Base-resolution methylomes of gliomas bearing histone H3.3 mutations reveal a G34 mutant-specific signature shared with bone tumors

Yuhei Sangatsuda,Hiromitsu Araki,Akihiko Yoshida,Daisuke Kuga,Yojiro Akagi,Masahiro Mizoguchi,Takeo Amemiya,Nobuhiro Hata,Ryusuke Hatae,Tatsuhiro Shibata,Koji Yoshimoto,Fumihito Miura,Yasuhito Arai,Takashi Ito,Koji Iihara,Yutaka Fujioka

doi:10.1038/s41598-020-73116-x

Yuhei Sangatsuda, Hiromitsu Araki + Show 14 more

Open Access

https://doi.org/10.1038/s41598-020-73116-x

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Abstract

Two recurrent mutations, K27M and G34R/V, in H3F3A, encoding non-canonical histone H3.3, are reported in pediatric and young adult gliomas, whereas G34W mutation is prevalent in bone tumors. In contrast to K27M mutation, it remains elusive how G34 mutations affect the epigenome. Here we performed whole-genome bisulfite sequencing of four G34R-mutated gliomas and the G34V-mutated glioma cell line KNS-42 for comparison with gliomas harboring K27M and no mutations in H3F3A and with G34W-mutated bone tumors. G34R-mutated gliomas exhibited lower global methylation levels, similar CpG island (CGI) methylation levels, and compromised hypermethylation of telomere-proximal CGIs, compared to the other two glioma subgroups. Hypermethylated regions specific to G34R-mutated gliomas were enriched for CGIs, including those of OLIG1, OLIG2, and canonical histone genes in the HIST1 cluster. They were notably hypermethylated in osteosarcomas with, but not without, G34W mutation. Independent component analysis revealed that G34 mutation-specific components shared a significant similarity between glioma and osteosarcoma, suggesting that G34 mutations exert characteristic methylomic effects regardless of the tumor tissue-of-origin. CRISPR/Cas9-mediated disruption of G34V-allele in KNS-42 cells led to demethylation of a subset of CGIs hypermethylated in G34R-mutated gliomas. These findings will provide a basis for elucidating epigenomic roles of G34 oncohistone in tumorigenesis.

Highlights

Two recurrent mutations, K27M and G34R/V, in H3F3A, encoding non-canonical histone H3.3, are reported in pediatric and young adult gliomas, whereas G34W mutation is prevalent in bone tumors
We used the post-bisulfite adaptor tagging (PBAT) method to conduct PCR-free wholegenome bisulfite sequencing (WGBS) of four, seven, and three cases of primary gliomas harboring G34R mutation (G34 subgroup), K27M mutation (K27 subgroup), and no mutation in the H3F3A gene (WT subgroup), respectively, as well as the G34V-mutated glioma cell line KNS-42 (Tables S1 and S2). Combining these WGBS datasets with those obtained from the International Cancer Genome Consortium (ICGC) database, we compared the mean CpG methylation levels among the three subgroups
G34 subgroup exhibited the lowest mean methylation level (55.8%), which was followed by those of K27 (63.6%) and WT (67.3%) subgroups (Fig. 1). This order was maintained in the promoters and gene bodies but not in CpG island (CGI); mean CGI methylation levels were comparable among the three subgroups (Fig. 1)

Summary

Introduction

K27M and G34R/V, in H3F3A, encoding non-canonical histone H3.3, are reported in pediatric and young adult gliomas, whereas G34W mutation is prevalent in bone tumors. Hypermethylated regions specific to G34R-mutated gliomas were enriched for CGIs, including those of OLIG1, OLIG2, and canonical histone genes in the HIST1 cluster. They were notably hypermethylated in osteosarcomas with, but not without, G34W mutation. CRISPR/Cas9-mediated disruption of G34V-allele in KNS-42 cells led to demethylation of a subset of CGIs hypermethylated in G34R-mutated gliomas. These findings will provide a basis for elucidating epigenomic roles of G34 oncohistone in tumorigenesis. A recent study demonstrated the utility of methylation profiling in auxiliary diagnosis of central nervous system tumors[10]

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