Analysis of repetitive element DNA methylation by MethyLight

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 (Sat) 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.

... 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

... large region of heterochromatin on the long arm of the Y chromosome. Satellite (Sat) repeats are composed of 170 bp DNA sequences and represent the main DNA component of every human centromere ( 24 ). Satellite 2 (Sat2) DNA sequences are found predominantly in juxtacentromeric heterochromatin of certain human chromosomes and are most abundant in the long juxtacentromeric heterochromatin region of chromosome (Chr) 1. Sat2 sequences are composed of variants of two tandem repeats of ATTCCATTCG followed by one or two copies of ATG ( 25 ). Both Chr1 Sat and Chr1 Sat2 sequences, as well as Sat repeats present throughout all the centromeres, are highly methylated in normal postnatal tissues, hypomethylated in sperm and often hypomethylated in various cancers ( 26 29 ). In addition, Sat2 sequences on Chr1 and Chr16 are also hypomethylated in the ICF (immunodeficiency, centromeric region instability and facial abnormalities) syndrome, which usually involves mutations in DNMT3B ( 30 , 31

... technology, a quantitative, TaqMan-based real-time PCR system to analyze DNA methylation profiles ( 35 ), by extending it to the analysis of highly repeated DNA sequences. We designed and applied MethyLight assays to examine the methylation levels of Alu, LINE-1, and Chr1 centromeric Sat and juxtacentromeric Sat2 repeat sequences. We evaluated repetitive element MethyLight measurements on a panel of normal and tumor DNA samples for which accurate HPLC-based global DNA methylation measurements were available. These data suggest that methylation of either interspersed or tandem repeats can be used as a surrogate marker for estimating global DNA methylation levels. The combination (mean) of Alu and Sat2 repeat methylation measurements yielded a particularly close correlation with global genomic 5-methylcytosine content measurements obtained by HPLC. Additionally, we exploited the high Alu copy number to design an Alu-based MethyLight control reaction to sensitively determine input DNA levels for

... the mean PMR or PUR values of the three measurements. The data were plotted as PMR or PUR mean values for each repetitive element versus HPLC-based global 5-methylcytosine measurements for each sample. The composite methylation measurements of Alu and Sat2 ( Figure 5H ) were determined by obtaining the mean between the triplicate ALU-M2 and SAT2-M1 PMR values, and then plotting the composite mean PMR value versus the HPLC-based global 5-methylcytosine measurement for each sample. Linear regression

...%. These designations were only included as an indication of global 5-methylcytosine content in various tissues and in cancer cells. Southern blot-based method of determining Chr1 Sat2 hypomethylation Chr1 Sat2 hypomethylation was determined by Southern blot analysis, comparison of band patterns in autoradiograms and quantitation of Phosphorimager results as described previously ( 33 ). The MethyLight PMR values were compared with the hypomethylation scores for each sample using ANOVA. RESULTS

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Gene: SAT2 - spermidine/spermine N1-acetyltransferase family member 2 - (NCBI)

Protein : 2q4v

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