Figure 4
Association between long interspersed nuclear element-1 (LINE-1) densities and methylation levels in malignant melanoma (MM). (A,B) Average levels of DNA methylation determined by methyl-CpG binding domain sequencing (MBD-seq) analysis in normal plasma cells (NPC; n = 3), monoclonal gammopathy of undetermined significance (MGUS; n = 3) and MM (n = 9) for (A) the whole genome and at the common breakpoints (CBPs, n = 80) and (B) the respective LINE-density regions. Correlation coefficients are shown above the box plots. ***P < 0.001. Note that average methylation levels were inversely correlated with the LINE-1 densities in MM, while they were positively correlated in NPC. (C) Average DNA-methylation levels at the sample-specific breakpoints in representative MM cases. Sample names and global LINE-1 methylation levels are indicated underneath, and the number of breakpoints in each sample is also indicated. Note that LINE-1 methylation levels at sample-specific CBPs were significantly downregulated in samples with global LINE-1 hypomethylation (MM3 and MM7), whereas MM4 exhibited the inverse pattern. (D) Frequencies of the indicated LINE-1 densities (0, 0.01 to 13.43, 13.44 to 26.35, 26.36 to 39.99 and ≥40.00 per 100,000 bp) at the sample-specific breakpoints and across the whole genome in three MM cases. Samples with global LINE-1 hypomethylation showed higher LINE-1 densities at the breakpoints (MM3 and MM7), while a sample without global hypomethylation (MM4) did not show that tendency. (E) Correlations between the levels of methylation of selected LINE-1 loci and those of global LINE-1 in MM samples (n = 73). Methylation in the 5' untranslated region (UTR) of a LINE-1 located at a CBP region on 12p13.2 is shown on the left and that of another LINE-1 located at a non-breakpoint region on 12q21.1 is shown on the right. Pearson's correlation coefficients with regression lines and their 95% confidence intervals are shown on the plots. ***P < 0.001. (F) There was no correlation between methylation levels within the gene body region of a selected LINE-1 and global LINE-1 methylation levels in MM samples (n = 73). Methylation levels in open reading frame 2 (ORF2) of a LINE-1 located at 12p12.3 were compared with global LINE-1 methylation in MM (n = 73). Pearson's correlation coefficient with regression line and its 95% confidence interval are shown on the plot.