Structure of the rat inhibin and activin betaA-subunit gene and regulation in an ovarian granulosa cell line.
We have isolated the rat inhibin and activin betaA-subunit gene, which is composed of three exons, and have characterized a 571-bp region upstream from the transcriptional start site that functions as a promoter in transient transfection studies in an ovarian granulosa cell line, GRMO2. Deletion analysis of the 571-bp promoter region has identified DNA sequences between -362 bp and -110 bp to be essential in mediating basal promoter activity and activation by forskolin (FSK) and/or 12-O-tetradecanoylphorbol-13-acetate (TPA). Within this region, a variant CRE (cAMP response element) has been identified at -120 bp. Point mutations in the variant CRE substantially reduce the ability of FSK and/or TPA to induce promoter activity in GRMO2 cells. A single nucleotide change in the variant CRE, which converts it to a consensus CRE, does not enhance promoter activity in response to FSK and/or TPA, but rather reduces promoter activity to the same extent as the other inactivating mutation in the variant CRE, suggesting that this element does not act as a classical CRE. Consistent with this, electrophoretic mobility shift assays performed using antibodies to a variety of cAMP and phorbol ester-responsive transcription factors indicate that the AP-1 family proteins jun-B and fos-B are present in the protein complex binding to the variant CRE. Overexpression of jun-B and fos-B in GRMO2 cells resulted in a robust activation of the betaA-subunit promoter. Our results suggest that this novel variant CRE sequence mediates both cAMP and phorbol ester regulation through its interactions with AP-1family proteins.
Ardekani
A M
AM
Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60208, USA.
Romanelli
J C
JC
Mayo
K E
KE
D028341:Activins; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000502:physiology; D005260:Female; D005786:Gene Expression Regulation / Q000502:physiology*; D014644:Genetic Variation / Q000235:genetics; D006107:Granulosa Cells / Q000502:physiology*; D007265:Inhibins / Q000235:genetics*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics / Q000502:physiology; D051381:Rats; D018808:Transcription Factor AP-1 / Q000378:metabolism
D009687:Nuclear Proteins; D018808:Transcription Factor AP-1; D028341:Activins; D007265:Inhibins; D000242:Cyclic AMP