Research Paper Volume 10, Issue 9 pp 2284—2315

Figure 3. Oxidative stress attenuated Sp1 binding to its GC-Box elements present in hPrdx6 gene promoter. Table 1. Evolutionary conserved Sp1 binding sequences in TATA-less Prdx6 promoters of mouse, rat and human cells. (A) Schematic illustration of 5’-proximal promoter region of Prdx6 containing Sp1 (GC-Box) binding sites showing primer location and sequences used in ChIP assay. (B and C) Oxidative stress (H₂O₂ or UVB)-induced reduction in DNA binding activity of Sp1 to hPrdx6 gene promoter containing GC-Box (Sp1 sites) in SRA-hLECs. ChIP assay was carried out by using ChIP-IT® Express and ChIP-IT® qPCR analysis kits (Active motif). Chromatin samples prepared from SRA-hLECs were exposed to varying concentrations of H₂O₂ (0, 50, 75 and 100µM) or UVB (0, 40, 80 and 120J/m²), and were subjected to ChIP assay with ChIP grade antibodies, anti-Sp1 (black bars) and control IgG (gray bars). The DNA fragments were used as templates for qPCR by using primer designed to amplify -342 to +30 region of the human Prdx6 gene promoter bearing GC-box (Sp1 sites). Histogram showed the amplified DNA by qPCR analysis: (B) Control (0) vs 50µM vs 75µM vs 100µM H₂O₂ treatment. (C) Control (0) vs 40J/m² vs 80J/m² vs 120J/m² UVB exposure. The data represent mean ± SD from three independent experiments (**p<0.05; *p<0.001). (D) Human Prdx6 promoter activity inhibited by mithramycin A (Mithra A), an inhibitor of Sp1, validated Sp1 regulation of hPrdx6 gene. Cells were transfected with CAT-hPrdx6 (-918/+30) or empty CAT vector construct and treated with Mithra-A at different concentrations for 24h. Data represent mean ± SD from three independent experiments (**p<0.05; *p<0.001).