Project 3: Mechanisms of Methyltransferase Dysregulation by Oncohistones

项目总结 摘要：本项目的主要目标是研究组蛋白的详细分子机制 与癌症相关的突变会扰乱染色质状态，导致疾病。该计划的下一阶段 是由我们最近与Allis实验室的工作推动的，揭示了推定的癌组蛋白的巨大扩大的景观 突变-数十种人类癌症中超过4200个错义突变。值得注意的是，突变发生在所有 四个核心组蛋白，以及在N末端和球状组蛋白折叠结构域中。非常大的 新发现的癌组蛋白数量使实验表征变得令人望而生畏。一个关键的挑战是 识别那些作为潜在癌症驱动因素的突变，而不是仅仅是 特定肿瘤(即乘客)的高突变负担。我们将与P01其他成员密切合作 团队来确定那些最有可能属于前一种类型的突变，因此在体内有价值 测试。从战略上讲，我们将使用新开发的高通量生化和 酵母遗传筛选工具(目标1)，有望识别改变染色质稳定性和/或染色体稳定性的突变 影响作用于染色质聚合物的反式作用因子的活性。“命中”的验证 筛选研究将形成目标2和目标3的核心，在那里我们将结合使用化学定义的 染色质模板与生化、蛋白质组和基因组学方法发展的机制 了解选择突变如何影响染色质状态。特别是，我们将研究如何在 核小体对称性，作为亚化学计量比掺入突变组蛋白的结果，影响关键过程 例如染色质重塑和转录。我们想像一下，许多在 这项工作的背景将在表观遗传学领域具有广泛的实用价值。归根结底，生化知识 在这个计划的过程中产生的基础将激励未来治疗癌症的努力 与癌组蛋白突变体有关。

PROJECT SUMMARY SUMMARY: The broad goal of this project is to study the detailed molecular mechanisms by which histone mutations associated with cancers perturb chromatin states, leading to disease. The next phase of this program is driven by our recent work with the Allis lab revealing a vastly expanded landscape of putative oncohistone mutations - over 4200 missense mutations in dozens of human cancers. Remarkably, the mutations occur in all four of the core histones and in both the N-terminal tails and globular histone fold domains. The very large number of newly identified oncohistones makes experimental characterization daunting. A key challenge is to identify those mutations that act as potential cancer drivers from those that are merely a consequence of the high mutation burden of a given tumor (i.e. passengers). We will work closely with other members of the P01 team to identify those mutations that most likely to fall into the former category and that, as such, merit in vivo testing. Strategically, we will approach this problem using newly developed high-throughput biochemical and yeast genetic screening tools (Aim 1) that are expected to identify mutations that alter chromatin stability and/or that affect the activity of trans-acting factors that operate on the chromatin polymer. Validation of the ‘hits’ from the screening studies will form the core of Aims 2 & 3 where we will combine the use of chemically-defined chromatin templates with biochemical, proteomic and genomic approaches to develop a mechanistic understanding of how select mutations impact chromatin state. In particular, we will study how breakdown in nucleosome symmetry, as a result of sub-stoichiometric incorporation of mutant histones, affects key processes such as chromatin remodeling and transcription. We imagine that many of the technologies developed in the context of this work will have broad utility in the epigenetics field generally. Ultimately, the biochemical knowledge base generated in the course of this program will motivate future therapeutic efforts for treating cancers associated with oncohistone mutants.