Single-cell analysis of immune cell fate decision making

免疫细胞命运决策的单细胞分析

• 批准号: 9335415
• 负责人: Hao Yuan Kueh
• 金额: $ 24.9万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9335415
• 关键词: Affect; Architecture; B-Lymphocytes; Behavior; Biological Models; Blood Cells; Cell Cycle; Cell Differentiation process; Cell Proliferation; Cells; Chromatin; Complement; Data; Decision Making; Deposition; Development; Developmental Gene; Enzymes; Epigenetic Process; Feedback; Future; Gene Expression; Gene Expression Profiling; Gene Expression Regulation; Genes; Genome; Genomics; Goals; Heritability; High-Throughput Nucleotide Sequencing; Histones; Image; Immune; Investigation; Length; MYC gene; Malignant Neoplasms; Mammalian Cell; Measurement; Measures; Mediating; Mediator of activation protein; Memory; Mentors; Modification; Molecular; Mouse Strains; Myelogenous; Phase; Population; Process; Proto-Oncogenes; Regenerative Medicine; Regulation; Regulator Genes; Reporter; Research; Role; Signal Transduction; Site; Specific qualifier value; Stem cells; System; T-Cell Activation; T-Cell Development; T-Lymphocyte; Technology; Testing; Tetanus Helper Peptide; Time; Work; base; discrete time; effective therapy; experimental study; gene induction; genome-wide; histone modification; insight; knock-down; leukemia; leukemia treatment; live cell imaging; macrophage; mathematical model; notch protein; novel therapeutics; overexpression; progenitor; public health relevance; single cell analysis; transcription factor

DESCRIPTION (provided by applicant): Mammalian stem and progenitor cells activate the expression of specific regulatory genes to establish and stabilize different cell fates, but the mechanisms and general principles controlling this process are not well understood. Cells utilize fate-specifying regulatory genes to establish and maintain distinct fate identities during development, but it is not clear how they activate and maintain the expression of these genes to establish fate identity. Here, I propose to study this question in the context of two systems: T-cell fate commitment, which is driven by the activation of the T-cell specific transcription factor Bcl11b (Aims 1 and 2); and macrophage development, which we recently found is driven by the cell-cycle length dependent accumulation of the myeloid transcription factor PU.1 (Kueh et al., 2013)1 (Aim 3). Using these two systems, I will test a number of widely debated ideas in the field of developmental gene regulation. First, I will test the idea that developmental signals directly activate the expression of regulatory genes to instruct cell fate (Aim 1). Next, I will tet two proposed classes of mechanisms for stabilizing regulatory gene expression and fate identity: cis-acting mechanisms involving stable and heritable epigenetic modifications at regulatory gene loci (Aim 2); and trans- acting mechanisms involving self-reinforcing positive feedback loops on regulatory gene expression (Aim 3). My main approach will be to use timelapse live-cell imaging to track the expression dynamics of Bcl11b during T-cell development, and PU.1 during macrophage development. As cell differentiation is a dynamic and intrinsically heterogeneous process, single-cell tracking by timelapse imaging will reveal insights that are difficult to obtain with conventional discrete time-point population measurements. To gain mechanistic insights, I will perturb the mechanisms under investigation, and measure their resultant effects using timelapse imaging. These perturbations will involve over-expression or knockdown of genes; for studies of cis-epigenetic mechanisms, I will also develop a CRISPr-based system for perturbing chromatin marks at specific sites in the genome. To better understand this experimental data, and generate predictions for future experiments, I will then use mathematical modeling to analyze the behavior and dynamics of the different regulatory mechanisms studied. Finally, I will complement these approaches with genome-wide measurements of gene expression states in developing cells using high throughput sequencing, which will provide a more global picture of developmental changes, and potentially yield new directions for future work. Through these studies, I hope to uncover fundamental insights into how mammalian cells establish and maintain their distinct fate identities. These insights will potentially help us develop new therapies for leukemia and other cancers, and help us better manipulate stem cells for regenerative medicine.

描述(申请人提供)：哺乳动物干细胞和祖细胞激活特定调控基因的表达，以建立和稳定不同的细胞命运，但控制这一过程的机制和一般原理尚不清楚。细胞利用决定命运的调控基因在发育过程中建立和维持不同的命运身份，但目前还不清楚它们如何激活和维持这些基因的表达来建立命运身份。在这里，我建议在两个系统的背景下研究这个问题：T细胞命运承诺，它是由T细胞特异性转录因子的激活驱动的 Bcl11b(目标1和2)；以及巨噬细胞发育，我们最近发现这是由髓系转录因子PU.1(Kueh等人，2013年)1(目标3)的细胞周期依赖积累推动的。使用这两个系统，我将测试发育基因调控领域中一些广受争议的想法。首先，我将测试发育信号直接激活调控基因表达以指示细胞命运的想法(目标1)。接下来，我将测试两类拟议的稳定调控基因表达和命运认同的机制：涉及调控基因座位稳定和可遗传的表观遗传修饰的顺式作用机制(目标2)；以及涉及调控基因表达的自我强化正反馈环的反式作用机制(目标3)。我的主要方法将是使用时间流逝活细胞成像来跟踪Bcl11b在T细胞发育过程中的表达动态，以及PU.1在巨噬细胞发育过程中的表达动态。由于细胞分化是一个动态和本质上不同的过程，通过时间流逝成像进行单细胞跟踪将揭示传统离散时间点种群测量难以获得的洞察力。为了获得机理上的洞察，我将扰乱正在研究的机理，并使用时间流逝成像来测量它们的综合影响。这些干扰将涉及基因的过度表达或敲除；为了研究顺式表观遗传机制，我还将开发一个基于CRISPR的系统，用于干扰基因组特定位置的染色质标记。为了更好地理解这些实验数据，并为未来的实验产生预测，我将使用数学建模来分析所研究的不同调控机制的行为和动力学。最后，我将使用高通量测序对发育中细胞的基因表达状态进行全基因组测量，以补充这些方法，这将提供发育变化的更全面的图景，并可能为未来的工作提供新的方向。通过这些研究，我希望揭示哺乳动物细胞如何建立和维持其独特的命运身份的基本见解。这些见解可能会帮助我们开发白血病和其他癌症的新疗法，并帮助我们更好地操纵干细胞用于再生医学。