text: Specific CpG sites in the IRF-4 promoter are methylated in hematopoietic cells
  In order to exactly map the methylation sites within the IRF-4 promoter, we treated DNA of Jurkat, CML-T1, U-937, K-562 and EM-2 cells as well as of SD-1, RPMI-8226 and BV-173 control cells with bisulfite, which chemically converts unmethylated cytosine to uracil, whereas it has no effect on methylated cytosine, i.e. in CpG (34). This technique is especially useful for detection of unknown methylation patterns. PCR amplification, cloning and sequencing of the bisulfite-treated DNA showed a specific methylation pattern of the analyzed 62 CpG sites in all cell lines (Figure 4 and Table 1). In general, the methylational status ranged from one cell line with a nearly non-methylated IRF-4 promoter (SD-1, IRF-4-positive) to a completely methylated IRF-4 promoter in CML-T1 (IRF-4-negative). Interestingly, the percentage of CpG methylation in the IRF-4 promoter from IRF-4-positive cells was very low (mean 24%) as compared with IRF-4-negative cells (mean 94%) (Figure 4A and Table 1). A 5'-region (R1) with 13 hypermethylated CpG sites (mean number of methylated clones 5.5 of 8 with 77% methylated CpGs) was found in most cells (except SD-1 and RPMI-8226) and a 3'-region (R3) of 6 hypomethylated CpG sites (mean number of methylated clones 1.7 of 8 with 33% methylated CpGs) was found in most cells (except CML-T1 and U-937) (Figure 4A and Table 1). 
  Intriguingly, a stretch of 13 CpG sites (#10-22; R2) was detected in between these regions, which were highly methylated in IRF-4-negative (mean number of methylated clones 7.1 of 8 with 89% methylated CpGs) but totally non-methylated in IRF-4-positive cells (Figure 4A and B). Furthermore, three CpG sites at the 5' end (#54, 56, 58) and two CpG motifs at the 3' end (#1, 2) showed this direct correlation between high methylation status and absence of IRF-4 expression. In addition, two CpG sites located in a NFkappaB (#48) and a SP1element (#45) are less methylated in IRF-4-positive than in IRF-4-negative cells (mean number of methylated clones: 1/8 versus 8/8). These results indicate the involvement of CpG methylation in the regulation of IRF-4 expression in leukemic cells. 
  