Genomic characterization of mouse hindlimb skeletal muscles with divergent slow myofiber content at an embryonic and adult time point [ATAC-Seq]

Here, we characterize the genomic landscape of four mouse hindlimb muscles that differ in slow myofiber content at two time points (E18.5 and adult) to identify novel enhancers that may underlie the expression of genes involved in myofiber development and physiology. RNA-seq analysis revealed that fast- and slow-biased muscles show divergent patterns of gene regulation beginning at a late embryonic time point and persisting through adulthood. Genes differentially expressed between adult fast- and slow-biased muscles were enriched with biological pathways unique to each myofiber type, with glycolytic pathways characterizing adult fast-biased muscles and mitochondrial biogenesis and lipid metabolism characterizing adult slow-biased muscles. By integrating differential expression analysis (RNA-seq) with differential accessibility analysis (ATAC-seq) we identified twelve conserved, muscle-specific candidate enhancers nearby differentially expressed genes that regulate cell metabolism and the genetic markers of myofiber type. Nine candidate enhancers significantly increased and three significantly decreased luciferase reporter activities in C2C12 cells, highlighting the dynamic role of tissue-specific enhancers and repressors on development and differentiation. However, further validation is needed to see if these enhancers regulate the expression of target genes and the effect on muscle myofiber content. Collectively, these results highlight the importance of conserved, skeletal muscle-specific enhancers on skeletal muscle development and adult myofiber phenotypes and provide additional genomic targets for further validation.