Regulatory Mechanisms of CD4+ T Cell Differentiation

在理解数百万已知的监管要素如何 在功能上对基因调控和表型有贡献。这种差距的持续存在是一种 这是一个重要的问题，因为在它被填满之前，识别基因仍然是极其困难的 数以千计的观察到的遗传与疾病表型相关的潜在机制。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靶基因。 表达和影响表型。这项特别提案的目标是在功能上 描述所有有助于CD4+T细胞分化的调控因素。为了做到这一点，我们 将确定调节免疫系统的因果调节机制。这样做的理由是 工作是，了解这些机制将是未来治疗方面努力的基础 调节免疫系统，并将建立一个发现平台来确定机制 在无数其他模型系统的基础上。具体地说，我们将描述三个互补 调节元件活性的组成部分：(一)调节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)调控元件对表型功能的贡献，即分化。 我们将通过三个具体目标实现这些目标。在目标1中，我们将量化所有 有证据表明小鼠CD4T细胞亚型之间活性不同的调节元件。 我们将使用一种基于捕获的高通量报告分析来实现这一点，它允许我们分析更大的 (&gt；500bp)来自特定基因组感兴趣区域的片段。在目标2中，我们将量化 利用结合高通量CRISPR/的新策略对靶基因的调控元件 基于Cas9的表观基因组编辑屏幕和目标高通量单细胞RNA测序。在……里面 目标3，我们将确定哪些调节元件对CD4T细胞是必要的或充分的 结合基于CRISPR/CAS9的高通量表观基因组编辑屏幕进行差异化 分化成特定的CD4T细胞亚型。每个目标都将提供所有 与CD4T细胞分化有关的调控元件。总之，AIMS将提供一个 全面、多层次和系统地了解基因调控元件 调节免疫系统。其结果将是一套可操作的目标，用于设计战略以 调节免疫系统的活动，以达到治疗的目的。因为该方法适用于任何 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，我们预计这个项目将既有近期的又有长期的 有利于确定监管要素对健康和疾病的贡献方式。

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.