Neuron-specific expression of the rat gonadotropin-releasing hormone gene is conferred by interactions of a defined promoter element with the enhancer in GT1-7 cells.
Neuroendocrine control of the reproductive cascade is mediated by GnRH, which in mammals is produced by a subset of neurons scattered throughout the hypothalamus and forebrain. Utilizing a cultured cell model of GnRH neurons (GT1-7 cells), two regulatory regions in the rat GnRH 5' flanking DNA were identified as essential for cell-type specificity: a 300-bp enhancer and a 173-bp conserved proximal promoter. Using transient transfections to compare expression in GT1-7 cells to a non-GnRH-expressing cell type (NIH 3T3), we show that the GnRH enhancer and the proximal promoter each play roles in conferring this specificity. Deletion of footprint 2 (FP2; -26 to -76) from the promoter when coupled to the GnRH enhancer diminishes reporter activity in GT1-7 cells more strongly than in NIH 3T3 cells. Furthermore, deletion of FP2 from the promoter when coupled to the heterologous Rous sarcoma virus 5'-long terminal repeat promoter abolishes the difference in reporter activity between GT1-7 and NIH 3T3 cells, suggesting that FP2 of the GnRH promoter is necessary for cell-specific expression. In addition, FP2 alone is sufficient to confer cell-specific expression and can interact with the GnRH enhancer to augment reporter gene expression specifically in GT1-7 cells. Finally, a 31-bp sequence from within FP2 (-63 to -33) synergistically activates transcription when coupled with the GnRH enhancer in GT1-7 cells but not in NIH 3T3 cells. Thus, this 31-bp region contains elements necessary for interaction between the GnRH enhancer and promoter. We show that two of five protein complexes that bind to the -63 to -33 region are GT1-7 cell specific, and both of them appear to be homeodomain proteins. The identification of a cell-specific element in the GnRH proximal promoter significantly advances our understanding of the transcriptional basis for neuron-specific GnRH gene expression.
Nelson
S B
SB
Department of Reproductive Medicine, University of California, San Diego, La Jolla 92093-0674, USA.
Lawson
M A
MA
Kelley
C G
CG
Mellon
P L
PL
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D007987:Gonadotropin-Releasing Hormone / Q000235:genetics*; D008156:Luciferases / Q000235:genetics; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000166:cytology / Q000502:physiology*; D011401:Promoter Regions, Genetic; D051381:Rats; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics
D007987:Gonadotropin-Releasing Hormone; D008156:Luciferases; D001616:beta-Galactosidase