Histone H3.3 K27M chromatin functions implicate a network of neurodevelopmental factors including ASCL1 and NEUROD1 in DIPG. Histone H3.3 K27M chromatin functions implicate a network of neurodevelopmental factors including ASCL1 and NEUROD1 in DIPG

Background: The histone variant H3.3 K27M mutation is a defining characteristic of diffuse intrinsic pontine glioma (DIPG)/diffuse midline glioma (DMG). This histone mutation is responsible for major alterations to histone H3 post-translational modification (PTMs) and subsequent aberrant gene expression. However, much less is known about the effect this mutation has on chromatin structure and function, including open versus closed chromatin regions as well as their transcriptomic consequences. Results: Recently, we developed isogenic CRISPR-edited DIPG cell lines that are wild- type for histone H3.3 that can be compared to their matched K27M lines. Here we show via ATAC-seq analysis that H3.3K27M glioma cells have unique accessible chromatin at regions corresponding to neurogenesis, NOTCH, and neuronal development pathways and associated genes that are overexpressed in H3.3K27M compared to our isogenic wild-type cell line. As to mechanisms, accessible enhancers and super-enhancers corresponding to increased gene expression in H3.3K27M cells were also mapped to genes involved in neurogenesis and NOTCH signaling, suggesting that these pathways are key to DIPG tumor maintenance. Motif analysis implicates specific transcription factors as central to the neuro-oncogenic K27M signaling pathway including, in particular, ASCL1 and NEUROD1. Conclusions: Altogether our findings indicate that H3.3K27M causes chromatin to take on a more accessible configuration at key regulatory regions for NOTCH and neurogenesis genes resulting in increased oncogenic gene expression, which is at least partially reversible upon editing K27M back to wild- type. Overall design: Total of four different cell lines each in duplicate (total of 8 samples sequenced). Lines notated as SU-DIPG-XVII/XIII-WT are isogenic to SU-SUPG-XVII/XIII and were edited using CRISPR-Cas9 (see Chen et al., 2020). Paired-end 150 sequencing was used.

研究背景：组蛋白变体H3.3 K27M突变是弥漫内生型桥脑胶质瘤（diffuse intrinsic pontine glioma, DIPG）/弥漫中线胶质瘤（diffuse midline glioma, DMG）的标志性特征。该组蛋白突变可引发组蛋白H3翻译后修饰（post-translational modifications, PTMs）的显著改变，并伴随异常基因表达。然而，该突变对染色质结构与功能的影响，包括染色质开放/闭合区域及其转录组学效应，目前尚不清楚。
研究结果：本研究前期构建了组蛋白H3.3为野生型的同基因CRISPR编辑DIPG细胞系，可与其匹配的K27M突变细胞系进行对照分析。本研究通过转座酶可及性测序（ATAC-seq）分析发现，相较于同基因野生型细胞系，H3.3K27M胶质瘤细胞在对应神经发生、NOTCH及神经元发育通路及其相关基因的区域存在独特的染色质可及性特征。从机制层面来看，H3.3K27M细胞中与基因表达上调相关的可及增强子与超级增强子，同样富集于神经发生与NOTCH信号通路相关基因，提示上述通路是维持DIPG肿瘤发生发展的关键。基序分析显示，特定转录因子是介导K27M突变致神经肿瘤发生信号通路的核心因子，其中尤以ASCL1与NEUROD1最为关键。
研究结论：综合上述结果表明，H3.3K27M突变可使NOTCH与神经发生基因的关键调控区域的染色质呈现更高的可及性，进而导致致癌基因表达上调，且该效应可通过将K27M编辑回野生型序列得到部分逆转。
实验设计：本研究共包含4种不同细胞系，每种设置2个生物学重复，总计8个测序样本。标注为SU-DIPG-XVII/XIII-WT的细胞系与SU-SUPG-XVII/XIII为同基因细胞系，通过CRISPR-Cas9编辑获得（详见Chen等，2020）。测序采用150bp双端测序策略。