DAXX-dependent H3.3 deposition maintains myoblast cell identity independently of other histone chaperone complexes

H3.3 histone chaperone DAXX regulates heterochromatin silencing; however, its function in transcription regulation remains understudied. Here, we show that <i>Daxx</i> knockout (KO) myoblasts have impaired differentiation and fusion. Transcriptomic analysis revealed a loss in myogenic gene expression and broad transcription dysregulation in <i>Daxx</i> KO myoblasts. Chromatin immunoprecipitation followed by sequencing demonstrated a marked reduction in H3.3 deposition at myogenic loci in <i>Daxx</i> KO myoblasts, which was further linked to decreased H3K27ac. Intriguingly, the double KO of <i>Daxx</i> and <i>Hira</i> resulted in distinct transcriptomic alterations than those of single KOs, demonstrating that DAXX and HIRA have both overlapping and unique roles in H3.3 incorporation. Our findings establish DAXX as a critical regulator of myogenic gene expression and muscle cell identity through a distinct mechanism from that of HIRA and highlighted an unanticipated plasticity in the deposition loci for DAXX and HIRA in myoblasts.