An Androgen Response Element in a Far Upstream Enhancer Region Is Essential for High, Androgen-Regulated Activity of the Prostate-Specific Antigen Promoter

textabstractProstate-specific antigen (PSA) is expressed at a\udhigh level in the luminal epithelial cells of the prostate\udand is absent or expressed at very low levels in\udother tissues. PSA expression can be regulated by\udandrogens. Previously, two functional androgenresponse\udelements were identified in the proximal\udpromoter of the PSA gene. To detect additional,\udmore distal control elements, DNaseI-hypersensitive\udsites (DHSs) upstream of the PSA gene were\udmapped in chromatin from the prostate-derived\udcell line LNCaP grown in the presence and absence\udof the synthetic androgen R1881. In a region 4.8 to\ud3.8 kb upstream of the transcription start site of the\udPSA gene, a cluster of three DHSs was detected.\udThe middle DNAseI-hypersensitive site (DHSII, at\ud;24.2 kb) showed strong androgen responsiveness\udin LNCaP cells and was absent in chromatin\udfrom HeLa cells. Further analysis of the region encompassing\udDHSII provided evidence for the presence\udof a complex, androgen-responsive and cellspecific\udenhancer. In transient transfected LNCaP\udcells, PSA promoter constructs containing this upstream\udenhancer region showed approximately\ud3000-fold higher activity in the presence than in the\udabsence of R1881. The core region of the enhancer\udcould be mapped within a 440-bp fragment. The\udenhancer showed synergistic cooperation with the\udproximal PSA promoter and was found to be composed\udof at least three separate regulatory regions.\udIn the center, a functionally active, high-affinity androgen\udreceptor binding site (GGAACATATTGTATC)\udcould be identified. Mutation of this element\udalmost completely abolished PSA promoter activity.\udTransfection experiments in prostate and nonprostate\udcell lines showed largely LNCaP cell specificity\udof the upstream enhancer region, although\udsome activity was found in the T47D mammary\udtumor cell line