FIG. 6.

TNF-α gene activation requires inducer-specific precise helical phasing between activator binding sites. 68-41 T cells were transfected with the wild-type −200 TNF-α promoter-Luc reporter or with the indicated mutant reporter constructs containing insertions between activator binding sites that disrupt and restore helical phasing as indicated and with the Renilla luciferase (Luc.) control plasmid. After the cells were stimulated with ionomycin or Sendai virus, luciferase activity was measured and divided by Renilla luciferase activity to normalize transfection efficiency and fold induction was calculated. The histograms show the results of four independent experiments. Error bars represent the standard errors of the mean. Note that we observed a strong correlation between the effects of the insertion mutants on basal and induced levels of transcription (data not shown), consistent with the fact that both the basal transcription complex and enhanceosome contribute to the induction of the TNF-α gene.