Cloning and characterization of mouse glutamine:fructose-6-phosphate amidotransferase 2 gene promoter

Abstract

Glutamine:fructose-6-phosphate amidotransferase (GFAT) is the first and rate-limiting enzyme of the hexosamine biosynthesis pathway, which plays an important role in glucose toxicity and cellular insulin resistance. Thus, the mechanisms by which GFAT expression is regulated under physiological and pathological conditions are of interest in connection with diabetes. In this study, we cloned the 5′-flanking region of the mouse GFAT2 gene and characterized its promoter activity. Sequence analysis revealed several putative regulatory elements Sp1, a CCAAT box, AP-1 and AP-2, but no TATA box. Transfection experiments showed that the 5′-flanking region between −2462 to +38 relative to the transcription start site of the GFAT2 gene drives transcription in NIH3T3 cells and that the fragment from −141 to −9 has the highest transcription activity. Reporter assays using deletion and mutant variants suggested that the Sp1 sites at positions −83 to −78 and −22 to −17 both play an important role in the basal promoter activity of the mouse GFAT2 gene. Electrophoretic mobility shift assay showed DNA-protein binding at both Sp1 sites. We also compared the promoter activities of mouse GFAT1 and GFAT2 in several cell lines.