Single‑Cell Transcriptomic Analysis Reveals the Temporal Dynamics of MyoD‑Mediated Fibroblast‑to‑Myocyte Transdifferentiation
Alimujiang Tudiyusufu1, WAN Xin1, GU Yingying1, HU Xiaodi1, ZHU Dahai1,QIN Yan2, ZHANG Yong1*, ZENG Ping2*
MyoD is a myogenic lineage-specific transcription factor essential for skeletal muscle cell fate determination. Ectopic expression of MyoD in fibroblasts is sufficient to induce transdifferentiation into myoblasts. However, the temporal regulatory features of MyoD‑mediated transdifferentiation, particularly the systematic downregulation of fibroblast‑associated genes, remain poorly understood. This bioinformatic reanalysis of publicly available multi-omics datasets aimed to characterize, at single‑cell resolution, the temporal dynamics and molecular mechanism underlying MyoD‑mediated transdifferentiation of MEFs (mouse embryonic fibroblasts) into skeletal muscle cells. This article analyzed publicly available GEO datasets including scRNA‑seq (single‑cell RNA‑sequencing) data, MyoD CUT&RUN data, and chromatin accessibility (SHARE‑seq) data from MyoD‑mediated MEF transdifferentiation. Pseudo‑time analysis using Monocle2 revealed three sequential stages along the transdifferentiation trajectory: early myoblasts (Cdh15+/Mef2a+), myocytes (Myog+/Actc1+), and terminally differentiated myocytes (Myh3+/Ckm+). At 24 h post‑induction, early myoblasts represented approximately 60% of cells, whereas by 72 h, terminally differentiated myocytes accounted for 60% of the population. Integrative analysis of MyoD CUT&RUN and SHARE‑seq data showed that MyoD directly binds to E‑box elements in promoters of skeletal muscle differentiation genes, leading to chromatin opening and activation of myogenic gene expression. In contrast, silencing of MEF‑related genes correlated with reduced chromatin accessibility at their promoter regions but lacked direct MyoD binding, suggesting an indirect, E‑box‑independent repression mechanism. Collectively, this study delineates an ordered three‑stage transdifferentiation process from MEFs to terminally differentiated myocytes and demonstrates that MyoD employs distinct epigenetic pathways to activate myogenic cell fate and repress fibroblast identity. The finding reveals a dual regulatory mechanism underlying transcription factor‑driven cell reprogramming.
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