Research Paper Volume 10, Issue 9 pp 2284—2315

Sumoylation-deficient Prdx6 repairs aberrant Sumoylation-mediated Sp1 dysregulation-dependent Prdx6 repression and cell injury in aging and oxidative stress

Figure 6. Cells overexpressing Sumo1 showed reduced Sp1 binding to its responsive elements in Prdx6 promoter. (A) Schematic illustration of Prdx6 gene promoter. (B) Gel-shift mobility assay showing that Sumo1 reduced the Sp1 DNA–binding activity of Prdx6 gene promoter. Gel-shift mobility assay was carried out using nuclear extracts isolated from mLECs transfected with pEGFP-Vector (Lanes 1 and 2) or pEGFP-Sumo1 (Lanes 3 and 4) incubated with 32p-labeled wild type probe (Lanes 1 and 3) or its mutant (Lanes 2 and 4). A diminished Cm1 band was observed in cells overexpressing Sumo1 (Lane 3) in comparison to vector control (Lane 1). No binding occurred in mutant probes (Lanes 2 and 4). (C) Histogram represents densitometry analysis of DNA-protein complex formed in gel-shift assay. Lane 1 vs lane 3, *p < 0.001. (D) ChIP assay showing Sumo1 overexpression significantly suppressed Sp1-DNA binding in Prdx6 gene promoter in dose-dependent manner. mLECs were transiently transfected with different concentrations of pEGFP-Sumo1 (0, 1, 2 and 4 μg). 72h later ChIP assay was carried out with anti-Sp1 and control IgG antibodies. Pulled DNA fragments were subjected to PCR analysis for Sp1 binding cis-elements of Prdx6 promoter. The product was analyzed through agarose-gel. Data represent three experiments. (E) Senp1 overexpression dramatically enhanced Sp1-DNA binding in concentration-dependent -manner. mLECs were transfected with increasing concentrations of pFlag-Senp1 (0, 0.5, 1 and 2μg) for 72h. ChIP analysis was carried out using chromatin samples prepared from transfected LECs with a ChIP grade antibody, anti-Sp1 and control IgG. The DNA fragments were used as templates for PCR by using primers designed to amplify −208 to +27 region of the Prdx6 promoter bearing Sp1 binding sites as shown. PCR product was analyzed through agarose gel as shown.