FIG. 7.

Activity of a p300/CBP-dependent transcriptional activator is reduced in Mediator-depleted nuclear extracts. (A) Schematic representation of the mechanism of transcriptional activation by Gal4-SRC2(PID). Gal4-SRC2(PID) directly recruits endogenous p300/CBP in HNE to promoters assembled into chromatin via the PID of SRC2. (B) Analysis of transcriptional activation by Gal4-SRC2(PID) with control and Mediator-depleted HNEs. The 2Gal-pS2 (top panel) and 2Gal-E4 (bottom panel) plasmid templates containing two Gal4 UAS sites upstream of the pS2 and E4 promoters, respectively, were assembled into chromatin in the presence or absence of Gal4-SRC2(PID), as indicated. The chromatin samples were subjected to in vitro transcription analysis, and the resulting RNA products were analyzed by primer extension. The relative transcription for each promoter is indicated. (C) Summary of multiple experiments like those in panel B showing the effects of Mediator depletion on activated transcription by Gal4-SRC2(PID) with the human pS2 and adenovirus E4 promoters. The data are plotted as fold activation over basal to account for the modest effects that Mediator depletion has on basal transcription (Fig. 4). Each bar represents the mean + the standard error from three or more separate determinations.