TRANSCRIPTIONAL REGULATION OF T-CELL RECEPTOR ALPHA GENE

T细胞受体α的组织特异性转录调控 链基因需要基因片段的生产性重排， TCR α/δ染色体基因座，产生活性或 “可接近的”染色质结构，以及远端3' TCR的募集， α增强子 该增强子可以调节其同源的Valpha启动子 根据精确的组合， 可变区、连接区和恒定区在重排过程中的变化。 在 此外，增强子功能似乎部分受附近的 消音器元件，将其活动限制在适当的阶段， T淋巴细胞发育。 最小的TCR α增强子已被定位 大约116个碱基对的DNA，并由四种蛋白质调节： T细胞限制性Lef-1、Ets-1和CBF（核心结合因子）和a 与cAMP相互作用的普遍存在的CREB蛋白家族成员 响应元素。 TCR α增强子在其他免疫系统中的重要性 TCR α基因激活的关键方面已经被证明是 表明从α/δ染色体上缺失该元件 基因座阻止T细胞中的生产性基因重排。 言下之意 增强子是转录未重排的 基因座，其发生在基因重排之前，和/或调节 染色质结构内的基因座，使其成为可访问的 V（D）J重组酶。 在这项建议中，我们计划研究 使用体外转录和染色质的α增强子功能 组装系统，准确地复制组织特异性和增强子， 依赖性TCR α基因表达。 首先，实验旨在 描述DNA拓扑结构和核小体结构， 产生功能性增强子，因为这在使用裸DNA时不会发生 体外模板。 第二，特定蛋白质 调节增强子活性将通过检查它们是否可以 远距离调节启动子结构。 第三，阻遏蛋白 在缺乏活性免疫调节剂的情况下， 增强子将被纯化并表征其DNA结合位点 以及与其他转录因子在体外相互作用的能力。 第四，由于对Valpha启动子调控知之甚少，我们 计划确定组成转录的基本因子 起始复合物上的TATA-少的序列，并分析关系 启动子和增强子的功能。 最后我们将 评估TCR α沉默物在调节谱系特异性 通过分析该元件是否可以调节TCR α转录， 体外启动子或增强子活性。 从一个 在这些体外实验中对TCR α增强子的分析应该 特别是与健康有关的问题，因为TCR α/δ染色体位点是许多异常易位的位点 与多种白血病和淋巴瘤有关。 的 这些细胞中癌基因的激活被认为是由于它们的 转移到活性TCR α增强子附近。

Tissue-specific transcriptional regulation of the T cell receptor alpha chain gene requires the productive rearrangement of gene segments within the TCR alpha/delta chromosomal locus, the generation of an active or "accessible" chromatin structure, and the recruitment of a distal 3' TCR alpha enhancer. This enhancer can regulate its cognate Valpha promoter from distances as great as 69 kb, depending upon the precise combination of variable, joining, and constant regions during rearrangement. In addition, enhancer function appears to be controlled, in part, by nearby silencer elements that restrict its activity to the appropriate stage of T lymphocyte development. The minimal TCR alpha enhancer has been mapped to approximately 116 base pairs of DNA and is regulated by four proteins: T cell-restricted Lef-1, Ets-1, and CBF (Core Binding Factor) and a member of the ubiquitous CREB family of proteins that interacts with cAMP response elements. The importance of the TCR alpha enhancer in other critical aspects of TCR alpha gene activation has been demonstrated by showing that a deletion of this element from the alpha/delta chromosomal locus prevents productive gene rearrangement in T cells. The implication is that the enhancer is required for transcription of the unrearranged locus, which occurs prior to gene rearrangement, and/or to modulate chromatin structure within the locus so that it becomes accessible to V(D)J recombinases. In this proposal, we plan to study the mechanism of alpha enhancer function using in vitro transcription and chromatin assembly systems that accurately reproduce tissue-specific and enhancer- dependent TCR alpha gene expression. First, experiments are designed to characterize the DNA topology and nucleosomal structure required to generate a functional enhancer since this does not occur using naked DNA templates in vitro. Second, the mechanism by which specific proteins regulate enhancer activity will be analyzed by examining whether they can modulate promoter structure at a distance. Third, a repressor protein that inactivates TCR alpha gene expression in the absence of an active enhancer will be purified and characterized for its site of DNA binding and ability to interact with other transcription factors in vitro. Fourth, since very little is known about Valpha promoter regulation, we plan to identify the basal factors that compose the transcription initiation complex on this TATA-less sequence and to analyze the relation of these proteins to promoter and enhancer function. Finally, we will assess the role of the TCR alpha silencer in regulating lineage-specific transcription by analyzing whether this element can modulate TCR alpha promoter or enhancer activity in vitro. The information gained from an analysis of the TCR alpha enhancer in these in vitro experiments should be particularly relevant to health-related issues since the TCR alpha/delta chromosomal locus is the site of many aberrant translocations that are associated with a variety of leukemias and lymphomas. The activation of oncogenes in these cells is thought to result from their translocation into the vicinity of an active TCR alpha enhancer.