Figure 1.
Schematic description of the MIRA method.
The diagram shows the different steps of the procedure. After DNA isolation and sonication, the DNA fragments are incubated with the methyl-CpG binding protein complex (MBD2b and MBD3L1) and the bound methylated DNA is purified with glutathione-conjugated magnetic beads capturing the complex via GST-tagged MBD2b. After end repair and linker ligation (not shown in the picture), a genome amplification reaction is performed. The amplified DNA is hybridized to a microarray. After bioinformatics analysis, the detected hyper and/or hypomethylated peaks are confirmed with COBRA (combined bisulfite restriction analysis) and bisulfite sequencing.
Table 1.
Number of peak differences in different comparisons.
Figure 2.
Examples of DNA methylation patterns in untreated and in irradiated cells.
The Figure shows several randomly selected Signalmap snapshots of the microarray signals (green) for chromosomes 1, 3, 11, 19 and the X chromosome after the indicated treatments and recovery times. Above the treatment description are the CpG islands (indicated in black). A. Signalmap snapshots of HBEC (human bronchial epithelial cells) with the treatments 0 days, 0 Gray; 0 days, 4 Gray; 7 days, 0 Gray; 7 days, 0.1 Gray; 7 days, 1 Gray; 7 days, 4 Gray. B. Signalmap snapshots of HFB (human fibroblasts) with the treatments 0 days, 10 Gray; 7 days, 0 Gray; 7 days, 0.1 Gray; 7 days, 1 Gray; 7 days, 4 Gray; 7 days, 10 Gray.
Figure 3.
Apparently altered methylation patterns in certain genes.
The picture shows six Signalmap snapshots of the microarray signals (green) from apparently differentially methylated genes after the particular radiation treatments and recovery times. Above the treatment description are the CpG islands (black). The signals framed by the red and blue rectangles are significantly (*) different methylated pairs of a control and a treatment, as determined by bioinformatics analysis. A. Signalmap snapshots of the genes MBP (0 days, 4 Gray vs. 7 days, 4 Gray (red rectangles) and 7 days, 0 Gray vs. 7 days, 0.1 Gray (blue rectangles), CLEC18C (0 days, 4 Gray vs. 7 days, 4 Gray (red rectangles) and a Y chromosomal region (7 days, 0 Gray vs. 7 days, 0.1 Gray (red rectangles) of HBEC. B. Signalmap snapshots of the genes CLEC18A (7 days, 0 Gray vs. 7 days, 1 Gray, red rectangles), SDHALP1 (7 days, 0 Gray vs. 7 days, 4 Gray, red rectangles) and ZCCHC16 (7 days, 0 Gray vs. 7 days 1 Gray, red rectangles) of HFB cells.
Figure 4.
Methylation analysis of genes with suspected differentially methylated regions.
DNA was treated with sodium bisulfite and amplified with conversion-specific COBRA (combined bisulfite restriction analysis) PCR primers. After the treatment with CpG specific restriction enzymes (E), these digests and a mock restriction digest (-) were run on a 2% agarose gel together with a 100 bp marker for analysis. A methylated (methyl.) and an unmethylated (unmethyl.) bisulfite-treated DNA sample were used as controls. A. COBRA gels of the genes MBP, CLEC18C and a Y chromosomal region in the HBEC cells with gamma radiation treatments 0 days, 0 Gray; 0 days, 4 Gray; 7 days, 0 Gray; 7 days, 0.1 Gray; 7 days, 1 Gray and 7 days, 4 Gray. B. COBRA gels of genes CLEC18A, SDHALP1 and ZCCHC16 in the HFB cells with the gamma radiation treatments 0 days, 10 Gray; 7 days, 0 Gray; 7 days, 0.1 Gray; 7 days, 1 Gray; 7 days, 4 Gray and 7 days, 10 Gray.
Figure 5.
The Figure shows the result of the bisulfite sequencing analysis. Each circle represents one CpG in the PCR product. The black circles indicate a methylated CpG and white circles indicate an unmethylated CpG. The gray circles show CpGs with an uncertain methylation status. Each row represents a single clone. A. The gene MBP and a region of the Y chromosome are shown. B. The genes SDHALP1 and ZCCHC16 are shown. On the left side, the data for untreated (control) cells are shown. The panels on the right show data for the irradiated cells. The bottom line describes the total fraction of methylated CpGs for each treatment and control.