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 proteins (CRPs). Footprint analysis with the androgen receptor (AR) DNA-binding domain on the promoter-containing fragments reveals an AR-binding 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'-AGAAGAaaaTGTACA-3' and overlaps with the ATG translation start codon. A double-stranded oligonucleotide containing this sequence 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 start point and are located at an identical position (-142/ -120) in crp1 and crp2. The AR affinity for these two slightly different sequence motifs is relatively weak. The biological function of all three AR-binding sites as transcription control elements has been studied. The ARBSd/crp2 element clearly shows androgen-response element characteristics. The contribution of the common upstream element to the androgen-dependent control of reporter gene transcription is less clear. The transcription of a reporter gene construct containing the crp2 footprint fragment crp2F (-273/+88) is hormonally regulated as determined by transfection into the human breast cancer cell line T-47D. Androgens, but also glucocorticoids, efficiently stimulate steroid-dependent transcription of the chloramphenicol acetyltransferase gene. Mutation of the 5'-TGTACA-3' sequence in ARBSd/crp2 destroys the AR binding and abolishes the androgen-dependent synthesis of chloramphenicol acetyltransferase. A large fragment derived from intron 1 of the crp1 and crp2 gene can also provide the androgen-dependent transcription of chimeric constructs 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 androgen responsiveness.
Devos
A
A
Division of Biochemistry, Faculty of Medicine, University of Leuven, Belgium.
Claessens
F
F
Alen
P
P
Winderickx
J
J
Heyns
W
W
Rombauts
W
W
Peeters
B
B
D000728:Androgens / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D001943:Breast Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D015891:Cystatins; D018983:DNA Footprinting; D004252:DNA Mutational Analysis; D003850:Deoxyribonuclease I / Q000235:genetics / Q000378:metabolism; D004586:Electrophoresis / Q000379:methods; D005091:Exons; D017930:Genes, Reporter; D006801:Humans; D008297:Male; D011506:Proteins / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger / Q000187:drug effects; D051381:Rats; D017208:Rats, Wistar; D011944:Receptors, Androgen / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
C079958:Andpro protein, rat; D000728:Androgens; D015891:Cystatins; C484121:P22k15 protein, rat; D011506:Proteins; D012333:RNA, Messenger; D011944:Receptors, Androgen; D011994:Recombinant Proteins; D003850:Deoxyribonuclease I