The regulation of CD8 expression during the differentiation of CD4-CD8+ (CD8SP) and intestinal intraepithelial lymphocytes (IELs) is still poorly understood. Results from laboratories using transgenic approaches have described an enhancer (called E81) approximately 16 kb upstream of the CD8a gene that drives reporter gene expression in IELs and mature CD8SP cells but not in thymocytes at earlier stages of differentiation. In vitro transfection studies in our laboratory have defined an approximately 200 nt cis-acting element (called L2a) located 4-5 kb upstream of the CD8a gene that appears to be the target of negative regulation in hybridomas of CD8-positive T-cells fused with the thymic lymphoma cell line, BW5 147. The L2a element is a nuclear matrix associated region (or MAR). Interestingly, inclusion of both the E81 enhancer and a 4.3 kb Hindlil/Hindlil fragment containing the L2a element in the transgenic construct results in E8I enhancer function in CD4+CD8+ double-positive (DP) thymocytes as well as in CD8SP T-cells and IELs. We hypothesize that it is the L2a element that imparts to the E81 enhancer the ability to function in DP thymocytes. To test this hypothesis, transgenic studies using a human CD2 reporter gene and 4.3 kb HindIII/HmdIII fragments with various deletions will be used to localize the region that imparts DP thymocyte function upon the E81 enhancer. The effect of these cis-acting sequences on the activity of other CD8 enhancers will be evaluated using the same approach knock-out and knock-in studies using the Cre/loxP system will be performed to test the effect of deletion or modification of L2a element (or other cis-acting sequences) in situ upon CD8 expression in thymic subpopulations and peripheral T-cells. If the L2a element proves to harbor the cis-acting sequences as hypothesized, we will specifically modify sites within L2a shown to bind the MAR-binding proteins, SATBI and CDP/Cux, suggested by us to play positive and negative roles, respectively, in CD8a gene regulation. Complementary studies in which expression of SATB1 or CDP/Cux is compromised will be attempted to determine whether the effect observed is similar to that seen when each protein's binding site in L2a is abolished. Finally, chromatin cross-linking and immunoprecipitation studies will be performed to test whether SATB 1 and CDP/Cux bind to the L2a element with cell specificity consistent with the roles we have hypothesized for them in CD8 gene regulation. These studies will contribute to our knowledge of CD8 gene regulation during T-cell differentiation, and to the roles played by MARs and their cognate binding proteins in modulating the activities of cis-acting sequences in vivo.
