A GC-box located at position −87/−78 of the A3G promoter is important for transcriptional activity
The reporter studies shown in Figure 3 had demonstrated a drop in luciferase activity after deletion of the 30 nt at the 5′ end of the 180 bp core promoter. We therefore inspected the 30 bp sequence deleted in the 150 bp fragment and identified a GC-box at position −87/−78 (see Figure 1). The sequence TGGGCGGGAC, which is interrupted in the 150 bp fragment, represents a variant of the (G/T)GGGCGG(G/A)(G/A)(C/T) consensus motif recognized by Sp1 and Sp3 transcription factors. To analyze whether this putative Sp1/Sp3-binding site mediates transcriptional activity of the 180 bp core promoter, we introduced two point mutations which changed the sequence from TGGGCGGGAC to TGTTCGGGAC (mutations shown in bold). This resulted in a 71% reduction of the transcriptional activity compared to the unmodified 180 bp promoter (Figure 5A) and this value was only marginally higher than the luciferase activity of the 150 bp fragment, indicating that the identified motif is essential for basal activity of the A3G core promoter. To further examine the transcriptional potency of the 30 nt present in the 180 bp promoter, we cloned the region −114/−85 (containing all nucleotides which are deleted in the 150 bp fragment, designated E1) or the region −92/−63 (containing the putative Sp1/Sp3 motif, designated E2) into the vector pGL3-Promoter (see Figure 1). This vector contains a luciferase reporter gene under the control of an SV40 promoter without enhancer sequences, and putative transcriptionally active sequences can be cloned upstream of the SV40 promoter. Luciferase assays showed that the E1 element increased SV40 promoter activity only by ∼2-fold (Figure 5B). In contrast, the 30 nt of E2 enhanced the transcriptional activity of the SV40 promoter by ∼4.3-fold, indicating that the intact GC-box present in the E2 element was responsible for the strongly enhanced transcriptional activity of the SV40 promoter.
Figure 5. A GC-box mediates transcriptional activity of the 180 bp core promoter. (A) A3.01 T cells were transfected with reporter plasmid pGL3-Basic containing 180, 150 or 120 bp of the A3G promoter. The two G-to-T substitutions introduced into the GC-box of the 180 bp fragment (180mut) are specified. After 48 h, cells were harvested for luciferase assay. Firefly luciferase activities were normalized to coexpressed renilla luciferase activities. Mean values (±SD) of a representative experiment performed in triplicate are shown. (B) pGL3-Promoter reporter plasmids containing the regions E1 or E2 (see Figure 1) upstream of the SV40 promoter were transfected into A3.01 T cells. 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.