Cancer-derived UTX TPR mutations G137V and D336G impair interaction with MLL3/4 complexes and affect UTX subcellular localization

Kato, Hiroyuki; Asamitsu, Kaori; Sun, Weihao; Kitajima, Shojiro; Yoshizawa-Sugata, Naoko; Okamoto, Takashi; Masai, Hisao; Poellinger, Lorenz

doi:10.1038/s41388-020-1218-3

Cited by 23 publications

(27 citation statements)

References 59 publications

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“…Here we showed that substitution variants did not affect RBBP5 binding. However, especially the KDM6A mutation D336G has been shown to be predominantly cytoplasmic in HeLa cells caused by impaired binding to ASH2L in a pull-down experiment and consequential reduced nuclear import by the KMT2C/D complex (17). In a previous study we observed that KDM6A nuclear import was strongly decreased after double, but not single, knock down of KMT2C and KMT2D proteins (18).…”

Section: Discussionmentioning

confidence: 78%

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

Koch

Lang

Whongsiri

et al. 2021

Preprint

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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.

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Section: Discussionmentioning

confidence: 78%

“…We used the freely available, open source Fiji plugin ScatterJ (17). Processed images were converted into 8-bit grey scale tagged image le formats (.tiff) and opened in ScatterJ.…”

Section: Scatterplot Generation and Cellular Back Mappingmentioning

confidence: 99%

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

Koch

Lang

Whongsiri

et al. 2021

Preprint

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“…First, UTX demethylates a target protein and thereby induces mitochondrial stress in some way. This is possible, since an increasing number of nonhistone target proteins of histone modifiers have been identified and a significant fraction of UTX is present in the cytoplasm 9 . Secondly, GSK-J4 induces mitochondrial stress irrelevant to UTX function.…”

Section: Discussionmentioning

confidence: 99%

“…S5). Moreover, we previously showed that exogenous UTX expression in UTXnegative HeLa cells caused less colony formation 9 . Thus, even without the UTX mutations, anomalies affecting UTX expression levels such as haploinsufficiency and DNA methylation in the promoter region may be significant contributors to cancer formation 9 .…”

Section: Induction Of Apoptosis By Gsk-j4 Does Not Correlate With Atfmentioning

confidence: 97%

A KDM6 inhibitor potently induces ATF4 and its target gene expression through HRI activation and by UTX inhibition

Kitajima

Sun

Lee

et al. 2021

Sci Rep

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UTX/KDM6A encodes a major histone H3 lysine 27 (H3K27) demethylase, and is frequently mutated in various types of human cancers. Although UTX appears to play a crucial role in oncogenesis, the mechanisms involved are still largely unknown. Here we show that a specific pharmacological inhibitor of H3K27 demethylases, GSK-J4, induces the expression of transcription activating factor 4 (ATF4) protein as well as the ATF4 target genes (e.g. PCK2, CHOP, REDD1, CHAC1 and TRIB3). ATF4 induction by GSK-J4 was due to neither transcriptional nor post-translational regulation. In support of this view, the ATF4 induction was almost exclusively dependent on the heme-regulated eIF2α kinase (HRI) in mouse embryonic fibroblasts (MEFs). Gene expression profiles with UTX disruption by CRISPR-Cas9 editing and the following stable re-expression of UTX showed that UTX specifically suppresses the expression of the ATF4 target genes, suggesting that UTX inhibition is at least partially responsible for the ATF4 induction. Apoptosis induction by GSK-J4 was partially and cell-type specifically correlated with the activation of ATF4-CHOP. These findings highlight that the anti-cancer drug candidate GSK-J4 strongly induces ATF4 and its target genes via HRI activation and raise a possibility that UTX might modulate cancer formation by regulating the HRI-ATF4 axis.

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“…ESCs were aggregated in hanging drops in the absence of LIF to form EBs, and the outgrowths were kept in culture for spontaneous differentiation (Figure 3-S1a). During EB differentiation, UTX was expressed at similar levels between f/f and KI cells (Figure 3-S1b), suggesting that the loss of enzymatic activity did not affect MLL4 level, which dictates the stability of UTX protein 4,24 . As expected, global H3K4me1 and, to a lesser extent, H3K4me2 decreased in KI cells during EB differentiation (Figure 3-S1c).…”

Section: Escs Lacking Mll3/4 Enzymatic Activities Are Capable Of Diffmentioning

confidence: 94%

MLL3/MLL4 methyltransferase activities control early embryonic development and embryonic stem cell differentiation in a lineage-selective manner

Xie

Lee

McKernan

et al. 2020

Preprint

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Enhancers drive cell-type-specific gene transcription and are marked by H3K4me1. MLL4 (KMT2D), a major H3K4me1 methyltransferase with partial functional redundancy with MLL3 (KMT2C), is critical for enhancer activation and cell-type-specific gene induction during cell differentiation and development. However, the roles of MLL3/4-mediated enhancer H3K4me1 and MLL3/4 enzymatic activities in general in these processes remain unclear. Here, we report that MLL3/4 enzymatic activities are partially redundant during mouse development. Simultaneous elimination of both leads to embryonic lethality around E8.5. Using embryoid body (EB) differentiation as an in vitro model for early embryonic development, we show that Mll3 knockout MLL4 enzyme-dead embryonic stem cells (ESCs) are capable of differentiating towards the three germ layers but display severe cavitation defects, likely due to impaired induction of visceral endoderm. Importantly, MLL3/4-catalyzed H3K4me1 is dispensable for enhancer activation during early EB differentiation and lineage-specific neural differentiation. Together, these results suggest a critical, but enhancer H3K4me1-independent, role of MLL3/4 enzymatic activities in early embryonic development and ESC differentiation.

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Cancer-derived UTX TPR mutations G137V and D336G impair interaction with MLL3/4 complexes and affect UTX subcellular localization

Cited by 23 publications

References 59 publications

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

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

A KDM6 inhibitor potently induces ATF4 and its target gene expression through HRI activation and by UTX inhibition

MLL3/MLL4 methyltransferase activities control early embryonic development and embryonic stem cell differentiation in a lineage-selective manner

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