Figure 2.

Identification and functional analysis of AR‐binding motifs. A: Consensus motifs were extracted from the 5,571 ARBSs of the primary AR target genes and analyzed for frequent motifs. The top three motifs are shown. Of these, the 15‐bp motif 3 has a high homology to a consensus androgen‐responsive element (ARE), whereas motif 2 represents a novel 11 bp CG‐rich element and motif 1 a 15‐bp AT‐rich AR‐binding element. B: The ARBS in the 7^th intron of the MLPH gene harbors a motif 2 element followed by a motif 3 ARE element at a distance of 11 bp (see Supp. Fig. S4.). This ARBS with wild‐type major T allele of rs11891426:T>G in motif 2 was cloned into the enhancer site of the PGL3P firefly luciferase reporter vector as described in experimental procedures and the vector was designated “MLPH^T.” Its counterparts with mutations abolishing either motif 2 or 3 were constructed using site‐directed mutagenesis and referred to as “MLPH^TM2^mutated” and “MLPH^TM3^mutated.” The firefly luciferase reporter vectors together with the renilla luciferase transfection control vector PGL4.73 were transfected into DUCaP cells and stimulated by androgen. The androgen‐induced changes in luciferase activity were measured after treatment with 1 nM R1881 or vehicle for 48 hr using a dual luciferase assay. The firefly luciferase activity was normalized to the transfection control renilla luciferase activity and compared relative to the activity of the unstimulated MLPH^T vector. Reporter gene assay values represent mean+SD of at least three independent experiments. Statistical differences were calculated using the unpaired t‐test. *P < 0.05.