An Androgen Response Element in a Far Upstream Enhancer Region Is Essential for High, Androgen-Regulated Activity of the Prostate-Specific Antigen Promoter
Prostate-specific antigen (PSA) is expressed at a\ud high level in the luminal epithelial cells of the prostate\ud and is absent or expressed at very low levels in\ud other tissues. PSA expression can be regulated by\ud androgens. Previously, two functional androgenresponse\ud elements were identified in the proximal\ud promoter of the PSA gene. To detect additional,\ud more distal control elements, DNaseI-hypersensitive\ud sites (DHSs) upstream of the PSA gene were\ud mapped in chromatin from the prostate-derived\ud cell line LNCaP grown in the presence and absence\ud of the synthetic androgen R1881. In a region 4.8 to\ud 3.8 kb upstream of the transcription start site of the\ud PSA gene, a cluster of three DHSs was detected.\ud The middle DNAseI-hypersensitive site (DHSII, at\ud ;24.2 kb) showed strong androgen responsiveness\ud in LNCaP cells and was absent in chromatin\ud from HeLa cells. Further analysis of the region encompassing\ud DHSII provided evidence for the presence\ud of a complex, androgen-responsive and cellspecific\ud enhancer. In transient transfected LNCaP\ud cells, PSA promoter constructs containing this upstream\ud enhancer region showed approximately\ud 3000-fold higher activity in the presence than in the\ud absence of R1881. The core region of the enhancer\ud could be mapped within a 440-bp fragment. The\ud enhancer showed synergistic cooperation with the\ud proximal PSA promoter and was found to be composed\ud of at least three separate regulatory regions.\ud In the center, a functionally active, high-affinity androgen\ud receptor binding site (GGAACATATTGTATC)\ud could be identified. Mutation of this element\ud almost completely abolished PSA promoter activity.\ud Transfection experiments in prostate and nonprostate\ud cell lines showed largely LNCaP cell specificity\ud of the upstream enhancer region, although\ud some activity was found in the T47D mammary\ud tumor cell line