ACTIVATION OF THE TSG-6 GENE BY NF-IL6 REQUIRES 2 ADJACENT NF-IL6 BINDING-SITES

Tumor necrosis factor (TNF)-stimulated gene 6 (TSG-6) encodes a protei n expressed during inflammation. We have previously shown that transcr iption factors of the NF-IL6 and AP-1 families cooperatively modulate activation of the TSG-6 gene by TNF or interleukin I (IL-1) through a promoter region that contains an NF-IL6 site (-106 to -114) and an AP- I element (-126 to -119). In this study we report the identification o f an additional NF-IL6 site (NF-IL6) located at positions -92 to -83. Footprinting and electrophoretic mobility shift assay suggested that NF-IL6 binds with higher affinity to the newly identified NF-IL6 site than to the earlier identified promoter-distal NF-IL6 site and that t he two sites cooperate in binding NF-IL6. TNF and IL-I stimulate speci fic binding of nuclear proteins to the NF-IL6 site more efficiently t han to the promoter-distal NF-IL6 site. Moreover, a mutation in the NF -IL6 site abolished transactivation of the TSG-6 promoter by NF-IL6 d espite the presence of the intact promoter-distal NF-IL6 site. A mutat ion in the promoter distal NF-IL6 site also greatly decreased activati on of the TSG-6 promoter by NF-IL6. We conclude that the two NF-IL6 si tes are functionally interdependent in the activation of the TSG-6 gen e.