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Mutational mechanisms of EZH2 inactivation in myeloid neoplasms

Mutational mechanisms of EZH2 inactivation in myeloid neoplasms
Mutational mechanisms of EZH2 inactivation in myeloid neoplasms
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.
0887-6924
3206-3214
Chase, Andrew
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Score, Joannah
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Lin, Feng
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Bryant, Catherine
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Waghorn, Katherine
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Yapp, Sarah
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Carreno-tarragona, Gonzalo
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Aranaz, Paula
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Villasante, Aranzazu
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Ernst, Thomas
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Cross, Nicholas C. P.
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Chase, Andrew
a40a09c2-3073-4655-ba0b-a802e34914b5
Score, Joannah
376ca13f-2b1b-43b5-aefa-da1d82291615
Lin, Feng
9067ee54-a57f-4432-b390-ec81fd8aa73c
Bryant, Catherine
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Waghorn, Katherine
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Yapp, Sarah
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Carreno-tarragona, Gonzalo
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Aranaz, Paula
a8649d20-cc22-4dda-8101-0e9fb393f0b8
Villasante, Aranzazu
c50d6585-1d97-448c-bbd4-fab872c905a3
Ernst, Thomas
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Cross, Nicholas C. P.
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Chase, Andrew, Score, Joannah, Lin, Feng, Bryant, Catherine, Waghorn, Katherine, Yapp, Sarah, Carreno-tarragona, Gonzalo, Aranaz, Paula, Villasante, Aranzazu, Ernst, Thomas and Cross, Nicholas C. P. (2020) Mutational mechanisms of EZH2 inactivation in myeloid neoplasms. Leukemia, 34 (12), 3206-3214. (doi:10.1038/s41375-020-0816-y).

Record type: Article

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.

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Accepted/In Press date: 26 March 2020
e-pub ahead of print date: 22 April 2020

Identifiers

Local EPrints ID: 439698
URI: http://eprints.soton.ac.uk/id/eprint/439698
ISSN: 0887-6924
PURE UUID: 3dd7ffa0-6006-473b-b62a-5868e58e17e6
ORCID for Andrew Chase: ORCID iD orcid.org/0000-0001-6617-9953
ORCID for Nicholas C. P. Cross: ORCID iD orcid.org/0000-0001-5481-2555

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Date deposited: 30 Apr 2020 16:30
Last modified: 28 Apr 2022 05:13

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Contributors

Author: Andrew Chase ORCID iD
Author: Joannah Score
Author: Feng Lin
Author: Catherine Bryant
Author: Katherine Waghorn
Author: Sarah Yapp
Author: Gonzalo Carreno-tarragona
Author: Paula Aranaz
Author: Aranzazu Villasante
Author: Thomas Ernst

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