Epigenetic profiling and enzymatic regulation of H3K23me3 during cellular differentiation

项目总结 真核DNA与组蛋白蛋白包装在一起形成核小体，核小体又浓缩成 构成不同功能形式的染色质的高级结构。人们普遍认为 染色质结构状态，如异染色质(高度浓缩/转录不活跃)或 常染色质(去凝集/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和 发育性疾病。表征这些表观遗传“开”/“关”的分子机制 通路需要确定新的组蛋白修饰状态，并开发新的方法 这使得对与不同染色质区域结合的因素进行全面研究和无偏见筛选成为可能。对这件事 最后，我们最近发现了一种新的组蛋白修饰，H3K23me3，使用了一种特殊的，完全异色的 在模式生物Tetrahymena thermophila中的核，并将其描述为中心周围的一个“标志” 染色质对于在减数分裂过程中保持基因组的完整性很重要。在这项提案中，我们的目标是 通过研究与催化和去除相关的酶学来继续我们最初的发现 H3K23me3，以及表征这个异染色质标记在人类和其他生物中的生物学作用 在哺乳动物中，它也可能参与神经发生。同样值得注意的是，我们将 综合分析哺乳动物H3K23me3相关染色质的表观蛋白质组 对我们的新技术rCRISPR-CHAP-MS进行了改进，该技术提供了对 大分子蛋白质在体内特定基因组位置与染色质的相互作用。在这样做的时候，我们将测试 假设H3K23me3通过与H3K23me3结合蛋白的相互作用，有助于准确定位， 在减数分裂和细胞分化过程中保护和保持异染色质形成的部位。测试我们的 假设并朝着我们的短期目标努力，我们将追求以下三个目标：(1)描述 H3K23me3在四膜虫中的酶学和生物学影响，(2)H3K23me3在四膜虫中的作用 以及(3)H3K23me3在哺乳动物神经元发育中的作用。我们的 H3K23me3等异染色组蛋白PTM的分子基础研究 表观遗传沉默应该有助于解决我们理解跨代遗传的长期目标 表观遗传修饰物，并可能引入与干扰相关的人类疾病的治疗靶点 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.