Down-regulation of interferon regulatory factor 4 gene expression in leukemic cells due to hypermethylation of CpG motifs in the promoter region

Abstract

Although the bcr-abl translocation has been shown to be the causative genetic aberration in chronic myeloid leukemia (CML), there is mounting evidence that the deregulation of other genes, such as the transcription factor interferon regulatory factor 4 (IRF-4), is also implicated in the pathogenesis of CML. Promoter methylation of CpG target sites or direct deletions/insertions of genes are mechanisms of a reversible or permanent silencing of gene expression, respectively. Therefore, we investigated whether IRF-4 promoter methylation or mutation may be involved in the regulation of IRF-4 expression in leukemia cells. Whereas promoter mutations or structural rearrangements could be excluded as a cause of altered IRF-4 expression in hematopoietic cells, the IRF-4 promoter methylation status was found to significantly influence IRF-4 transcription. First, treatment of IRF-4-negative lymphoid, myeloid and monocytic cell lines with the methylation-inhibitor 5-aza-2-deoxycytidine resulted in a time- and concentration-dependent increase of IRF-4 mRNA and protein levels. Second, using a restriction-PCR-assay and bisulfite-sequencing we identified specifically methylated CpG sites in IRF-4-negative but not in IRF-4-positive cells. Third, we clearly determined promoter methylation as a mechanism for IRF-4 down-regulation via reporter gene assays, but did not detect an association of methylational status and mRNA expression of DNA methyltransferases or methyl-CpG-binding proteins. Together, these data suggest CpG site-specific IRF-4 promoter methylation as a putative mechanism of down-regulated IRF-4 expression in leukemia.

Figure 1

Correlation of IRF-4 mRNA expression and nucleotide changes in the IRF-4 promoter. (A) RT–PCR of different hematopoietic cell lines. (B) nucleotide changes in the promoter of IRF-4-positive or -negative cells in comparison to the germline sequence at positions 98, 111 and 1063 (GenBank accession no. U52683), representing nucleotide −1081, −1068 and −116 (30) or −1094, −1081 and −129 (31), respectively.

Figure 2

Expression of IRF-4 in hematopoietic cells after treatment with AzadC. Representative cells and experiments are shown. (A) RT–PCR after incubation of CML-T1 and EM-2 with 0.5, 1 and 3 µM AzadC for 72 h; (B) RT–PCR after treatment of CML-T1 and EM-2 with 3 µM AzadC for 24, 48 and 72 h; (C) immunoblotting after treatment of CML-T1 and LAMA-84 with 1 and 3 µM AzadC for 72 h; (D) RT–PCR after treatment of BV-173 for 24, 48 and 72 h and RPMI-8226 for 72 h with 3 µM AzadC.

Figure 3

Restriction-PCR-assay of the IRF-4 promoter in hematopoietic cells. (A) Simplified structure of the CpG sites in the human IRF-4 promoter region including exon 1. Each CpG motif is shown as circle and numbered (above, labeling beginning from the 3′ end moving upstream). Restriction sites, fragments (F1, F2) of the restriction-PCR-assay and regions amplified after bisulfite treatment for sequencing are shown below. The numbers in italics refer to the sequence data by Grossman et al. (30). (B and C) Restriction-PCR-assay. Restriction of DNA with EcoRI (E, no recognition site), HpaII (H, sensitive), Bsp143II (BII, sensitive), Bsh1236I (BI, sensitive) and MspI (M, resistant) and subsequent PCR amplification. Three representative IRF-4-negative (K-562, CML-T1, EM-2) and two IRF-4-positive cell lines (SD-1, BV-173) are shown. IRF-4 expression is denoted on the right. (B) PCR-fragment 1 (F1); (C) PCR-fragment 2 (F2).

Figure 4

Methylation pattern of CpG sites in the IRF-4 promoter region. Methylation is detected via sequencing of bisulfite-treated DNA from hematopoietic cells. (A) Schematic figure of the IRF-4 promoter region (see Figure 3A) for each cell line. Each CpG motif is shown as circle and numbered (above, labeling beginning from the 3′ end moving upstream), white circles mean no methylated clone (from eight), gray circles mean one to four methylated clones and black circles mean five to eight methylated clones. IRF-4 expression is denoted on the right. Below, CpG sites maybe responsible for methylation-dependent IRF-4 silencing (arrows); R1, region with generally high methylation; R2, region with correlation between methylation and IRF-4 expression; R3, region with generally low methylation. Putative binding sites for transcription factors are shown above (5× AP1, 3× Sp1, 2× Ets-1, 2× κB, 1× NF-3, 1× CD28RE, 1× PU.1). (B) Schematic figure of specific region 2 (R2): CpG sites from #10 to #22 are shown above. Number of methylated clones (from eight) is shown for each cell line.

Figure 5

Investigation of putative mechanisms for IRF-4 deregulation. (A–C) Influence of in vitro methylation on the activity of an IRF-4 promoter-reporter gene construct. (A) Control of complete methylation via restriction. Digestion of the construct before (lanes 2, 4 and 6) and after methylation (lanes 1, 3 and 5) with respective methylation-sensitive (HpaII, lanes 1 and 2) and -resistant endonucleases (MspI, lanes 3 and 4). (B and C) Reporter gene assays with non-methylated control (co) and in vitro methylated (met) IRF-4 promoter constructs and SD-1 (B) or Jurkat cells (C). The promoter activity is displayed as fold increase to non-methylated construct (via ratio of firefly to renilla luciferase). (D) Expression of DNMT and MBP mRNA in various hematopoietic cells. RT–PCR of various hematopoietic cells: SD-1 (lane 1), RPMI-8226 (lane 2), BV-173 (lane 3), U-937 (lane 4), CML-T1 (lane 5), LAMA-84 (lane 6), Jurkat (lane 7), EM-2 (lane 8) and K-562 (lane 9). β-Actin is used as reference.

Figure 6

Expression of ICSBP in hematopoietic cells after treatment with AzadC. Representative experiments are shown. (A) RT–PCR after treatment of CML-T1 and EM-2 with 3 µM AzadC for 24, 48 and 72 h; (B) RT–PCR after treatment of LAMA-84 and U-937 with 3 µM AzadC for 24, 48 and 72 h.