KDM6A mutations promote acute cytoplasmic DNA release, DNA damage response and mitosis defects

Abstract Background KDM6A, encoding a histone demethylase, is one of the top ten mutated epigenetic cancer genes. The effect of mutations on its structure and function are however poorly characterized. Methods Database search identified nonsense and missense mutations in the N-terminal TPR motifs and the C-terminal, catalytic JmjC domain, but also in the intrinsically disordered region connecting both well-structured domains. KDM6A variants with cancer-derived mutations were generated using site directed mutagenesis and fused to eGFP, which served as an all-in-one affinity and fluorescence tag to study demethylase activity by an ELISA based assay in vitro, complex assembly by Co-immunoprecipitation and localization by microscopy in cellulo. Results Independent of the mutation and demethylase activity, all KDM6A variants were detectable in the nucleus. KDM6A truncations displayed changes in complex assemblies: affecting (1) known interactions with the COMPASS complex component RBBP5 and (2) KDM6A-DNA associated assemblies with the nucleolar protein Nucleophosmin. Furthermore, we observed a severe cellular phenotype characterized by multiple acute effects on nuclear integrity, namely, release of nuclear DNA into the cytoplasm, increased level of DNA damage indicators RAD51 and p-γH2A.X, and hence mitosis defects. Conclusion These observations reveal novel effects of pathogenic variants pointing at new specific functions of KDM6A as well as at a dominant negative effect of KDM6A truncation variants. The underlying mechanisms and affected pathways have to be investigated in future research to understand how tumor cells cope with and benefit from KDM6A truncations.