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。我们提出的研究，以阐明调节的ThPOK沉默，一个关键的顺式元件，是必要的谱系特异性ThPOK表达，测试Zfp 281，一个潜在的新的调节ThPOK表达的功能意义，并剖析的作用，ThPOK在外周T细胞，使用一种新的小鼠品系（OB 11线），其中ThPOK是选择性关闭在外周CD 4 T细胞。目标1。ThPOK消音器的功能解剖。这一目标有两个目标：1）为了测试NFAT和Egr因子控制沉默子功能的新假设，通过定位功能相关的NFAT和Egr共有基序，测试这些位点的突变是否影响沉默子的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)为了表征成熟CD 4 T细胞中ThPOK表达所需的100 bp调控基序，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。 目标二。分析Zfp 281在控制ThPOK转录和T细胞发育/功能中的作用。Y1 H筛选揭示Zfp 281作为ThPOK转录的潜在新调节子。多个额外的证据表明Zfp 281在ThPOK调节和T细胞发育/功能中的作用。为了直接测试这一点，我们将产生并充分表征条件性T细胞特异性Zfp 281敲除小鼠。此外，我们将使用Zfp 281-GFP报告小鼠评估Zfp 281在胸腺发育过程中在单细胞水平上的表达，并确定ThPOK沉默子和远端启动子元件内突变Zfp 281共有基序的功能后果。将Zfp 281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性进展。 目标3：ThPOK在外周T细胞功能中的作用在外周CD 4 T细胞中选择性缺乏ThPOK的小鼠表现出IL-9、IL-17和IL 2 R的表达增加。我们建议阐明这些基因的去抑制的潜在机制，并确定下调ThPOK是否对促进正常Th分化过程中细胞因子的表达很重要。首先，我们将调查是否去阻遏基因的ThPOK调节的直接目标，如果是这样，是否在靶位点的表观遗传重塑的结果ThPOK的损失。其次，我们将评估在不同Th极化条件下是否存在对ThPOK的选择性需求。具体来说，我们将测试是否存在或组成型表达的ThPOK优先影响某些Th极化程序，以及是否ThPOK表达差异调节Th极化特定的谱系。

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.