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

描述(由申请人提供)：ThPOK在胸腺发育中发挥关键作用，并可能在成熟的T细胞功能中发挥作用。我们建议利用一种新的小鼠品系(OB11系)来阐明ThPOK沉默的调控，Zfp281是ThPOK表达的潜在新的调节因子，并分析ThPOK在外周T细胞中的作用，其中ThPOK在外周T细胞中选择性地关闭。目的1.对ThPOK消声器进行功能剖析。本研究的目的有两个：1)验证NFAT和Egr因子控制沉默功能的新假设，通过定位功能相关的NFAT和Egr共识基序，测试这些位点的突变是否影响RUNX结合和沉默的表观遗传状态，以及确定NFAT和Egr因子的异位表达是否可以直接拮抗沉默。2)鉴定在成熟的CD4T细胞中ThPOK表达所需的100bp的调控基序，该基序似乎介导了染色体间的相互作用。我们将确定该基序的存在/不存在是否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。目的2.分析Zfp281在ThPOK转录调控和T细胞发育/功能中的作用。Y1H屏幕显示Zfp281是一种潜在的ThPOK转录新调节因子。多条额外的证据表明，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，我们将生成一个条件性T细胞特异性Zfp281基因敲除小鼠并对其进行充分鉴定。此外，我们将使用Zfp281-GFP报告小鼠在胸腺发育过程中在单细胞水平上评估Zfp281的表达，并确定ThPOK沉默因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/功能的新调节因子将代表着一项重要的概念进步。目的3.明确ThPOK在外周T细胞功能中的作用。外周CD4T细胞中ThPOK选择性缺失的小鼠表现出IL-9、IL-17和IL2R的表达增加。我们建议阐明这些基因下调的潜在机制，并确定ThPOK下调是否是促进正常Th分化过程中细胞因子表达的重要因素。首先，我们将研究下调基因是否是ThPOK调控的直接靶点，如果是，ThPOK缺失是否会导致靶点的表观遗传重塑。其次，我们将评估在不同的Th极化条件下是否存在对ThPOK的选择性要求。具体地说，我们将测试ThPOK的缺失或结构性表达是否优先影响特定的Th极化程序，以及ThPOK的表达是否在Th极化期间对特定谱系进行差异调节。

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.