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

描述（由申请人提供）：ThPOK在胸腺发育中起关键作用，并可能在成熟T细胞功能中起关键作用。我们建议研究阐明ThPOK沉默子的调控，这是谱系特异性ThPOK表达所必需的关键顺式元件，测试Zfp281的功能意义，这是一种潜在的ThPOK表达的新调节剂，并使用一种新的小鼠品系（OB11系）来分析ThPOK在外周血CD4 T细胞中选择性关闭的作用。目的1。ThPOK消声器的功能解剖。本研究有两个目的：1)通过定位功能相关的NFAT和Egr共识基序，检验NFAT和Egr因子控制沉默者功能的新假设，测试这些位点的突变是否影响沉默者的Runx结合和表观遗传状态，并确定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调控基序，该基序可能介导染色体间相互作用。我们将确定该基序的存在/缺失是否会影响消声器的表观遗传状态，并使用3C和FISH方法来研究其在ThPOK位点核重定位中的潜在作用。目标2。Zfp281在控制ThPOK转录和T细胞发育/功能中的作用分析。Y1H筛选显示Zfp281可能是ThPOK转录的新调控因子。多个额外的证据表明Zfp281在ThPOK调控和T细胞发育/功能中发挥作用。为了直接测试这一点，我们将生成并充分表征条件T细胞特异性Zfp281敲除小鼠。此外，我们将使用Zfp281- gfp报告小鼠来评估胸腺发育过程中单细胞水平上Zfp281的表达，并确定ThPOK沉默子和远端启动子元件中Zfp281共识基序突变的功能后果。将Zfp281定义为T细胞发育/功能的新调节剂将代表一个重要的概念进步。目标3。确定ThPOK在外周T细胞功能中的作用。小鼠外周血CD4 T细胞选择性缺乏ThPOK， IL-9、IL-17和il - 2r的表达增加。我们建议阐明这些基因下调的潜在机制，并确定ThPOK下调是否对促进正常Th分化过程中细胞因子的表达重要。首先，我们将研究低表达基因是否是ThPOK调控的直接靶点，如果是，ThPOK的缺失是否会导致靶位点的表观遗传重塑。其次，我们将评估在不同的极化条件下，ThPOK是否存在选择性要求。具体来说，我们将测试ThPOK的缺失或组成性表达是否优先影响某些Th极化程序，以及ThPOK表达是否在特定谱系的Th极化过程中受到差异调节。

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.