Abstract
Within the human TNF promoter we have identi?ed two sites at positions −189 and −101 that show C/EBP specific binding of nuclear proteins from cells of the human monocytic line Mono Mac 6. Supershift analysis with anti C/EBP antibodies revealed that the complexes formed consist of both C/EBPα and C/EBPβ. When studying reporter constructs with a 5′-deletion series of the TNF promoter in cotransfection experiments with a C/EBPβ expression plasmid, a construct with the −1064 TNF fragment gave 26-fold transactivation, the −630 fragment showed 23-fold transactivation and the −107 fragment (containing the −101 C/EBP binding motif) still gave 16-fold transactivation. Mutagenesis of the −101 site in the −630 construct resulted in a reduction of C/EBP driven transactivation from 26-fold to 7-fold. Finally, when Mono Mac 6 cells were transfected with these constructs, stimulation by LPS induced a 19-fold transactivation in the −630 wild type construct, while the −630 construct carrying the −101 mutation was transactivated only 4-fold. Hence, the data indicate that the −101 C/EBP motif is crucial for TNF gene expression in human monocytes.
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