text: Increase of IRF-4 expression in hematopoietic cells after demethylating treatment
  We next analyzed whether promoter methylation could be responsible for down-regulation of IRF-4 expression. A region including exon1 in the IRF-4 promoter exhibited a large number of CpG-rich sequences (Figure 3A). Several chemical substances such as 5-aza-2-deoxycytidine (AzadC) or 5-azacytidine (AzaC) inhibit de novo and maintenance methylation, and thus can be used to discern promoter methylation (32,33). We used AzadC to generate unmethylated DNA. A 72 h AzadC-treatment resulted in a concentration-dependent activation of IRF-4 transcription in Jurkat and CML-T1 T-cells as well as in U-937, K-562 and EM-2 cell lines (Figure 2A). IRF-4 transcription was induced in a time-dependent manner and was observed as early as 24 h after treatment with AzadC and increased over time until 72 h (Figure 2B). Time and strength of the appearance of IRF-4 transcripts varied among cell lines, i.e. CML-T1 responded strongest to AzadC-treatment (data not shown). In line with this, AzadC-treatment of CML-T1 and LAMA-84 cells also translated in an induction of IRF-4 protein expression (Figure 2C). Accordingly, treatment of the IRF-4-positive cell line BV-173, SD-1 and RPMI-8226 with AzadC had no effect on IRF-4 expression (Figure 2D). There was no difference in the effects of AzaC versus AzadC, as both increased the IRF-4 mRNA level in CML-T1 cells as well (data not shown). This implied that promoter methylation may control IRF-4 expression, but an alternative explanation may be activation of positive transcriptional regulators of IRF-4 by AzadC (or AzaC). 
  