Identification of the deoxyribonucleic acid-binding site of a regulatory protein involved in prostate-specific and androgen receptor-dependent gene expression.
In this study, we used a 5'-flanking region (-426/+28) of the rat prostatic probasin (rPB) gene shown to be sufficient to direct prostate-specific expression in transgenic mice in identifying the exact DNA-binding site of a putative prostate-specific transcription factor. Chloramphenicol acetyl transferase (CAT)-reporter gene analyses revealed that the construct pCAT PB -244/+52 was equally well induced by androgens in both prostatic LNCaP and nonprostatic COS-1, MCF-7, HEC-1, and HEP-1 cell lines, indicating that although the probasin gene region -244/+52 was important for androgen regulation, it was not regulated in a prostate-specific manner. Further studies suggested that the region -278/-240 was most crucial for prostate-specific expression. The sequence -426/-279 could be considered a silencer area, especially in nonprostatic cells. In deoxyribonuclease I footprinting, a protected 12-bp region was found between the nucleotides -251 and -240 only with nuclear extracts of prostatic origin. Deletion of this area decreased androgen induction significantly (P < 0.05) in transient transfections of prostatic cells compared with the wild-type reporter construct. Glucocorticoids were incapable of increasing the induction of the pCAT PB -278/+52 reporter construct compared with that of pCAT PB -244/+52 in the prostatic cell line LNCaP, suggesting that the putative prostate-specific protein acts as an inducer only when androgen and androgen receptor are present.
Patrikainen
L
L
Biocenter Oulu and World Health Organization Collaborating Centre for Research on Reproductive Health, University of Oulu, Finland.
Shan
J
J
Porvari
K
K
Vihko
P
P
D000727:Androgen-Binding Protein / Q000235:genetics*; D000818:Animals; D001665:Binding Sites; D001943:Breast Neoplasms; D019556:COS Cells; D004247:DNA / Q000378:metabolism*; D016889:Endometrial Neoplasms; D005260:Female; D015870:Gene Expression; D006801:Humans; D008113:Liver Neoplasms; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D016297:Mutagenesis, Site-Directed; D011467:Prostate / Q000378:metabolism*; D011471:Prostatic Neoplasms; D051381:Rats; D011944:Receptors, Androgen / Q000502:physiology*; D014407:Tumor Cells, Cultured
D000727:Androgen-Binding Protein; D011944:Receptors, Androgen; C027519:probasin; D004247:DNA