PMA-responsive 5' flanking sequences of the human TNF gene

In order to define functional domains involved in the control of TNF-gene transcription, 5'-flanking sequences of the TNF-gene were analysed by TNF-promoter deletion mutants linked to the CAT-gene and by gel retardation assays. To this, three TNF-promoter fragments of different length were fused to the CAT reporter gene and transiently transfected into several human cell lines. We found that a 315 bp long fragment encompassing positions -285 bp to +30 bp with respect to the TNF mRNA cap site is sufficient to function as a PMA inducible promoter/enhancer in all cell lines tested. By further deletion analysis of this clone we could narrow the PMA-inducible element within a 185 bp long sequence (-285 to -110 bp). In addition, we investigated specific interactions between nuclear proteins and TNF promoter sequences using gel-retardation assays. Besides several constitutive binding activities, we could demonstrate in several cell lines a nuclear protein that is induced by PMA and binds to the fragment of the TNF promoter containing the PMA-inducible element.