while strong island probe methylation was not altered between Using DNA methylation of different ReDS revealed that gestations (Fig. 5C; 1st trimester, 11.15% ± 0.61; 2nd trimester, tissue to tissue DNA methylation was most variable at LINE- 11.21% ± 0.49 and term 11.82% ± 0.63). Mean LINE-1 DNA 1, non-islands and weak CpG islands and that the pattern of methylation did not change throughout gestation, although vari- DNA methylation across tissues was similar at these three ReDS. ation did decrease (Fig. 5B; 1st trimester, 62.69% ± 7.00; 2nd Correlations between LINE-1, weak CpG island and non-island trimester, 62.52% ± 4.60 and term, 62.54% ± 2.78). Thus, villi methylation also suggest that these measures of dispersed DNA gain in DNA methylation throughout gestation occurs at specific methylation may be associated with each other. Studies exam- ReDS. ining the genomic distribution of REs have demonstrated an enrichment of LINE-1 in low GC content regions and on the X Discussion chromosome,14 in addition to an underrepresentation near genes, where Alu elements are overrepresented.41 LINE-1s may be sites of The phrase “global DNA methylation” has been widely and de novo DNA methylation in the developing embryo from which indiscriminately used in the literature to describe DNA methyla- DNA methylation silencing is spread TSS.42 However, this spread tion measured by a variety of techniques. Arguably, the only true into CpG island promoters may be buffered by Alus, which are global measures of DNA methylation assess genome-wide total enriched near TSSs associated with CpG islands.31 We observed 5-mC content. However, both LINE-1 and Alu DNA methyla- lower levels of DNA methylation in CpG island promoters close tion are commonly used as surrogates for “global DNA methyla- to TSS across all tissue types and increased DNA methylation at tion,” implying that the status of the genome is being examined CpGs in island promoters further away from TSS. Kang et al. comprehensively. In this study, we examined representative mem- proposed a model of counteracting forces in the spread of DNA bers of five targets for DNA methylation (ReDS)—LINE-1, Alu, methylation toward promoters that leads to a transitional area of strong CpG island promoters, weak CpG island promoters and DNA methylation bordering CpG island promoters.31 However, non-island promoter CpGs—in placental villi, fetal organ tissues Kang et al. use a definition for CpG islands that falls in between and adult blood. We have shown that the assessment of LINE-1 the weak and strong CpG islands identified in our study. The and Alu DNA methylation is affected by assay method, evolu- spreading model of DNA methylation from LINE-1 sequences tionary age composition of REs ©and 2tiss0ue 1typ2e. ALddaitionnaldly, essuggBestsiaonesxpclaniaetionnfocr ethe.correlation we observed between distinct inter-tissue patterns of DNA methylation were observed LINE-1 and weak CpG island DNA methylation. at each of the ReDS. The subdivision of Illumina Infinium Studies using HPLC have determined that the placenta has HumanMethylation27 BeadChip array probDesinoto sntronog,twedak i sthte rloiwbestu5-tmeC.content in comparison to other normal tis- and non-island probes, showed that the gestational age-related sues.36,43,44 Thus, the placenta is often described as hypomethyl- gain in villi DNA methylation observed in other studies35-37 pre- ated compared with other fetal and adult tissues. Specific regions dominantly occurred at weak CpG island and non-island regions. have also been shown to have decreased DNA methylation in the LINE-1 and Alu sequences account for about 17% and 11% of placenta, including Alu,45 LINE-1 and regions on the X chro- the human genome, respectively,14 and only a subset of these can mosome.46 Our study confirmed previous reports that LINE-1s be interrogated by any given technique. Although many stud- are hypomethylated in the placenta vs. other tissues. Some active ies use both LINE-1 and Alu as surrogate measures for “global LINE-1 and Alu elements may play a functional role in the pla- DNA methylation,” few have examined the question of whether centa, contributing to its invasive and proliferative properties. LINE-1 and Alu DNA methylation correlate with each other Placental-specific gene expression has been shown from both and with total 5-mC levels. Choi et al. reported a correlation of LINE-121 and Alu47 sequences and may be more common in the LINE-1 with Alu DNA methylation in neuroendocrine tumors placenta due to the lower levels of LINE-1 and Alu DNA meth- but not in control samples.38 Given that REs may be sensitive ylation. Syncytin is a classic example of another type of trans- sites in the genome for changes in DNA methylation, the cor- posable element expressed exclusively in the placenta throughout relation of LINE-1 and Alu DNA methylation may be stronger gestation48 and plays a critical role in the fusion of pluripotent under pathological conditions. This theory is supported by two cytotrophoblast into differentiated syncytiotrophoblast cells.49 additional studies in cancer cells39,40 as well as our negative find- Interestingly, we found that there were not large differences in ings in control human tissues. Wang et al. found no correlation the DNA methylation of placenta compared with somatic tis- of total 5-mC, measured by Methylamp with mean LINE-1 sues at strong CpG islands or close to a TSS, in support of pre- DNA methylation in human nervous tissue.30 However, a study vious findings on the X chromosome.46 Since LINE-1 and Alu by Weisenberger et al. in blood identified a strong association of elements make up almost 30% of the genome,14 REs may bias both LINE-1 and Alu DNA methylation with total 5-mC con- genome-wide tissue comparisons of DNA methylation based on tent measured by HPLC.25 Some of this study-to-study variation total 5-mC content. may be attributed to the use of different methods for measuring Although REs are widespread elements that can be rapidly RE DNA methylation as well as total 5-mC content. Our com- and inexpensively assessed, our results suggest that LINE-1s parison of RE DNA methylation obtained by Pyrosequencing and Alus are sequences in the genome with distinct trends in vs. array probes suggests that the assessment of LINE-1 and Alu DNA methylation. In fact, LINE-1 DNA methylation may be DNA methylation is assay-dependent and thus may contribute to more closely associated with weak CpG island methylation than how these ReDS correlate with a genome-wide measure of 5-mC. “global DNA methylation.” Despite our findings, it should not 660 Epigenetics Volume 7 Issue 6