Oxidative stress-induced formation of a FA-BRG1–promoter complex. (A) FANCD2 does not bind to the promoters of antioxidant genes in unstressed cells. Untreated normal lymphoblasts were subjected to a ChIP assay using Abs against FANCD2 or BRG1. PCR amplification was performed using primers specific for the promoters of indicated antioxidant or housekeeping genes. (B) FANCD2 is recruited to the GPX1and TXNRD1 promoter regions after H₂O₂ treatment. Normal cells were treated with 100μM H₂O₂ for 2 hours then released for the indicated time intervals. Proteins were extracted at different time points, followed by a ChIP assay using Abs against FANCD2 or BRG1. Precipitated samples were then subjected to PCR using primers for the promoters of GPX1 or TXNRD1. Representative images (top) and quantifications (botton) were shown. The intensity of the DNA bands was quantified using ImageJ software (NIH). Results are means ± SD of 3 independent experiments. (C) FA-BRG1-promoter complex was absent in FA cells. FA-A cells were treated with 100μM H₂O₂ for 2 hours then released for the indicated time intervals. Proteins were extracted at different time points, followed by a ChIP assay using Abs against FANCD2 or BRG1. Precipitated samples were then subjected to PCR using primers for the promoters of GPX1 or TXNRD1. (D) Oxidative stress induces accumulation of acetylated histone in the promoters of antioxidant genes of both normal and FA cells. Normal and FA-A cells were treated with or without 100μM H₂O₂ for 2 hours followed by release into fresh medium. Cells were then subjected to ChIP assay using Abs specific for acetylated histone H3K9/14 (Ac-H3K9/14) or methylated histone H3K9 (Me-H3K9) followed by PCR using primers for the promoter regions of GPX1, TXNRD1, or β-tubulin. Representative images (top) and quantifications (bottom) are shown. The intensity of the DNA bands was quantified using ImageJ software (NIH). Results are means ± SD of 3 independent experiments. (E) Repair kinetics of oxidative damage in BRG1-bound antioxidant gene promoter. FA-A and gene-corrected cells were treated with or without H₂O₂ for 2 hours and released into fresh medium for up to 24 hours. ChIP assay using Abs against BRG1 was performed, and the bound DNA fragments were subjected to the Fpg cleavage/PCR-based DNA repair assay using primers specific for the promoter of (left) GPX1 or (right) TXNRD1. Percentage of intact DNA represents the ratio of PCR products after Fpg cleavage to those present in uncleaved DNA.