Epigenetic profiling and enzymatic regulation of H3K23me3 during cellular differentiation

细胞分化过程中 H3K23me3 的表观遗传分析和酶促调控

• 批准号: 9239607
• 负责人: Sean D. Taverna
• 金额: $ 38.78万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-03-01 至 2021-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9239607
• 关键词: Address; Animal Model; Binding Proteins; Biological; Caenorhabditis elegans; Cell Differentiation process; Cell Nucleus; Cells; ChIP-seq; Chromatin; Chromatin Structure; Complex; DNA; DNA Binding; DNA Damage; DNA Sequence; Development; Disease; Enzymatic Biochemistry; Epigenetic Process; Euchromatin; Event; Fingers; Gene Silencing; Genetic Recombination; Genetic Transcription; Genome; Genome Stability; Genomic Instability; Genomics; Germ Cells; Goals; Heritability; Heterochromatin; Higher Order Chromatin Structure; Histone H3; Histones; Human; In Vitro; Infertility; Link; Lysine; Maintenance; Malignant Neoplasms; Mammals; Meiosis; Methodology; Methylation; Methyltransferase; Mitotic; Modeling; Modification; Molecular; Mus; N-terminal; Neuronal Differentiation; Neurons; Nucleosomes; Pathway interactions; Pattern; Phenotype; Play; Positioning Attribute; Post-Translational Protein Processing; Proteins; Proteomics; Rattus; Reader; Regulation; Reporting; Research Proposals; Resolution; Role; Site; Sterility; Technology; Testing; Tetrahymena; Tetrahymena thermophila; Tissues; Work; epigenetic profiling; follow-up; genome integrity; histone methylation; histone modification; human disease; human embryonic stem cell; in vivo; insight; mammalian genome; member; mutant; neurodevelopment; neurogenesis; neuron development; new technology; novel; relating to nervous system; repaired; therapeutic target; transgenerational epigenetic inheritance

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.

项目概要 真核 DNA 与组蛋白一起包装形成核小体，核小体又凝结成 构成染色质不同功能形式的高阶结构。人们普遍认为 染色质结构状态，例如异染色质（高度浓缩/转录失活）或 常染色质（解压缩/转录活性）可以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和 发育性疾病。表征调节这些表观遗传“开”/“关”的分子机制 途径需要识别新的组蛋白修饰状态并开发新的方法 允许对与不同染色质区域结合的因素进行全面研究和公正筛选。对此 最后，我们最近发现了一种新的组蛋白修饰，H3K23me3，使用专门的、完全异染色质的 模型生物嗜热四膜虫的细胞核，并将其表征为中心周的“标记” 染色质对于减数分裂过程中维持基因组完整性非常重要。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来跟进我们最初的发现 H3K23me3，以及表征该异染色质标记在人类和其他动物中的生物学作用 哺乳动物，它也可能参与神经发生。还值得注意的是，我们将 使用综合分析哺乳动物中 H3K23me3 相关染色质的表蛋白质组 我们对 rCRISPR-ChAP-MS 新技术进行了改进，该技术可用于分析 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 H3K23me3 通过与 H3K23me3 结合蛋白相互作用，有助于查明， 在减数分裂和细胞分化过程中保护并维持异染色质形成位点。来测试我们的 假设并努力实现我们的短期目标，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) 表征 H3K23me3 在四膜虫中的作用 哺乳动物减数分裂，以及 (3) 表征 H3K23me3 在哺乳动物神经元发育中的作用。我们的 研究 H3K23me3 等异染色质组蛋白 PTM 如何促进的分子基础 表观遗传沉默应该有助于实现我们理解跨代遗传的长期目标 表观遗传修饰剂，并可能引入与破坏相关的人类疾病的治疗靶点 基因沉默或异染色质途径。