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

• 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
• 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

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在胸腺发育中起着关键作用，并且可能在成熟的T细胞功能中起着关键作用。我们提出的研究旨在阐明THPOK消音器的调节，这是谱系特定Thpok表达所必需的关键顺式元素，测试ZFP281的功能意义，ZFP281是THPOK表达的潜在新调节剂，并使用新颖的小鼠菌株（OB11 line）选择了THPOK在外围T细胞中Thpok在Thep plime的作用。目标1。thpok消音器的功能解剖。该目标有两个目标：1）测试新的假设，即NFAT和EGR因子控制消音器的功能，通过映射功能相关的NFAT和EGR共识基序，测试这些位点的突变是否影响Runx结合状态和表观遗传状态，并确定NFAT和Egr Egragogrognize silsine silencIs silencInd nfat的表达。 2）表征成熟CD4 T细胞中ThPOK表达所必需的100 bp调节基序，并似乎介导染色体相互作用。我们将确定该基序的存在/不存在是否会影响消音器的表观遗传状态，并使用3C和FISH方法来解决其在THPOK基因座的核重点中的潜在作用。 AIM 2。分析ZFP281在控制THPOK转录和T细胞开发/功能中的作用。 Y1H屏幕显示ZFP281是THPOK转录的潜在新调节剂。多个其他证据表明，ZFP281在THPOK调节中的作用以及T细胞的开发/功能。为了直接测试这一点，我们将生成并充分表征有条件的T细胞特异性ZFP281敲除鼠标。此外，我们将使用ZFP281-GFP报告基因小鼠在胸腺发育过程中评估单细胞水平的ZFP281表达，并确定突变ZFP281在Thpok Silencer和远端启动子元素中突变的ZFP281共识基序的功能后果。将ZFP281定义为T细胞开发/功能的新型调节剂将代表重要的概念进步。 AIM 3。定义THPOK在周围T细胞功能中的作用。在外周CD4 T细胞中选择性缺乏THPOK的小鼠表现出IL-9，IL-17和IL2R的表达增加。我们建议阐明这些基因消除这些基因的潜在机制，并确定THPOK的下调对于在正常TH分化过程中促进细胞因子表达是否重要。首先，我们将研究过压缩的基因是否是THPOK调控的直接靶标，如果是，THPOK的丢失是否导致靶基因座的表观遗传重塑。其次，我们将评估在不同的极化条件下对THPOK是否有选择性要求。具体而言，我们将测试THPOK的缺失或本构表达是否优先影响某些极化程序，以及在TH对特定谱系期间THPOK表达是否受到差异调节。