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

J Koch,M J Hoffmann,P Whongsiri,A Lang,W A Schulz,A Greife

doi:10.1186/s12860-021-00394-2

J Koch, M J Hoffmann + Show 4 more

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https://doi.org/10.1186/s12860-021-00394-2

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Abstract

BackgroundKDM6A, 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.MethodsDatabase 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 these two well-structured domains. KDM6A variants with cancer-derived mutations were generated using site directed mutagenesis and fused to eGFP serving as an all-in-one affinity and fluorescence tag to study demethylase activity by an ELISA-based assay in vitro, apoptosis by FACS, complex assembly by Co-immunoprecipitation and localization by microscopy in urothelial cells and apoptosis by FACS.ResultsIndependent of the mutation and demethylase activity, all KDM6A variants were detectable in the nucleus. Truncated KDM6A variants displayed changes in complex assemblies affecting (1) known interactions with the COMPASS complex component RBBP5 and (2) KDM6A-DNA associated assemblies with the nuclear protein Nucleophosmin. Some KDM6A variants induced 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 mitosis defects. These damaging effects were correlated with increased cell death.ConclusionThese observations reveal novel effects of pathogenic variants pointing at new specific functions of KDM6A 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.

Highlights

KDM6A, encoding a histone demethylase, is one of the top ten mutated epigenetic cancer genes
Generation of amino acid substitution and truncated variants of KDM6A based on the mutational landscape of KDM6A in tumor tissues and cell lines Based on 64,668 tested samples from 44 tissue types, 2496 unique mutations are listed for KDM6A in COSMIC v92 (GRCh 38, November 2020)
As nonsense mutations make up nearly one quarter of all point mutations and generate truncated variants with partial losses of the intrinsically disordered region (IDR) and Jumonji C domain (JmjC), we established a set of truncated variants, each with an eGFP-tag fused to the N-terminus: ΔTPR, ΔIDR, ΔJmjC, Tetratricopeptide repeat (TPR) (= ΔIDR/ΔJmjC) and JmjC (= ΔTPR/ΔIDR) (Fig. 1C)

Summary

Introduction

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 poorly characterized. The KDM6A protein has three functionally and structurally relevant regions conserved among several vertebrates. These include the eight N-terminal tetratricopeptide repeats (TPRs, aa 92–385 in human), which represent a common protein interaction motif [13]. The well-characterized C-terminal and highly conserved, catalytically active Jumonji C domain (JmjC) is important for histone H3K27me2/3-recognition, binding and demethylation [14]. A high number of other chromatin-associated proteins, such as Nucleophosmin, p300 and KMT2A-D, possess such flexible sequences, which seem to be common and crucial motifs for interactions with proteins and DNA/ RNA [16]

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