Dissecting dual roles of MyoD during lineage conversion to mature myocytes and myogenic stem cells

Abstract

The generation of myotubes from fibroblasts upon forced MyoD expression is a classic example of transcription factor-induced reprogramming. We recently discovered that additional modulation of signaling pathways with small molecules facilitates reprogramming to more primitive induced myogenic progenitor cells (iMPCs). Here, we dissected the transcriptional and epigenetic dynamics of mouse fibroblasts undergoing reprogramming to either myotubes or iMPCs using a MyoD-inducible transgenic model. Induction of MyoD in fibroblasts combined with small molecules generated Pax7+ iMPCs with high similarity to primary muscle stem cells. Analysis of intermediate stages of iMPC induction revealed that extinction of the fibroblast program preceded induction of the stem cell program. Moreover, key stem cell genes gained chromatin accessibility prior to their transcriptional activation, and these regions exhibited a marked loss of DNA methylation dependent on the Tet enzymes. In contrast, myotube generation was associated with few methylation changes, incomplete and unstable reprogramming, and an insensitivity to Tet depletion. Finally, we showed that MyoD's ability to bind to unique bHLH targets was crucial for generating iMPCs but dispensable for generating myotubes. Collectively, our analyses elucidate the role of MyoD in myogenic reprogramming and derive general principles by which transcription factors and signaling pathways cooperate to rewire cell identity.

Integrative molecular roadmap for direct conversion of fibroblasts into myocytes and myogenic progenitor cells

“…Direct reprogramming via MyoD+F/R/C triggers extensive molecular transformations. One notable pathway that was uniquely up-regulated following F/R/C treatment is Notch, which was similarly reported to be unique to iMPC reprogramming in a recent study (27). This pathway has been extensively investigated in the context of satellite cell quiescence and activation and shown to be critical for their stemness and self-renewal (34,(49)(50)(51).…”

“…S2, B and C). Last, we wished to compare our transcriptome analysis to a recently published work that dissected iMPC formation via a transgenic MyoD-reprogrammable mouse strain ( 27 ). To this end, we compared their RNA-seq dataset for MyoD+F/R/C reprogramming during defined time points to our own.…”

“…For each fuzzy cluster, genes were annotated by gene ontology (GO) terms using the STRING v11 database. Bulk RNA-seq data from Yagi et al ( 27 ) were downloaded from Gene Expression Omnibus (GSE169489) and processed in the same way as mentioned before using the SUSHI framework ( 60 ).…”

The Art of Reprogramming for Regenerative Medicine

“…The combination of MYOD overexpression and treatment with three chemical molecules CHIR99021, Forskolin and RepSOX could convert mouse fibroblasts into expandable induced PAX7 + myogenic progenitor cells (iMPCs) ( Bar-Nur et al, 2018 ). Mechanically, the characteristics of fibroblasts cell fate would be lost gradually, which is prior to the acquirement of stem cell properties during the converting process where Tet regulated DNA demethylation plays a critical role ( Yagi et al, 2021 ).…”

Small molecules facilitate single factor-mediated sweat gland cell reprogramming

“…The reprogrammed cells not only express the SG cell markers cytokeratin (CK)5, CK10, CK18, CK19, CK14, CEA and aquaporin 5 (AQP5), but also facilitate the restoration of SGs in vivo. The enforced expression of lineage-specific factors has been extensively used in the reprogramming strategy [ 6 , 7 ]; however, no single factor has been sufficient for cellular reprogramming [ 8 , 9 ]. Combinatorial approaches for direct lineage conversion must be constructed [ 10 ].…”