Identification of a tumor necrosis factor-responsive element in the tumor necrosis factor α gene

The regulation by tumor necrosis factor α (TNF) of its own promoter has been investigated by transient transfection and nuclear protein binding assays. In human K652 erythroleukemia cells TNF produced an 8-10-fold activation of the human TNF promoter linked to the chloramphenicol acetyltransferase gene. The TNF-responsive element was localized to the -125 to -82 region by examining the TNF activation in 5'deletion or site-directed mutants of the TNF promoter and by demonstrating that the -125 to -82 fragment confers TNF responsiveness to the thymidine kinase promoter. This region contains a palindrome, 5′ TGAGCTCA 3′, that resembles the consensus binding sequences for the transcription factors, activator protein-1 (AP-1), cyclic AMP-responsive element binding protein (CREB), and activation transcription factor (ATF). An internal deletion in the palindrome abolished the TNF responsiveness, whereas known AP-1 and CREB/ATF elements were unresponsive to TNF. In band shift analyses a nuclear factor from U937 cells specifically bound to the -125 to -82 TNF-responsive fragment in or near the palindromic sequence. Oligonucleotides containing AP-1 or CREB/ATF sites did not effectively compete for the binding, indicating that the U937 cell factor is different from these factors. Anti-c-fos antiserum did not affect binding of the U937 cell factor, whereas anti-c-jun antiserum did block its binding, indicating that either c-jun or a protein antigenically related to c-jun is a component of the factor. These results suggest that the TNF-responsive element is not activated by AP-1 or CREB in U937 cells and that a novel DNA binding factor is important for constitutive and inducible TNF gene expression.