Sp1 and Sp3 regulate transcriptional activity of the facilitative glucose transporter isoform-3 gene in mammalian neuroblasts and trophoblasts.
The murine facilitative glucose transporter isoform 3 (Glut 3) is developmentally regulated and is predominantly expressed in neurons and trophoblasts. Employing the primer extension and RNase protection assays, the transcription start site (denoted as +1) of the murine Glut 3 gene was localized to 305 base pairs (bp) 5' to the ATG translation start codon. Transient transfection assays in N2A, H19-7 neuroblasts, and HRP.1 trophoblasts using sequential 5'-deletions of the murine Glut 3-luciferase fusion gene indicated that the -203 to +237 bp region with reference to the transcriptional start site contained promoter activity. Repressor function was limited to the -137 to -130 bp region within the transcriptional activation domain. The nuclear factors Sp1 and Sp3 bound this GC-rich region in N2A, H19-7, and HRP.1 cells. Dephosphorylation of Sp1 was essential for Glut 3 DNA binding. The related Sp3 protein also bound this same region of mouse Glut 3 in all three cell lines. Mutations of the Sp1-binding site employed in transient transfection and mobility shift assays confirmed the nature of the DNA-binding proteins, while supershift assays with anti-Sp1 and anti-Sp3 IgGs characterized the differences in the two DNA-binding proteins. Co-transfection of the Glut 3-luciferase fusion gene with or without mutations of the Sp1-binding site along with the Sp1 or Sp3 expression vectors in Drosophila SL2 cells confirmed a reciprocal effect, with Sp1 suppressing and Sp3 activating Glut 3 gene transcription.
Divisions of Neonatology and Developmental Biology, the Department of Pediatrics, University of Pittsburgh School of Medicine, Magee-Womens Research Institute, Pittsburgh, Pennsylvania 15213, USA.
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Rajakumar
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Thamotharan
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RK
Menon
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Devaskar
D000818:Animals; D001483:Base Sequence; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005260:Female; D005786:Gene Expression Regulation; D051274:Glucose Transporter Type 3; D051379:Mice; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000096:biosynthesis* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D019319:Okadaic Acid / Q000494:pharmacology; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D051705:Sp3 Transcription Factor; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D014327:Trophoblasts / Q000166:cytology / Q000378:metabolism*
D004268:DNA-Binding Proteins; D051274:Glucose Transporter Type 3; D009004:Monosaccharide Transport Proteins; D009419:Nerve Tissue Proteins; D020033:Protein Isoforms; C495689:Slc2a3 protein, mouse; C495690:Slc2a3 protein, rat; D016329:Sp1 Transcription Factor; C497122:Sp3 protein, mouse; C497123:Sp3 protein, rat; D014157:Transcription Factors; D051705:Sp3 Transcription Factor; D019319:Okadaic Acid