Structural Biology of Human H3K9 Methyltransferases

Lysine to methionine (K-to-M) mutations in genes encoding histone H3 are thought to drive a subset of pediatric brain and bone cancers. These high-frequency K-to-M mutations occur at sites of methylation on histone H3, and tumors containing the mutant histones exhibit a global loss of specific histone methylation marks. Previous studies showed that K-to-M mutant histones, also known as oncohistones, are potent orthosteric inhibitors of specific Su(var)3-9, Enhancer-ofzeste, Trithorax (SET) domain methyltransferases. However, the biochemical and biophysical details of the interaction between K-to-M mutant histones and the respective SET domain methyltransferases are currently unknown. Here, we use the histone H3K9-directed methyltransferase G9a as a model to explore the mechanism of inhibition by K-to-M oncohistones. X-ray cocrystal structures revealed that the K9M residue of histone H3 occupies the active site cavity of G9a, and kinetic analysis indicates competitive inhibition of G9a by histone H3K9M. Additionally, we find that the cofactor S-adenosyl methionine (SAM) is necessary for stable interaction between G9a and H3K9M histone. Consistent with the formation of a ternary complex, we find that the inhibitory peptide is uncompetitive with regard to SAM. These data and others indicate that K-to-M oncohistones promote global loss of specific lysine methylation through sequestration and inhibition of SAM-bound SET domain methyltransferases.C ovalent modifications to both DNA and histone proteins turn chromatin into a dynamic information hub that integrates diverse biochemical stimuli to regulate genomic DNA access to the transcription machinery and ultimately, establish and maintain cellular phenotypes. Moreover, there is increasing appreciation that alterations of the chromatin landscape, including DNA and histone modifications, are involved in the pathogenesis of cancer. Nowhere is this finding better supported than with the groundbreaking discoveries of high-frequency somatic mutations in histones that are drivers of tumorigenesis.Monoallelic missense mutations in genes encoding for histone H3 were recently found in bone and brain tumors that affect children and young adults. Approximately 80% of diffuse intrinsic pontine glioma contain a lysine 27-methionine (K27M) mutation (1, 2), and 95% of chondroblastoma samples contain a K36M mutation in genes encoding either histone H3.1 or H3.3 (3). The K27M and K36M mutations in histone H3 are the known lysine to methionine ("K-to-M") histone H3 missense mutations found in human disease thus far. Although these oncohistones represent a small population of total histone H3 in tumor cells (3-17% of histone H3), they remarkably lead to global loss of the associated methylation mark on the WT complement of histone H3. The nearly invariant nature of the K-to-M mutation strongly suggests that this specific amino acid substitution imparts a unique gain of function to the mutant histone. We and others previously showed that K-to-M oncohistones transform the histone H3 prote...

Section: Resultssupporting

confidence: 88%

Section: Methodsmentioning

confidence: 99%

S -adenosyl methionine is necessary for inhibition of the methyltransferase G9a by the lysine 9 to methionine mutation on histone H3

Jayaram

Hoelper

Jain

et al. 2016

“…Similarly, the wild-type enzyme G9a can dimethylate H3K9 but is unable to perform the di-to trimethylation reaction. Yet, when tyrosine 1067 of G9a (analogous to Y641 of EZH2) is mutated to phenylalanine, the enzyme now gains the ability to trimethylate H3K9 (21). The tolerance for multiple Y641 mutations in EZH2 suggests that a release of steric crowding may allow greater access for proper alignment of the larger dimethyl lysine as the substrate for the di-to trimethylation reaction.…”

Section: Resultsmentioning

confidence: 99%

Coordinated activities of wild-type plus mutant EZH2 drive tumor-associated hypertrimethylation of lysine 27 on histone H3 (H3K27) in human B-cell lymphomas

Sneeringer

Scott

Kuntz

et al. 2010

623

535

EZH2, the catalytic subunit of the PRC2 complex, catalyzes the mono- through trimethylation of lysine 27 on histone H3 (H3K27). Histone H3K27 trimethylation is a mechanism for suppressing transcription of specific genes that are proximal to the site of histone modification. Point mutations of the EZH2 gene (Tyr641) have been reported to be linked to subsets of human B-cell lymphoma. The mutant allele is always found associated with a wild-type allele (heterozygous) in disease cells, and the mutations were reported to ablate the enzymatic activity of the PRC2 complex for methylating an unmodified peptide substrate. Here we demonstrate that the WT enzyme displays greatest catalytic efficiency ( k cat / K ) for the zero to monomethylation reaction of H3K27 and diminished efficiency for subsequent (mono- to di- and di- to trimethylation) reactions. In stark contrast, the disease-associated Y641 mutations display very limited ability to perform the first methylation reaction, but have enhanced catalytic efficiency for the subsequent reactions, relative to the WT enzyme. These results imply that the malignant phenotype of disease requires the combined activities of a H3K27 monomethylating enzyme (PRC2 containing WT EZH2 or EZH1) together with the mutant PRC2s for augmented conversion of H3K27 to the trimethylated form. To our knowledge, this is the first example of a human disease that is dependent on the coordinated activities of normal and disease-associated mutant enzymatic function.

“…The human SET7/9 methyltransferase normally monomethylates H3K4; however, when the SET7/9 Y245 residue (the equivalent of EZH2 Y641) is mutated to alanine, the mutant can no longer modify an H3K4me0 substrate but, instead, gains the ability to di-and trimethylate an H3K4me1 peptide substrate (37). Similarly, exchange of the Y641 equivalent in the H3K9 methyltransferase G9a (Y1067) for phenylalanine converts the enzyme from a mono-and dimethyltransferase to a trimethyltransferase (38).…”

Section: Discussionmentioning

confidence: 99%

Mutation of A677 in histone methyltransferase EZH2 in human B-cell lymphoma promotes hypertrimethylation of histone H3 on lysine 27 (H3K27)

McCabe

Graves

Ganji

et al. 2012

434

425

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.