Dissecting the role of ThPOK in thymic development and T cell differentiation

剖析 ThPOK 在胸腺发育和 T 细胞分化中的作用

基本信息

批准号：
8704657
负责人：
Dietmar J Kappes
金额：
$ 33.92万
依托单位：
RESEARCH INST OF FOX CHASE CAN CTR
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-08-01 至 2018-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8704657
关键词：
Address Affect Beryllium Binding Biological Assay CD4 Positive T Lymphocytes CD8B1 gene Cell physiology Cells Consensus Cytokine Receptor Gene Cytokine Receptors Data Defect Development Dissection Distal Ectopic Expression Elements Epigenetic Process Exhibits Family Feedback Gene Silencing Genes Genetic Transcription Health Human IL2RA gene Immune system In Vitro Individual Interleukin-17 Interleukin-9 Investigation Knockout Mice Length Lymphomagenesis Maps Mature T-Lymphocyte Mediating Molecular Mouse Strains Mus Mutate Mutation Nuclear Pathway interactions Peripheral Phenotype Play Process Regulation Reporter Role Signal Transduction Site Staging T cell differentiation T-Cell Development T-Cell Lymphoma T-Lymphocyte Testing Thymus Gland Transfection base blocking factor cytokine derepression in vivo interest member mouse model mutant novel overexpression programs promoter public health relevance receptor expression research study response thymocyte

项目摘要

DESCRIPTION (provided by applicant): ThPOK plays a key role in thymic development, and potentially in mature T cell function. We propose studies to elucidate regulation of the ThPOK silencer, a key cis element that is necessary for lineage specific ThPOK expression, test the functional significance of Zfp281, a potential new regulator of ThPOK expression, and dissect the role of ThPOK in peripheral T cells, using a novel mouse strain (OB11 line) in which ThPOK is selectively turned off in peripheral CD4 T cells. Aim 1. Functional dissection of the ThPOK silencer. This aim has two objectives: 1) To test the novel hypothesis that NFAT and Egr factors control silencer function, by mapping functionally relevant NFAT and Egr consensus motifs, testing whether mutation of these sites affects Runx binding and epigenetic state of the silencer, and determining whether ectopic expression of NFAT and Egr factors can directly antagonize silencing. 2) To characterize a 100 bp regulatory motif that is required for ThPOK expression in mature CD4 T cells, and seems to mediate interchromosomal interactions. We will determine whether presence/absence of this motif affects the epigenetic state of the silencer, and use 3C and FISH approaches to address its potential role in nuclear repositioning of the ThPOK locus. Aim 2. Analysis of the role of Zfp281 in control of ThPOK transcription and T cell development/function. A Y1H screen revealed Zfp281 as a potential new regulator of ThPOK transcription. Multiple additional lines of evidence suggest a role for Zfp281 in ThPOK regulation, and T cell development/function. To test this directly, we will generate and fully characterize a conditional T cell-specific Zfp281 knockout mouse. Additionally, we will use Zfp281-GFP reporter mice to assess Zfp281 expression at the single-cell level during thymic development, and determine functional consequences of mutating Zfp281 consensus motifs within the ThPOK silencer and distal promoter elements. Defining Zfp281 as a novel regulator of T cell development/function would represent an important conceptual advance. Aim 3. Defining the role of ThPOK in peripheral T cell function. Mice that selectively lack ThPOK in peripheral CD4 T cells exhibit increased expression of IL-9, IL-17 and IL2R. We propose to elucidate the underlying mechanism for derepression of these genes and determine whether downmodulation of ThPOK is important for promoting cytokine expression during normal Th differentiation. First, we will investigate whether derepressed genes are direct targets of ThPOK regulation, and if so, whether loss of ThPOK results in epigenetic remodeling of target loci. Secondly, we will assess whether there is a selective requirement for ThPOK under different Th polarization conditions. Specifically, we will test whether the absence or constitutive expression of ThPOK preferentially affects certain Th polarization programs, and whether ThPOK expression is differentially regulated during Th polarization to particular lineages.

描述（由申请人提供）：ThPOK 在胸腺发育中发挥关键作用，并可能在成熟 T 细胞功能中发挥关键作用。我们提出研究阐明 ThPOK 沉默子（谱系特异性 ThPOK 表达所必需的关键顺式元件）的调节，测试 Zfp281（ThPOK 表达的潜在新调节因子）的功能意义，并剖析 ThPOK 在外周 T 细胞中的作用，使用一种新型小鼠品系（OB11 系），其中 ThPOK 在外周 CD4 T 细胞中选择性关闭。目标 1. ThPOK 消音器的功能剖析。这一目标有两个目标：1) 通过绘制功能相关的 NFAT 和 Egr 共有基序，测试这些位点的突变是否影响 Runx 结合和沉默子的表观遗传状态，并确定 NFAT 和 Egr 因子控制沉默子功能的新假设。 NFAT和Egr因子的异位表达是否可以直接拮抗沉默。 2) 表征成熟 CD4 T 细胞中 ThPOK 表达所需的 100 bp 调节基序，并且似乎介导染色体间相互作用。我们将确定该基序的存在/不存在是否会影响沉默子的表观遗传状态，并使用 3C 和 FISH 方法来解决其在 ThPOK 基因座核重新定位中的潜在作用。目标 2. 分析 Zfp281 在控制 ThPOK 转录和 T 细胞发育/功能中的作用。 Y1H 筛选显示 Zfp281 是 ThPOK 转录的潜在新调节因子。多个额外证据表明 Zfp281 在 ThPOK 调节和 T 细胞发育/功能中发挥作用。为了直接测试这一点，我们将生成并全面表征条件 T 细胞特异性 Zfp281 敲除小鼠。此外，我们将使用 Zfp281-GFP 报告小鼠评估胸腺发育过程中单细胞水平的 Zfp281 表达，并确定 ThPOK 沉默子和远端启动子元件内 Zfp281 共有基序突变的功能后果。将 Zfp281 定义为 T 细胞发育/功能的新型调节剂将代表一个重要的概念进步。目标 3. 明确 ThPOK 在外周 T 细胞功能中的作用。外周 CD4 T 细胞选择性缺乏 ThPOK 的小鼠表现出 IL-9、IL-17 和 IL2R 表达增加。我们建议阐明这些基因去抑制的潜在机制，并确定 ThPOK 的下调对于促进正常 Th 分化过程中细胞因子的表达是否重要。首先，我们将研究去抑制基因是否是 ThPOK 调节的直接目标，如果是，ThPOK 的缺失是否会导致目标位点的表观遗传重塑。其次，我们将评估不同Th极化条件下ThPOK是否存在选择性要求。具体来说，我们将测试 ThPOK 的缺失或组成型表达是否优先影响某些 Th 极化程序，以及 ThPOK 表达在特定谱系的 Th 极化过程中是否受到差异调节。