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）：替代的 CD4/CD8 谱系定型是由 ThPOK 的差异表达决定的，而 ThPOK 的表达又在转录水平上受到控制。我们假设差异 ThPOK 表达受到 TCR 信号传导的指导性调节。我们的长期目标是确定 TCR 信号传导与差异 ThPOK 表达之间的联系途径。初步结果表明，ThPOK 表达由 2 个交替启动子控制，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的目标，以阐明 TCR 信号传导与 ThPOK 转录之间的联系。目标 1：以交替的远端和近端启动子为中心的非重叠报告盒在 CD4 胸腺细胞中显示优先表达，表明存在 2 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. 使用体外和体内报告基因检测绘制重要的转录控制元件图谱，2. 使用 DNase I 超敏反应 (DHS) 方法定义发育过程中 ThPOK 位点染色质可及性的变化，以及 3. 识别候选元件远端盒的调节因素并测试其功能意义。目标 2：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 CD4 谱系特异性表达。在此目标中，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 CD4 谱系特异性控制机制的依赖性，2. 确定 CD4 承诺中每个启动子的功能要求，使用其中一个或另一个启动子被特异性失活的敲入小鼠，以及 3.使用单细胞 PCR 和报告基因敲入小鼠，比较不同特异性和亲和力的胸腺细胞之间单细胞水平的 ThPOK 表达，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。目标 3：ThPOK 和 Gata-3 都是 CD4 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 Gata-3-/- 小鼠的发育缺陷（反之亦然），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。项目叙述：控制 CD4 和 CD8 T 细胞谱系定型的机制对于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 细胞淋巴瘤发生的有效诱导剂，并且在大部分人 T 细胞淋巴瘤中过度表达。因此，了解 ThPOK 表达的控制也与淋巴瘤发生机制直接相关。