Large-scale determination of the methylation status of retrotransposons in different tissues using a methylation tags approach

Abstract

A technique for simultaneous determination of the methylation status of numerous loci containing retroelements (REs) is reported. It is based on the observation that methylated and unmethylated areas in the genome are usually extended, and therefore the methylation of particular methyl-sensitive restriction endonuclease recognition sites might reflect the methylation status of DNA regions around them. The method includes dot-blot hybridization of repeat flanking sequences arrayed on a solid support with specifically amplified flanking regions of presumably unmethylated repeats. A multitude of flanking regions of REs adjacent to unmethylated restriction sites are amplified simultaneously, providing a complex hybridization probe. The technique thus allows the determination of the methylation status of restriction sites, which serve as tags of the methylation status of the surrounding regions. The validity of the technique was confirmed by various means, including bisulfite sequencing. The technique was successfully applied to the identification of methylation patterns of the regions surrounding 38 human-specific HERV-K(HML-2) long terminal repeats in cerebellum- and lymph node-derived genomic DNAs. The described technique can be readily adapted to the use of DNA microarray technology.

Figure 1

Scheme of the method. After restriction and adaptor ligation (stage 1), an unmethylated pool of RE flanks is selectively PCR amplified (stage 2) and hybridized (stage 3) with an (micro)array of the whole pool of the same RE-flanking sequences. Methylated and non-methylated HpaII (HhaI) restriction sites are shown by short vertical lines with black circles or without circles, respectively. REs and oligonucleotide adaptors are shown as green and black-and-white boxes, respectively. Primer positions and orientations are indicated by arrows.

Figure 2

Dot-blot hybridization with cerebellum (A and B) and lymph node (C) DNA-derived probes. LTRs are numbered from left to right, and from top to bottom. In (A) and (C), position A1 corresponds to LTR1, A2 to LTR2, etc., B1 to LTR13, etc., C1 to LTR25, etc., D1 to LTR37, and D2 to LTR38. Spots D3 in (A) and (C) and A11 in (B) correspond to 50 ng of phage λ DNA. Designation of spots in (B) corresponds to that in (A). Rows A and B in (B) represent the same materials spotted in duplicate.

Figure 3

PCR through HpaII sites of interest. (A) Cerebellum- and lymph node-derived DNAs were digested by the methyl-sensitive restriction endonuclease HpaII and amplified with primers specific to either side of the HpaII site (e.g. P1 and P2, or P3 and P2, where P2 is the primer against the LTR sequence). Methylated or unmethylated CpG/CCGG sites are designated by filled and empty circles, respectively. (B) Gel electrophoresis of the PCR products after amplification for the LTR28 (lanes 1–3), LTR30 (lanes 4–6) and LTR14 (lanes 7–9) flanks through adjacent HpaII sites. Lanes 2, 5 and 8, and 3, 6 and 9 represent PCR products generated by amplification of HpaII-digested DNAs from cerebellum and lymph node, respectively. Lanes 1, 4 and 7 correspond to the amplified DNA fragments from native placenta. Lane 10, length marker.

Figure 4

Results of bisulfite sequencing. For each of three LTR-flanking sequences, four clones were sequenced. The methylation results obtained for each of the clones are schematically presented at the bottom of the figure as lines of circles. Filled (black) and empty circles designate methylated and unmethylated CpGs, respectively.