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. 2005 Dec 2;33(21):6823-36.
doi: 10.1093/nar/gki987. Print 2005.

Analysis of Repetitive Element DNA Methylation by MethyLight

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Free PMC article

Analysis of Repetitive Element DNA Methylation by MethyLight

Daniel J Weisenberger et al. Nucleic Acids Res. .
Free PMC article

Abstract

Repetitive elements represent a large portion of the human genome and contain much of the CpG methylation found in normal human postnatal somatic tissues. Loss of DNA methylation in these sequences might account for most of the global hypomethylation that characterizes a large percentage of human cancers that have been studied. There is widespread interest in correlating the genomic 5-methylcytosine content with clinical outcome, dietary history, lifestyle, etc. However, a high-throughput, accurate and easily accessible technique that can be applied even to paraffin-embedded tissue DNA is not yet available. Here, we report the development of quantitative MethyLight assays to determine the levels of methylated and unmethylated repeats, namely, Alu and LINE-1 sequences and the centromeric satellite alpha (Satalpha) and juxtacentromeric satellite 2 (Sat2) DNA sequences. Methylation levels of Alu, Sat2 and LINE-1 repeats were significantly associated with global DNA methylation, as measured by high performance liquid chromatography, and the combined measurements of Alu and Sat2 methylation were highly correlative with global DNA methylation measurements. These MethyLight assays rely only on real-time PCR and provide surrogate markers for global DNA methylation analysis. We also describe a novel design strategy for the development of methylation-independent MethyLight control reactions based on Alu sequences depleted of CpG dinucleotides by evolutionary deamination on one strand. We show that one such Alu-based reaction provides a greatly improved detection of DNA for normalization in MethyLight applications and is less susceptible to normalization errors caused by cancer-associated aneuploidy and copy number changes.

Figures

Figure 1
Figure 1
An Alu consensus DNA sequence determined from the sequences of young and old individual Alu repeats. Old Alu sequences (Alu-J, SluSp, AluSx, AluSq and AluSc) and young Alu sequences (AluY, AluSb2, AluYb8, AluYa5 and AluYa8) were compared in order to generate an Alu consensus sequence for the purpose of designing an Alu-based MethyLight control reaction. Since the goal was to design a methylation-independent reaction of as many individual Alu repeats as possible, all CpG dinucleotides became a part of the consensus sequence. Old Alu sequences are the predominant form in human cells, and this is also reflected in the Alu consensus sequence. The continuous and dashed lines underneath the consensus sequence panels indicate the MethyLight PCR amplicon locations within the consensus sequence for the Alu control reaction (ALU-C4), two reactions toward the methylated consensus sequence (ALU-M2 and ALU-M3) and one reaction toward the unmethylated Alu consensus sequence (ALU-U3).
Figure 2
Figure 2
Strategy for designing an Alu-based MethyLight control reaction against the Alu consensus DNA sequence in Figure 1. Since CpGs in Alu repeats can either be methylated (red) or unmethylated (green), one cannot distinguish if a CpG dinucleotide was subjected to evolutionary deamination of a methylated CpG (yellow) or was an unmethylated CpG deaminated due to reaction with bisulfite, as both events result in a TpG after bisulfite-specific PCR. However, if the bisulfite PCR primers are designed for amplification of the strand opposite to that which was evolutionarily deaminated, a TpA sequence results (see Methylation-Independent Sequence, shaded in black), which is distinct from the PCR products from an unmethylated CpG (CpA or TpG) after bisulfite conversion.
Figure 3
Figure 3
Evaluation of the performance of the Alu-based control reaction compared with a single-copy control reaction. Serial 1:25 dilutions of bisulfite-converted, M.SssI-treated DNA were used to compare the Alu and COL2A1 control reactions by real-time PCR. The fluorescence is plotted versus the PCR cycle number for both reactions and each sample dilution is indicated.
Figure 4
Figure 4
Evaluation of MethyLight reactions toward the methylated and unmethylated versions of Alu, LINE-1, Satα and Sat2 sequences on a panel of DNA samples. Levels of methylation (in black) are expressed as PMR using DNA treated with M.SssI as a methylated reference. Levels of unmethylated DNA (in white) are expressed as PUR in which a WGA-DNA sample was used as an unmethylated reference. Each value represents the mean of 3–6 methylation measurements, except for the ALU-M3 reaction, which is the average of two measurements. Error bars indicate the standard error of the mean and have been omitted for the ALU-M3 reaction, as indicated by an asterisk, since we have only two PMR measurements for this reaction. We detected PMR or PUR values of <0.01 due to cross-reactivity of the methylated or unmethylated primers to either unmethylated or methylated template DNA, respectively, except for the ALU-M5 reaction, which gave slightly higher PMR values on the WGA-PBL DNA sample.
Figure 5
Figure 5
Correlation of MethyLight-based measurements of each repetitive element with HPLC-based global DNA methylation measurements for the samples described in Table 2. PMR values for the methylated Alu sequences (A–D), LINE-1 (E), Satα (F), Sat2 (G), are correlated with HPLC measurements. The correlation between the composite mean PMR values of ALU-M2 and SAT2-M1 reactions and global methylation measurements is shown in (H). The correlation of PUR values for the unmethylated Alu, LINE-1 and Satα sequences with HPLC-based 5-methylcytosine measurements are shown in (I–K). The HPLC data represent the mean of 2–3 measurements and the MethyLight data represent the mean of three measurements. The MethyLight and HPLC-based methylation data were correlated using linear regression analysis for each repetitive element.
Figure 6
Figure 6
MethyLight data (PMR) versus Southern blot-based Chr1 Sat2 hypomethylation densitometry scores. A score of 0, no hypomethylation; 1, small amounts of hypomethylation; 2, moderate hypomethylation; 3, strong hypomethylation on 7 normal tissues, two ICF cell lines, one control cell line and 20 cancer tissue samples (Wilms tumors and ovarian carcinomas). The data points are indicated by the squares (hypomethylation score = 0, upward triangles (hypomethylation score = 1), downward triangles (hypomethylation score = 2) and diamonds (hypomethylation score = 3). Mean PMR values are indicated by the horizontal bars. The significance of the association of both types of data after ANOVA analysis is shown.

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