Glut 3, the most efficient facilitative glucose transporter, is solely expressed in murine brain neurons (N) and placental trophoblasts (T), and ensures glucose supply to fuel neurotransmission and fetal growth. To determine the molecular mechanisms which control Glut 3 expression in N and T, we undertook primer extension assays and established the transcription start site (TSS) of the mouse Glut 3 gene upstream from the ATG translation start codon at -305 bp in the N2A murine neuroblasts, and at -125 bp in the HRP.1 rat placental T. We then cloned the -203 to +243 bp (numbered per the TSS in N) mouse Glut 3 DNA which contains putative binding sites and successive 5′-deletions, upstream to a promoterless and enhancerless luciferase reporter gene. These deletional constructs were transfected into the N2A, and HRP.1 cells and the reporter gene activity assessed. The -203 to +243 bp DNA exhibited an increase (promoter), where as the -177 to +243 bp DNA showed an inhibition of the reporter gene activity (repression) which disappeared in the-104 to +243 bp DNA fragment. Mobility shift (EMSA) and DNAse 1 footprinting assays established that the -143 to -123 bp mouse Glut 3 DNA duplex specifically bound a nuclear protein from N2A and HRP.1 cells. Computer analysis for potential DNA binding motifs and competition EMSA using oligonucleotides with a consensus sequence for canonical transcription factors suggested, while supershift assays established the nuclear factor Sp1 to bind this DNA element. We conclude that 1] alternate TSS situated ≈180 bp apart initiate Glut 3 transcription in N vs. T, and 2] Sp1 represses the promoter-driven Glut 3 transcriptional activity in N and T. We speculate that changes in Sp1 DNA-binding activity modulate Glut 3 gene expression and function in N and T.
{Supported by NIH-HD33997}
