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生畏的“表观遗传挑战”。一个单一的基因组是如何解释的， 一千种不同的细胞命运细胞外信号是如何快速而有力地开启细胞内的选择基因的？ 三十亿个碱基对的基因组？表观遗传学机制是平衡血细胞分化和 对病原体或组织损伤的细胞反应的速度和范围-定义免疫、耐受性 感染期间的存活率。理解“解决”这些表观遗传挑战的机制至关重要 是研究组蛋白，即包装和调节基因组的蛋白质。这个项目的重点是揭示 组蛋白和组蛋白翻译后修饰（PTM）在哺乳动物中的功能。特别 感兴趣的是组蛋白变体H3.3，其代表基因组中H3的15个拷贝中的2个，但富含 动态调节的染色质，如增强子、启动子和基因体。此外，H3.3是唯一 H3，其以不依赖DNA合成的方式表达。由于这些原因，我们专注于研究 H3.3残基和修饰在造血发育和免疫细胞功能中的功能， 系统反映了哺乳动物复杂的发育和快速的细胞反应，与健康高度相关。 和疾病 初步实验集中在H3.3S31ph的共转录修饰和功能缺失上 该标记的缺失消除了巨噬细胞系（RAW264.7s）对LPS应答的能力。来测出 其他H3.3残基和修饰是这种快速转录反应所必需的，我已经开发了一种 BMDM中的新型敲除和置换系统（Aim 1）。早期的研究结果表明， 赖氨酸残基与精氨酸（H3.3K4R，H3.3K36R）的结合导致刺激诱导的转录降低，而 其它（H3.3K9R、H3.3K27R）则无影响。为了验证这些结果的功能相关性，我们有 显示了H3.3对于体内免疫应答的需要。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的功能， 造血发育中的关键残基（Aim 2）。最初的实验表明， 造血干细胞存活和巨噬细胞分化。组蛋白的靶向无偏筛选 “阅读器、写入器和擦除器”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产生广泛的影响， 疾病，以及细菌感染和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.