FANCD2 ubiquitination is required for the formation of FA-BRG1–promoter complex. (A) Reconstitution of the FA-D2 cells with WT FANCD2 or the nonubiquitinated FANCD2-K561R mutant. FANCD2-deficient PD20 cell were transduced with retrovirus-expressing empty vector, WT-FANCD2, or the FANCD2-K561R mutant followed by puromycin selection. Stable cell lines were treated with or without H₂O₂ followed by Western analysis using Abs against FANCD2, FANCA, BRG1, or β-actin. (B) Oxidative stress induces chromatin loading of BRG1 and FA proteins in FANCD2-corrected cells. Cells described in panel A were treated with or without H₂O₂ followed by chromatin fractionation. Chromatin extracts were then analyzed by Western blotting with Abs against BRG1, FANCA, FANCC, FANCD2, FANCG, or FOXO3a. Histone H2A was included as a loading control. (C) FANCD2 ubiquitination is required for the formation of the FA-BRG1-DNA complex. Cells described in panel A were transduced with retrovirus expressing Flag-tagged FANCA, followed by cell sorting for GFP. Sorted cells were then treated with or without H₂O₂ for 2 hours and released for the indicated hours. ChIP assays using Abs against Flag or BRG1 were followed by PCR amplification using primers specific for the promoter of GPX1. (D) FANCD2 ubiquitination is required for the protection of antioxidant gene promoter DNA from oxidative damage. Cells described in panel C were treated with or without H₂O₂ for 2 hours and released into fresh medium for an additional 12 hours. ChIP assay using Ab against BRG1 was performed, and bound DNA fragments were subjected to the Fpg cleavage/PCR-based DNA-repair assay using primers specific for the promoter of GPX1. The percentage of intact DNA represents the ratio of PCR products after Fpg cleavage to those present in uncleaved DNA.