FIG. 9.

CBP and p300 proteins mediate LPS induction of TNF-α. (A) Inhibition of CBP and p300 impairs TNF-α transcription induced by LPS. J774 cells were cotransfected with 2 μg of −200 TNF-α luciferase reporter and 2 μg of the vectors expressing wild-type or mutant (Δ2–36) forms of E1A 12S. Wild-type E1A represses CBP and p300 activity, while the mutant form does not. Histograms of uninduced (UN) or LPS-induced cells are shown, representing at least three independent experiments. Cotransfection of an empty vector with luciferase reporter yielded results essentially identical to those obtained with E1A(Δ2–36) (data not shown). Transfection efficiency was normalized as described in the legend to Fig. 2, and error bars represent the standard errors of the means. (B) The CRE/κ3/Ets sequence functions as a CBP- and p300-dependent element. J774 cells were cotransfected with (CRE/κ3)² −39 TNF-α luciferase reporter and vectors expressing wild-type or mutant E1A 12S and analyzed as described above. Histograms of uninduced and LPS-induced cells are shown, representing at least three independent experiments; error bars represent the standard errors of the means. Cotransfection of empty vector with luciferase reporter yielded results essentially identical to those obtained with E1A(Δ2–36) (data not shown). (C) LPS potentiates transcriptional activity of CBP and p300. J774 cells were cotransfected with a Gal4-dependent luciferase reporter (2 μg) and vectors expressing full-length CBP or p300 fused to the Gal4 DNA-binding domain (0.2, 0.7, or 2 μg) or the Gal4 DNA-binding domain alone (2 μg). The fold induction of LPS-induced activity relative to uninduced activity is shown. The total amount of DNA was kept constant with empty vector. Assays were quantified as described above. (D) Model of an LPS-specific TNF-α enhancer complex. A diagram of TNF-α promoter elements and the cognate transcription factors recruited upon LPS stimulation is shown. These factors are known to interact, constitutively or inducibly, with CBP and p300 proteins, which are required for LPS induction of TNF-α gene expression.