Figure 7.
Enhancement of retinoid-dependent promoter activity by transfected PCAF. (A) NIH-3T3 (1.5 × 105) cells were cotransfected with the RARE–Tk–luciferase reporter and pcx–PCAF (+, 50 ng; ++, 200 ng; or +++, 400 ng), and luciferase activity was measured following 24 hr of 1 μm all trans-RA treatment. Shaded and solid bars represent luciferase activity with and without RA, respectively. Values represent the average of triplicate cultures ± s.d. (B) Transfection was performed as in A but with additional expression vectors for RXRβ and RARα (40 ng of each), and with pcx–PCAF (+, 50 ng; ++, 200 ng; and +++, 400 ng) or pcx–PCAF mutants (ΔN2 and ΔC, 400 ng). Values represent the average of four independent assays ±s.d. (C) Transfection was performed as in B, but with 400 ng each of control (pcx), PCAF, or HAT deletions (ΔHAT1 and ΔHAT2). Values represent the average of triplicates ±s.d. (D) Detection of transfected PCAF and endogenous p300. Nuclear extracts from 6 × 105 NIH-3T3 cells transfected with indicated amounts (ng) of pcx–PCAF or deletion PCAFs tested in B and C were immunoblotted with anti-M2-Flag antibody to detect PCAF or with anti-p300 antibody. Asterisks indicate the position of the wild-type or mutant PCAFs. (E) Pool of NIH-3T3 cells stably transfected with the RARE–Tk–luciferase reporter was transiently transfected with pcx–PCAF or pcx–ΔC plus an IL-2R vector, and treated with 1 μm all trans-RA. Cells were sorted by anti-IL-2R antibody panning and luciferase activity was measured as in A. Values represent the average of triplicates ± s.d.