A composite hormone response element regulates transcription of the rat GHRH receptor gene.
To further elucidate the molecular mechanisms underlying the transcriptional regulation of the GHRH receptor (GHRH-R) gene, hormonal regulation of the promoter activity of this gene was examined. An approximately 3-kb genomic fragment spanning the promoter region of the gene was sequenced and the transcription start site was determined by RT-PCR and RNase protection assay. A major start site was localized at -105 (relative to the translation initiation codon, ATG), and a pit-1 binding sequence characteristic of pituitary specific genes was found at -155 to -146. Deletion and mutation studies demonstrated this site to be functional. In the presence of dexamethasone, the GHRH-R promoter (from -2935 to -11) directed luciferase expression in MtT-S cells, a somatotropic cell line, but not in the PC12 cells that normally do not express GHRH-R. While T(3), all trans-RA, and 9cis-RA alone weakly enhanced the reporter gene expression, each of these substances was found to act as a synergistic enhancer in the presence of dexamethasone. Additional deletion and mutation analyses demonstrated a functional RA response element at -1090 to -1074. Two functional glucocorticoid response elements and a T(3) response element were found in an 80-bp 5'-flanking sequence of the pit-1 site. Interestingly, it is suggested that the 6-bp half-site AGGACA (from -209 to -204) functions as a 3'-half-site of T(3) response element as well as a 5'-half-site of one of the glucocorticoid response elements.
Nogami
Haruo
H
Department of Neuroendocrinology, Basic Medical Sciences, University of Tsukuba, Tsukuba 305-8575, Japan. hnogami@md.tsukuba.ac.jp
Hiraoka
Yoshiki
Y
Matsubara
Maki
M
Nonobe
Eriko
E
Harigaya
Toshio
T
Katayama
Masateru
M
Hemmi
Noriaki
N
Kobayashi
Shuzo
S
Mogi
Koichi
K
Aiso
Sadakazu
S
Kawamura
Koki
K
Hisano
Setsuji
S
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter / Q000235:genetics; D005938:Glucocorticoids / Q000494:pharmacology / Q000502:physiology*; D007124:Immunoenzyme Techniques; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D051381:Rats; D011966:Receptors, LHRH / Q000235:genetics / Q000502:physiology*; D020218:Response Elements / Q000235:genetics / Q000502:physiology*; D014158:Transcription, Genetic; D014284:Triiodothyronine / Q000235:genetics / Q000494:pharmacology; D015854:Up-Regulation / Q000235:genetics
D003062:Codon; D005938:Glucocorticoids; D011966:Receptors, LHRH; D014284:Triiodothyronine