs. Large White) identified breed-specific DARs in myonuclei, linking MEF2-mediated regulation to myofiber hypertrophy. Pseudo-temporal analysis of myogenesis showed dynamic accessibility changes in key myogenic genes (e.g., MYF5, MYH1 ). This study unveils cell-type-resolved chromatin landscapes underlying myofiber specification, tissue heterogeneity, and breed-specific muscle development in pigs.