cis-acting elements of the rat aromatase promoter required for cyclic adenosine 3',5'-monophosphate induction in ovarian granulosa cells and constitutive expression in R2C Leydig cells.
The cytochrome P450 aromatase gene is transcriptionally regulated by FSH and steroids in granulosa cells of developing ovarian follicles. To characterize the molecular mechanisms by which this regulation occurs, the promoter of the rat aromatase gene has been analyzed by 1) mapping the transcriptional start site, 2) constructing deletion mutant reporter genes for transfection assays, and 3) determining DNA-protein interactions by gel shift assays. Transient transfection assays indicated that promoter sequences between -176 and -31 basepairs (bp) were required for cAMP inducibility of reporter constructs in primary cultures of granulosa cells and for expression in rat R2C Leydig cells, which constitutively express high amounts of aromatase mRNA. Nuclear extract proteins from granulosa cells and R2C Leydig cells specifically bound a labeled -176/13-bp fragment and were competed by cold competitor fragment as well as by a shorter region (-90/-66 bp) containing an AGGTCA hexameric motif. Competition was inhibited by mutation of the central GGs and was affected by adjacent 5' contextual sequences. A possible candidate for the binding activity observed in granulosa cells and R2C cell nuclear extracts binds an oligonucleotide containing the aromatase AGGTCA motif and is an orphan member of the steroid/thyroid hormone superfamily. These results are the first to characterize cis-acting DNA elements in the aromatase promoter, identify a region that confers cAMP inducibility in granulosa cells and constitutive expression in R2C cells, and localize a hexameric sequence within this region that binds at least one member of the orphan receptor class of transcription factors.
Fitzpatrick
S L
SL
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
Richards
J S
JS
D000818:Animals; D001141:Aromatase / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D001667:Binding, Competitive; D002460:Cell Line; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction; D005260:Female; D005822:Genetic Vectors; D006107:Granulosa Cells / Q000201:enzymology*; D007985:Leydig Cells / Q000201:enzymology*; D008297:Male; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000502:physiology*; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D014158:Transcription, Genetic; D014162:Transfection
D009687:Nuclear Proteins; D012333:RNA, Messenger; D000242:Cyclic AMP; D001141:Aromatase