Modulating MyoD1 dosage activates alternate cell fate beyond myogenic differentiation.

Transcription factor (TF) expression and dosage regulate developmental cell fate decisions. Increased TF dosage has been predicted to enhance expression of high-affinity target genes but also increase the binding of lower-affinity loci. The relative importance of high- versus lower-affinity TF binding in guiding cell fate decisions remains unclear. To test the roles of TF dosage, we examined the effects of increasing the dosage of MyoD1, the "master regulator of myogenesis", on skeletal muscle differentiation. Unexpectedly, increased MyoD1 dosage inhibited canonical myogenesis and redirected myoblast differentiation towards forming spontaneously contracting myotubes. This novel phenotype was driven by the MyoD1-dose-dependent upregulation of non-myogenic genes, including cell adhesion genes whose ectopic expression also inhibited classical myogenic differentiation and enabled myotube contraction. Live-cell single-molecule imaging showed that elevated MyoD1 dosage increased total chromatin binding and CUT&RUN profiling demonstrated that this increase occurred via preferential binding to lower-affinity loci. Integration of CUT&RUN, ATAC-seq and RNA-seq experiments revealed that increased MyoD1 binding correlated to the upregulation of otherwise lowly expressed genes. These findings suggest that increased MyoD1 dosage induced a selective gene regulatory expansion from high- to lower-affinity cis-regulatory elements, activating a broader ensemble of target genes, revealing a TF dose-dependent mechanism that can trigger distinct developmental programs.