Project 2: Elucidating Mechanisms of Chromatin Dysregulation by Oncohistones

项目摘要(ALLIS) 全基因组测序技术使人类能够史无前例地发现体细胞突变 人类癌症中的染色质和表观遗传修饰物，提供癌症表观基因组之间的机械联系 和基因改变。表观遗传调节器中致癌基因突变的总数导致了 癌症表观基因组背后的“驱动突变”的新观点。没有比这更能说明这一点的了 核心组蛋白高频(50%-95%)错义突变的经典发现，如组蛋白H3 儿童脑胶质瘤中赖氨酸27突变为蛋氨酸(H3K27M)，H3赖氨酸36突变为蛋氨酸(H3K36M) 软骨母细胞瘤和未分化肉瘤的突变。在之前的授权期内，我们已经展示了 这些突变直接阻止了翻译后的一些关键的调节组蛋白的编写 修饰(PTM)通过改变染色质组织、转录和某些 2例细胞命运和分化。最近，我们扩大了对 癌症中的组蛋白突变。我们描述了一幅意想不到的广阔的新癌组蛋白景观 突变发生在大约4%的癌症中。这些突变不仅在H3N末端尾部发现， 这是经典的癌组蛋白的位置，但也在球状结构域和所有四个核心组蛋白中。我们的 初步数据表明，这些突变中的至少一个子集会影响染色质的一个或多个属性 染色质依赖的过程，包括核小体稳定性、组蛋白PTMS和细胞分化。我们 因此，假设新的癌组蛋白突变将影响组蛋白PTM和染色质的格局 以上下文依赖的方式组织，导致基因表达失调和对细胞命运的影响 和肿瘤的发生。这项工作的目标是严格测试这些假设，以获得一套全面的 使用包括遗传学(条形码)在内的多学科方法的癌症相关组蛋白突变 癌组蛋白文库、小鼠模型、条形码细胞系)、表观遗传学(芯片序列、ATAC序列、DNA甲基化 分析)、转录学(RNA-seq)和化学生物学(“设计染色质”，小分子抑制剂)。 具体地说，我们将1)定义新的肿瘤组蛋白作用的分子机制及其对 染色质和基因表达；2)确定这些分子变化如何转化为表型 细胞分化和肿瘤同种异体移植模型，并探索挽救分化的药物策略 以及3)将我们的研究扩展到动物模型和不同的细胞环境中，以测试小说的作用 癌组蛋白在肿瘤发生发展中的作用。总而言之，这些方法将阐明 并对组蛋白和染色质结构在肿瘤中的作用提供了重要的见解。 肿瘤发生学。我们的发现有望为药物干预开辟新的途径。 癌症治疗的异常表观遗传途径。为了促进这一提议的成功，一个世界级的 研究团队已经组建，他们是癌症、染色质和化学生物学方面的专家。

PROJECT SUMMARY (ALLIS) Genome-wide sequencing technologies have allowed an unprecedented discovery of somatic mutations in chromatin and epigenetic modifiers in human cancers, providing mechanistic links between cancer epigenomes and genetic alterations. The collective number of oncogenic mutations in epigenetic regulators has led to the emerging view of “driver mutations” underlying cancer epigenomes. Nowhere is this better illustrated than with the now classical findings of high-frequency (50-95%) missense mutations in core histones, such as histone H3 lysine 27 to methionine (H3K27M) mutation in pediatric gliomas, and H3 lysine 36 to methionine (H3K36M) mutations in chondroblastomas and undifferentiated sarcomas. During the prior grant period, we have shown that these mutations directly prevent the ‘writing’ of some critical regulatory histone post-translational modifications (PTMs) to promote oncogenesis through altered chromatin organization, transcription, and in some cases cell fate and differentiation. More recently, we have extended our understanding of the landscape of histone mutations in cancers. We characterized an unexpectedly broad landscape of novel oncohistone mutations that occur in roughly 4% of all cancers. These mutations are found not only in the H3 N-terminal tail, which is the site of classical oncohistones, but also in the globular domain and in all four core histones. Our preliminary data suggest that a least a subset of these mutations affect one or more properties of chromatin and chromatin-dependent processes including nucleosome stability, histone PTMs, and cellular differentiation. We therefore hypothesize that novel oncohistone mutations will impact the landscape of histone PTMs and chromatin organization in a context dependent manner, leading to dysregulation of gene expression and effects on cell fate and tumorigenesis. The goal of this work is to rigorously test these hypotheses for a comprehensive set of cancer-associated histone mutations using a multidisciplinary approach that include genetics (barcoded oncohistone libraries, mouse models, barcoded-cell lines), epigenetics (ChIP-seq, ATAC-seq, DNA-methylation profiling), transcriptomics (RNA-seq), and chemical biology (“designer chromatin”, small molecule inhibitors). Specifically, we will 1) define molecular mechanisms by which novel oncohistones act and their impact on chromatin and gene expression; 2) determine how these molecular changes translate into phenotypes using cellular differentiation and tumor allograft models, and explore pharmacologic strategies to rescue differentiation blockade; and 3) extend our studies into animal models and diverse cellular contexts to test the roles of novel oncohistones in tumorigenesis and development. Together, these approaches will shed light on the function of newly discovered oncohistones and provide important insight into the role of histones and chromatin structure in tumorigenesis. Our findings are expected to pave new avenues towards intervening pharmacologically the aberrant epigenetic pathways for cancer therapeutics. To facilitate the success of this proposal, a world-class team of investigators, experts in cancer, chromatin and chemical biology, have been assembled.