In this study, we analyzed the DNA methylation of several representative repetitive elements: LINE-1, Alu, and Sat-α. LINE-1 is an abundant retrotransposon that makes up approximately 20% of the mammalian genome. It encodes a reverse transcriptase and is able to amplify and transpose itself within the genome. Alu is one of the short interspersed elements that comprise approximately 10% of the total DNA. Alu does not encode a functional protein, but depends on the machinery of active LINE-1 for transposition [30, 31]. Sat-α is a member of the tandemly repeated sequence family, members of which are located at the centromeres of all primate chromosomes . Their presence and spread cause several inherited diseases through the induction of genomic diversity [21, 33]. To avoid their inappropriate activation, transcription of repetitive elements is regulated by epigenetic mechanisms, including DNA methylation . Dysregulation of repetitive elements, especially LINE-1, due to hypomethylation, has recently been observed in various tumors [24–27, 34–37]. Moreover, insertion of LINE-1 leads to activation of several oncogenes [38, 39]. We found strong positive correlations between methylation levels of the all repetitive elements analyzed including local-specific LINE-1 (Figure 1A; see Additional file 3, Table S3, Figure 4E), which suggests the existence of a key factor inducing global hypomethylation of repetitive elements.
Methylation within the promoter regions of protein-coding genes has been found to be generally lower in MM than MGUS or NPC [15, 16], and it declines progressively during malignant progression in plasma cells. In the present study, we found a similar decline in the methylation of repetitive elements in MGUS and MM samples. Bollati et al. also observed lower levels of repetitive-element methylation in MM, but they did not analyze methylation levels in MGUS . Reduced methylation of repetitive elements has also been reported in precancerous lesions in various organs [41, 42], suggesting that global hypomethylation is an early event during tumorigenesis in a number of malignancies. Although methylation of repetitive elements was generally down-regulated in MM compared with NPC, there was substantial case-to-case variability, and the level of methylation, especially of LINE-1, was strongly associated with the degree of copy-number loss and genomic breaks. Similar findings have been reported for other types of malignancy, suggesting that global hypomethylation is associated with chromosomal instability [26, 27, 35, 36]. Consistent with that idea, recent evidence suggests that hypomethylation-induced activation of repetitive elements is directly associated with the chromosomal instability seen in cancer [21, 38, 39, 43, 44]. In the context of those earlier reports, our findings indicate that, in malignant cells, LINE-1 may be more active and exhibit a greater potential to induce genomic alterations than other repetitive elements.
We found that LINE-1 density was greater at the 80 CBPs than elsewhere, which suggests that hypomethylation of LINE-1 may be an important factor affecting genomic breaks. For example, the 14q32.33 locus, which exhibits frequent chromosomal translocations and rearrangements in MM and shows very high LINE-1 density, is a site that exhibits possible vulnerability in MM. Conceptually similar to the CBPs described in this study, common fragile sites (CFSs) are highly unstable regions of the genome . Our data indicate that one well-known CFS, FRA6E/PARK2, is located at chromosome 6q26 , and the surrounding area is also a CBP in MM. A number of CFSs are reportedly located in regions in which there is a high density of both repetitive elements and CBPs [47, 48]. In addition, genomic loci at 12p13, where we investigated local LINE-1 methylation levels and which is a CBP, are frequently deleted, and are associated with a poor prognosis in MM . Chromosomal aberrations at 12p13 have also been reported in other types of hematological malignancy . We also identified two CBPs at 13q14, a critical region that is often deleted in MM and other lymphoid disorders, and in which enrichment of LINE-1 and repetitive elements has been reported .
Our results demonstrate that DNA-methylation levels at CBPs and regions with high LINE-1 densities decline during the development of MM. Detailed analysis of selected LINE-1 loci revealed that reductions in methylation within the 5' UTR, but not the gene body region, were significantly associated with global hypomethylation. These results indicate that hypomethylation in the 5' UTR of LINE-1 at CBP regions is deeply involved in the development of MM. However, our findings may not support a simple hypothesis that hypomethylation at LINE-1 loci is a determinant of genomic vulnerability at that position, because methylation levels of both CBP-associated and CBP-independent LINE-1 loci positively correlated with global methylation. Collectively, our results suggest that both higher LINE-1 density and hypomethylation in the 5' UTR may be critical factors inducing genomic vulnerability in MM. By contrast, we also observed that a small number of MM cases exhibited frequent genomic breaks, despite relatively high global LINE-1 methylation levels. These cases may be indicative of an underlying mechanism other than LINE-1 hypomethylation, and further study will be needed to understand the complexity of genomic vulnerability in malignant cells.
In addition to LINE-1, Sat-α is reportedly hypomethylated and transcriptionally active in various tumors [23, 52]. Although we found the strongest correlation between copy-number aberrations and LINE-1 hypomethylation, further investigation of the significance of other repetitive elements is needed. We observed a stronger association between repetitive-element methylation and chromosomal aberrations than did Bollati et al. in their earlier study . This may be attributable to differences in the technologies used to detect copy-number alterations; whereas we performed aCGH that was specialized for comprehensive and sensitive genomic analysis, Bollati et al. used fluorescence in situ hybridization to detect specific chromosomal aberrations.
Although we demonstrated a novel association between the density and hypomethylation of LINE-1 and genomic alterations in MM, there are several limitations to this study. First, the number of benign control samples was small, and as a result, the statistical power was not sufficient to find significant associations. Second, because we could not obtain control samples of normal DNA from the patients with MM, our results may have been partially influenced by inter-individual copy-number variations. In addition, as described above, we could not rule out the involvement of physiological class-switch rearrangements at the IGH locus. Third, and most importantly, because a longitudinal study was not performed to analyze the molecular changes during the development and progression of MM, the direct causal relationship between LINE-1 hypomethylation and genomic vulnerability remains to be validated in a future functional study.
Despite the aforementioned limitations, we found that LINE-1 hypomethylation is associated with a poor prognosis in MM. Even after stratification and adjustment for several confounders, the association remained statistically significant, suggesting LINE-1 hypomethylation as an independent prognostic factor. Moreover, our findings are consistent with similar results obtained in other malignancies, and is supported by several reports in which a poorer prognosis was observed in MM with non-hyperdiploidy [23, 25, 36, 37].. Our data suggest that non-hyperdiploidy, which is indicative of genomic and chromosomal loss, is associated with LINE-1 hypomethylation .