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 deve lopmentally regulated and is predominantly expressed in neurons and tr ophoblasts. Employing the primer extension and RNase protection assays , the transcription start site (denoted as +1) of the murine Glut 3 ge ne was localized to 305 base pairs (bp) 5' to the ATG translation star t 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 activit y. Repressor function was limited to the -137 to -130 bp region within the transcriptional activation domain. The nuclear factors Spl and Sp 3 bound this GC-rich region in N2A, H19-7, and HRP.1 cells. Dephosphor ylation of Sp1 was essential for Glut 3 DNA binding. The related Sp3 p rotein also bound this same region of mouse Glut 3 in all three cell l ines. Mutations of the Sp1-binding site employed in transient transfec tion and mobility shift assays confirmed the nature of the DNA-binding proteins, while supershift assays with anti-Sp1 and anti-Sp3 IgGs cha racterized the differences in the two DNA-binding proteins. Co-transfe ction of the Glut 3-luciferase fusion gene with or without mutations o f the Sp1-binding site along with the Spl or Sp3 expression vectors in Drosophila SL2 cells confirmed a reciprocal effect, with Sp1 suppress ing and Sp3 activating Glut 3 gene transcription.