DAXX Directs H3.4-to-H3.3 Histone Replacement During Spermatogenesis [Cut & Run]

During spermatogenesis, extensive chromatin remodeling and histone replacement reshape the male germline epigenome. While HIRA is known to mediate transcription-coupled incorporation of histone variant H3.3, we identify DAXX as a key histone chaperone directing genome-wide, transcription-dependent replacement of H3.4 (H3T) with H3.3 on autosomes during male meiosis. Simultaneously, DAXX also directs transcription-independent H3.4-to-H3.3 replacement on the sex chromosomes during meiotic sex chromosome inactivation (MSCI). These distinct, chromosome-specific modes of DAXX-mediated H3.3 deposition are essential for epigenomic integrity in the male germline. Loss of DAXX disrupts this process, leading to widespread transcriptional dysregulation in haploid round spermatids and male infertility. Overall design: RNA-seq analysis using DAXX-ctrl and DAXX-cKO 4wk FACS sorted pachytene spermatocytes (PS) and round spermatids (RS);CUT&Run analysis on H3.1/H3.2, H3.3, H3t, H3K27ac, and H3K9me3 using DAXX-ctrl and DAXX-cKO 4wk sorted pachytene spermatocytes (PS) and round spermatids (RS).