TRANSCRIPTIONAL REGULATION OF T-CELL RECEPTOR ALPHA GENE

T细胞受体α的组织特异性转录调控 链基因需要对基因片段进行生产性重排 TCR阿尔法/德尔塔染色体基因座，一个活跃或 “可接近”的染色质结构和远端3‘TCR的募集 阿尔法增强剂。该增强子可以调节其同源Valpha启动子 从69kb的距离，取决于精确的组合 在重排过程中的可变区、连接区和恒定区。在……里面 此外，增强子功能似乎在一定程度上受附近基因控制 将其活动限制在适当阶段的消音器元素 T淋巴细胞发育。最小TCRα增强子已被映射 大约116个碱基对的DNA，由四种蛋白质调控： T细胞限制性Lef-1、Ets-1和CBF(核心结合因子)和a 与cAMP相互作用的广泛存在的CREB蛋白家族的成员 响应要素。TCRα增强子在其他疾病中的重要性 TCRα基因激活的关键方面已经被证明是 表明该元素从阿尔法/德尔塔染色体中删除 基因座阻止T细胞中的生产性基因重排。言下之意 是增强子是转录未重排基因所必需的 发生在基因重排之前的基因座，和/或调节 轨迹内的染色质结构，以便它可以被 V(D)J重组酶。在这项建议中，我们计划研究 利用体外转录和染色质实现α-增强子功能 准确复制组织特异性和增强剂的组装系统- 依赖TCRα基因表达。首先，实验的目的是 描述所需的DNA拓扑和核小体结构 产生功能增强子，因为这不是使用裸DNA发生的 体外模板。第二，特定蛋白质的作用机制 调节增强子活性将通过检查它们是否可以 远距离调控启动子结构。第三，一种抑制蛋白 在缺乏活性的情况下使TCRα基因的表达失活 将提纯增强子并对其DNA结合部位进行鉴定 以及在体外与其他转录因子相互作用的能力。 第四，由于对Valpha启动子的调控知之甚少，我们 计划确定构成转录的基本因素 无TATA序列上的起始复合体及其关系分析 这些蛋白质的启动子和增强子功能。最后，我们会 评估TCRα沉默因子在调节谱系特异性中的作用 通过分析该元件是否能调节TCRα转录 体外启动子或增强子活性。从一家公司获得的信息 这些体外实验中对TCRα增强子的分析应该 与健康相关的问题特别相关，因为TCR Alpha/Delta染色体是许多异常易位的场所 与多种白血病和淋巴瘤有关。这个 这些细胞中癌基因的激活被认为是由于它们的 易位到活性TCRα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.