The histone H3G34R mutation disrupts the epigenome via catalytic inactivation of the ASH1 H3K36 methyltransferase [ChIP-seq]

The recurrent mutation of histone variant H3.3 at glycine-34 (H3.3G34) defines a type of pediatric glioma. Characteristic changes to the epigenome associated with the disease are thought to be the consequence of altered methylation of the adjacent lysine-36 (K36) residue, but the complexity of this regulatory pathway in humans, combined with a multi-component disease etiology, has limited our understanding of how H3.3G34 mutations contribute to oncogenesis. Here we use Neurospora crassa to show that the most common mutation associated with this tumor, glycine to arginine (G34R), drives aberrant heterochromatin formation and abnormal growth by inhibiting the H3K36 methyltransferase ASH1. Inactivation of ASH1, either directly or with H3G34R, drives spurious intergenic DNA methylation, redistribution of H3K27 methylation, and derepression of silent genes. We provide evidence that these defects are largely due to aberrant activity of RNA polymerase II (RNAPII)-associated SET-2, and propose targeted SET-2 inhibition as a therapeutic strategy for H3.3G34R gliomas. Overall design: We analyzed the distribution of histone H3 lysine 27 methylation (H3K27me2/3), H3K36me2/3, and H3K9me3 in Neurospora crassa by chromatin immunoprecipitation. Strains were grown, crosslinked, lysed, modified nucleosomes were immunopurified, and associated DNA was sequenced. A total of five genetic backgrounds are included here. Each background and each mark was done in replicate. Data sets with wild-type strains and related mutant backgrounds are deposited in GEO submision GSE118495.

组蛋白变体H3.3的甘氨酸34位复发性突变（H3.3G34）是一类儿童胶质瘤的标志性特征。该疾病相关的表观基因组特征性改变，既往被认为是相邻赖氨酸36（K36）残基甲基化状态异常的结果；但由于该调控通路在人类体内的复杂性，加之该病存在多组分致病机制，我们对H3.3G34突变如何驱动肿瘤发生的认知始终受限。本研究利用粗糙脉孢菌（Neurospora crassa）模型，证实该肿瘤最常见的突变类型——甘氨酸突变为精氨酸（G34R）——通过抑制H3K36甲基转移酶ASH1，诱导异常异染色质形成与细胞生长异常。无论是直接抑制ASH1，还是通过H3G34R突变间接使其失活，均可引发异常的基因间区域DNA甲基化、H3K27甲基化分布重塑，以及沉默基因的去阻遏。本研究证实，上述表观遗传缺陷主要源于与RNA聚合酶II（RNA polymerase II, RNAPII）结合的SET-2活性异常，并提出靶向抑制SET-2可作为H3.3G34R胶质瘤的治疗策略。整体实验设计：本研究通过染色质免疫共沉淀（chromatin immunoprecipitation）技术，分析了粗糙脉孢菌中组蛋白H3赖氨酸27甲基化（H3K27me2/3）、H3K36me2/3以及H3K9me3的分布情况。实验流程包括菌株培养、交联裂解、修饰型核小体免疫纯化，以及对其结合的DNA进行测序。本数据集共包含5种遗传背景样本，每种遗传背景对应每种组蛋白修饰均设置了生物学重复。野生型菌株及相关突变体背景的数据集已提交至基因表达综合数据库（Gene Expression Omnibus, GEO），登录号为GSE118495。