Skip to main content
. 1999 Sep 28;96(20):11566–11571. doi: 10.1073/pnas.96.20.11566

Figure 4.

Figure 4

E1A and EBNA2 transactivate MYC transcription through the PRF interferon-regulated element. (A) EBNA2 binds p300 in vivo. Western blot of EBNA2 and EBNA2ΔCBF coimmunoprecipitated with N-15 anti-p300 antibody from IRF1/2−/− MEF cells as in Fig. 2A. EBNA2 (E) and EBNA2ΔCBF (Δ) interact with full-length p300 (lanes 4 and 5) and truncation constructs (lanes 7 and 8 and 11 and 12) containing the region between amino acids 347 and 596. The shortest p300 deletion construct (lanes 14 and 15), ending at amino acid 347, does not coimmunoprecipitate EBNA2. No immunoprecipitation is present in lanes without EBNA2/EBNA2ΔCBF (−) overexpression (lanes 3, 6, 10, and 13). Lanes 1 and 2 are positive control lanes and lane 9 is a negative control immunoprecipitation with anti-FLAG antibody. (B) KSHV vIRF, EBV EBNA2, and adenovirus E1A transactivate the pBB-Luc reporter but not the pΔPRFBB-Luc reporter. A mutant E1A, E1AΔ2–36, that does not bind p300/CBP does not activate pBB-Luc. Human T lymphotrophic virus (HTLV) I Tax protein activates both pBB-Luc and pΔPRFBB-Luc through NF-κB sites. Viral protein expression plasmids (2 μg each) were cotransfected with reporter plasmids (2 μg) into IRF1/2−/− cells. (C) EBNA2 is cotransactivated by CBP and P/CAF but not by p300. Increasing amounts of each coadaptor were transfected together with EBNA2 expression plasmid and pBB-Luc reporter plasmid. At CBP and P/CAF amounts greater than 1 μg and 100 ng expression plasmid, respectively, transcriptional squelching is present. Coadaptor amounts were 1 ng, 5 ng, 10 ng, 50 ng, 100 ng, 1 μg, and 2 μg of expression plasmids. (D) E1A is cotransactivated by P/CAF but not by either CBP or p300. P/CAF squelching occurs at 1 μg of expression plasmid. Coadaptor amounts as in Fig. 3C. (E) Coadaptor competition at the MYC promoter. Increasing amounts of CBP (1 ng, 10 ng, 50 ng, 100 ng, and 1 μg) reverse p300 (10 ng) induced repression of vIRF (1 μg) and EBNA2 (2 μg) transactivation of MYC. (F) Increasing amounts of P/CAF (1 ng, 10 ng, 50 ng, 100 ng, and 1 μg) reverse p300 (1 ng) -induced repression of EBNA2 (2 μg) and E1A (1 μg) transactivation of MYC.