Characterization of protein complexes formed on the repressor elements of the human tumor necrosis factor alpha gene

Human tumor necrosis factor alpha (TNF-alpha) is an important cytokine responsible for pleiotropic effects in vivo. The expression of TNF-alpha is under both positive and negative regulation. Previously we showed that a 108 bp region (-280 to -172) in the TNF-alpha promoter represses TNF-alpha transcription in U937 cells. We also demonstrated that a smaller region of the promoter spanning base pairs -254 and -230 is sufficient for repressor function. This 25 bp TNF-alpha repressor site (TRS) contains a 10 bp sequence homologous to the binding site of activator protein AP-2, yet it does not bind the AP-2 protein. In this study, we demonstrate that this 10 bp core sequence is an essential element for the repressor function of the TRS. Using gel retardation analysis with the 108 bp repressor element and the TRS as probes, multiple specific DNA binding complexes have been identified from U937 nuclear extracts. The complexes B, C, and D on the 108 bp probe and the three major complexes on the 25 bp TRS probe are also present in Jurkat and Mono Mac 6 cells, and their abundance in these cell lines seems to correlate with their postulated repressor function. We have demonstrated that the major TRS binding proteins, with estimated MWs of 30-60 kD, copurify on a heparin agarose column and on a DNA affinity column conjugated with the 10 bp core sequence.