Somatic missense mutations in histone genes, often referred to as ‘oncohistones’, have been identified in diverse types of human cancers. The functional and mechanistic impact of most oncohistones remains unknown. To address this gap, we developed CHANCLA, a modular platform for high-throughput functional screening of oncohistones using multiomic phenotypic readouts. We used CHANCLA to systematically measure the impact of 303 human oncohistones on cellular proliferation, differentiation, histone-specific post-translational modifications, and chromatin accessibility. Integrative multiomic analyses revealed discrete oncohistone molecular classes that promote proliferation, block lineage-specific differentiation, and physically remodel the chromatin landscape by altering specific histone modifications and reducing nucleosome stability. Structural mapping and computational modeling studies uncovered that functionally convergent mutations are clustered at key nucleosome interfaces, particularly H2B-H4, and that chromatin accessibility-promoting mutations are linked to mono-nucleosome destabilization. Leveraging this multiomic resource, we discovered that the H3.3-Q5H mutant histone is a bona fide human oncohistone that accelerates lung adenocarcinoma growth in vivo. Mechanistically, we found that H3.3-Q5H expression leads to suppression of promoter-associated H3K4me3 and expansion of repressive H3K27me3 domains, resulting in increased KRAS signaling and gene expression programs associated with epithelial-to-mesenchymal transition. Together, this work provides a multiomic functional atlas of cancer-associated histone mutations, identifies structural and mechanistic principles governing chromatin reprogramming by oncohistones, and establishes CHANCLA as a modular platform for systematic discovery of mechanisms and vulnerabilities associated with these genetic lesions.
