CBP/p300 is involved in multiple signaling pathways leading to the induction of TNF-α transcription. The −200 TNF-α Luc (A) or −105 IFN-β Luc (B) reporter plasmids were cotransfected with an expression vector for wild-type E1A 12S (E1A 12S) or mutant E1A 12S (E1AΔ2–36) protein or the expression vector alone (vector) in 68–41 T cells. Histograms of luciferase activity are shown, demonstrating inhibition of TNF-α transcription upon induction by anti-CD3 or virus (A) or inhibition of IFN-β transcription upon virus induction (B) by wild-type E1A 12S. The results are an average of three independent experiments, and the error bars represent SEM. Extracts were normalized to Renilla luciferase activity as described in Materials and Methods.

CBP/p300 is involved in induction of the CRE/κ3-NFAT element in response to multiple stimuli or by exogenous NFATp. The −39 TNF-α (CRE/κ3-NFAT)² Luc (A) or −40 IFN-β (CRE/κ3-NFAT)² CAT (B and C) reporter plasmids were cotransfected with an expression vector for wild-type E1A 12S (E1A 12S) or mutant E1A 12S (E1AΔ2–36) protein or the expression vector alone (vector) into 68–41 T cells (A) or HeLa cells (B and C). (A) Histograms of luciferase activity upon induction by anti-CD3 or virus (V) demonstrating inhibition of CRE/κ3-NFAT reporter activity by wild-type E1A 12S. (B and C) Histograms of CAT activity in response to increasing amounts of exogenous NFATp (30, 100, or 300 ng) in the presence of fixed amounts of wild-type or mutant E1A 12S (0.1 μg) (B) or in response to increasing amounts of wild-type or mutant E1A 12S (0.1, 0.3, or 1.0 μg) in the presence of fixed amounts of exogenous NFATp (100 ng) (C), demonstrating inhibition of NFATp-mediated CRE/κ3-NFAT reporter activity by wild-type E1A 12S. The results in A—C are an average of three independent experiments, and the error bars represent SEM. For luciferase assays, extracts were normalized to Renilla luciferase activity; for CAT assays, extracts were normalized to β-galactosidase activity.

Gene dosage-dependent requirement for CBP in TNF-α gene transcription in response to activation via the T cell receptor. An autoradiograph of RNase protection assays is shown. Levels of endogenous TNF-α (A) and IFN-β (B) mRNA induced by various stimuli from wild-type mice (+/+) or from heterozygous littermates (+/−) lacking one copy of the CBP allele were measured. The γ-actin gene was used as a control for RNA loading and processing. (A) In CBP heterozygous mice, induction of TNF-α mRNA upon stimulation by α-CD3 after 1 or 3 h is inhibited relative to levels observed in wild-type mice, whereas induction of TNF-α mRNA upon stimulation by virus remains unaffected. Results from two CBP^+/− and two wild-type CBP^+/+ littermates are shown. (B) Virus induction of IFN-β mRNA in CBP^+/− mice is not reduced relative to levels observed in wild-type littermates. In the experiment displayed, when total mRNA levels are normalized to γ-actin, the relative levels of virus-inducible IFN-β mRNA derived from the CBP^+/− mouse relative to the levels observed in the wild-type littermate are equivalent. The percentage of T and B cells in spleens from CBP^+/− and wild-type CBP^+/+ littermates was equivalent as determined by FACS analysis (data not shown). We note that the CBP^+/− mice used in the experiments displayed were in a 129/Sv background and are representative of three individual CBP^+/− and wild-type CBP^+/+ littermates. When the mice were backcrossed into a BL6 background, inhibition of anti-CD3 induction of TNF-α was attenuated (data not shown), suggesting a strain-specific modifier. It should be noted that such a strain-specific backcrossing effect previously has been observed with regard to heterozygote lethality in p300^+/− mice ().

Transcriptional activation of CBP in response to α-CD3 and virus stimulation of T cells. A histogram of the fold induction of luciferase activity of a minimal promoter containing five copies of the Gal4-binding site fused to luciferase (1 μg) is shown. Increasing amounts (130 ng, 400 ng, 1.3 μg, and 4 μg) of vector expressing the Gal4 DNA-binding domain fused to full-length CBP (Gal4-CBP) or 4 μg of vector expressing the Gal4 DNA-binding domain (Gal4-DBD) was transfected into 68–41 T cells and stimulated with α-CD3 or virus as indicated. Because Gal4-CBP expression depends on the simian virus 40 (SV40) promoter, and the SV40 promoter-driven expression of exogenous β-galactosidase is not potentiated by anti-CD3 or virus (data not shown), we conclude that the observed effects on Gal4-CBP transcriptional activity are posttranslational. The results are an average of three independent experiments, and the error bars represent SEM. Extracts were normalized to Renilla luciferase activity.