Epigenetic Mechanisms of Oncohistone Detoxification

 描述(由申请者提供)：本培训计划中建议的研究将集中于研究通过操纵组蛋白翻译后修饰来减轻癌组蛋白在细胞中的毒性的机制。在真核细胞中，DNA以染色质的形式存储，染色质由包裹在组蛋白周围的DNA组成的重复单位组成，称为核小体。从每个组蛋白延伸出来的是翻译后以各种方式修饰的多肽尾巴，包括乙酰化、甲基化、糖基化和泛素化。这些修饰(PTM)调节对DNA的访问，DNA反过来控制转录、复制和其他细胞过程。重要的是，这些表观遗传过程的错误调控与癌症有关，最近我们的团队和其他人也表明，在癌细胞中发现了核心组蛋白的突变。特别是，组蛋白3(H3)的突变与一种致命的儿童脑瘤有关。在大多数弥漫性脑桥胶质母细胞瘤(DIPGs)中，H3中的赖氨酸27在少数核小体中突变为蛋氨酸(H3K27M)。这种突变是一种甲基转移酶，是多梳抑制物复合体2(PRC2)的有效抑制物。一般来说，PRC2对H3K27的甲基化与基因沉默有关，因此，由于H3K27M导致的H3K27me3水平的降低导致基因组程序的中断，从而扰乱细胞的发育。由于PRC2能够感知染色质状态并基于这些信号调节其甲基转移酶输出，我们假设PRC2抑制可能受到同一H3尾部(即在顺式)上现有的PTM的影响。事实上，我们发现PTMS，特别是H3的多乙酰化，已经被发现可以减弱H3K27M多肽的抑制作用。因此，我们推测，有意操纵与PRC2相关的PTM水平可以用来减弱H3K27M的致病性。本项目探索的策略是使用组蛋白脱乙酰酶(HDAC)抑制剂在H3K27M突变细胞中实现H3乙酰化水平的提高，并研究这种操作影响PTM交叉对话的机制。本文描述的工作使用化学生物学方法来探索与H3K27M“解毒”相关的特定机制，以及更好地理解“癌组蛋白”总体上的表观遗传错误调节。本项目的具体目的是：(1)筛选HDAC抑制剂对表达突变型H3K27M细胞中甲基转移酶活性的影响。(2)研究HDAC对H3K27M表达细胞甲基转移酶活性的影响机制。(3)检测HDAC抑制剂对表达H3K27M突变体的胶质母细胞瘤细胞致病性的影响。这项研究应用化学生物学工具来阐明通过HDAC抑制剂操纵细胞中的乙酰化/甲基化串扰来影响这种癌症突变的机制。进行这些基础研究的长期目标是阐明这些相互关联的PTM通路，并为未来治疗程序的发展提供信息。

 DESCRIPTION (provided by applicant): The proposed research in this training plan will focus on investigating the mechanisms by which the toxicity of oncohistones in cells can be abated by manipulating histone post-translational modifications. In eukaryotic cells, DNA is stored as chromatin, which consists of repeating units called nucleosomes made up of DNA wrapped around histone proteins. Extending out from each histone are peptide tails that are post-translationally modified in a variety of ways, including by acetylation, methylation, glycosylation and ubiquitylation. These modifications (PTMs) modulate access to the DNA, which in turn controls transcription, replication, and other cellular processes. Importantly, misregulation of these epigenetic processes has been linked to cancer, and recently it has also been shown by our group and others that mutations in the core histone proteins are found in cancer cells. In particular, mutations in histone 3 (H3) are associated with a deadly form of pediatric brain tumor. In a majority of diffuse pontine glioblastomas (DIPGs), lysine 27 in H3 is mutated to methionine (H3K27M) in a small percentage of nucleosomes. This mutation acts as a potent inhibitor of the polycomb repressor complex 2 (PRC2), which is a methyltransferase. In general, methylation of H3K27 by PRC2 is associated with gene silencing, so reduced levels of H3K27me3 due to H3K27M lead to disruption of genomic programs and thus perturbation of cellular development. Since PRC2 is able to sense chromatin states and modulate its methyltransferase output based on those signals, we hypothesized that PRC2 inhibition could be impacted by existing PTMs on the same H3 tail (i.e. in cis). Indeed, we found that PTMs, particularly polyacetylation of H3, have been found to diminish the inhibitory effect of H3K27M peptides. Therefore, we postulate that the deliberate manipulation of PTM levels relevant to PRC2 could be used to abate the pathogenicity of H3K27M. The strategy explored in this project is to use histone deacetylase (HDAC) inhibitors to achieve elevated levels of acetylation of H3 in H3K27M mutant cells and to study the mechanisms by which this manipulation impacts the PTM cross-talks. The work described herein employs a chemical biology approach to probe specific mechanisms associated with H3K27M "detoxification" as well as to provide a better understanding of epigenetic misregulation by "oncohistones" in general. The specific aims of this project are: (1) To screen HDAC inhibitors for their effect on methyltransferase activity in cells expressing the mutant H3K27M. (2) To characterize mechanisms by which HDACs impact methyltransferase activity in cells expressing H3K27M. (3) To determine the effect of HDAC inhibitors on the pathogenicity of glioblastoma cells that express the H3K27M mutant. This research applies chemical biology tools to elucidate mechanisms by which this cancerous mutation is affected by manipulating the acetylation/methylation cross-talk in cells via HDAC inhibitors. The long term goal of performing these fundamental studies is to illuminate these interconnected PTM pathways and inform the future development of therapeutic procedures.