IDENTIFICATION OF A FUNCTIONAL ANDROGEN-RESPONSE ELEMENT IN THE EXON 1-CODING SEQUENCE OF THE CYSTATIN-RELATED PROTEIN GENE CRP2

Two hormone-responsive segments, one in the region of the promoter and one in intron 1, are identified in two homologous androgen-regulated and differentially expressed rat genes encoding the cystatin-related p roteins (CRPs). Footprint analysis with the androgen receptor (AR) DNA -binding domain on the promoter-containing fragments reveals an AR-bin ding site downstream of the transcription start point in the crp2 gene (ARBSd/crp2, +40/+63). It displays an androgen response element-like sequence motif 5'-AGAAGAaaaGTACA-3' and overlaps with the ATG translat ion start codon. A double-stranded oligonucleotide containing this seq uence forms a DNA-protein complex with the full-length AR synthesized by vaccinia, as seen in band shift assays. Additional AR-binding sites , ARBSu/crp1 and ARBSu/crp2, occur 5' upstream of the transcription st art point and are located at an identical position (-142/-120) in crp1 and crp2. The AR affinity for these two slightly different sequence m otifs is relatively weak. The biological function of all three AR-bind ing sites as transcription control elements has been studied. The ARBS d/crp2 element clearly shows androgen-response element characteristics . The contribution of the common upstream element to the androgen-depe ndent control of. reporter gene transcription is less clear. The trans cription of a reporter gene construct containing the crp2 footprint fr agment crp2F (-273/+88) is hormonally regulated as determined by trans fection into the human breast cancer cell line T-47D. Androgens, but a lso glucocorticoids, efficiently stimulate steroid-dependent transcrip tion of the chloramphenicol acetyltransferase gene. Mutation of the 5' -TGTACA-3' sequence in ARBSd/crp2 destroys the AR binding and abolishe s the androgen-dependent synthesis of chloramphenicol acetyltransferas e. A large fragment derived from intron 1 of the crp1 and crp2 gene ca n also provide the androgen-dependent transcription of chimeric constr ucts in T-47D cells. However, the induction measured is less than the one observed with crp2F (-273/+88), and this activity seems to reside in several subfragments that each display a low but consistent androge n responsiveness.