Multiple modes of PRC2 inhibition elicit global chromatin alterations in H3K27M pediatric glioma

A methionine substitution at lysine 27 on histone H3 variants (H3K27M) characterizes ~80% of diffuse intrinsic pontine gliomas (DIPG) and inhibits PRC2 in a dominant negative fashion. Yet, the mechanisms for this inhibition and abnormal epigenomic landscape have not been resolved. Using quantitative proteomics, we discovered that robust PRC2 inhibition requires levels of H3K27M greatly exceeding those of PRC2, seen in DIPG. While PRC2 inhibition requires interaction with H3K27M, we found this interaction on chromatin is transient with PRC2 largely being released from H3K27M. Unexpectedly, inhibition persisted even after PRC2 dissociated from H3K27M-chromatin suggesting a lasting impact on PRC2. Furthermore, allosterically activated PRC2 is particularly sensitive to K27M leading to a failure to spread H3K27me3 at distinct foci. In turn, levels of Polycomb antagonists such as H3K36me2 are elevated suggesting a more global, downstream effect on the epigenome. Together, these findings reveal the conditions required for H3K27M-mediated PRC2 inhibition and reconcile seemingly paradoxical effects of H3K27M on PRC2 recruitment and activity. Overall design: Role of PRC2 in H3K27M pediatric glioma

组蛋白H3变体第27位赖氨酸发生甲硫氨酸取代（H3K27M），存在于约80%的弥漫内生型桥脑胶质瘤（diffuse intrinsic pontine gliomas, DIPG）中，并以显性负性方式抑制多梳抑制复合体2（Polycomb Repressive Complex 2, PRC2）。然而，该抑制作用的机制以及异常表观基因组谱尚未被阐明。本研究通过定量蛋白质组学分析发现，要实现强效的PRC2抑制，H3K27M的表达水平需大幅超过PRC2，这一特征在DIPG中得以体现。尽管PRC2的抑制依赖于与H3K27M的相互作用，但我们观察到二者在染色质上的结合是瞬时的，PRC2大多会从H3K27M复合物上解离。出乎意料的是，即便PRC2从结合H3K27M的染色质上解离，抑制作用仍持续存在，这表明H3K27M对PRC2存在持久影响。此外，别构激活的PRC2对K27M尤为敏感，导致其无法在特定位点扩散H3K27三甲基化（H3K27me3）标记。相应地，诸如H3K36me2这类多梳蛋白拮抗剂的表达水平升高，提示该过程对表观基因组存在更广泛的下游效应。综上，本研究明确了H3K27M介导的PRC2抑制所需的条件，并调和了H3K27M在PRC2招募与活性方面看似矛盾的效应。整体实验设计：PRC2在H3K27M阳性儿童胶质瘤中的作用