A histone H2A and H2B point mutant library defines essential in vivo functions in Drosophila

Histone variants and post-translational modifications are critical for chromatin organization and gene regulation, yet the in vivo, residue-specific functions of histone H2A and H2B in metazoans remain poorly understood. Here, we present a Drosophila histone H2A and H2B point mutant library, generated using a φC31 integrase-based system to replace the endogenous histone gene cluster with engineered mutant arrays. This resource targets 30 conserved residues, with each mutant validated molecularly and phenotypically. Systematic analysis revealed that nearly half of the mutants are lethal, while others exhibit defects in fertility, DNA damage response, retrotransposon silencing, or immunity. Notably, H2BR96A disrupts Polycomb-mediated silencing of HOX genes, while H2BK11A impairs basal antimicrobial peptide production, linking histone structure to immune defense. This scalable library provides a powerful tool for dissecting H2A and H2B functions in vivo, accelerating discoveries in chromatin dynamics, development, genome stability, immunity, and epigenetic regulation.