Figure 2

DNA methylation changes at the promoters of Il2 and Csf2 during T cell activation and development. A. Schematic representation of the promoter regions of the Il2 and Csf2 genes, showing the nucleotide distances upstream from the transcription start sites (dark arrows), the locations of the CpG dinucleotides (vertical arrows) and NF-kB binding elements for reference. PCR primers that covered one or multiple CpG dinucleotides were designed for each inducible gene for the MeDIP assay and are shown as thick horizontal lines and denoted by capital letters. B. - F. The MeDIP assay, using a 5-methylcytosine-specific monoclonal antibody (Epiteck), and real-time PCR, was used to determine the DNA methylation levels of individual CpG dinucleotides located at the promoter regions of Il2 and Csf2 in CD4⁺ T cells. B. Basal DNA methylation levels at the promoter regions of Il2 and Csf2 in unstimulated CD4⁺ T cells (NS) isolated from both age groups of mice were examined; the data are shown as means and SEM. C. - F. The DNA methylation levels of the Il2 and Csf2 genes in CD4⁺ T cells upon PMA/I stimulation for 0, 0.5, 2 and 4 hours. The data were normalized to Gapdh, and were then normalized to the unstimulated samples, and are shown as the mean and SEM of three independent experiments. Every experiment was repeated at least 3 times