Transcriptional Control of Th-POK, a Key Regulator of Lineage Control

描述（由申请人提供）：交替的CD 4/CD 8谱系定型由ThPOK的差异表达决定，而ThPOK的差异表达又在转录水平上受到控制。我们推测ThPOK的差异表达受TCR信号传导的间接调控。我们的长期目标是确定TCR信号传导与ThPOK差异表达之间的联系途径.初步结果表明，ThPOK的表达是由2个交替启动子，这是优先激活在胸腺发育的不同阶段控制。本建议追求三个互补的目标，以阐明TCR信号转导和ThPOK转录之间的联系。目标1：非重叠的报告盒集中在交替的远端和近端启动子显示优先表达的CD 4胸腺细胞，表明存在2个不同的调节元件，可以介导的CD 4谱系特异性表达。为此，我们将1。使用体外和体内报告基因测定绘制重要的转录控制元件，2.使用DNA酶I超敏反应（DHS）方法定义发育过程中ThPOK基因座处染色质可及性的变化，以及3.鉴定远端盒的候选调节因子并测试其功能意义。目标二：我们假设远端和近端启动子可能分别控制ThPOK的瞬时TCR依赖性诱导和长期CD 4谱系特异性表达。在这个目标下，我们将1。使用仅一种或另一种机制活跃的小鼠测试远端和近端报告盒对TCR信号传导或CD 4谱系特异性控制机制的依赖性，2.使用其中一个或另一个启动子特异性失活的敲入小鼠，确定每个启动子在CD 4定型中的功能要求，以及3.使用单细胞PCR和报告基因敲入小鼠，在不同特异性和亲和力的胸腺细胞之间在单细胞水平上比较ThPOK表达，以测试竞争信号强度和谱系定型的动力学信号模型的关键方面。目标3：ThPOK和加塔-3都是CD 4承诺所必需的，但它们的功能关系仍不清楚。我们将测试ThPOK过表达是否可以纠正加塔-3-/-小鼠中的发育缺陷（反之亦然），在ThPOK上调之前是否需要加塔-3，以及加塔-3是否直接参与ThPOK转录的调节。项目叙述：控制CD 4和CD 8 T细胞谱系定型的机制对正常免疫系统的发育至关重要，因此对健康至关重要。此外，我们已经证明，ThPOK是一种有效的诱导T细胞淋巴瘤在小鼠中，并在相当大一部分的人T细胞淋巴瘤过表达。因此，理解ThPOK表达的控制也与淋巴瘤发生的机制直接相关。

DESCRIPTION (provided by applicant): Alternate CD4/CD8 lineage commitment is determined by differential expression of ThPOK, which is in turn controlled at the level of transcription. We postulate that differential ThPOK expression is regulated instructively by TCR signaling. Our long- term goal is to define the pathways linking TCR signaling with differential ThPOK expression. Preliminary results show that ThPOK expression is controlled by 2 alternate promoters, which are preferentially activated at different stages of thymic development. The present proposal pursues three complementary aims to elucidate the connection between TCR signaling and ThPOK transcription. Aim 1: Non-overlapping reporter cassettes centered on the alternate distal and proximal promoters show preferential expression in CD4 thymocytes, indicating the existence of 2 different sets of regulatory elements that can mediate CD4 lineage-specific expression. In this aim, we will 1. map important transcriptional control elements using in vitro and in vivo reporter assays, 2. define changes in chromatin accessibility at the ThPOK locus during development using the DNase I hypersensitivity (DHS) approach, and 3. identify candidate regulatory factors for the distal cassette and test their functional significance. Aim 2: We hypothesize that distal and proximal promoters may control transient TCR-dependent induction and long-term CD4 lineage-specific expression of ThPOK, respectively. In this aim we will 1. Test dependence of distal and proximal reporter cassettes on TCR signaling or CD4 lineage-specific control mechanisms using mice in which only one or the other mechanism is active, 2. Determine the functional requirement for each promoter in CD4 commitment, using knockin mice in which one or the other promoter is specifically inactivated, and 3. Compare ThPOK expression at the single-cell level between thymocytes of different specificities and affinities, using single-cell PCR and reporter knockin mice, to test key aspects of the competing signal strength and kinetic signaling models of lineage commitment. Aim 3: ThPOK and Gata-3 are both required for CD4 commitment, but their functional relationship remains unclear. We will test whether ThPOK overexpression can correct the developmental deficiency in Gata-3-/- mice (and vice versa), whether Gata-3 is required prior to ThPOK upregulation, and whether Gata-3 is directly involved in regulation of ThPOK transcription. Project Narrative: The mechanisms that control lineage commitment of CD4 and CD8 T cells are critical to development of a normal immune system and therefore essential to health. Furthermore, we have demonstrated that ThPOK is a potent inducer of T cell lymphomagenesis in mice, and is overexpressed in a substantial fraction of human T cell lymphomas. Hence understanding the control of ThPOK expression is also directly relevant to the mechanism of lymphomagenesis.