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

Oncogene volume 39, pages 3322–3335 (2020)Cite this article

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Abstract

The ubiquitously transcribed tetratricopeptide repeat on X chromosome (UTX) is a major histone H3 lysine 27 (H3K27) demethylase and the mixed-lineage leukemia (MLL) proteins are the H3K4 methyltransferases. UTX is one of the major components of MLL3- and MLL4-containing (MlLL3/4) complexes and likely has functions within the complexes. Although UTX is frequently mutated in various types of cancer and is thought to play a crucial role as a tumor suppressor, the importance of UTX interaction with MLL3/4 complexes in cancer formation is poorly understood. Here, we analyzed the ability of cancer-derived UTX mutant proteins to interact with ASH2L, which is a common core component of all the MLL complexes, and MLL3/4-specific components PTIP and PA1, and found that several single-amino acid substitution mutations in the tetratricopeptide repeat (TPR) affect UTX interaction with these components. Interaction-compromised mutants G137V and D336G and a TPR-deleted mutant Δ80-397 were preferentially localized to the cytoplasm, suggesting that UTX is retained in the nucleus by MLL3/4 complexes through their interaction with the TPR. Intriguingly, WT UTX suppressed colony formation in soft agar, whereas G137V failed. This suggests that interaction of UTX with MLL3/4 complex plays a crucial role in their tumor suppressor function. Preferential cytoplasmic localization was also observed for endogenous proteins of G137V and another mutant G137VΔ138 in HCT116 created by CRISPR-Cas9 gene editing. Interestingly, expression levels of these mutants were low and MG312 stabilized both endogenous as well as exogenous G137V proteins. These results reveal a novel mechanism of UTX regulation and reinforce the importance of UTX interaction with MLL3/4 complexes in cancer formation.

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Fig. 1: UTX TPR sequence and structure.
Fig. 2: Interaction of cancer-derived UTX missense mutants with ASH2L.
Fig. 3: Subcellular localization of mutant UTX proteins.
Fig. 4: Analysis of endogenous UTX proteins carrying G137V or G137V with a small deletion constructed by CRISPR gene editing.
Fig. 5: Analysis of G137V and G137VΔ138 interaction with endogenous PTIP.
Fig. 6: Stabilization of G137V and G137VΔ138 by MG132.
Fig. 7: Impairment of WT UTX functions by TPR mutations.

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Acknowledgements

We thank all the LP lab members for technical assistance. This work was supported by grants from the Singapore National Research Foundation (R-713-005-014-271 and R-713-000-162-511), the Singapore Ministry of Education under the Research Centres of Excellence Programme, and the Singapore Ministry of Health’s National Medical Research Council (Clinician Scientist Individual Research Grant, NMRC/CIRG/1389/2014) to LP, and Japan Society for the Promotion of Science KAKENHI (17K08638) to KA and (Scientific Research A, 23247031 and 26251004) to HM, and the Takeda Science Foundation to HM. In the midst of this project, Prof. Lorenz Poellinger passed away, to whom we dedicate this work.

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  1. Deceased: Lorenz Poellinger

Authors and Affiliations

  1. Cancer Science Institute of Singapore, National University of Singapore, 14 Medical Drive, Singapore, 117599, Republic of Singapore

    Hiroyuki Kato, Wendi Sun, Shojiro Kitajima & Lorenz Poellinger

  2. Department of Genome Medicine, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya, Tokyo, 156-8506, Japan

    Hiroyuki Kato, Naoko Yoshizawa-Sugata & Hisao Masai

  3. Department of Molecular and Cellular Biology, Nagoya City University Graduate School of Medical Science, Mizuho-ku, Nagoya, 467-860, Japan

    Hiroyuki Kato, Kaori Asamitsu & Takashi Okamoto

  4. The Institute for Advanced Biosciences, Keio University, Kakuganji 246-2, Mizukami, Tsuruoka, Yamagata, 997-0052, Japan

    Shojiro Kitajima

  5. Department of Cell and Molecular Biology, Karolinska Institutet, SE-171 77, Stockholm, Sweden

    Lorenz Poellinger

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Kato, H., Asamitsu, K., Sun, W. et al. Cancer-derived UTX TPR mutations G137V and D336G impair interaction with MLL3/4 complexes and affect UTX subcellular localization. Oncogene 39, 3322–3335 (2020). https://doi.org/10.1038/s41388-020-1218-3

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