FIGURE 4.

trans-Regulation of the CD8 cluster. A, to investigate trans- and cis-regulatory elements across the human and murine CD8 locus, we defined regions of interest based on bioinformatic approaches. We aligned the mouse and human CD8 genes (VISTA Genome Browser) and searched for CNS sites. CNS sites were defined as regions of >200 bp with >70% homology between human and mouse. Eight regions of interest (CNS1–CNS8) were defined based on the degree of sequence conservation and the presence of reported regulatory regions. C II–C IV are previously reported DNase hypersensitivity clusters with regulatory capacities; E8_I–E8_IV are previously defined enhancer elements (6). B, reporter constructs of CNS regions within the human CD8 cluster were generated and transfected into Jurkat T cells. CNS2 is syntenic to the murine CD8b promoter, and CNS7 and CNS8 are within previously reported regulatory elements. All constructs exhibited enhanced activity over an empty pGL3 plasmid (empty vector (EV)). C, CREMα reduced the activity of CNS2, whereas CREB enhanced promoter activity. Both CREMα and CREB did not show significant effects on either CNS7 or CNS8.