Expression of the gene for cytochrome P-450 17 alpha-hydroxylase/C17-20 lyase (CYP17) in porcine Leydig cells: identification of a DNA sequence that mediates cAMP response.
Regulation of CYP17 gene expression in porcine Leydig cells was investigated in primary culture. We previously reported the sequence of the 5' upstream and much of the pig gene. (Zhang et al. (1992) Biochim. Biophys. Acta. 1131, 345-348). DNase I footprinting assays identified a region between -193 and -174 that was bound by nuclear proteins. Examination of the DNA sequence in this region revealed putative Sp1 and AP-2 binding sites, but gel retardation assays using an oligonucleotide from -198 to -168 as a probe revealed two specific DNA-protein complexes that were not Sp1 or AP-2. The oligonucleotide was cloned into a reporter gene containing a minimal porcine CYP17 promoter and the resultant construct was transiently transfected into porcine Leydig cells. This chimeric construct had both basal and cAMP-induced transcriptional activities. Southwestern blot identified a prominent binding of a nuclear protein around 68 kDa and a weaker binding of a nuclear protein around 110 kDa. Sequences between -250/+1 are highly homologous to those sequences from human, bovine and rodent CYP17 gene, but the -193/-174 region has no homology to those genes. Other regions of the porcine CYP17 were also important for the basal and cAMP-mediated regulation. Luciferase expression vectors were prepared with 5' flanking DNA from the porcine CYP17 gene and were expressed in primary culture of porcine Leydig cells. The region between -587/-325 was important for basal transcription, and a region of DNA between -325 and -140 was important for cAMP regulation.
Zhang
P
P
Department of Endocrinology, Prince of Wales Hospital, Randwick, NSW, Australia.
Han
X G
XG
Mellon
S H
SH
Hall
P F
PF
D000311:Adrenal Glands / Q000166:cytology; D000446:Aldehyde-Lyases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000502:physiology; D003850:Deoxyribonuclease I / Q000378:metabolism; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D007985:Leydig Cells / Q000201:enzymology*; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D008297:Male; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000235:genetics; D051381:Rats; D012689:Sequence Homology, Nucleic Acid; D013254:Steroid 17-alpha-Hydroxylase; D013552:Swine; D014158:Transcription, Genetic; D014162:Transfection
D004268:DNA-Binding Proteins; D009841:Oligonucleotides; D003577:Cytochrome P-450 Enzyme System; D000242:Cyclic AMP; D008156:Luciferases; D013254:Steroid 17-alpha-Hydroxylase; D003850:Deoxyribonuclease I; D000446:Aldehyde-Lyases