DNA methylation analysis of 293T cells expressing wild-type RUNX1 and a chimera protein RUNX11-299-SPI11-102
Transcription factor (TF)-dependent DNA demethylation is associated with generation of specific DNA methylation profiles in normal cellular development and disease, although only a small fraction of TFs are known to promote DNA demethylation. We examined DNA methylation profiles of 293T mock, 293T wild type RUNX1, and 293T RUNX11-299–SPI11-102 cells by reduced representation bisulfite sequencing analysis. Genome-wide DNA demethylation was observed specifically at RUNX1-binding sites in 293T cells expressing a chimeric protein consisting of RUNX11-299 and SPI11-102, similar to 293T cells expressing wild type RUNX1. Overall design: 293T cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and penicillin/streptomycin (100 U/mL, 100 mg/mL). The cells were infected overnight with the lentivirus vectors at a multiplicity of infection of 7–15 in the presence of 8 mg/mL polybrene, followed by puromycin selection at 3 µg/mL for 10 days. Genomic DNA was extracted from the 293T cells and subjected to the reduced representation bisulfite sequencing.