Mutation of A677 in histone methyltransferase EZH2 in human B-cell lymphoma promotes hypertrimethylation of histone H3 on lysine 27 (H3K27).
Trimethylation of histone H3 on lysine 27 (H3K27me3) is a repressive posttranslational modification mediated by the histone methyltransferase EZH2. EZH2 is a component of the polycomb repressive complex 2 and is overexpressed in many cancers. In B-cell lymphomas, its substrate preference is frequently altered through somatic mutation of the EZH2 Y641 residue. Herein, we identify mutation of EZH2 A677 to a glycine (A677G) among lymphoma cell lines and primary tumor specimens. Similar to Y641 mutant cell lines, an A677G mutant cell line revealed aberrantly elevated H3K27me3 and decreased monomethylated H3K27 (H3K27me1) and dimethylated H3K27 (H3K27me2). A677G EZH2 possessed catalytic activity with a substrate specificity that was distinct from those of both WT EZH2 and Y641 mutants. Whereas WT EZH2 displayed a preference for substrates with less methylation [unmethylated H3K27 (H3K27me0):me1:me2 k(cat)/K(m) ratio = 9:6:1] and Y641 mutants preferred substrates with greater methylation (H3K27me0:me1:me2 k(cat)/K(m) ratio = 1:2:13), the A677G EZH2 demonstrated nearly equal efficiency for all three substrates (H3K27me0:me1:me2 k(cat)/K(m) ratio = 1.1:0.6:1). When transiently expressed in cells, A677G EZH2, but not WT EZH2, increased global H3K27me3 and decreased H3K27me2. Structural modeling of WT and mutant EZH2 suggested that the A677G mutation acquires the ability to methylate H3K27me2 through enlargement of the lysine tunnel while preserving activity with H3K27me0/me1 substrates through retention of the Y641 residue that is crucial for orientation of these smaller substrates. This mutation highlights the interplay between Y641 and A677 residues in the substrate specificity of EZH2 and identifies another lymphoma patient population that harbors an activating mutation of EZH2.
Cancer Epigenetics Discovery Performance Unit, Cancer Research, Oncology Research and Development, GlaxoSmithKline, Collegeville, PA 19426, USA. michael.t.mccabe@gsk.com
Michael T
MT
McCabe
Alan P
AP
Graves
Gopinath
G
Ganji
Elsie
E
Diaz
Wendy S
WS
Halsey
Yong
Y
Jiang
Kimberly N
KN
Smitheman
Heidi M
HM
Ott
Melissa B
MB
Pappalardi
Kimberly E
KE
Allen
Stephanie B
SB
Chen
Anthony
A
Della Pietra
Edward
E
Dul
Ashley M
AM
Hughes
Seth A
SA
Gilbert
Sara H
SH
Thrall
Peter J
PJ
Tummino
Ryan G
RG
Kruger
Martin
M
Brandt
Benjamin
B
Schwartz
Caretha L
CL
Creasy
D000409:Alanine / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D045744:Cell Line, Tumor; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000071221:Enhancer of Zeste Homolog 2 Protein; D015972:Gene Expression Regulation, Neoplastic; D005998:Glycine / Q000235:genetics; D006579:Heterozygote; D000076983:Histone Methyltransferases; D011495:Histone-Lysine N-Methyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006657:Histones / Q000378:metabolism*; D006801:Humans; D016393:Lymphoma, B-Cell / Q000201:enzymology* / Q000235:genetics*; D008239:Lysine / Q000378:metabolism*; D008745:Methylation; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D063151:Polycomb Repressive Complex 2; D013379:Substrate Specificity; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D004268:DNA-Binding Proteins; D006657:Histones; D050505:Mutant Proteins; D014157:Transcription Factors; D000076983:Histone Methyltransferases; C498409:EZH2 protein, human; D000071221:Enhancer of Zeste Homolog 2 Protein; D011495:Histone-Lysine N-Methyltransferase; D063151:Polycomb Repressive Complex 2; D008239:Lysine; D000409:Alanine; D005998:Glycine