GENETICS OF HUMAN LYMPHOCYTE ANTIGEN CD8

人类淋巴细胞抗原 CD8 的遗传学

• 批准号: 3149970
• 负责人: Paula B. Kavathas
• 金额: $ 25.07万
• 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/3149970
• 关键词: CD8 molecule; DNA binding protein; DNA footprinting; DNA methylation; T lymphocyte; gel mobility shift assay; gene expression; gene mutation; genetic regulatory element; human tissue; immunogenetics; tissue /cell culture; transfection

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.

人淋巴细胞表面分子CD 8的功能是 与T细胞一起沿着识别MHC I类的辅助受体分子 受体，并作为信号分子，通过其与 酪氨酸激酶p56 lck。 这是一个关键的分子， 和负选择在T细胞发育中的作用， 成熟的细胞毒性CD 8 + T细胞。 CD 8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。 的基因 编码共缔合并在细胞上表达的多肽 T淋巴细胞表面的α/α或α/β二聚体。 的 该建议的目的是阐明 特异性调节CD 8 α基因的表达， 还协同调节人淋巴细胞中的连锁CD 8 β基因 发展 基因之间的区域可能含有调节基因。 影响两种基因表达的因素， 他们的推动者。 了解CD 8 α基因是如何调节的， 提供了一个分析转录机制的机会， 在T细胞发育期间和在CD 8+细胞毒性细胞中的调节。 为了实现这一目标，我们将描述启动子， CD 8 α基因的增强子/沉默子，并定义组织特异性 负责CD 8基因表达的调节因子。 通过 DNA酶I超敏反应图谱我们发现了一个T细胞特异性增强子 位于CD 8 α基因的最后一个内含子， 加强活动。 最小增强子为691 bp， 已知DNA结合蛋白的位点。 与增强剂相关的是 沉默元件，其使增强子活性降低80%。 的 反向重复序列与沉默元件重叠的位置表明 染色质结构在沉默活性中的潜在作用。 我们 具体目标是（i）确定调控元件和DNA结合 对于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CD 8 α基因连接的特异性超敏位点，和（iv）与 将我们对超敏感位点的搜索扩展到基因间区域 在CD 8基因和CD 8 α基因的3'之间。 功能分析 将通过转染细胞中的瞬时表达研究进行 代表不同的T细胞亚群以及非淋巴系。 调控元件将通过突变分析和能力来定义。 结合蛋白质，如通过EMSA、DNA酶I足迹法测定， 甲基化干扰技术。 基因调控的缺失可能是肿瘤细胞形成的一个因素。 了解CD 8 α基因是如何调节的将提供一个 测试药物调节基因表达能力的基础 淋巴细胞特异性基因，从而抑制淋巴细胞生长 肿瘤的