Multiple elements in the distal part of the 1.2 kb 5'-flanking region of the rat GnRH receptor gene regulate gonadotrope-specific expression conferred by proximal domain.
Several lines of evidence indicate that the number of GnRH receptors (GnRH-R) and therefore, gonadotrope responsiveness to GnRH, is highly dependent upon the level of GnRH-R mRNA. To explore this aspect of regulation, we have isolated a 3.3 kb fragment encompassing the promoter region of the rat GnRH-R gene. Primer extension and RNase protection assays allowed the identification of five major transcriptional start sites located within the 110 bp region upstream of the translation start codon. Transfection experiments using the CAT reporter gene demonstrated that the 1261 bp 5' flanking region is required to direct high efficient expression in the gonadotrope-derived alphaT3-1 cell line thus contrasting with mouse in which the only 500 bp proximal sequence appeared to be sufficient. Another difference between rat and mouse was apparent in the 183 bp region of the rat promoter which induced a 3-fold stimulation of thymidine kinase promoter activity in both alphaT3-1 and CHO cells. Subsequent deletion analysis of the region residing between -1261 and -519 revealed the presence of multiple regulatory domains that contributed to the cell-specific activity. However, despite this efficiency in the context of the wild-type promoter, they failed to induce the activity of the minimal thymidine kinase (TK) promoter in the absence of the proximal 600 bp promoter region. Accordingly, a composite TK promoter containing the entire 1.2 kb promoter induced a 10-fold increase in the activity of the TK promoter in alphaT3-1 cells. Taken together these data suggest that distal regulatory regions are critical and require cooperation with proximal specific-promoter elements for activating basal R-GnRH gene expression in gonadotrope cells.
Pincas
H
H
Endocrinologie Cellulaire et Moléculaire de la Reproduction, Université Pierre et Marie Curie, Centre National de la Recherche Scientifique-URA 1449, Paris, France.
Forraï
Z
Z
Chauvin
S
S
Laverrière
J N
JN
Counis
R
R
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009928:Organ Specificity; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011966:Receptors, LHRH / Q000235:genetics*; D020218:Response Elements / Q000235:genetics*; D016415:Sequence Alignment; D017384:Sequence Deletion; D014158:Transcription, Genetic / Q000235:genetics; D014162:Transfection
D012333:RNA, Messenger; D011966:Receptors, LHRH