PROJECT 4: ELUCIDATING MECHANISMS OF HISTONE H3K36 DYSREGULATION BY ONCOHISTONES

摘要：已知的组蛋白H3K36导向的甲基转移酶对于正常的动物发育是必不可少的，并且在人类癌症中经常是失控的。我们已经发现，高频组蛋白H3突变(癌组织蛋白)利用正常的基于染色质的调节机制，包括H3K36甲基化，来驱动肿瘤的发生。以前，我们发现H3.3G34癌组织蛋白选择性地阻断SETD2介导的H3K36甲基化，并影响导致细胞异常分化和增殖的基因增强子的活性。此外，我们发现H3K36M癌蛋白通过竞争性抑制NSD1/2和SETD2酶来促进全基因组组蛋白和DNA甲基化的变化。我们将利用和扩展我们的初步发现，通过H3.3 G34和H3 K36M癌组织蛋白通过错误调节H3K36甲基化实现促肿瘤基因表达程序来确定机制。我们将采用整合生化、基因组和分子方法的多学科方法来增强我们对K36M和G34癌组织蛋白的理解，并将我们的理解应用于诊断和治疗应用。具体地说，我们将：i)利用基于细胞的系统和患者肿瘤样本，通过组蛋白突变来确定染色质景观的变化；以及ii)表征有助于建立肿瘤发生的调控不当的发育程序。这些研究将为开发旨在改善NSD1/2和组蛋白H3突变对人类癌症的致病影响的治疗策略提供指导。我们还将扩展我们的发现，以阐明NSD1和H3K36M突变改变鳞状细胞癌染色质景观的机制(目标1)。我们将确定G34突变改变染色质修饰和基因表达的机制，并定义H3.3伴侣通路在介导G34表型中的作用(目标2)。此外，我们还发现H3K36甲基化与PRC2活性相反，从而防止了多梳介导的基因抑制。我们现在将利用这些生化发现来确定EZH2 H3K36结合口袋在不同肿瘤发生模型中的功能(目标3)。我们研究的预期结果将引导我们形成新的理论，并为这些癌组织蛋白提供关键的机制见解，这些癌组织蛋白可以很容易地在体内癌症模型(项目1)和体外化学平台(项目3)中进行测试。为了实现这些目标，项目4还需要与这两个核心进行密切互动。

SUMMARY: The known histone H3K36-directed methyltransferases are essential for normal animal development and are frequently dysregulated in human cancers. We have found that high-frequency histone H3 mutations (oncohistones) exploit normal chromatin-based regulatory mechanisms, including H3K36 methylation, to drive tumorigenesis. Previously, we found that H3.3 G34 oncohistones selectively block SETD2-mediated H3K36 methylation and affect the activity of gene enhancers that results in aberrant cellular differentiation and proliferation. Moreover, we found that the H3 K36M oncohistone promotes genome-wide changes in histone and DNA methylation through competitive inhibition of NSD1/2 and SETD2 enzymes. We will leverage and extend our preliminary findings to define the mechanisms by H3.3 G34 and H3 K36M oncohistones achieve pro-tumorigenic gene expression programs through misregulation of H3K36 methylation. We will employ a multi-disciplinary approach that integrates biochemical, genomic, and molecular methods to enhance our understanding of K36M and G34 oncohistones and apply our understanding toward diagnostic and therapeutic applications. Specifically, we will: i) identify the changes in chromatin landscape by histone mutations using cell-based systems and patient tumor samples; and ii) characterize misregulated developmental programs that help establish tumorigenesis. These studies will provide guidance for the development of therapeutic strategies designed to ameliorate the pathogenic effects of NSD1/2 and histone H3 mutations in human cancers. We will also extend our findings to elucidate the mechanisms by which NSD1 and H3 K36M mutations alter the chromatin landscape in squamous cell carcinomas (Aim 1). We will define the mechanisms of by which G34 mutations alter chromatin modifications and gene expression, and we will define the role of H3.3 chaperone pathways in mediating G34 phenotypes (Aim 2). Additionally, we have found that H3K36 methylation opposes PRC2 activity, thus preventing Polycomb-mediated gene repression. We will now leverage these biochemical findings to determine the function of the EZH2 H3K36-binding pocket in different tumorigenesis models (Aim 3). Expected results from our study will lead us formulate novel theories and provide crucial mechanistic insights of these oncohistones which can be readily tested in in vivo cancer models (Project 1,2) and in vitro chemistry platforms (Project 3). To accomplish these aims, Project 4 also requires close interactions with both Cores.