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

项目摘要 复杂的生物体面临着令人望而生畏的“表观遗传学挑战”。单个基因组是如何被解释为指示 一千种截然不同的细胞命运？细胞外信号如何快速而有力地打开细胞中的选择基因 30亿个碱基对基因组？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--定义免疫力、耐受性、 以及在感染期间存活。对于理解“解决”这些表观遗传挑战的机制至关重要 是对组蛋白的研究，组蛋白是包装和调节基因组的蛋白质。这个项目的重点是揭示 哺乳动物中组蛋白和组蛋白翻译后修饰(PTM)的功能。特别的 感兴趣的是组蛋白变异体H3.3，它代表基因组中H3的15个拷贝中的2个，但富含 动态调节染色质，如增强子、启动子和基因体。此外，H3.3是唯一 H3以不依赖DNA合成的方式表达。出于这些原因，我们一直专注于研究 H3.3残基及其修饰在造血发育和免疫细胞功能中的作用 系统反映了复杂的哺乳动物发育和快速的细胞反应，与健康高度相关。 和疾病。 初步实验重点是共转录修饰H3.3S31ph的功能，以及丢失 这一标记的缺失使巨噬细胞系(RAW264.7s)丧失了对内毒素的反应能力。为了检查哪一项 这种快速转录反应还需要其他H3.3残基和修饰，我已经开发出一种 BMDM中的新敲除和替换系统(目标1)。早期的研究结果表明，某些基因突变 赖氨酸残基为精氨酸(H3.3K4R，H3.3K36R)导致刺激诱导的转录减少，而 其他(H3.3K9R、H3.3K27R)则不起作用。为了验证这些结果的功能相关性，我们有 表明H3.3对李斯特菌的体内免疫应答是必需的。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H3.3的功能 造血发育中的关键残留物(目标2)。初步实验表明，H3.3在 造血干细胞存活和巨噬细胞分化。组蛋白的靶向和无偏筛选 “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节途径联系起来 以及各种因素。这些研究将共同阐明表观遗传机制如何调控细胞分化。 以及细胞反应的速度和范围。通过提高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途径突变患者的直接影响。

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.