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Biochemical validation of EHMT1 missense mutations in Kleefstra syndrome


Kleefstra syndrome (KS) (9q34 deletion syndrome) is a rare autosomal dominant disorder characterized by intellectual disability, frequently coupled with a spectrum of complex physical and clinical manifestations. As the euchromatic histone methyltransferase-1 gene (EHMT1, GLP, or KMT1D) within the 9q34 region is deleted or mutated in most of the individuals with KS, its absence or defect in one allele is speculated to cause the major symptoms of the syndrome. Most of the EHMT1 mutations are frameshift or nonsense mutations, but two individuals with KS were reported to possess EHMT1 missense mutations. These two mutations have been predicted to cause a defective enzymatic function, but precise biochemical validation was not conducted. Therefore, we validated these two mutations by performing in vitro histone methyltransferase (HMT) activity assay and found that C1073Y and R1197W mutations severely affected the HMT activity. Additionally, the same amino-acid substitutions in mouse GLP induced impairment of in vivo GLP function. Furthermore, these two EHMT1 mutants showed defective heterocomplex formation with G9a (partner HMT) which is essential for their in vivo HMT function. Conclusively, our biochemical characterization clearly demonstrates that the previously reported two missense mutations of EHMT1 deteriorate HMT activity and GLP function, which presumably cause KS.

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  1. 1.

    Kleefstra T, Brunner HG, Amiel J, Oudakker AR, Nillesen WM, Magee A, et al. Loss-of-function mutations in euchromatin histone methyl transferase 1 (EHMT1) cause the 9q34 subtelomeric deletion syndrome. Am J Hum Genet. 2006;79:370–7.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Kleefstra T, van Zelst-Stams WA, Nillesen WM, Cormier-Daire V, Houge G, Foulds N, et al. Further clinical and molecular delineation of the 9q subtelomeric deletion syndrome supports a major contribution of EHMT1 haploinsufficiency to the core phenotype. J Med Genet. 2009;46:598–606.

    CAS  Article  PubMed  Google Scholar 

  3. 3.

    Kleefstra T, Kramer JM, Neveling K, Willemsen MH, Koemans TS, Vissers LE, et al. Disruption of an EHMT1-associated chromatin-modification module causes intellectual disability. Am J Hum Genet. 2012;91:73–82.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Willemsen MH, Vulto-van Silfhout AT, Nillesen WM, Wissink-Lindhout WM, van Bokhoven H, Philip N, et al. Update on Kleefstra Syndrome. Mol Syndromol. 2012;2:202–12.

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Shinkai Y. Regulation and function of H3K9 methylation. Subcell Biochem. 2007;41:337–50.

    PubMed  Google Scholar 

  6. 6.

    Kimura H. Histone modifications for human epigenome analysis. J Hum Genet. 2013;58:439–45.

    CAS  Article  PubMed  Google Scholar 

  7. 7.

    Shinkai Y, Tachibana M. H3K9 methyltransferase G9a and the related molecule GLP. Genes Dev. 2011;25:781–8.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Tachibana M, Ueda J, Fukuda M, Takeda N, Ohta T, Iwanari H, et al. Histone methyltransferases G9a and GLP form heteromeric complexes and are both crucial for methylation of euchromatin at H3-K9. Genes Dev. 2005;19:815–26.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Tachibana M, Sugimoto K, Nozaki M, Ueda J, Ohta T, Ohki M, et al. G9a histone methyltransferase plays a dominant role in euchromatic histone H3 lysine 9 methylation and is essential for early embryogenesis. Genes Dev. 2002;16:1779–91.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Tachibana M, Matsumura Y, Fukuda M, Kimura H, Shinkai Y. G9a/GLP complexes independently mediate H3K9 and DNA methylation to silence transcription. EMBO J. 2008;27:2681–90.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Shimazu T, Barjau J, Sohtome Y, Sodeoka M, Shinkai Y. Selenium-based S-adenosylmethionine analog reveals the mammalian seven-beta-strand methyltransferase METTL10 to be an EF1A1 lysine methyltransferase. PLoS ONE 2014;9:e105394.

    Article  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Wu H, Min J, Lunin VV, Antoshenko T, Dombrovski L, Zeng H, et al. Structural biology of human H3K9 methyltransferases. PLoS ONE 2010;5:e8570.

    Article  PubMed  PubMed Central  Google Scholar 

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We thank Dr. Masoud Vedadi for providing the hGLP catalytic domain (982–1266 aa) expression vector and Dr. Yasuo Tsunaka for discussion on GLP structural data analysis.


This research was supported by a RIKEN internal research fund.

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Correspondence to Yoichi Shinkai.

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The authors declare that they have no conflict of interest.

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Yamada, A., Shimura, C. & Shinkai, Y. Biochemical validation of EHMT1 missense mutations in Kleefstra syndrome. J Hum Genet 63, 555–562 (2018).

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