GENETICS OF HUMAN LYMPHOCYTE ANTIGEN CD8

人类淋巴细胞抗原 CD8 的遗传学

• 批准号: 2071071
• 负责人: Paula B. Kavathas
• 金额: $ 25.68万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-07-01 至 1997-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2071071
• 关键词: GENETICS; HUMAN; LYMPHOCYTE; ANTIGEN; CD8

The human lymphocyte cell surface molecule, CD8, functions as a coreceptor molecule for recognition of MHC class I along with the T cell receptor and as a signalling molecule through its association with the tyrosine kinase p56lck. This is a critical molecule for both positive and negative selection in T cell development and for function of the mature cytotoxic CD8+ T cells. the CD8 gene region, located on human chromosome 2 is composed of the linked alpha and beta genes. The genes encode polypeptides that co-associate and are expressed on the cell surface of T lymphocytes as alpha/alpha or alpha/beta dimers. the objective of this proposal is to elucidate the molecular mechanisms that specifically regulate the expression of the CD8alpha gene and that may also coordinately regulate the linked CD8beta gene in human lymphocyte development. The region between the genes may contain regulatory elements that influence expression of both genes within the context of their promoters. Understanding how the CD8alpha gene is regulated should provide an opportunity to analyze mechanisms for transcriptional regulation during T cell development and in the CD8+ cytotoxic cell. To achieve this objective, we will characterize the promoter and enhancer/silencers of the CD8alpha gene and define the tissue specific regulatory factors that are responsible for CD8 gene expression. by DNase I hypersensitivity mapping we found a T cell specific enhancer located in the last intron of the CD8alpha gene that gave 100 fold enhancement of activity. The minimal enhancer was 691 bp and had seven sites for known DNA binding proteins. Linked to the enhancer was a silencer element which decreased enhancer activity by 80 percent. The location of an inverted repeat overlapping the silencer element suggests a potential role for chromatin structure in silencing activity. Our specific aims are to (i) identify the regulatory elements and DNA binding proteins important for enhancer and promoter activity, (ii) to determine the molecular basis for silencing activity, (iii) to determine the functional significance of DNA fragments containing two additional T cell specific hypersensitive sites linked to the CD8alpha gene and (iv) to extend our search for hypersensitive sites to the intergenic region between the CD8 genes and 3' of the CD8alpha gene. functional analysis will be performed by transient expression studies in transfected cells representing different T cell subsets as well as in non-lymphoid lines. Regulatory elements will be defined by mutational analysis and by ability to bind proteins as determined by EMSA, DNase I footprinting, and methylation interference techniques. Abnormalities in gene regulation can be a factor in tumor cell formation. Understanding how the CD8alpha gene is regulated would provide a foundation for testing drugs for their ability to modulate expression of lymphocyte specific genes and thereby inhibit the growth of lymphocyte tumors.

人类淋巴细胞表面分子CD8的功能是 识别MHC-I类分子和T细胞的辅助受体分子 受体，并作为信号分子，通过与 酪氨酸激酶p56lck。这是一种关键的分子，对两者都是积极的 在T细胞发育过程中的负选择和对T细胞的功能 成熟的细胞毒性CD8+T细胞。CD8基因区，位于人类 2号染色体由相连的α基因和β基因组成。基因 编码共同结合的多肽并在细胞上表达 T淋巴细胞表面为α/α或α/β二聚体。这个 这一建议的目的是阐明 特异性地调节CD8α基因的表达，这可能 也协调调节人淋巴细胞中连锁的CD8β基因 发展。基因之间的区域可能包含调控基因 影响这两个基因表达的因素 他们的推动者。了解CD8α基因是如何调控的 提供了分析转录的机制的机会 T细胞发育和CD8+细胞毒细胞的调控。 为了实现这一目标，我们将对启动者和 CD8α基因的增强子/抑制子，并定义组织特异性 负责CD8基因表达的调节因素。通过 DNase I超敏图谱我们发现了一个T细胞特异性增强子 位于CD8基因的最后一个内含子，该内含子使100倍 活跃性增强。最小增强子为691bp，有7个 已知的DNA结合蛋白的位置。与增强剂相关联的是一个 消音器元件，使增强剂活性降低80%。这个 与消音器元件重叠的反向重复的位置表明 染色质结构在沉默活性中的潜在作用。我们的 具体目标是：(I)确定调控元件和DNA结合 对增强子和启动子活性重要的蛋白质，(Ii)确定 沉默活性的分子基础，(Iii)确定 含有两个额外T细胞的DNA片段的功能意义 与CD8α基因和(Iv)相关联的特定超敏部位 将我们对过敏性位点的搜索扩展到基因间隔区 CD8基因和CD8α基因的3‘之间。功能分析 将通过在转基因细胞中进行瞬时表达研究来实现 代表不同的T细胞亚群以及在非淋巴系中。 监管要素将通过突变分析和能力来定义 结合由EMSA、DNase I足迹确定的蛋白质，以及 甲基化干扰技术。 基因调控的异常可能是肿瘤细胞形成的一个因素。 了解CD8α基因是如何调控的将提供一种 测试药物调节基因表达能力的基础 从而抑制淋巴细胞的生长 肿瘤。