Cancer-associated histone mutations were shown to enhance histone exchange and nucleosome sliding.
Major Finding: Cancer-associated histone mutations were shown to enhance histone exchange and nucleosome sliding.
Approach: This work involved systematic functional characterization of a comprehensive mutant histone library.
Impact: This study provides foundation for the investigation of oncogenic histones and their role in cancer.
Mutations in all four core histones have been observed across many human cancer types, and although several mutations affecting histone posttranslational modifications have been studied, the majority of high-frequency histone mutations remain uncharacterized. To systematically investigate cancer-associated histone mutations, Bagert, Mitchener, and colleagues created a library encompassing a multitude of mutant histones, each containing a point mutation in the globular or tail domain. In addition to applying systematic alanine scanning mutagenesis across the histone proteins, mutants in which specific missense mutations were overrepresented in patient data were generated. Mutant histones were assembled into DNA-barcoded mononucleosomes and tested for differences in various high-throughput in vitro biochemical assays, revealing oncohistone mutants that enhanced dimer exchange and decreased thermal stability. In a humanized yeast model, in which all wild-type yeast histones are replaced with humanized histones, the identified mutant histones were also shown to decrease yeast growth. Because mutant histones are often incorporated along with wild-type histones to form heterotypic rather than homotypic mutant nucleosomes in cellular contexts, asymmetric mononucleosomes were assembled and similarly assayed for dimer exchange and thermal stability, revealing that mutant histones could exert these effects even in the presence of wild-type histones. In addition to histone exchange and nucleosome stability, fundamental chromatin remodeling processes also involve nucleosome sliding: the translocation of a nucleosome along DNA. Cancer-associated histone mutations were capable of increasing or decreasing rates of nucleosome sliding as evidenced by the results of a restriction enzyme–based chromatin remodeling assay. To investigate transcriptional changes induced by mutant histones, C3H10T1/2 murine mesenchymal progenitor cells (MPC) were engineered to express wild-type or mutant histone 2B, leading to downregulation of lineage commitment genes during MPC differentiation and causing transcriptional upregulation of genes involved in cancer-associated pathways, including the PI3K–AKT, WNT, and RAP1 pathways. Taken together, this research supports a model in which oncogenic mutant histones may promote cancer by perturbing chromatin remodeling processes and provides a foundation for further high-throughput analysis of the oncogenic histone mutation landscape.
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