Epigenetic profiling and enzymatic regulation of H3K23me3 during cellular differentiation

项目摘要 真核DNA与组蛋白一起包装形成核小体，核小体又浓缩成 构成染色质不同功能形式的更高级结构。人们普遍认为， 染色质结构状态，如异染色质（高度浓缩/转录失活）或 常染色质（去致密/转录活性）可以表观遗传方式繁殖（即， 与表型的可遗传变化相关，但与DNA序列的变化无关）。重要的是， 染色质结构的错误调节和组蛋白的翻译后修饰与癌症有关， 发育性疾病。表征调节这些表观遗传“开”/“关”的分子机制 途径需要鉴定新的组蛋白修饰状态，并开发新的方法学 这允许对与不同染色质区域结合的因子进行全面研究和无偏筛选。本 最后，我们最近发现了一种新的组蛋白修饰，H3 K23 me 3，使用专门的，完全异染色质的， 细胞核中的模式生物四膜虫嗜热，并将其作为一个“标记”的臂间 在减数分裂过程中对保持基因组完整性很重要的染色质。在本提案中，我们的目标是 通过研究与催化和去除相关的酶学，继续我们最初的发现。 H3 K23 me 3，以及表征这种异染色质标记物在人类和其他哺乳动物中的生物学作用。 哺乳动物，在那里它也可能参与神经发生。值得注意的是，我们将 全面分析哺乳动物中H3 K23 me 3相关染色质的表观蛋白质组， 我们的新技术rCRISPR-ChAP-MS的改进，它提供了分析 体内特定基因组位置染色质上的大分子蛋白质相互作用。在此过程中我们将测试 假设H3 K23 me 3通过与H3 K23 me 3结合蛋白的相互作用，有助于精确定位， 在减数分裂和细胞分化过程中保护异染色质形成的位点并使其永久化。来测试我们 假设和工作对我们的短期目标，我们将追求以下三个目标：（1）表征 H3 K23 me 3在四膜虫中的酶学和生物学作用，（2）表征H3 K23 me 3在四膜虫中的作用， （3）研究H3 K23 me 3在哺乳动物神经元发育中的作用。我们 研究异染色质组蛋白PTM如H3 K23 me 3如何有助于 表观遗传沉默应该有助于解决我们的长期目标，了解跨代遗传， 表观遗传修饰剂，并可能为与破坏的 基因沉默或异染色质途径。

PROJECT SUMMARY Eukaryotic DNA is packaged with histone proteins to form nucleosomes, which in turn condense into higher-ordered structures that constitute the different functional forms of chromatin. It is well accepted that chromatin structural states such as heterochromatin (highly condensed/ transcriptionally inactive) or euchromatin (decondensed/ transcriptionally active), can be propagated in an epigenetic fashion (i.e. associated with heritable changes in phenotype that are not related to changes in DNA sequence). Importantly, mis-regulation of chromatin structure and posttranslational modifications on histones are linked to cancer and developmental diseases. Characterizing the molecular mechanisms regulating these epigenetic “on”/ “off” pathways requires identification of new histone modification states, and development of new methodologies that permit comprehensive studies and unbiased screens of factors bound to distinct chromatin regions. To this end, we recently found a new histone modification, H3K23me3, using a specialized, entirely heterochromatic nucleus in the model organism Tetrahymena thermophila, and characterized it as a “mark” of the pericentric chromatin that is important for maintaining genome integrity during meiosis. In this proposal, our goal is to follow up on our initial discovery by studying the enzymology associated with catalyzing and removing H3K23me3, as well as characterizing the biological role of this heterochromatin marker in humans and other mammals, where it may also be involved in neurogenesis. It is also worthwhile to note that we will comprehensively analyze the epiproteome of H3K23me3-associated chromatin in mammals using improvements on our new technology termed rCRISPR-ChAP-MS, which provides for the analysis of macromolecular protein interactions on chromatin at a defined genomic position in vivo. In doing so we will test the hypothesis that H3K23me3, through interactions with H3K23me3 binding proteins, helps to pinpoint, protect, and perpetuate sites of heterochromatin formation during meiosis and cell differentiation. To test our hypothesis and work towards our short term goal, we will pursue the following three Aims: (1) Characterize the enzymology and biological impact of H3K23me3 in Tetrahymena, (2) Characterize the role of H3K23me3 in mammalian meiosis, and (3) Characterize the role of H3K23me3 in mammalian neuronal development. Our study of the molecular underpinnings of how heterochromatic histone PTMs like H3K23me3 contribute to epigenetic silencing should help address our long term goal of understanding transgenerational inheritance of epigenetic modifiers, and may introduce therapeutic targets for human diseases associated with disrupted gene silencing or heterochromatin pathways.