Abstract
EZH2, a component of the polycomb repressive complex 2, catalyses the trimethylation of histone H3 lysine 27, a chromatin mark associated with transcriptional repression. EZH2 loss-of-function mutations are seen in myeloid neoplasms and are associated with an adverse prognosis. Missense mutations in the SET/CXC domain abrogate catalytic activity as assessed by in vitro histone methylation assays, but missense mutations clustering in the conserved DI and DII regions retain activity. To understand the role of DI and DII mutations, we initially developed a cell-based histone methylation assay to test activity in a cellular context. Murine induced pluripotent stem cells lacking EZH2 were transiently transfected with wild type or mutant EZH2 (n = 15) and any resulting histone methylation was measured by flow cytometry. All DI mutations (n = 5) resulted in complete or partial loss of methylation activity whilst 5/6 DII mutations retained activity. Next, we assessed the possibility of splicing abnormalities induced by exon 8 mutations (encoding DII) using RT-PCR from primary patient samples and mini-gene assays. Exon 8 mutations resulted in skipping of exon 8 and an out-of-frame transcript. We have therefore shown that mutations within regions encoding EZH2 domains DI and DII are pathogenic by loss of function and exon skipping, respectively.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Ernst T, Chase AJ, Score J, Hidalgo-Curtis CE, Bryant C, Jones AV, et al. Inactivating mutations of the histone methyltransferase gene EZH2 in myeloid disorders. Nat Genet. 2010;42:722–6.
Nikoloski G, Langemeijer SM, Kuiper RP, Knops R, Massop M, Tonnissen ER, et al. Somatic mutations of the histone methyltransferase gene EZH2 in myelodysplastic syndromes. Nat Genet. 2010;42:665–7.
Antonysamy S, Condon B, Druzina Z, Bonanno JB, Gheyi T, Zhang F, et al. Structural context of disease-associated mutations and putative mechanism of autoinhibition revealed by X-ray crystallographic analysis of the EZH2-SET domain. PLoS ONE. 2013;8:e84147.
Wu H, Zeng H, Dong A, Li F, He H, Senisterra G, et al. Structure of the catalytic domain of EZH2 reveals conformational plasticity in cofactor and substrate binding sites and explains oncogenic mutations. PLoS ONE. 2013;8:e83737.
Lee CH, Yu JR, Kumar S, Jin Y, LeRoy G, Bhanu N, et al. Allosteric Activation Dictates PRC2 Activity Independent of Its Recruitment to Chromatin. Mol Cell. 2018;70:422–34.
Villasante A, Piazzolla D, Li H, Gomez-Lopez G, Djabali M, Serrano M. Epigenetic regulation of Nanog expression by Ezh2 in pluripotent stem cells. Cell Cycle. 2011;10:1488–98.
Matsuda T, Cepko CL. Electroporation and RNA interference in the rodent retina in vivo and in vitro. Proc Natl Acad Sci USA. 2004;101:16–22.
Kishore S, Khanna A, Stamm S. Rapid generation of splicing reporters with pSpliceExpress. Gene. 2008;427:104–10.
Cross NC, Melo JV, Feng L, Goldman JM. An optimized multiplex polymerase chain reaction (PCR) for detection of BCR-ABL fusion mRNAs in haematological disorders. Leukemia. 1994;8:186–9.
Raponi M, Kralovicova J, Copson E, Divina P, Eccles D, Johnson P, et al. Prediction of single-nucleotide substitutions that result in exon skipping: identification of a splicing silencer in BRCA1 exon 6. Hum Mutat. 2011;32:436–44.
Fraile-Bethencourt E, Diez-Gomez B, Velasquez-Zapata V, Acedo A, Sanz DJ, Velasco EA. Functional classification of DNA variants by hybrid minigenes: Identification of 30 spliceogenic variants of BRCA2 exons 17 and 18. PLoS Genet. 2017;13:e1006691.
Laible G, Wolf A, Dorn R, Reuter G, Nislow C, Lebersorger A, et al. Mammalian homologues of the Polycomb-group gene Enhancer of zeste mediate gene silencing in Drosophila heterochromatin and at S. cerevisiae telomeres. EMBO J. 1997;16:3219–32.
Jiao L, Liu X. Structural basis of histone H3K27 trimethylation by an active polycomb repressive complex 2. Science. 2015;350:aac4383.
Margueron R, Justin N, Ohno K, Sharpe ML, Son J, Drury WJ 3rd, et al. Role of the polycomb protein EED in the propagation of repressive histone marks. Nature. 2009;461:762–7.
Zehir A, Benayed R, Shah RH, Syed A, Middha S, Kim HR, et al. Mutational landscape of metastatic cancer revealed from prospective clinical sequencing of 10,000 patients. Nat Med. 2017;23:703–13.
Bartels S, Schipper E, Hasemeier B, Kreipe H, Lehmann U. Routine clinical mutation profiling using next generation sequencing and a customized gene panel improves diagnostic precision in myeloid neoplasms. Oncotarget. 2016;7:30084–93.
Seiwert TY, Zuo Z, Keck MK, Khattri A, Pedamallu CS, Stricker T, et al. Integrative and comparative genomic analysis of HPV-positive and HPV-negative head and neck squamous cell carcinomas. Clin Cancer Res. 2015;21:632–41.
Shirahata-Adachi M, Iriyama C, Tomita A, Suzuki Y, Shimada K, Kiyoi H. Altered EZH2 splicing and expression is associated with impaired histone H3 lysine 27 tri-Methylation in myelodysplastic syndrome. Leuk Res. 2017;63:90–7.
Findlay GM, Daza RM, Martin B, Zhang MD, Leith AP, Gasperini M, et al. Accurate classification of BRCA1 variants with saturation genome editing. Nature. 2018;562:217–22.
Acknowledgements
We are grateful to Professor Manuel Serrano for providing us with the murine Ezh2 knockout iPS cell line Ezh2 Δ/Δ clone 10. This study was funded by Bloodwise Specialist Programme Grant nos. 13002 and 18007 to NCPC and AC.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Chase, A., Score, J., Lin, F. et al. Mutational mechanisms of EZH2 inactivation in myeloid neoplasms. Leukemia 34, 3206–3214 (2020). https://doi.org/10.1038/s41375-020-0816-y
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41375-020-0816-y
This article is cited by
-
Tumor-suppressive functions of protein lysine methyltransferases
Experimental & Molecular Medicine (2023)
-
Immunohistochemical loss of enhancer of Zeste Homolog 2 (EZH2) protein expression correlates with EZH2 alterations and portends a worse outcome in myelodysplastic syndromes
Modern Pathology (2022)