A new model for understanding a brain tumor epigenetic driver

摘要 弥漫性桥脑胶质瘤(DIPG)是一种侵犯脑干的幼儿侵袭性肿瘤， 尽管放射治疗可以减缓肿瘤的生长，但大多数患有DIPG的儿童在确诊后两年内死亡。 为了开发更有效的治疗方法，有必要了解分子和细胞机制， 驾驶DIPG。大约80%的DIPG肿瘤携带组蛋白3(H3)基因之一的点突变，该基因 导致用赖氨酸27取代蛋氨酸(H3K27M)。H3K27M突变体蛋白的表达 致癌因素，促进细胞增殖，并导致基因异常表达。染色质是一种动态的 DNA和组蛋白的聚合体，H3赖氨酸27(H3K27-ME3)的三甲基化与 转录抑制。H3K27-ME3由EZH2催化，H3K27M突变组蛋白抑制 EZH2活性，导致整个基因组中H3K27-ME3的广泛丢失。然而，H3K27-ME3的这一损失 与转录去抑制没有密切的关系。例如，在表达H3K27M的细胞中 突变后，在丢失H3K27-ME3的基因中，只有大约4%的基因表达增加。是否会改为H3K27M 通过改变基因组组织的“高层次”方面来影响基因表达，例如 染色质具有特定的核“隔间？”内核膜的片层在转录上是一种 压抑的核隔间。片层相关结构域(LAD)是大的、特定的基因组区域 位于核周，LAD内的基因转录活性较低。H3K27-ME3高度 丰富在LAD和非LAD基因组区域之间的“边界”，提示该染色质的作用 在调节高级基因组组织的这一方面的修饰。在初步研究中，我们有 在携带H3K27M突变的DIPG细胞中映射LAD，发现LAD结构是唯一的 与其他类型的癌症和正常细胞相比，受到干扰。鉴于我们对生物化学的理解 H3K27M突变蛋白，EZH2的功能，以及我们对LAD边界结构的了解，我们提出 验证以下假设的实验：通过抑制EZH2并导致基因组范围的变化 H3K27-ME3水平，H3K27M突变蛋白破坏正常的LAD结构，这种破坏 核室相关基因组结构是DIPG基因异常表达的基础。这 这项工作对目前对H3K27M致癌效应的理解提出了挑战。而不是在当前的基础上 模型解释了H3K27M致癌功能在局部染色质水平的状态变化，获得了结果 测试一种新的范式，即H3K27M导致了更大规模的基因组组织变化 原子核。H3K27M等组蛋白突变代表了一类新的致癌驱动因素 癌症种类繁多，既有罕见的，也有流行的。因此，了解H3K27M如何影响核 隔室相关基因组组织可以为更广泛的类别提供基础知识 癌组蛋白突变。

ABSTRACT Diffuse intrinsic pontine glioma (DIPG) is an aggressive tumor of young children that infiltrates the brainstem, and although radiation therapy can slow tumor growth, most children with DIPG die within two years of diagnosis. To develop more effective therapies, it is necessary to understand the molecular and cellular mechanisms that drive DIPG. Approximately 80% of DIPG tumors carry a point mutation in one of the histone 3 (H3) genes that causes a substitution of lysine 27 for methionine (H3K27M). Expression of H3K27M mutant protein is an oncogenic driver, increasing cell proliferation, and causes aberrant gene expression. Chromatin is a dynamic polymer of DNA and histone proteins, and the trimethylation of H3 lysine 27 (H3K27-me3) correlates with transcriptional repression. H3K27-me3 is catalyzed by EZH2, and the H3K27M mutant histone protein inhibits EZH2 activity, causing widespread loss of H3K27-me3 across the genome. However, this loss of H3K27-me3 does not closely correlate with transcriptional de-repression. For example, in cells expressing the H3K27M mutation, only about 4% of the genes that lose H3K27-me3 increase their expression. Does H3K27M instead affect gene expression by altering “higher-order” aspects of genome organization, such as the interaction of chromatin with specific nuclear “compartments?” The lamina of the inner nuclear membrane is a transcriptionally repressive nuclear compartment. Lamina associated domains (LADs) are large, specific genomic regions located at the nuclear periphery, and genes within LADs exhibit low transcriptional activity. H3K27-me3 is highly enriched at the “borders” between LADs and non-LAD genomic regions, suggesting a role for this chromatin modification in regulating this aspect of higher-order genome organization. In Preliminary Studies, we have mapped LADs in DIPG cells that carry the H3K27M mutation, finding that the LAD architecture is uniquely disrupted as compared to other types of cancer and normal cells. Given our biochemical understanding of H3K27M mutant protein, the function of EZH2, and our knowledge of LAD border structure, we propose experiments that test the following hypothesis: By inhibiting EZH2 and causing genome-wide changes in H3K27-me3 levels, the H3K27M mutant protein disrupts normal LAD structures, and that such disruption of nuclear compartment-associated genome organization underlies the abnormal gene expression of DIPG. This work challenges the current understanding of H3K27M oncogenic effects. Instead of building upon the current model that explains H3K27M oncogenic function at the level of local chromatin state changes, results obtained test a new paradigm, that H3K27M causes a much larger-scale change to the organization of the genome in the nucleus. Mutations in histone proteins such as H3K27M represent an emerging class of oncogenic drivers in a wide diversity of cancer, both rare and prevalent. Thus, understanding how H3K27M affects nuclear compartment-associated genome organization may provide foundational knowledge to the broader class of oncohistone mutations.