Mechanical Signaling Regulates DNA Methylation to Maintain Muscle Stem Cell Quiescence

Skeletal muscle stem cells (MuSCs) reside within a mechanically dynamic niche where they integrate biophysical and biochemical cues to maintain quiescence. Here, we show that substrate stiffness and RhoA-dependent signaling regulate MuSC fate. MuSCs cultured on soft matrices or depleted of RhoA exhibit altered morphology, diminished actomyosin organization, and undergo premature activation. Loss of RhoA reshapes the DNA methylation landscape, leading to widespread changes in gene expression and alternative splicing. Dnmt3a was among the genes transcriptionally downregulated following loss of RhoA signaling. Mechanistically, RhoA maintains Dnmt3a expression by promoting SP1 occupancy at its promoter. Importantly, loss of Dnmt3a in quiescent MuSCs is sufficient to drive activation, identifying Dnmt3a as a key epigenetic effector downstream of mechanical signaling. Together, these findings define a mechanotransduction-epigenetic axis in which RhoA maintains stem cell quiescence by preserving DNA methylation programs through Dnmt3A.