CCAAT/enhancer binding protein is involved in the expression of the tumour necrosis factor gene in human monocytes.
Within the human TNF promoter we have identified 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/EBP alpha and C/EBP beta. When studying reporter constructs with a 5'-deletion series of the TNF promoter in cotransfection experiments with a C/EBP beta 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.
Wedel
A
A
Institute for Immunology, University of Munich, Germany.
Sulski
G
G
Ziegler-Heitbrock
H W
HW
D022762:CCAAT-Enhancer-Binding Proteins; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D009000:Monocytes / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000235:genetics*
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D014409:Tumor Necrosis Factor-alpha