Mutation analysis of HNF-4 binding sites in the human glucose-6-phosphatase promoter.
HNF-4, a member of the nuclear receptor superfamily, binds to HNF-4 response elements (HRE), consisting of a direct repeat of the hexameric half-sites spaced by 1 nt (direct repeat 1) and activates a number of genes, which play central roles in fatty acids and glucose metabolism. Glucose-6-phosphatase (G6Pase) catalyzes the terminal step in the gluconeogenic and glycogenolytic pathways. A previous study has shown that HNF-4 binds to two DR1s in the regions A (located between -266 and -234) and B (located between -306 and -274) on the human G6Pase promoter. We found that the region B contains the one more DR1 element, composed of the two half-sites, designated half-sites a and b, the latter of which overlaps with the previously identified DR1 consisting of two half-sites, designated half-sites b and c. In this study, electrophoretic mobility shift assay (EMSA) using point mutations in each half-site a, b, or c indicated that HNF-4 binds to the combination of half-sites a and b, but not to half-sites b and c. Furthermore, mutational analysis demonstrated that, in the context of the human G6Pase promoter, the half-sites a and b, but not the half-sites b and c, are required for the stimulatory effect of HNF-4. These results suggested that the DR1 element containing the half-sites a and b is a functional HRE that mediates the induction of hG6Pase promoter activity by HNF-4.
Hirota
Keiko
K
Center of Tsukuba Advanced Research Alliance, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan.
Aoyama
Hisanori
H
Fukamizu
Akiyoshi
A
D001483:Base Sequence; D001665:Binding Sites; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000378:metabolism*; D017353:Gene Deletion; D005952:Glucose-6-Phosphatase / Q000235:genetics*; D051557:Hepatocyte Nuclear Factor 4; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D010750:Phosphoproteins / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D014157:Transcription Factors / Q000378:metabolism*
D004268:DNA-Binding Proteins; D051557:Hepatocyte Nuclear Factor 4; D010750:Phosphoproteins; D014157:Transcription Factors; D005952:Glucose-6-Phosphatase