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

描述（由申请人提供）：交替的CD4/CD8谱系承诺是由ThPOK的差异表达决定的，而ThPOK又在转录水平上受到控制。我们假设ThPOK的差异表达是由TCR信号指示调节的。我们的长期目标是确定TCR信号与ThPOK差异表达之间的联系途径。初步结果表明，ThPOK的表达受2个交替启动子控制，它们在胸腺发育的不同阶段被优先激活。本研究旨在阐明TCR信号和ThPOK转录之间的联系。目的1：以远端和近端交替启动子为中心的非重叠报告磁带在CD4胸腺细胞中优先表达，表明存在两组不同的调节元件可以介导CD4谱系特异性表达。为此，我们将1。利用体外和体内报告基因分析绘制重要的转录控制元件；2 .使用DNase I超敏（DHS）方法定义发育过程中ThPOK位点染色质可及性的变化；确定远端盒的候选调节因子并测试其功能意义。目的2：我们假设远端启动子和近端启动子可能分别控制瞬时tcr依赖性诱导和长期CD4谱系特异性表达的ThPOK。为此，我们将1。使用仅激活其中一种机制的小鼠，测试远端和近端报告磁带对TCR信号或CD4谱系特异性控制机制的依赖性。2 .确定CD4承付中每个启动子的功能需求，使用敲入小鼠，其中一个或另一个启动子被特异性失活；比较不同特异性和亲和的胸腺细胞在单细胞水平上的ThPOK表达，使用单细胞PCR和报告基因敲入小鼠，以测试谱系承诺的竞争信号强度和动力学信号模型的关键方面。目的3:ThPOK和Gata-3都是CD4承诺所必需的，但它们的功能关系尚不清楚。我们将测试ThPOK过表达是否可以纠正Gata-3-/-小鼠的发育缺陷（反之亦然），在ThPOK上调之前是否需要Gata-3，以及Gata-3是否直接参与ThPOK转录的调节。控制CD4和CD8 T细胞谱系承诺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是小鼠T细胞淋巴瘤形成的有效诱导剂，并且在很大一部分人T细胞淋巴瘤中过表达。因此，了解对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.