Abstract
BackgroundDNA methylation has been linked to genome regulation and dysregulation in health and disease respectively, and methods for characterizing genomic DNA methylation patterns are rapidly emerging. We have developed/refined methods for enrichment of methylated genomic fragments using the methyl-binding domain of the human MBD2 protein (MBD2-MBD) followed by analysis with high-density tiling microarrays. This MBD-chip approach was used to characterize DNA methylation patterns across all non-repetitive sequences of human chromosomes 21 and 22 at high-resolution in normal and malignant prostate cells.ResultsExamining this data using computational methods that were designed specifically for DNA methylation tiling array data revealed widespread methylation of both gene promoter and non-promoter regions in cancer and normal cells. In addition to identifying several novel cancer hypermethylated 5' gene upstream regions that mediated epigenetic gene silencing, we also found several hypermethylated 3' gene downstream, intragenic and intergenic regions. The hypermethylated intragenic regions were highly enriched for overlap with intron-exon boundaries, suggesting a possible role in regulation of alternative transcriptional start sites, exon usage and/or splicing. The hypermethylated intergenic regions showed significant enrichment for conservation across vertebrate species. A sampling of these newly identified promoter (ADAMTS1 and SCARF2 genes) and non-promoter (downstream or within DSCR9, C21orf57 and HLCS genes) hypermethylated regions were effective in distinguishing malignant from normal prostate tissues and/or cell lines.ConclusionsComparison of chromosome-wide DNA methylation patterns in normal and malignant prostate cells revealed significant methylation of gene-proximal and conserved intergenic sequences. Such analyses can be easily extended for genome-wide methylation analysis in health and disease.
Highlights
DNA methylation has been linked to genome regulation and dysregulation in health and disease respectively, and methods for characterizing genomic DNA methylation patterns are rapidly emerging
Development and refinement of methyl-binding domain (MBD)-Chip and associated computational analyses We previously showed that MBD2-MBD polypeptidebound magnetic beads could be used to efficiently and quantitatively capture methylated DNA fragments [9]
HpaII (5’-CCGG-3’ recognition sites) methyltransferases or with M.SssI (5’-CG-3’ recognition sites) methyltransferase or mock to produce genomic DNA that contains 0, 6, 10, or 37 methylated CpGs within a 262 bp GSTP1 R.AluI fragment. Subjecting these R.AluI digested DNAs to MBD2-MBD enrichment and quantifying the amount of enriched GSTP1 promoter DNA by real time PCR revealed that the degree of enrichment was proportional to the number of methylated CpGs in a nonlinear fashion (Figure 1B)
Summary
DNA methylation has been linked to genome regulation and dysregulation in health and disease respectively, and methods for characterizing genomic DNA methylation patterns are rapidly emerging. We have developed/refined methods for enrichment of methylated genomic fragments using the methyl-binding domain of the human MBD2 protein (MBD2-MBD) followed by analysis with high-density tiling microarrays This MBD-chip approach was used to characterize DNA methylation patterns across all non-repetitive sequences of human chromosomes 21 and 22 at high-resolution in normal and malignant prostate cells. Characterizing DNA methylation patterns genome-wide and with high-resolution can yield many insights into human health and disease and could provide novel DNA-based biomarkers for detection and risk stratification of various human health disorders. Such DNA based biomarkers are already entering clinical use for detection of various cancers including prostate cancer [4]. Even among the MBD polypeptides, just the MBD domain of the human MBD2 protein (MBD2-MBD) has exquisitely high affinity and specificity for 5meC, and previous reports have used this reagent to sensitively and detect methylated DNA from as few as 5 cell equivalents [9]
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