CONTROL OF PURINE CATABOLISM IN THE THYMUS

该项目旨在确定一类新的人类基因调控 与常规增强剂不同但协同作用的元件 与传统的增强剂，以引起强大的基因激活， T细胞发育转染、生物化学和转基因分析 人腺苷脱氨酶（ADA）基因第一内含子中的顺式元件具有 发现了被称为“促进因子”的关键控制序列 这个调查员。目前，促进者由两个lkb定义 片段，其双侧侧接常规的300 bp T细胞增强子。 它们的存在可以通过增强子显著增加基因表达， 但它们的作用仅在转基因小鼠中可检测到。促进者 防止插入位点依赖性基因抑制， 基因表达严格地与基因拷贝数成比例增加。 两名主持人必须出席其职能： 缺失的，表达的细胞类型特异性得以维持，但 转基因受到导致表达差的位置效应的影响。 一个潜在的重要线索，他们的机制是， 缺失消除了DNA酶I超敏感位点在相邻的 增强子结构域，也完全阻止基因的任何增加， 增加基因拷贝数。数以百计的非易化基因 副本以等于或小于仅一个或 两个方便的副本。类似大小的人类DNA片段 代替缺少的主持人部分， 序列负责。引导者序列被假设为 通过与核因子相互作用在T细胞发育过程中发挥作用 负责组织染色质结构：它们可能是代表性的 一种新的调控元件， 染色体内的调节域。 这个应用程序将测试这一假设，即辅导员组成 与ADA基因增强子合作的特定可定义元件。 具体目的是（1）鉴定负责以下的特定序列： 促进者活动，以及（2）确定 控制这些控制序列如何与增强子合作。坚强 转基因分析中每个易化片段的作用提供了一个清晰的 意味着使用突变识别其单个元素和属性 技术.长期目标是确定潜在的能力， 与其他增强子和可转移基因相互作用的促进剂，以及 确定促进者机制， 与特定的主持人元素互动。详细研究 辅导员将提供深入了解一个关键的监管区域， 控制T细胞发育所必需的嘌呤代谢， 增加我们对染色质可及性是如何调节的理解。 我们在未来使用促进者元素的能力可能会大大提高 基因的设计能够纠正遗传疾病或对抗 病毒或肿瘤疾病。

This project seeks to identify a novel class of human gene regulatory elements that are different from conventional enhancers, but cooperate with a conventional enhancer to cause powerful gene activation in developing T-cells. Transfection, biochemical, and transgenic analyses of cis-elements in the human adenosine deaminase (ADA) gene first intron have uncovered crucial control sequences that have been termed "facilitators" by this investigator. The facilitators are presently defined by two l kb segments that bilaterally flank a conventional 300 bp T-cell enhancer. Their presence can drastically increase gene expression by the enhancer, but their effects are only detectable in transgenic mice. Facilitators prevent insertion site-dependent gene suppression and they cause thymocyte gene expression to increase strictly in proportion to gene copy number. Both facilitators must be present for their function: with either segment deleted, cell-type specificity of expression is maintained, but the transgenes are subject to positional effects that cause poor expression. A potentially important clue for their mechanism is that facilitator deletion abolishes DNase I hypersensitive site formation over the adjacent enhancer domain and also completely prevents any increase in gene expression from increased gene copy number. Hundreds of unfacilitated gene copies are expressed at a level equal to or less than that of only one or two facilitated copies. Similar size human DNA fragments could not substitute for a missing facilitator segment indicating that specific sequences were responsible. Facilitator sequences are hypothesized to function during T-cell development by interacting with nuclear factors responsible for organizing chromatin structure: they may be representative of a novel clams of regulatory elements that enable the formation of regulatory domains within chromosomes. This application will test the hypothesis that facilitators are composed of specific definable elements that cooperate with the ADA gene enhancer. The specific aims are (1) to identify specific sequences responsible for facilitator activity, and (2) to determine the rules or constraints that govern how these control sequences cooperate with the enhancer. The strong effect of each facilitator segment in transgenic analyses provides a clear means to identify its individual elements and properties using mutational techniques. Long range goals are to determine the potential ability of faciIitators to interact with other enhancers and transferable genes, and to determine facilitator mechanisms by identifying nuclear components that interact with the specific facilitator elements. Detailed study of the facilitators will provide insight into a crucial regulatory region that controls purine catabolism necessary for T-cell development, and should increase our understanding of how chromatin accessibility is regulated. Our ability to use facilitator elements in the future may greatly improve the design of genes capable of correcting inherited illnesses or combating viral or neoplastic diseases.