Misawa H, Yamaguchi M
Laboratory of Endocrinology and Molecular Metabolism, Graduate School of Nutritional Sciences, University of Shizuoka, 52-1 Yada, Shizuoka, 422-8526, Japan.
Biochem Biophys Res Commun. 2000 Mar 5;269(1):270-8. doi: 10.1006/bbrc.2000.2275.
The characterization of the binding of nuclear protein on the 5'-flanking region of the rat regucalcin gene was investigated. Nuclear extracts from rat liver and H4-II-E hepatoma cells were used for oligonucleotide competition gel mobility shift assay. An oligonucleotide between position -523 and -506 in the 5'-flanking region of the rat regucalcin gene, which contains a nuclear factor I (NF1) consensus motif TTGGC(N)(6)CC, competed with the probe for the binding of the nuclear proteins from rat liver and H4-II-E cells. The mutation of TTGGC in the consensus sequence caused an inhibition of the binding of nuclear factors. The presence of Bay K 8644, insulin, and phorbol esters could stimulate the binding of the nuclear factors to the TTGGC region of the rat regucalcin gene in H4-II-E cells. The specific mutation introduced in this region, which was ligated to a luciferase reporter gene, reduced significantly the effects of Bay K 8644, insulin, and phorbol esters in stimulating the regucalcin gene transcriptional activity in H4-II-E cells. These results suggest that the specific nuclear factor binds to the NF1-like sequence, which can stimulate the transcriptional activity, in the promoter region of regucalcin gene in liver cells.
