H3K27M in Gliomas Causes a One-step Decrease in H3K27 Methylation and Reduced Spreading Within the Constraints of H3K36 Methylation [ChIP-seq]

The discovery of H3K27M mutations in pediatric gliomas marked a new chapter in cancer epigenetics. Numerous studies have investigated the effect of this mutation on H3K27 trimethylation, but only recently have we started to realize its additional effects on the epigenome. Here, we use isogenic glioma H3K27M(+/-) cell lines to investigate H3K27 methylation and its interaction with H3K36 and H3K9 modifications. We describe a “Step Down” effect of H3K27M on the distribution of H3K27 methylation: me3 is reduced to me2, me2 is reduced to me1, while H3K36me2/3 delineate the boundaries for the spread of H3K27me marks. We also observe a replacement of H3K27me2/3 silencing by H3K9me3. Using a computational simulation, we explain our observations by reduced effectiveness of PRC2 and constraints imposed on deposition of H3K27me by antagonistic H3K36 modifications. Our work further elucidates the effect of H3K27M in gliomas, as well as general principles of deposition of H3K27 methylation. Overall design: WGBS, RNA-seq, and ChIP-seq for histone H3 post-translational modifications in high-grade glioma cell lines.

儿童胶质瘤中H3K27M突变的发现，开启了癌症表观遗传学研究的崭新篇章。既往诸多研究已探讨该突变对H3K27三甲基化的影响，但直至近年，学界才逐渐认识到其对表观基因组的额外调控作用。本研究利用同基因背景的H3K27M阳性/阴性胶质瘤细胞系，探究H3K27甲基化模式及其与H3K36、H3K9修饰的相互作用。我们揭示了H3K27M对H3K27甲基化分布的"阶梯式下调"效应：H3K27三甲基化（me3）水平下调为二甲基化（me2）水平，二甲基化（me2）进一步下调为一甲基化（me1）水平；同时H3K36me2/3可界定H3K27me修饰的扩散边界。此外，我们还观察到H3K27me2/3介导的基因沉默被H3K9me3所替代。通过计算模拟实验，我们将上述观测结果归因于多梳抑制复合体2（Polycomb Repressive Complex 2，PRC2）功能减弱，以及拮抗型H3K36修饰对H3K27me沉积施加的约束。本研究进一步阐明了H3K27M在胶质瘤中的作用机制，以及H3K27甲基化沉积的通用规律。实验整体设计：对高级别胶质瘤细胞系开展全基因组亚硫酸氢盐测序（Whole Genome Bisulfite Sequencing，WGBS）、RNA测序（RNA-seq）及组蛋白H3翻译后修饰染色质免疫共沉淀测序（Chromatin Immunoprecipitation Sequencing，ChIP-seq）。