Epigenetic control of myogenic identity of human muscle stem cells in Duchenne Muscular Dystrophy

In Duchenne Muscular Dystrophy (DMD), the absence of the subsarcolemmal dystrophin protein leads to repeated myofiber damages inducing cycles of muscle regeneration that is driven by muscle stem cells (MuSCs). With time, MuSC regenerative capacities are overwhelmed, leading to fibrosis and muscle atrophy. Whether MuSCs from DMD muscle have intrinsic alterations or are primed by their degenerative/regenerative environment is still debated. We investigated cell behavior and gene expression in human using MuSCs derived from DMD or healthy muscles. We found that proliferation, differentiation and fusion were not altered in DMD-MuSCs, but with time, they lost the expression of the myogenic marker CD56 twice as fast as healthy MuSCs. The rapid drift towards a fibroblast-like cell identity was observed at the clonal level, and resulted from the altered expression of epigenetic enzymes required to maintain the myogenic cell fate. Indeed, the reexpression of CBX3, SMC3, H2AFV and H3F3B prevented the MuSC identity drift. Amongst the epigenetic changes, a closing of chromatin at the gene encoding the transcription factor MEF2B caused a down-regulation of its expression and a loss of the myogenic fate. Thus, MEF2B is a key mediator of the myogenic identity in human MuSCs, that is altered in DMD pathology. Overall design: To identify potential targets of CBX3, H2A.Z2, H3.3 and SMC3 in MuSCs, ATAC sequencing was performed before and after lentiviral transduction of DMD human CD56pos MuSCs (Muscle Stem Cells). Five conditions were analyzed: (1) pure DMD-CD56pos cells harvested before the infection (Day 0); (2) empty-CD56pos and (3) empty-CD56neg cells resulting from DMD-CD56pos cells transfected empty lentivirus and cultured for 15,20 and 25 days (for about 10 population doublings at 25 days); (4) 4V-CD56pos and (5) 4V-CD56neg cells resulting from DMD-CD56pos cells transfected with CBX3, H2AZ2, H3F3B and SMC3 lentiviruses and cultured for 25 days (10 population doublings).