Structure of the distal human gonadotropin releasing hormone (hGnrh) gene promoter and functional analysis in Gt1-7 neuronal cells.
To assess potential species-specific expression of gonadotropin releasing hormone (GnRH), the distal human (h) GnRH promoter was cloned, characterized and tested in gene transfer studies. The nucleotide sequence of approximately 3.8 kb of 5'-flanking region was determined. Homology to the rat (r) GnRH sequence was observed in the proximal promoter region between -551 h (-424 r) and the transcriptional start site and within multiple distal promoter regions. In contrast, there was little similarity in the sequences between -1131/-551 h and -1031/-424 r. A deletion panel of 5'-flanking hGnRH promoter constructs was made and tested in transient transfection assays in GnRH-producing mouse GT1-7 neuronal cells. The largest hGnRH promoter construct (-3832/+5 h) exhibited high levels of reporter activity, similar to that observed with the largest rGnRH construct (-3026/+116 r). However, in contrast to the rat gene, deletion of distal promoter sequences of the hGnRH promoter to -1971, -1131 or -551 did not result in a decrease in luciferase reporter activity. Further truncation to -350 resulted in a 3-fold decrease in luciferase activity. There was no preferential use of the putative upstream hGnRH start site in neuronal cells. DNase I protection assays showed unique protection patterns with nuclear extracts from GT1-7 and Gn10 neuronal cells and the hGnRH and rGnRH promoter fragments. These data suggest the presence of different cis-acting elements and transacting factors that mediate species-specific neuronal GnRH expression.
Kepa
J K
JK
Department of Medicine, University of Colorado Health Sciences Center and Research Service, Veterans Affairs Medical Center, Denver 80220, USA.
Spaulding
A J
AJ
Jacobsen
B M
BM
Fang
Z
Z
Xiong
X
X
Radovick
S
S
Wierman
M E
ME
D000818:Animals; D001483:Base Sequence; D046508:Culture Techniques; D004247:DNA; D018983:DNA Footprinting; D017353:Gene Deletion; D007987:Gonadotropin-Releasing Hormone / Q000235:genetics*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D051381:Rats; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection
D007987:Gonadotropin-Releasing Hormone; D004247:DNA