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 knockout (KO) myoblasts have impaired differentiation and fusion. Transcriptomic analysis revealed a loss in myogenic gene expression and broad transcription dysregulation in KO myoblasts. Chromatin immunoprecipitation followed by sequencing demonstrated a marked reduction in H3.3 deposition at myogenic loci in KO myoblasts, which was further linked to decreased H3K27ac. Intriguingly, the double KO of and 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.