AE associates with the POLE and OGG1 promoters and promotes DNA damage accumulation. (A) Graphic of the 5′ regulatory regions of the OGG1 and POLE genes. Black ovals represent RUNX1-binding sites; arrows indicate orientation. Numbers represent distance between the indicated regions, in base pairs. (B) RUNX1-binding sites were amplified by PCR after chromatin immunoprecipitation. The RUNX1 site present in the p14^ARF promoter was used as a positive control. Water was used as a negative control. One percent of lysate input used for immunoprecipitation (IP) is amplified as an additional control. Control IP has no Ab and specific IP was with anti-HA Ab (AE contains the HA epitope). (C) MIT and AE cells were stained for the DNA damage marker γH2A.X; nuclei are stained with DAPI. (D) Cells positive for γH2A.X were scored in 4 control, 4 AE, and 3 MLL-AF9 cultures. (E) Percentage of cells with DNA damage in 3 pairs of AE and control MIT cultures during the first 5 weeks after transduction. (F) Cells from samples in panel E were cultured under 2.6% oxygen starting at week 2 after transduction. Percentage of cells positive for γH2A.X was scored at weeks 3 and 5 after transduction. Error bars represent SD. (G) Amount of reactive oxygen species was measured by flow cytometry in AE and MIT cultures stained with H₂DCFDA probe. MIT cells treated with 300 μM H₂O₂ for 20 minutes were used as a positive control. The staining was repeated multiple times with no consistent difference between the 2 cultures.