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

Th-POK 的转录控制，是谱系控制的关键调节因子

• 批准号: 7590440
• 负责人: Dietmar J Kappes
• 金额: $ 49.1万
• 依托单位: RESEARCH INST OF FOX CHASE CAN CTR
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7590440
• 关键词: Address; Affinity; Binding; Biological Assay; CD8B1 gene; Cells; Chromatin; Complement; Data; Defect; Deoxyribonuclease I; Dependence; Development; Distal; Ectopic Expression; Elements; Engineering; Genetic Transcription; Goals; Health; Human; Hypersensitivity; Immune system; In Vitro; Kinetics; Ligands; Link; Lymphomagenesis; Maps; Mediating; Modeling; Molecular Genetics; Mus; Pathway interactions; Play; Regulation; Regulatory Element; Reporter; Role; Signal Transduction; Specificity; Staging; T-Cell Lymphoma; T-Lymphocyte; Testing; Transact; Transcriptional Regulation; Transgenes; Up-Regulation; in vivo; overexpression; promoter; thymocyte; transcription factor

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.

描述（由申请人提供）：替代CD4/CD8谱系承诺取决于THPOK的差分表达，而THPOK又在转录级别受到控制。我们假设差异THPOK表达受TCR信号传导的指导调节。我们的长期目标是定义将TCR信号与差异THPOK表达联系起来的途径。初步结果表明，ThPOK表达受2个替代启动子的控制，这些启动子在胸腺发育的不同阶段优先激活。本提案追求三个互补旨在阐明TCR信号传导与THPOK转录之间的联系。 AIM 1：以替代远端和近端启动子为中心的非重叠的报道盒在CD4胸腺细胞中显示优先表达，表明存在2种不同的调节元件，这些元件可以介导CD4谱系特异性表达。在此目的中，我们将1。使用体外和体内报告基因测定法，2。使用DNase I超敏（DHS）方法在开发过程中定义染色质可及性的变化，并确定远端CASSETTE和测试其功能意义。目标2：我们假设远端和近端启动子可以分别控制THPOK的瞬时TCR依赖性诱导和长期CD4谱系特异性表达。在此目的中，我们将1。使用小鼠使用小鼠对TCR信号传导或CD4谱系特异性控制机制的测试依赖性，其中只有一种或另一种机制是活跃的小鼠，2。确定CD4承诺中每个启动子的功能需求。使用单细胞PCR和报告基因敲击蛋白小鼠的不同特异性和亲和力的胸腺细胞测试竞争信号强度和谱系承诺的动力学信号传导模型的关键方面。 AIM 3：CD4承诺都需要THPOK和GATA-3，但它们的功能关系尚不清楚。我们将测试THPOK的过表达是否可以纠正GATA-3 - / - 小鼠（反之亦然）的发育缺陷，是否需要GATA-3，是否需要GATA-3，以及GATA-3是否直接参与THPOK转录的调节。项目叙述：控制CD4和CD8 T细胞谱系承诺的机制对于正常免疫系统的发展至关重要，因此对健康至关重要。此外，我们已经证明ThpoK是小鼠T细胞淋巴瘤发生的有效诱导剂，并且在大量人类T细胞淋巴瘤中过表达。因此，了解THPOK表达的控制也与淋巴作用的机理直接相关。