Garefalaki, Anna; (2002) Identification of regulatory regions that determine expression of the murine CD8 locus. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The coreceptors CD4 and CD8 play a crucial role during thymocyte development and T cell effector function and their expression is developmentally regulated. To determine the molecular mechanisms underlying the regulation of CD8 gene expression, the murine CDS gene locus was cloned and analysed for deoxyribonuclease (DNaseI) hypersensitivity. Such analysis revealed three thymocyte specific DNaseI hypersensitive regions (cluster II, III and IV). To characterise the role of different clusters of hypersensitive sites in CD8 gene regulation in the context of the endogenous chromosomal location, we deleted selected regions from the mouse genome by homologous recombination. Deletion of cluster III (either all three sites or just sites 1 and 2), which is located in the intergenic region between the CD8α and β genes and directs expression of a reporter transgene in mature CD8 T cells only, affected expression of CD8αα homodimers on intraepithelial (IEL) T cells, both on γδTCR and αβTCR subsets. Surprisingly, none of the thymocyte or peripheral αβTCR CD8αβ T cell subsets were affected by this mutation, which indicated differential activation of these elements within the various T cell subsets. Deletion of cluster II, which is located immediately upstream of the CD8α gene, had two main effects: it affected the levels of expression of the CD8 gene and caused an abnormal subset distribution in the thymus with fewer DP and CDS SP cells and an increase in cells with a CD4 SP phenotype. Staining with several maturation markers, showed that the increased numbers of thymocytes falling within the CD4 SP gate were immature cells. It is concluded that removal of regulatory sequences present in cluster II disturbs the normal developmental chromatin remodelling of the CD8 locus and as a consequence the expression of the CD8α gene.

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