Cloning and partial characterization of the mouse glutamine:fructose-6-phosphate amidotransferase (GFAT) gene promoter.
Glutamine:fructose-6-phosphate amidotransferase (GFAT) is the enzyme that is rate limiting in the synthesis of glucosamine and hexosamines. Glucosamine has been proposed to contribute to the glucotoxicity of diabetes. Evidence that the gene encoding GFAT is transcriptionally regulated prompted us to clone and characterize its promoter. The position of the mouse GFAT promoter relative to the translational start site was located by primer extension and found to be 149 bp upstream of the translational start site. A 1.9 kb SacI fragment of the GFAT gene was found to contain the promoter and 88 bp of sequence downstream of the transcriptional start site. This promoter segment could drive expression of a luciferase reporter gene, could confer correct transcriptional initiation to the reporter and could confer the EGF-responsiveness previously observed in the native gene. The mouse GFAT promoter lacks a canonical TATA box and has several GC boxes within a highly GC-rich region. Deletional analysis of the promoter indicated that a proximal element extending to -120 relative to the transcriptional start site could confer reporter expression at a level of 57% of the 1.9 kb construct. Detailed analysis of this proximal region by DNase I footprinting, electrophoretic mobility shift assays and site-directed mutagenesis indicated that Sp1 binds to three elements in this proximal promoter segment and plays a vital role in regulation of transcription from this gene.
Sayeski
P P
PP
Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
Wang
D
D
Su
K
K
Han
I O
IO
Kudlow
J E
JE
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005944:Glucosamine / Q000096:biosynthesis; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / Q000737:chemistry / Q000235:genetics*; D006595:Hexosamines / Q000096:biosynthesis; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D016329:Sp1 Transcription Factor / Q000378:metabolism; D016385:TATA Box; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D006595:Hexosamines; D016329:Sp1 Transcription Factor; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D004815:Epidermal Growth Factor; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); D005944:Glucosamine