Figure 5.

 The transcriptional coactivator p300 functions synergistically with ER to enhance ligand-dependent transcription with chromatin templates in vitro. (A) Synthesis and purification of human p300. His₆-tagged, human p300 was synthesized in Sf9 cells by using a baculovirus expression vector and purified by nickel chelate chromatography. The protein was subjected to 8% polyacrylamide–SDS gel electrophoresis and staining with Coomassie Blue R-250. (B) Purified, exogenous p300 stimulates ligand-dependent transcription by ER. pERE was assembled into chromatin with ER and E₂, as noted, and the samples were subjected to in vitro transcription analysis in the presence or the absence of p300. The final concentrations of ER and E₂ in the transcription reactions were 4.5 and 30 nm, whereas p300 varied from 0 to 30 nm. (C) Transcriptional enhancement by p300 requires a chromatin template. Chromatin assembly–in vitro transcription reactions were performed as in B. (Right, non-chromatin) pUC118 competitor DNA was added to the assembly reactions prior to pERE, as in Fig. 2D. The final concentrations of ER, E₂, and p300 in the transcription reactions were 4.5, 30, and 15 nm. (D) Anti-estrogens inhibit transcriptional synergism between ER and p300. Chromatin assembly–in vitro transcription reactions were performed as in B, with the indicated components. The final concentrations of factors and ligands in the transcription reactions were ER (4.5 nm), p300 (15 nm), E₂ (15 nm), and anti-estrogens (3 μm).