Characterization and hormonal regulation of the promoter of the rat prostaglandin endoperoxide synthase 2 gene in granulosa cells. Identification of functional and protein-binding regions.
Prostaglandin endoperoxide synthase isoform 2 (PGS-2) mRNA and protein are transiently induced by gonadotropins in granulosa cells of preovulatory follicles prior to ovulation. To better understand the hormonal regulation of the rat PGS-2 (rPGS-2) gene in these cells, genomic clones containing rPGS-2 as well as up to 6 kilobases of 5'-flanking DNA were isolated by screening a rat liver genomic library with a labeled 5'-fragment of the mouse PGS-2 cDNA. Primer extension analysis using ovarian follicular mRNA identified the presence of a single rPGS-2 transcription initiation site located 144 nucleotides upstream of the ATG translation initiation codon. To test for promoter activity within the 5'-flanking region of the rPGS-2 gene, a genomic fragment, -2698/32 (1 = cap site), as well as a series of 5'-deletion mutants, were fused upstream of the chloramphenicol acetyltransferase (CAT) reporter gene and transfected into primary cultures of granulosa cells. Forskolin (7.5 microM), follicle-stimulating hormone (500 ng/ml) and luteinizing hormone (500 ng/ml) induced CAT activity following transfection with the -2698/32PGS.CAT, whereas gonadotropin-releasing hormone (10(-6) M) and interleukin-1 beta (30 ng/ml) had no effect. Deletion mutants delineated the region spanning from -192 to -54 of the transcription start site to be essential for both basal and forskolin-regulated expression of the reporter gene. The same DNA fragment (-192/-54) exhibited specific binding to granulosa cell nuclear extract proteins as analyzed by electrophoretic mobility shift assays. Additional specific bands were observed in extracts prepared from granulosa cells exposed to an ovulatory dose of gonadotropin. Collectively, these results provide the first structural and functional evidence that the transcriptional regulation of the rat PGS-2 gene by gonadotropins and forskolin in granulosa cells involves 5'-flanking DNA sequences, specifically a region between -192 and -54 of the transcription initiation site.
Sirois
J
J
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
Levy
L O
LO
Simmons
D L
DL
Richards
J S
JS
D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D006063:Chorionic Gonadotropin / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism; D004958:Estradiol / Q000494:pharmacology*; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology; D006107:Granulosa Cells / Q000187:drug effects / Q000201:enzymology*; D007016:Hypophysectomy; D007527:Isoenzymes / Q000235:genetics*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism; D016376:Oligonucleotides, Antisense; D006080:Ovarian Follicle / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D015183:Restriction Mapping; D017384:Sequence Deletion; D014158:Transcription, Genetic
D006063:Chorionic Gonadotropin; D004268:DNA-Binding Proteins; D007527:Isoenzymes; D009687:Nuclear Proteins; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D005576:Colforsin; D004958:Estradiol; D005640:Follicle Stimulating Hormone; D004247:DNA; D011451:Prostaglandin-Endoperoxide Synthases; D015500:Chloramphenicol O-Acetyltransferase