REsEaRch papER REs Ea Rch papER because LINE-1 and Alu sequences account for close to 30% of In this study, we use the term representative dispersed the total number of base pairs in the human genome;11 about sequences (ReDS) to describe subsets of five targets—LINE-1, 12% of all CpG dinucleotides fall within LINE-1s while about Alu, strong CpG islands, weak CpG islands and non-islands— 25% fall within Alu sequences.12,13 Alu and LINE-1 transpos- which were examined to determine “dispersed DNA methyla- able elements integrated into the ancestral genome more than 80 tion” in a set of placental chorionic villi at three gestational ages and 150 million years ago respectively, and over time, lineages (1st trimester villi, 2nd trimester villi and term villi) and in four have diverged significantly from the original DNA sequences.14 somatic tissues (fetal brain, fetal kidney, fetal muscle and adult Alu and LINE-1s amplify by a copy-and-paste mechanism that blood; Fig. 1). Alu and LINE-1 DNA methylation was assessed by reverse transcribes the repeat sequence into a new location.11 The Pyrosequencing of consensus sequences in addition to examining reverse transcriptase machinery is not robust: whereas the short groups of probes on the Illumina Infinium HumanMethylation27 Alu sequences of about 300 bps can be entirely transcribed,15 full BeadChip array that map to REs. DNA methylation of three LINE-1s are much longer (up to ~6,000 bps) and thus are 5' categories of CpG islands was also examined using probes from truncated at many insertion sites.16 Phylogenetic analyses divide the Illumina Infinium HumanMethylation27 BeadChip array: the existing 500,000 copies of LINE-1 and 1,000,000 copies (1) strong island probes (map to high density CpG islands); (2) of Alu14 each into three large subfamilies based on evolutionary weak island probes (map to intermediate density CpG islands) age.15,17,18 REs can have diverse effects on local genomic envi- and; (3) non-island probes (map to CpGs outside of islands) (see ronment including altering gene expression and acting as sites Methods for a full description of each assay).9 Assessment of RE for crossover, leading to duplications or deletions (reviewed in DNA methylation was compared between assays and correlation Kazazian, 200419). Full-length LINE-1 elements contain a sense of DNA methylation was assessed between the five ReDS. Our and antisense promoter close to their 5' end, in addition to a sense analyses have highlighted that DNA methylation at CpG sites promoter in the 3' UTR20 that can act as alternative promoters.21 in LINE-1 and Alu generally follow patterns distinct from other It is hypothesized that the host genome methylates RE DNA9,22 genomic sequences in the control tissues examined here. as a defense mechanism to limit detrimental transcription. However, these DNA methylation patterns differ based on the Results evolutionary age of the RE exam©ined.232Gi0ven 1the2ir coLpyanunmbder es Bioscience. and the diversity of subfamily sequences, it is unlikely that dif- Evolutionary age and assay method affect the assessment of ferent techniques used to measure RE DNA methylation, such LINE-1 and Alu DNA methylation. Characteristics that may as Pyrosequencing,1,24 MethyLight25 and CODBRoA1 mneaosurte tdhe isbitasrthiebasusesstmeen.t of RE DNA methylation include evolution- same population of Alu or LINE-1 elements. ary age,13,23 genomic location13,31 and assay method. DNA meth- There is evidence that REs are particularly prone to changes ylation of LINE-1 and Alu sequences assessed by Pyrosequencing in DNA methylation with exposure to environmental toxins. In was compared with subsets of probes from the Illumina Infinium mice, feeding mothers the xenoestrogen bisphenol A (BPA) dur- HumanMethylation27 BeadChip array that we identified as ing pregnancy results in hypomethylation of the RE upstream mapping to REs. Subfamily consensus sequences are used to of the Avy gene26 and a coordinated change in coat color in the design LINE-1 and Alu Pyrosequencing primers and thus mul- offspring.27 Furthermore, supplying the mother with a methyl tiple primer sets are available for each family. Here, we used a donor-rich diet partially counteracts the effects of BPA-induced primer set that measures four CpGs in the 5' CpG island pro- hypomethylation.26 In humans, several studies have reported moter of the LINE-1H consensus sequence and three CpGs in small reductions in LINE-1 and/or Alu DNA methylation in the body of the AluSx consensus sequence, since these primer sets adults exposed to benzene,28 particulate air pollution,3,29 DDT4 have been widely used in recent years.1,3,4,28,29,32 While there are and PCBs.4 about 130,000 copies of AluSx in the human genome,15 there are Since REs are present throughout the genome and sensitive only about 1,200 copies of LINE-1H,33 and up to 70% of these to environmental exposures, measuring the DNA methylation are expected to be 5' truncated.16 Thus, the LINE-1 and Alu status of Alu and LINE-1 sequences is an attractive method to Pyrosequencing assays sample only a small portion of each sub- rapidly and economically assess the DNA methylation status of family and are only representative of these dispersed sequences. many CpGs.1 There is however, little evidence in direct support RE array probes cover a variety of evolutionary age groups but, of LINE-1 and Alu DNA methylation as surrogates for genome- due to the design of the array, the population we could exam- wide DNA methylation25,30 or DNA methylation of other groups ine was biased toward REs incorporated into gene promoters. of genomic sequences. The self-replication of REs in addition to A variable number of array probes mapped to each of the Alu their evolutionary age and parasitic relationship with the human and LINE-1 subfamilies: old Alu (AluJ; n = 153), intermediate genome makes LINE-1 and Alu DNA methylation particularly Alu (AluS; n = 392), young Alu (AluY; n = 78) and old LINE-1 interesting yet difficult to study. In order to design and compare (LINE-1M; n = 192), intermediate LINE-1 (LINE-1P; n = experiments, there is a need to understand what LINE-1 and Alu 26), young LINE-1 (LINE-1H; n = 4). This sample represents DNA methylation assays measure, how RE DNA methylation approximately 0.52% of all AluJ, 0.11% of all AluS, 0.08% of relates to other DNA methylation assays and the conditions that all AluY and 0.23% of all LINE-1H, 0.01% of all LINE-1P and influence LINE-1 and Alu assessment. 0.03% of all LINE-1M. www.landesbioscience.com Epigenetics 653