Cell-of-origin and Developmental Trajectories Cooperate to Determine Chromatin Landscapes in Histone-Mutant Diffuse Midline Gliomas

Diffuse midline gliomas (DMGs) are universally fatal pediatric brain tumors associated with mutations in genes encoding either histone H3.1 or histone 3.3, often substitution of methionine for lysine 27 (H3K27M). H3K27 is a critical determinant of chromatin state via methylation by Enhancer-of-Zeste-Homolog-2 (EZH2). Previous reports have suggested that the pathologically low levels of H3K27me3 found in histone-mutant DMGs result primarily from H3K27M inhibiting EZH2 directly, but recent reports have called this model into question. To better understand the chromatin landscape of DMGs, we applied CUT&RUN to patient-derived DMG cell lines. Remarkably, we find that the PRC2 activity is similar in DMGs and embryonic stem cells, suggesting a primitive cell-of-origin, despite transcriptionally active regions maintaining markers of both stem cells and differentiated cells. We also show that exogenous expression of H3.3M at physiological levels has little effect on H3K27me3 levels, that H3K27M can colocalize with H3K27me3 in vivo and that the H3.3K27M oncohistone does not show evidence of sequestering PRC2 components. Our results suggest that chromatin landscapes in DMGs are a consequence of a stem-like chromatin state that is retained despite activation of differentiation programs. Our findings have implications for understanding DMG gliomagenesis and therapeutic approaches centered on epigenome-modifying agents. We used Cleavage under targets and Release using nuclease (Cut-and-Run), a chromatin profiling strategy in which antibody-targeted controlled cleavage by micrococcal nuclease releases specific protein-DNA complexes into the supernatant for paired-end DNA sequencing.弥漫性中线胶质瘤（Diffuse midline gliomas, DMGs）是一类均为致命性的儿童脑肿瘤，其发生与编码组蛋白H3.1或组蛋白H3.3的基因发生突变密切相关，最常见的突变类型为赖氨酸27被甲硫氨酸取代（H3K27M）。组蛋白H3K27位点可通过zeste增强子同源物2（Enhancer-of-Zeste-Homolog-2, EZH2）介导的甲基化修饰，成为调控染色质状态的关键决定因素。既往研究认为，组蛋白突变型DMGs中检测到的病理性低水平H3K27me3，主要源于H3K27M对EZH2的直接抑制，但近期相关研究对这一模型提出了质疑。 为更深入解析DMGs的染色质调控图谱，本研究对患者来源的DMG细胞系应用了靶向切割与释放核酸酶法（Cleavage under targets and Release using nuclease, CUT&RUN）。值得注意的是，本研究发现DMGs与胚胎干细胞中的多梳抑制复合体2（PRC2）活性水平相近，提示其细胞起源具有原始干细胞特性，尽管其转录活跃区域同时兼具干细胞与分化细胞的标志物特征。本研究还证实：在生理水平外源表达H3.3M对H3K27me3水平几乎无影响；H3K27M可在体内与H3K27me3共定位；且致癌组蛋白H3.3K27M并未表现出螯合PRC2复合体组分的证据。本研究结果表明，DMGs的染色质调控图谱源于一类保留干细胞样染色质状态的细胞，即便分化程序已被激活。上述发现对于解析DMG的胶质瘤发生机制以及以表观基因组修饰剂为核心的治疗策略具有重要参考价值。 本研究采用的靶向切割与释放核酸酶法（CUT&RUN）是一种染色质谱分析策略，通过抗体靶向定位靶标后，利用微球菌核酸酶进行可控切割，将特异性蛋白-DNA复合物释放至上清液中，用于双端DNA测序。