Silencing of Sp1 and Sp3 reduces A3G promoter activity
To confirm the role of Sp1 and Sp3 in regulation of A3G promoter activity, we silenced their translation via RNA interference. Functionality of the siRNAs directed against Sp1 or Sp3 was confirmed by western blot analysis: protein levels of Sp1 as well as the long and short isoforms of Sp3 were strongly reduced in the presence of 150 or 300 ng specific siRNA (Figure 7A). An unspecific control siRNA had no influence (Figure 7A). We then cotransfected the luciferase reporter plasmid containing the 180 bp A3G promoter together with 100 ng siRNA using an optimized protocol for the cotransfection of plasmid plus siRNA. This resulted in a 31–43% reduction of luciferase activity in the presence of Sp1- or Sp3-specific siRNA compared to the control siRNA. In contrast, no influence on transcriptional activity of the 150 bp fragment, which does not contain the Sp1/Sp3-binding motif, was observed. Thus, both Sp1 and Sp3 factors are mediating transcriptional activity of the A3G promoter.
Figure 7. Silencing of Sp1 and Sp3 reduces A3G promoter activity. (A) HeLa cells were transfected with 150 and 300 ng of unspecific, Sp1-specific or Sp3-specific siRNA. After 48 h, cells were harvested and Sp1 and Sp3 proteins were detected by western blot analysis. As loading control, protein levels of tubulin are shown. (B) HeLa cells were cotransfected with reporter plasmid pGL3-Basic containing 180 or 150 bp of the A3G promoter and siRNA. Hundred nanogram of unspecific siRNA (control), Sp1-specific siRNA (Sp1), Sp3-specific siRNA (Sp3) or a mixture of 50 ng Sp1-specific plus 50 ng Sp3-specific siRNA (Sp1+Sp3) were used. Firefly luciferase activities after 48 h were normalized to coexpressed renilla luciferase activities. Mean values (±SD) of a representative experiment performed in triplicate are shown.