Figure 1.

AR and p160-CBP/p300 coactivators are preferentially recruited to the PSA enhancer to mediate androgen activation of PSA promoter in LNCaP cells. (A) DHT induces the recruitment of AR and p160-CBP/p300 coactivators preferentially at the PSA enhancer. A diagram of the 6-kb upstream regulatory region of AR target gene PSA with androgen-responsive enhancer and elements indicated above and the various fragments amplified in the ChIP assay marked below as A to P. The amplification efficiency of the primer pairs for the different fragments was assessed by PCR under the same cycling condition by using two different volumes (1× and 4×) of diluted LNCaP cell genomic DNA prepared during the ChIP procedures. Indicated antibodies were used to perform ChIP assay with LNCaP cells treated with 10 nM DHT for 30 min or 4 h, or mock-treated (C) for 4 h, to measure chromatin occupancy of AR, ACTR, TIF2, and CBP along the 6-kb sequence of PSA. Input chromatin DNA taken before immunoprecipitation from different samples was analyzed by PCR and found to be identical (data not shown). (B) LNCaP cell transfection was performed to analyze the ability of PSA enhancer and/or promoter to mediate androgen transcriptional induction and coactivation function. LNCaP cells in hormone-depleted media were cotransfected with either the 630-bp PSA proximal promoter-Luc reporter (a) or the 5.85-kb entire PSA upstream regulatory sequence-Luc (b) and the ACTR or TIF2 expression plasmids. Transfected cells were then treated with 10 nM DHT (filled bars) or mock-treated (open bars) for 24 h before measuring luciferase activities.