Fig 4. DNA methylation at enhancers partially accounts for the transcriptome plasticity of MuSCs upon heterotopic transplantation.

(A) PCA analysis of TA pre-graft, EOM pre-graft and EOM post-graft MuSCs DNA methylation at promoters fails to separate samples based on anatomical location. (B) PCA analysis of TA pre-graft, EOM pre-graft and EOM post-graft MuSCs DNA methylation at enhancers separates samples based on anatomical location and demonstrates that EOM MuSCs after grafting resemble TA MuSCs at an epigenetic level in the enhancer context. (C) Density plots of the methylation difference between post-graft EOM MuSCs and pre-graft EOM MuSCs at enhancers associated with EOM or TA upregulated genes. EOM enhancers became hypermethylated in EOM MuSCs after grafting into the TA environment, while TA enhancers were hypomethylated. * p < 0.05 by Student’s t test. (D) Enhancer methylation and gene expression levels of selected EOM and TA DEGs that are responsive to grafting. (E) DNA methylation level across the HoxA gene cluster in pre-graft EOM, pre-graft TA and post-graft EOM samples. The HoxA region was highly methylated in TA MuSCs but not in EOM MuSCs. Notably, DNA methylation was gained across this region when EOM MuSCs were grafted into TA muscle.