An E-box region within the prostaglandin endoperoxide synthase-2 (PGS-2) promoter is required for transcription in rat ovarian granulosa cells.
The prostaglandin endoperoxide synthase-2 (PGS-2) gene encodes an isoform of prostaglandin synthase that is transiently induced by protein kinase A (luteinizing hormone/cAMP) and protein kinase C (gonadotropin-releasing hormone) agonists in granulosa cells of ovulating follicles. The promoter of the rat PGS-2 gene contains a CAAT enhancer-binding protein consensus site (CAAT box) which can confer hormone inducibility to a PGS-2.CAT reporter gene, as well as a putative E-box region. To determine if the E-box region was involved in hormone induced trans-activation of the rat PGS-2 gene, constructs with the CAAT box and E-box regions (-192 PGS-2.CAT), only the putative E-box (-110 PGS-2.CAT), or neither region (-52 PGS-2.CAT) were transiently transfected into rat granulosa cell cultures. CAT activity was induced in both the -192 and -110 PGS-2*CAT vectors by luteinizing hormone (10-fold) and gonadotropin-releasing hormone (6-fold), whereas CAT activity of the -52 PGS-2.CAT construct did not differ from the promoterless vector (pCAT-Basic). Deletion of 1 base pair from the E-box within the -110 PGS-2.CAT construct, as well as point mutations within the CAAT box, E-box, or both regions of the -192 PGS-2.CAT construct, demonstrated that the E-box is critical for basal transcription, and that regions, in addition to the CAAT box, are involved in hormone induction of the PGS-2 gene. An oligonucleotide spanning the rat PGS-2 E-box bound two specific protein complexes which were supershifted in the presence of antibody specific for the upstream stimulatory factor. Thus, in rat granulosa cells, the PGS-2 E-box region appears to interact with upstream cis-acting elements other than the CAAT box to confer hormonal regulation of the gene. The E-box region of the rat PGS-2 promoter does not contain ATF/CRE activity found in the human and mouse PGS-2 promoters, but is critical for basal transcription of the PGS-2 gene in rat granulosa cells and binds the upstream stimulatory factor (as do E-box regions of other genes regulated in the ovary).
Morris
J K
JK
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030, USA.
Richards
J S
JS
D000818:Animals; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D004247:DNA; D004268:DNA-Binding Proteins / Q000235:genetics; D005260:Female; D006107:Granulosa Cells / Q000201:enzymology*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics; D011401:Promoter Regions, Genetic; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D012689:Sequence Homology, Nucleic Acid; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D051720:Upstream Stimulatory Factors
D022762:CCAAT-Enhancer-Binding Proteins; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D014157:Transcription Factors; D051720:Upstream Stimulatory Factors; D004247:DNA; D011451:Prostaglandin-Endoperoxide Synthases; D015500:Chloramphenicol O-Acetyltransferase