Activation-Dependent Transcriptional Regulation of the Human fas Promoter Requires NF-κB p50-p65 Recruitment

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Abstract
Fas (CD95) and Fas ligand (CD95L) are an interacting receptor-ligand pair required for immune homeostasis. Lymphocyte activation results in the upregulation of Fas expression and the acquisition of sensitivity to FasL-mediated apoptosis. Although Fas upregulation is central to the preservation of immunologic tolerance, little is known about the molecular machinery underlying this process. To investigate the events involved in activation-induced Fas upregulation, we have examined mRNA accumulation, faspromoter activity, and protein expression in the Jurkat T-cell line treated with phorbol myristate acetate and ionomycin (P/I), pharmacological mimics of T-cell receptor activation. Although resting Jurkat cells express Fas, Fas mRNA was induced approximately 10-fold in 2 h upon P/I stimulation. Using sequential deletion mutants of the human fas promoter in transient transfection assays, we identified a 47-bp sequence (positions −306 to −260 relative to the ATG) required for activation-driven fas upregulation. Sequence analysis revealed the presence of a previously unrecognized composite binding site for both the Sp1 and NF-κB transcription factors at positions −295 to −286. Electrophoretic mobility shift assay (EMSA) and supershift analyses of this region documented constitutive binding of Sp1 in unactivated nuclear extracts and inducible binding of p50-p65 NF-κB heterodimers after P/I activation. Sp1 and NF-κB transcription factor binding was shown to be mutually exclusive by EMSA displacement studies with purified recombinant Sp1 and recombinant p50. The functional contribution of the κB-Sp1 composite site in P/I-inducible fas promoter activation was verified by using κB-Sp1 concatamers (−295 to −286) in a thymidine kinase promoter-driven reporter construct and native promoter constructs in Jurkat cells overexpressing IκB-α. Site-directed mutagenesis of the critical guanine nucleotides in the κB-Sp1 element documented the essential role of this site in activation-dependentfas promoter induction.