Dissecting the Role of Myf6 in Facilitating Fibroblast Conversion into Myogenic Progenitor and Differentiated Cells [CUT&Run]

Conversion of fibroblasts into skeletal muscle cells by overexpression of the transcription factor MyoD has been widely employed over past decades in a multitude of experimental settings. However, the potential of additional myogenic regulatory factors such as Myf5, Myf6 and Myog to elicit a myogenic conversion has been significantly less studied. To address this disparity, we set out to investigate the role of MRFs in fibroblast conversion into skeletal muscle stem and differentiated cells. Surprisingly, we found that Myf6, a transcription factor associated with postnatal late-stage myogenesis, efficiently converted fibroblasts into myotubes alone, or alternatively into expandable Pax7-expressing induced myogenic progenitor cells (iMPCs) in the presence of defined small molecules. To investigate reprogramming dynamics in a tightly regulated system, we generated an inducible Myf6 overexpression transgenic mouse model, which enabled dissection of transcription and epigenetic dynamics unique to iMPC formation. Mechanistically, we characterized the chromatin binding of Myf6, as well as its protein binding partners, during iMPC reprogramming and conventional transdifferentiation, revealing shared and different targets and binding partners. To further investigate its role, we surprisingly found that endogenous Myf6 is dispensable for iMPC formation by MyoD and small molecules. In accordance with this observation, we demonstrated that subjecting MyoD-KO fibroblasts to reprogramming with Myf6 and small molecules gave rise to expandable MyoD-KO iMPCs, indicating that MyoD is similarly dispensable for iMPC formation and self-renewal. Multi-omics analyses of Myf6-derived MyoD-KO iMPCs uncovered upregulated genes that compensated for the lack of MyoD, whose downregulation during iMPC production mitigated Pax7 expression, highlighting their roles as reprogramming regulators. Collectively, these results illustrate a redundancy between MRFs’ function in myogenic cellular conversions, highlighting an unexpected potency for Myf6 in the induction of a muscle stem cell fate in vitro.