GENETICS OF HUMAN LYMPHOCYTE ANTIGEN CD8

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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.