A Comprehensive Analysis of Allelic Methylation Status of CpG Islands on Human Chromosome 21q

Abstract

Approximately half of all human genes have CpG islands (CGIs)around their promoter regions. Although CGIs usually escape methylation, those on Chromosome X in females and those in the vicinity of imprinted genes are exceptions: They have both methylated and unmethylated alleles to display a “composite” pattern in methylation analysis. In addition, aberrant methylation of CGIs is known to often occur in cancer cells. Here we developed a simple HpaII-McrBC PCR method for discrimination of full, null, incomplete, and composite methylation patterns, and applied it to all computationally identified CGIs on human Chromosome 21q. This comprehensive analysis revealed that, although most CGIs (103 out of 149)escape methylation, a sizable fraction (31 out of 149)are fully methylated even in normal peripheral blood cells. Furthermore, we identified seven CGIs showing the composite methylation, and demonstrated that three of them are indeed methylated monoallelically. Further analyses using informative pedigrees revealed that two of the three are subject to maternal allele-specific methylation. Intriguingly, the other CGI is methylated in an allele-specific but parental-origin-independent manner. Thus, the cell seems to have a broader repertoire of methylating CGIs than previously thought, and our approach may contribute to uncover novel modes of allelic methylation

Comparative analysis of DNA methylation in transgenic mice with unstable CGG repeats from FMR1 gene

“…McrBC and HpaII assay similar to that of Yamada et al 22 was used for evaluating DNA methylation. McrBC cleaves only if the CpG dinucleotides within its half sites are methylated.…”

Re-expression of DNA methylation-silenced CD44 gene in a resistant NB4 cell line: rescue of CD44-dependent cell death by cAMP

“…24 Three restriction enzymes exhibiting complementary methylation sensitivity were used: the methylation-sensitive restriction enzyme HpaII, its methylation-insensitive isoschizomer MspI (control), both acting at CCGG sites, as well as McrBC, a restriction enzyme that cleaves only methylated DNA (in contrast to HpaII) at G/A m cytosines. Of note, this restriction enzyme cannot digest the C m CGG HpaII specific sites.…”

Evaluation of a Quantitative DNA Methylation Analysis Technique using Methylation-Sensitive/Dependent Restriction Enzymes and Real-Time PCR

“…This technique has three basic steps: (1) the digestion of a DNA sample of interest with several methylationsensitive restriction enzymes (MSREs) and a methylation-dependent restriction enzyme (MDRE); (2) the designing of primers to specific genomic regions; (3) a real-time PCR amplification reaction to monitor the formation of the PCR product. A similar strategy combining a MSRE and MDRE has been successfully used previously; 11 however, the method was limited to a non-quantitative assessment. Although this strategy investigates fewer CpGs than the bisulfite-sequencing technique, by generating data in a single day this method is substantially faster and more economical.…”