Describing the Epigenetic Mechanisms in Control of Hematopoietic Development and Rapid Inflammatory Responses

描述控制造血发育和快速炎症反应的表观遗传机制

• 批准号: 10490961
• 负责人: Andrew Daman
• 金额: $ 4.68万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-31 至 2024-01-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10490961
• 关键词: Animal Model; Arginine; Bacterial Infections; Base Pairing; Biological Models; Biological Process; Biology; Blood; Blood Cells; Bone Marrow; Cell Differentiation process; Cell Line; Cell Lineage; Cell physiology; Cells; Chromatin; Collaborations; Complex; DNA biosynthesis; Data; Defect; Development; DiGeorge Syndrome; Disease; Enhancers; Epigenetic Process; Face; Foundations; Future; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Models; Genetic Transcription; Genome; Goals; Health; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Heterochromatin; High-Throughput Nucleotide Sequencing; Histone H3; Histones; Human; Immune; Immune response; Immunity; Individual; Infection; Inflammatory Response; Knock-out; Knowledge; Lentivirus; Link; Listeria; Lysine; Malignant Neoplasms; Maps; Modeling; Modification; Mus; Mutation; Myeloid Cells; Organism; Pathway interactions; Patients; Phenotype; Post-Translational Protein Processing; Process; Proteins; Reader; Regulation; Regulatory Pathway; Research; Role; Serine; Signal Transduction; Speed; Spleen; System; T-Lymphocyte; Tail; Testing; Tissues; Training; Variant; Vertebrates; Yeasts; adult stem cell; base; cell type; epigenetic regulation; exhaustion; experimental study; extracellular; fly; histone modification; in vivo; insight; interest; macrophage; mammalian genome; mouse model; mutant; novel; pathogen; progenitor; promoter; response; screening; stem cell survival; therapeutic target

Project Summary Complex organisms face daunting “epigenetic challenges”. How is a single genome interpreted to instruct over one thousand distinct cell fates? How do extracellular signals rapidly and robustly turn on select genes in the three billion base-pair genome? Epigenetic mechanisms underlie balanced blood cell differentiation and the speed and scope of cellular responses to pathogens or tissue damage—features that define immunity, tolerance, and survival during infection. Critical to understanding the mechanisms that “solve” these epigenetic challenges is the study of histones, proteins that package and regulate the genome. The focus of this project is to reveal the function of histones and histone post-translational modifications (PTMs) in mammalian organisms. Of particular interest is Histone variant H3.3, which represents 2 of 15 copies of H3 in the genome but is enriched in dynamically regulated chromatin such as enhancers, promotors and gene bodies. Additionally, H3.3 is the only H3 that is expressed in a DNA synthesis independent fashion. For these reasons we have focused on studying the function of H3.3 residues and modifications in hematopoietic development and immune cell function as these systems reflect complex mammalian development and rapid cellular responses, and are highly relevant to health and disease. Preliminary experiments focused on the function of co-transcriptional modification H3.3S31ph, and loss of this mark abrogates the ability of a macrophage cell line (RAW264.7s) to respond to LPS. To examine which other H3.3 residues and modifications are required for this rapid transcriptional response, I have developed a novel knockout and replacement system in BMDMs (Aim 1). Early results have shown that mutation of certain lysine residues to arginine (H3.3K4R, H3.3K36R) leads to decreased stimulation-induced transcription, whereas others (H3.3K9R, H3.3K27R) have no effect. To validate the functional relevance of these results, we have shown the requirement of H3.3 for in vivo immune response to listeria. Our results will inform ongoing studies to define dedicated mechanisms for rapid transcription. Additionally, we will use this model of knockout and replacement to determine the function of H3.3 and key residues in hematopoietic development (Aim 2). Initial experiments shown the requirement for H3.3 in hematopoietic stem cell survival, and macrophage differentiation. Targeted and unbiased screening of histone “readers, writers, and erasers” will enable us to link H3.3 mutant phenotypes to chromatin regulatory pathways and factors. Together these studies will elucidate how epigenetic mechanisms can regulate cellular differentiation and the speed and scope of cellular responses. By advancing basic knowledge of the epigenetic mechanisms regulating these cellular processes, the proposed research will have broad implications for basic biology and disease, as well as direct implications in bacterial infection and patients with H3.3 pathway mutations.

项目概要 复杂的生物体面临着令人畏惧的“表观遗传挑战”。如何解释单个基因组来指导 一千种不同的细胞命运？细胞外信号如何快速而有力地开启细胞中的选定基因 三十亿个碱基对基因组？表观遗传机制是平衡血细胞分化和 细胞对病原体或组织损伤反应的速度和范围——定义免疫、耐受性、 和感染期间的存活率。对于理解“解决”这些表观遗传挑战的机制至关重要 是对组蛋白、包装和调节基因组的蛋白质的研究。该项目的重点是揭示 组蛋白和组蛋白翻译后修饰 (PTM) 在哺乳动物中的功能。特别是 人们感兴趣的是组蛋白变体 H3.3，它代表基因组中 15 个 H3 拷贝中的 2 个，但富含 动态调节的染色质，例如增强子、启动子和基因体。此外，H3.3 是唯一 H3 以独立于 DNA 合成的方式表达。由于这些原因，我们集中精力学习 H3.3残基的功能以及造血发育和免疫细胞功能中的修饰 系统反映了复杂的哺乳动物发育和快速的细胞反应，并且与健康高度相关 和疾病。 初步实验重点关注共转录修饰H3.3S31ph的功能，以及丢失 该标记的消失会消除巨噬细胞系 (RAW264.7s) 对 LPS 的反应能力。要检查哪个 这种快速转录反应需要其他 H3.3 残基和修饰，我开发了一种 BMDM 中的新型敲除和替换系统（目标 1）。早期结果表明某些突变 赖氨酸残基变为精氨酸（H3.3K4R、H3.3K36R）会导致刺激诱导的转录减少，而 其他（H3.3K9R、H3.3K27R）没有影响。为了验证这些结果的功能相关性，我们有 显示了 H3.3 对李斯特菌体内免疫反应的要求。我们的结果将为正在进行的研究提供信息 定义快速转录的专用机制。 此外，我们将使用这种敲除和替换模型来确定 H3.3 和 造血发育中的关键残留物（目标 2）。初步实验表明，H3.3 的要求 造血干细胞存活和巨噬细胞分化。组蛋白的靶向和公正筛选 “读者、作者和橡皮擦”将使我们能够将 H3.3 突变表型与染色质调控途径联系起来 和因素。这些研究将共同​​阐明表观遗传机制如何调节细胞分化 以及细胞反应的速度和范围。通过推进表观遗传机制的基础知识 调节这些细胞过程，拟议的研究将对基础生物学和 疾病，以及对细菌感染和 H3.3 通路突变患者的直接影响。