Modulating MyoD1 dosage activates alternate cell fate beyond myogenic differentiation

Transcription factor (TF) upregulation accompanies many cellular state transitions, yet how increased TF abundance impacts gene regulation remains unclear. Two broad models are often invoked, whereby higher TF levels amplify the expression of pre-existing target genes, or, by mass-action binding, expand genome engagement and regulation to lower-affinity sites. We sought to elucidate how these two regulatory modes contribute to cell differentiation in a well characterized myogenic system by upregulating the expression of the myogenic TF MyoD1 in C2C12 myoblasts. Unexpectedly, elevated MyoD1 levels impaired myoblast fusion (a hallmark of myogenic differentiation), yet enabled robust contraction in myotubes that did form. Live-cell single-molecule imaging and CUT&RUN profiling revealed that elevated MyoD1 dosage increased total genome-wide chromatin binding and broadened genome occupancy by preferentially engaging lower-affinity sites. Integrating CUT&RUN with RNA-seq experiments linked expanded MyoD1 binding to upregulation of cell adhesion genes. Cell mixing and fractionated RNA-seq experiments supported a two-population model in which an adhesion-gene-upregulated, unfused myoblast population supported contraction of myotubes formed by fusion-competent cells. Ectopic expression of several individual MyoD1-upregulated cell adhesion genes was sufficient to recapitulate the “off script” myotube contraction phenotype. Together, these results support a MyoD1 dose-dependent “spillover” model, in which increased TF abundance broadens cis-regulatory engagement and produces distinct cell differentiation outcomes. Significance Statement Transcription factor dosage is a central control knob in many biological and disease-associated cell state transitions, but its range of gene regulatory consequences are poorly understood. By inducibly elevating MyoD1 expression in C2C12 cells, we uncovered a paradoxical outcome: elevated MyoD1 levels suppressed myoblast fusion yet enabled robust myotube contraction. Elevated MyoD1 levels expanded MyoD1 genome occupancy through engagement of low-affinity sites and upregulated adhesion genes whose individual ectopic expression was sufficient to recapitulate the contraction phenotype. Mixing experiments indicated a non-cell-autonomous contribution from an adhesion-gene-high subpopulation that supported contraction of myotubes. Our results demonstrate that TF dosage can expand regulatory scope and yield qualitatively different differentiation outcomes.