Lysine methylation at chromatin and cellular responses to stress

染色质赖氨酸甲基化和细胞对应激的反应

• 批准号: 9366601
• 负责人: Erin Green
• 金额: $ 29.09万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE COUNTY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-15 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9366601
• 关键词: Address; Aging; Binding; Biochemical; Biological Assay; Cell Maintenance; Cell Survival; Cells; Cellular Stress; ChIP-seq; Chromatin; Chromatin Structure; Cues; DNA Packaging; Data; Defect; Disease; Environment; Environmental Risk Factor; Enzymes; Epigenetic Process; Foundations; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Genes; Genetic; Genome; Genome Stability; Genomics; Grant; Histone H2A; Histones; Homeostasis; Human; In Vitro; Investigation; Knowledge; Link; Lysine; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Methylation; Methyltransferase; Modification; Molecular; Molecular Analysis; Molecular Target; Mutation; Neurodevelopmental Disorder; Oncogenic; Orthologous Gene; Oxidative Stress; PHD Finger; Pathologic; Pathologic Processes; Pathology; Pathway interactions; Pattern; Play; Positioning Attribute; Post-Translational Protein Processing; Proteins; Proteomics; Reader; Regulation; Research; Resolution; Role; SET Domain; Saccharomyces cerevisiae Proteins; Series; Signal Transduction; Site; Stem cells; Stimulus; Stress; Stress Response Signaling; Stress Tests; Structure; Substrate Specificity; System; Tertiary Protein Structure; Testing; Work; Yeasts; base; biological adaptation to stress; epigenetic regulation; epigenomics; experimental study; genetic analysis; histone methylation; histone methyltransferase; histone modification; human disease; insight; mutant; novel; preference; programs; protein protein interaction; response; sensor; transcriptome sequencing; tumorigenesis

Project Summary Cell survival in the presence of fluctuating environmental signals is critically dependent on rapid changes in gene expression. Chromatin-modifying enzymes are key regulators of genome reprogramming during stress, and aberrant regulation or mutation of these enzymes results in disrupted gene expression programs and inappropriate responses to cellular stress. Such consequences contribute to pathological processes including oncogenesis and aging of human cells. Despite critical roles for chromatin modifiers in these pathways, there are still substantial gaps in our knowledge regarding novel sites of histone modification and their effects on genome regulation, particularly in the presence of diverse stresses encountered in the environment. Our preliminary work has uncovered that the Saccharomyces cerevisiae protein Set4, a potential ortholog of the human protein MLL5, is important for cell survival in oxidative stress and that it is an active histone methyltransferase. The central hypothesis of our work is that Set4 is a stress- regulated methyltransferase that activates a defined gene expression program in response to stress through its lysine methylation activity. Three specific aims are proposed. In Aim I, we will define the substrate specificity of Set4 both in vitro and in cells using biochemical and mass spectrometry based approaches. We will test whether Set4 primarily targets histones, or if it also has additional, non-histone methyl-lysine substrates that contribute to the oxidative stress response. In Aim II, molecular and genetic analysis will be used to determine pathways that regulate Set4 itself in response to oxidative, and other, stresses. Mechanisms by which Set4 controls gene expression will be elucidated through RNA-sequencing analysis and chIP- sequencing of Set4 and its cognate methyl mark in a series of mutants under stress. Aim III will test the hypothesis that the PHD finger of Set4 is required for Set4-depdent stress responses by promoting its localization to chromatin. Biochemical and proteomic assays will determine the histone or non-histone binding partner of the PHD finger of Set4, and targeted molecular and genomic experiments will assess the role of the PHD finger in the localization and activity of Set4 at chromatin. These research aims will provide substantial insight in to the function of a novel epigenetic modifier that we expect to be applicable to its potential human ortholog MLL5, which has been implicated in stem cell maintenance, tumorigenesis and neurodevelopmental disorders. Furthermore, this work will uncover new links between environmental stress and chromatin-based regulation of gene expression, which will be critical to our understanding of how misregulation of the genome by aberrant stress signaling contributes to human disease.

项目概要 在环境信号波动的情况下，细胞的存活严重依赖于快速 基因表达的变化。染色质修饰酶是基因组的关键调节因子 应激期间的重新编程以及这些酶的异常调节或突变会导致 基因表达程序被破坏以及对细胞应激的不适当反应。这样的 后果导致病理过程，包括人类的肿瘤发生和衰老 细胞。尽管染色质修饰剂在这些途径中发挥着关键作用，但仍然存在大量的 我们关于组蛋白修饰的新位点及其对基因组影响的知识空白 监管，特别是在环境中遇到各种压力的情况下。我们的 初步工作发现酿酒酵母蛋白 Set4 是一种潜在的 人类蛋白 MLL5 的直系同源物对于氧化应激下的细胞存活很重要，并且 活性组蛋白甲基转移酶。我们工作的中心假设是 Set4 是一个压力- 受调节的甲基转移酶，激活特定的基因表达程序以响应 通过其赖氨酸甲基化活性来应激。提出了三个具体目标。在目标一中，我们将 使用生化和质量定义 Set4 的体外和细胞内底物特异性 基于光谱测量的方法。我们将测试 Set4 是否主要针对组蛋白，或者是否 还具有额外的非组蛋白甲基赖氨酸底物，有助于氧化应激 回复。在目标 II 中，将使用分子和遗传分析来确定以下途径： 调节 Set4 本身以应对氧化应激和其他应激。 Set4 的机制 控制基因表达的因素将通过 RNA 测序分析和 ChIP 来阐明 对一系列应激突变体中的 Set4 及其同源甲基标记进行测序。目标 III 将 检验 Set4 的 PHD 手指对于 Set4 相关的应激反应所必需的假设： 促进其定位于染色质。生化和蛋白质组学测定将确定 Set4 PHD 指的组蛋白或非组蛋白结合伴侣，以及靶向分子和 基因组实验将评估 PHD 手指在定位和活性中的作用 染色质上的 Set4。这些研究目标将为深入了解 我们期望适用于其潜在的人类直系同源物 MLL5 的新型表观遗传修饰剂， 与干细胞维持、肿瘤发生和神经发育有关 失调。此外，这项工作将揭示环境压力与 基于染色质的基因表达调控，这对于我们理解基因表达至关重要 异常应激信号对基因组的错误调节如何导致人类疾病。