Regulatory Mechanisms of CD4+ T Cell Differentiation

CD4 T细胞分化的调控机制

• 批准号: 9754852
• 负责人: GREGORY E CRAWFORD
• 金额: $ 99.22万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-21 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9754852
• 关键词: Adaptive Immune System; Attenuated; Autoimmune Diseases; Autoimmunity; Automobile Driving; Biological Assay; Biological Models; Biological Process; CD4 Positive T Lymphocytes; CRISPR/Cas technology; Cells; Chromatin; Collaborations; Complex; DNase I hypersensitive sites sequencing; Data; Development; Disease; Disease model; Elements; Engineering; Equilibrium; Event; Fill-It; Flow Cytometry; Foundations; Future; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genome; Genomic Segment; Genomics; Goals; Health; Helper-Inducer T-Lymphocyte; Homeostasis; Human; Hypersensitivity; Immune; Immune response; Immune system; Immunity; Immunomodulators; In Vitro; Malignant Neoplasms; Mammalian Cell; Maps; Measures; Mediator of activation protein; Mus; Patients; Phenotype; Population; Publications; Publishing; Recording of previous events; Regulator Genes; Regulatory Element; Regulatory T-Lymphocyte; Reporter; Reporter Genes; Supporting Cell; System; T cell differentiation; T-Lymphocyte; Therapeutic; Therapeutic Intervention; Treatment Efficacy; Work; base; cell type; design; disease phenotype; epigenome; epigenome editing; experience; genetic association; genome-wide; in vivo Model; interest; new therapeutic target; novel strategies; pathogen; screening; single-cell RNA sequencing

There is a fundamental gap in understanding how the millions of known regulatory elements functionally contribute to gene regulation and phenotypes. Continued existence of that gap is an important problem because, until it is filled, it will remain extremely difficult to identify the genetic mechanisms underlying the thousands of observed genetic associations with disease phenotypes. Our long-term goal is to understand how and to what extent gene regulatory elements alter target gene expression and impact phenotypes. The objectives of this particular proposal are to functionally characterize all regulatory elements contributing to the differentiation of CD4+ T cells. In doing so, we will identify the causal regulatory mechanisms that modulate the immune system. The rationale for this work is that understanding those mechanisms will be the foundation for future efforts to therapeutically modulate the immune system, and will establish a discovery platform for determining the mechanisms underlying countless other model systems. Specifically, we will characterize three complementary components of regulatory element activity: (i) the capacity of regulatory elements to drive expression of a reporter gene, (ii) the effect of each regulatory element on the expression of one or more target genes, and (iii) the contributions of regulatory elements to phenotypic function, namely differentiation. We will accomplish those goals across three specific aims. In Aim 1, we will quantify the activity of all regulatory elements that have evidence of differential activity between subtypes of mouse CD4 T cells. We will do so using a capture-based high-throughput reporter assay that allows us to assay larger (>500 bp) fragments from specific genomic regions of interest. In Aim 2, we will quantify the effects of regulatory elements on target genes using a novel strategy that combines high-throughput CRISPR/ Cas9-based epigenome editing screens and targeted high-throughput single-cell RNA-sequencing. In Aim 3, we will determine which regulatory elements are necessary or sufficient for CD4 T cell differentiation using high-throughput CRISPR/Cas9-based epigenome-editing screens combined differentiation into particular CD4 T cell subtypes. Each aim will provide functional characterization of all of the regulatory elements implicated in CD4 T cell differentiation. Together, the aims will provide a comprehensive, multi-layered, and systematic understanding of the ways that gene regulatory elements modulate the immune system. The result will be an actionable set of targets for designing strategies to modulate immune system activity for therapeutic benefit. Because the approach is general to any model system, the same strategy can be readily transferred to diverse systems including differentiation and disease models. Therefore, we expect that this project will have both immediate and long-term benefit for determining the ways that regulatory elements contribute to health and disease.

在理解数以百万计的已知监管要素如何 在功能上有助于基因调控和表型。这种差距的持续存在是 重要的问题，因为在它被填补之前，识别基因仍然极其困难 数以千计的观察到的与疾病表型的遗传关联的潜在机制。我们的 长期目标是了解基因调控元件如何以及在多大程度上改变目标基因 表达和影响表型。该特定提案的目标是在功能上 描述了有助于 CD4+ T 细胞分化的所有调控元件。这样做，我们 将确定调节免疫系统的因果调节机制。这样做的理由 工作的重点是了解这些机制将成为未来治疗努力的基础 调节免疫系统，并将建立一个发现平台来确定其机制 无数其他模型系统的基础。具体来说，我们将描述三个互补的特征 调控元件活动的组成部分：（i）调控元件驱动表达的能力 报告基因，(ii) 每个调控元件对一个或多个靶标表达的影响 基因，以及（iii）调节元件对表型功能（即分化）的贡献。 我们将通过三个具体目标来实现这些目标。在目标 1 中，我们将量化所有的活动 有证据表明小鼠 CD4 T 细胞亚型之间活性存在差异。 我们将使用基于捕获的高通量报告分析来实现这一点，该分析使我们能够分析更大的样本 来自特定感兴趣基因组区域的 (>500 bp) 片段。在目标 2 中，我们将量化以下效果： 使用结合高通量 CRISPR/ 的新策略对靶基因进行调控元件 基于 Cas9 的表观基因组编辑筛选和靶向高通量单细胞 RNA 测序。在 目标 3，我们将确定哪些调控元件对于 CD4 T 细胞是必要或充分的 使用基于高通量 CRISPR/Cas9 的表观基因组编辑筛选相结合进行分化 分化为特定的 CD4 T 细胞亚型。每个目标都将提供所有功能特征 与 CD4 T 细胞分化有关的调节元件。这些目标共同将提供一个 全面、多层次、系统地了解基因调控元件的作用方式 调节免疫系统。结果将是一套可操作的目标，用于设计战略 调节免疫系统活性以获得治疗效果。因为该方法对于任何 模型系统，相同的策略可以很容易地转移到不同的系统，包括差异化 和疾病模型。因此，我们预计该项目将具有近期和长期的效益。 有助于确定监管因素对健康和疾病的影响方式。