Two proximal activating protein-1-binding sites are sufficient to stimulate transcription of the ovine follicle-stimulating hormone-beta gene.
FSH is an important regulator of mammalian gametogenesis and the female reproductive cycle. Although little is known about the transcriptional regulation of the beta-subunit (the rate-limiting subunit of FSH synthesis), sequence analysis of the ovine FSHbeta promoter has revealed a number of potential activating protein-1 (AP-1; Jun/Fos)-binding sites. To determine whether the gene encoding the beta-subunit of ovine FSH (oFSHbeta) is responsive to AP-1 transcriptional complexes, chimeric constructs containing deleted portions of the oFSHbeta promoter fused to a luciferase reporter were transiently transfected along with c-Jun and c-Fos expression constructs into JAR cells. Analysis of these deletion constructs revealed that the proximal promoter of oFSHbeta is highly stimulated by c-Jun and c-Fos proteins (typically 20-fold with a reporter construct containing oFSHbeta sequences from -215 to +759 bp). This stimulation was lost when a similar construct containing sequences from -84 to +759 bp was tested. Transcriptional start site analysis using reverse transcription-PCR verified that the transcriptional initiation of the -215-bp deletion construct, with or without cotransfected c-Jun/c-Fos, was the same as that observed in vivo. Computer analysis of oFSHbeta sequences from -215 to +1 bp identified four putative AP-1-like elements, located at -155, -120, -83, and -10 bp. Gel retardation experiments using oligonucleotides corresponding to the four putative AP-1-like sites revealed that only -120 and -83 sites in oFSHbeta bound AP-1 proteins in vitro. Site-directed mutagenesis of the -120 and -83 sites showed that each element was required for stimulation by c-Jun and c-Fos proteins as well as 12-O-tetradecanoyl phorbol-13-acetate in transient transfection assays. Finally, immunocytochemical dual labeling was used to show that more than 75% of all FSHbeta-containing cells in ovine pituitary sections from cycling ewes contained nuclear c-Jun, JunB, JunD, and Fos proteins. These data, taken together, show that oFSHbeta transcription can be stimulated by c-Jun and c-Fos proteins via two functionally linked AP-1-like sites in the oFSHbeta proximal promoter and that these sites are likely to be important regulators of FSH production in vivo.
Strahl
B D
BD
Department of Biochemistry, North Carolina State University, Raleigh 27695-7622, USA.
Huang
H J
HJ
Pedersen
N R
NR
Wu
J C
JC
Ghosh
B R
BR
Miller
W L
WL
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D002822:Choriocarcinoma; D016384:Consensus Sequence; D017931:DNA Primers; D005260:Female; D005640:Follicle Stimulating Hormone / Q000096:biosynthesis* / Q000235:genetics*; D037201:Follicle Stimulating Hormone, beta Subunit; D005914:Globins / Q000096:biosynthesis; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D016760:Proto-Oncogene Proteins c-fos / Q000096:biosynthesis; D016755:Proto-Oncogene Proteins c-jun / Q000096:biosynthesis; D011817:Rabbits; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D017384:Sequence Deletion; D012756:Sheep; D013552:Swine; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured; D014594:Uterine Neoplasms
D017931:DNA Primers; D037201:Follicle Stimulating Hormone, beta Subunit; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D011993:Recombinant Fusion Proteins; D018808:Transcription Factor AP-1; D005640:Follicle Stimulating Hormone; D005914:Globins; D008156:Luciferases