Single cell analysis of hematopoietic cell fate determination

造血细胞命运决定的单细胞分析

• 批准号: 8768311
• 负责人: Hao Yuan Kueh
• 金额: $ 9万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 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 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8768311
• 关键词: Affect; Architecture; B-Lymphocytes; Behavior; Biological Models; Blood Cells; Cell Cycle; Cell Differentiation process; Cell Fate Control; Cell Proliferation; Cells; Chromatin; Complement; Data; Deposition; Development; Developmental Gene; Enzymes; Epigenetic Process; Feedback; Future; Gene Expression; Gene Expression Profiling; Gene Expression Regulation; Genes; Genome; Genomics; Goals; Hematopoietic; High-Throughput Nucleotide Sequencing; Histones; Image; Immune; Investigation; Length; Life; Macrophage Activation; 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; cellular imaging; effective therapy; gene induction; genome-wide; histone modification; insight; leukemia; macrophage; mathematical model; notch protein; progenitor; proto-oncogene protein Spi-1; public health relevance; research study; single cell analysis; stem; 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（由申请人提供）：哺乳动物干细胞和祖细胞激活特异性调控基因的表达，以建立和稳定不同的细胞命运，但控制该过程的机制和一般原理尚不清楚。细胞在发育过程中利用指定命运的调控基因来建立和维持不同的命运身份，但目前尚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，这是由T细胞特异性转录因子的激活驱动的 Bcl 11b（目的1和2）;和巨噬细胞发育，我们最近发现巨噬细胞发育是由髓样转录因子PU.1的细胞周期长度依赖性积累驱动的（Kueh等人，2013）1（目标3）。使用这两个系统，我将测试一些在发育基因调控领域广泛争论的观点。首先，我将测试的想法，发育信号直接激活调控基因的表达，以指导细胞的命运（目标1）。接下来，我将泰特两类稳定调控基因表达和命运同一性的机制：顺式作用机制，涉及调控基因位点的稳定和可遗传的表观遗传修饰（目标2）;反式作用机制，涉及调控基因表达的自我加强正反馈回路（目标3）。我的主要方法是使用时间推移活细胞成像来跟踪T细胞发育过程中Bcl 11b的表达动态，以及巨噬细胞发育过程中PU.1的表达动态。由于细胞分化是一个动态的和内在的异质性的过程，单细胞跟踪时间推移成像将揭示的见解，是难以获得与传统的离散时间点群体测量。为了获得机械的见解，我将扰动调查中的机制，并使用时间推移成像测量其结果。这些干扰将涉及基因的过度表达或敲低;对于顺式表观遗传机制的研究，我还将开发一种基于CRISPr的系统，用于干扰基因组中特定位点的染色质标记。为了更好地理解这些实验数据，并为未来的实验做出预测，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，这将提供更全面的发育变化情况，并可能为未来的工作提供新的方向。通过这些研究，我希望能够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。这些见解可能有助于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。