Novel Egr/NF-AT composite sites mediate activation of the CD95 (APO-1/Fas)ligand promoter in response to T cell stimulation

Expression of the CD95 (APO-1/Fas) ligand (CD95L) in activated T cells is a major cause of activation-induced T cell apoptosis. The transcription fact ors NF-AT and Egr-3 (a member of the immediate-early transcription factors involved in cellular growth and differentiation) have been implicated in ac tivation of the CD95L promoter upon T cell activation. On the basis of DNas e I footprinting, electrophore tic mobility shift assay, antibody supershif t analysis and transfection studies, we have identified two novel Egr-bindi ng sites 5' upstream of the previously identified Egr site. Mutation analys is of each Egr site shows that all three sites are important for full CD95L promoter activity. Strikingly, all Egr sites, including the previously ide ntified Egr site, are adjacent to or overlap with DNA sequences homologous to NF-AT binding sites and confer T cell activation-induced, cyclosporin A- sensitive transcriptional activity. Antibody supershift analysis revealed t hat NF-AT and Egr proteins are the components of inducible DNA-binding comp lexes formed on the two novel Egr sites. Cotransfection experiments showed that Egr-1, Egr-3 and NF-AT display a cooperative and synergistic activatio n of transcription mediated by these three Egr/NF-AT composite regulatory e lements. These findings provide further insight into the mechanisms involve d in the regulation of the CD95L expression in response to T cell activatio n.