Protein lysine 43 methylation by EZH1 promotes AML1-ETO transcriptional repression in leukemia

Liping Dou, Fei Yan, Jiuxia Pang, Dehua Zheng, Dandan Li, Li Gao, Lijun Wang, Yihan Xu, Jinlong Shi, Qian Wang, Lei Zhou, Na Shen, Puja Singh, Lili Wang, Yonghui Li, Yvchi Gao, Tao Liu, Chongjian Chen, Aref Al-Kali, Mark R. LitzowYoung In Chi, Ann M. Bode, Chunhui Liu, Haojie Huang, Daihong Liu, Guido Marcucci, Shujun Liu, Li Yu

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Abstract

The oncogenic fusion protein AML1-ETO retains the ability of AML1 to interact with the enhancer core DNA sequences, but blocks AML1-dependent transcription. Previous studies have shown that post-translational modification of AML1-ETO may play a role in its regulation. Here we report that AML1-ETO-positive patients, with high histone lysine methyltransferase Enhancer of zeste homolog 1 (EZH1) expression, show a worse overall survival than those with lower EZH1 expression. EZH1 knockdown impairs survival and proliferation of AML1-ETO-expressing cells in vitro and in vivo. We find that EZH1 WD domain binds to the AML1-ETO NHR1 domain and methylates AML1-ETO at lysine 43 (Lys43). This requires the EZH1 SET domain, which augments AML1-ETO-dependent repression of tumor suppressor genes. Loss of Lys43 methylation by point mutation or domain deletion impairs AML1-ETO-repressive activity. These findings highlight the role of EZH1 in non-histone lysine methylation, indicating that cooperation between AML1-ETO and EZH1 and AML1-ETO site-specific lysine methylation promote AML1-ETO transcriptional repression in leukemia.

Original languageEnglish (US)
Article number5051
JournalNature communications
Volume10
Issue number1
DOIs
StatePublished - Dec 1 2019

Bibliographical note

Funding Information:
treatments, the whole cellular lysates were prepared by harvesting the cells in 1× cell lysis buffer (20 mM HEPES [pH 7.6], 150 mM NaCl, and 0.1% NP40 supplemented with 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 mM PMSF, 1 mM NaF, 1 mM benzimedin, and protease inhibitors (protease inhibitor cocktail set III; Calbiochem-Novabiochem, San Diego, CA)37–39. Approximately 200 μg nuclear extract or 1 mg total protein lysate was precleared with 70 μL of slurry of protein A or G Dynabeads (Upstate Biotechnology) for 2 h at 4 °C. Dynabeads (70 μL) were coated with 2 to 5 μg antibodies at 4 °C overnight. The wash buffer (1× TBS, 0.1% Tween® 20) was used to elute proteins. The immunoprecipitates and whole cell lysates were subjected to Western blotting using established methods3,37. The Western blot was quantified using the Image J Software from the U.S. National Institutes of Health (NIH).

Funding Information:
In vivo leukemogenesis assays. C57BL/6J mice, athymic nude mice, and NOD/ SCID/γcnull immunodeficient NOG mice (4–6 weeks old, male) were purchased from Harlan Laboratories (Madison, WI) and Charles River (Beijing, China). All animal experiments were approved by the Institutional Animal Care and Use Committees of University of Minnesota and the Chinese PLA General Hospital, and were performed in accordance with the U.S. National Institutes of Health (NIH) Guide for Care and Use of Laboratory Animals. NOD/SCID/γcnull immunodeficient NOG mice were maintained under specific pathogen-free conditions at the animal facility of Chinese PLA General Hospital.

Funding Information:
This work was partially supported by grants from the National Institutes of Health/ National Cancer Institute (R01CA149623, R21CA155915, and R03CA186176), the Hormel Foundation, the Beijing Nova Program (2011114), National Natural Science Foundation of China (30800482, 30971297, 81102242, 81000221, 81270610, 81470010, 81170518, 81870117, and 90919044), the Beijing Natural Science Foundation of China (7102147, 7172200, and 7132217), Hainan Provincial Natural Science Foundation of China (818MS157), the Jilin Province Science and Technology Development Plan (20190201252JC), National Public Health Grand Research Foundation (201202017), the funding of the public health project (Z111107067311070), and the National 973 Project of China (2005CB522400). We thank T.T. Lam, K. Wilczak, and J. Kanyo at the MS and Proteomics Resource at Yale University for the high-resolution mass spectrometry data. We also thank Dr. Clara Nervi for SKNO-1 cell line.

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