Histone H3.3 subvariant H3mm15 is required for normal spermatogenesis by regulating gene expression

Histone H3 variants play a fundamental role in chromatin regulation, contributing to gene expression. While the functional diversity of H3.3 subvariants remains largely unexplored, we identified H3mm15 as a testis-expressed H3.3 subvariant that regulates meiotic transcription. Using RNA sequencing and variant-specific reverse-transcription quantitative PCR, we demonstrate here that H3mm15 is expressed predominantly in the testis. Loss of H3mm15 leads to seminiferous tubule disorganization and altered expression of spermatogenesis-related genes. Chromatin profiling reveals that H3mm15 is relatively enriched at promoter-proximal regions compared with H3.3, despite its lower overall chromatin incorporation and lower nucleosomal stability. Functional assays show that H3mm15 has a more moderate impact on transcriptional responses to stimuli than H3.3. Although H3mm15 forms a less stable nucleosome, its reduced stability does not confer enhanced transcription by RNAPII in vitro. Our findings suggest the existence of an H3.3 sub-barcode mechanism, in which structurally distinct H3.3 subvariants differentially regulate chromatin states and contribute to gene regulation during spermatogenesis.