Regulation of human B19 parvovirus promoter expression by hGABP (E4TF1) transcription factor

The genetic expression of human B19 parvovirus is only dependent on one promoter in vivo and in vitro. This is the P6 promoter, which is located on the left side of the genome and is a single-stranded DNA molecule. This led us to investigate the regulation of the P6 promoter and the possible resulting variability of the nucleotide sequence. After analysis of the promoter region of 17 B19 strains, only 1.5% variability was found. More exciting was the finding of mutations that were clustered around the TATA box and defined a highly conserved region (nucleotides 113-210) in the proximal part of the P6 promoter. HeLa and UT7/Epo cell extracts were found to protect this region, which contained a core motif for Ets family proteins, with YY1 and Sp1 binding sites on either side. Gel mobility shift assays performed with nuclear proteins from HeLa and UT7/Epo cells identified DNA-binding proteins specific for these sites. By supershift analysis, we demonstrated the binding of the hGABP (also named E4TF1) protein to the Ets binding site and the fixation of Sp1 and YY1 proteins on their respective motifs. In Drosophila SL2 cells, hGABPalpha and -beta stimulated P6 promoter activity, and hGABPalpha/hGABPbeta and Sp1 exerted synergistic stimulation of this activity, an effect diminished by YY1.     

The position of the fast-inactivation gate during lidocaine block of voltage-gated Na+ channels

We recently identified three areas of Sp1 binding located between -568 and -453 of the 5' flanking region of the murine alpha2(I) collagen promoter which are necessary for optimal activity. We now identify two additional regions of Sp1 binding located at -371 to -351 (region 4) and at -690 to -613 (region 5), which when mutated increased promoter activity in transfected rat hepatic stellate cells indicating they contain negative regulatory elements. AP-2 bound to region 4 while YY1 bound most strongly to region 5. AP-2 decreased Sp1 binding to region 4 and had a dual effect on Sp1 binding to region 5 decreasing and increasing Sp1 binding at low and high concentrations of AP-2, respectively. YY1 enhanced Sp1 binding to both regions. AP-2 inhibited or enhanced the stimulatory effect of a transfected Sp1 expression vector on the alpha2(I) collagen promoter in Drosophila cells at low or high AP-2 expression, respectively. YY1 enhanced or inhibited the activation of the promoter by low or high Sp1 expression, respectively. This study identifies two negative regulatory elements in the murine alpha2(I) collagen promoter and shows that AP-2 and YY1 interact with Sp1 at these sites and can inhibit the activating action of Sp1.