Activation of the TSG-6 gene by NF-IL6 requires two adjacent NF-IL6 binding sites

Tumor necrosis factor (TNF)-stimulated gene 6 (TSG-6) encodes a protein expressed during inflammation. We have previously shown that transcription factors of the NF-IL6 and AP-1 families cooperatively modulate activation of the TSG-6 gene by TNF or interleukin 1 (IL-1) through a promoter region that contains an NF-IL6 site (-106 to -114) and an AP-1 element (-126 to -119). In this study we report the identification of 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 the two sites cooperate in binding NF-IL6. TNF and IL-1 stimulate specific binding of nuclear proteins to the NF-IL6* site more efficiently than 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 despite the presence of the intact promoter-distal NF-IL6 site. A mutation in the promoter-distal NF-IL6 site also greatly decreased activation of the TSG-6 promoter by NF-IL6. We conclude that the two NF-IL6 sites are functionally interdependent in the activation of the TSG-6 gene.