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A novel methyl-binding domain protein enrichment method for identifying genome-wide tissue-specific DNA methylation from nanogram DNA samples

ABSTRACT: Growing evidence suggests that DNA methylation plays a role in tissue-specific differentiation. Current approaches to methylome analysis using MBD-enrichment are restricted to large (≥1 µg) DNA samples, limiting the analysis of small tissue samples. Here we present a technique which enables characterization of genome wide tissue-specific methylation patterns from nanogram quantities of DNA. We have developed a methodology utilizing MBD2b/MBD3L1-enrichment for methylated DNA, kinase pre-treated ligation-mediated PCR amplification (MeKL) and hybridization to the comprehensive high-throughput array for relative methylation (CHARM) customized NimbleGen 2.1M mouse tiling arrays (Feinberg_MM8_Me_HX1), which we termed MeKL-chip. The kinase-modification in combination with the addition of PEG have increased the ligation-mediated PCR amplification over 20-fold, enabling >400-fold amplification of starting DNA. We have shown that MeKL-chip can be applied to as little as 20 ng DNA, enabling comprehensive analysis of small DNA samples. Applying MeKL-chip to the mouse retina (a limited tissue source) and brain, 2498 tissue-specific differentially methylated regions (T-DMRs) were characterized. The top five T-DMRs (Rgs20, Hes2, Nfic, Cckbr and Six3os1) were validated by pyrosequencing. MeKL-chip enables genome-wide methylation analysis of nanogram quantities of DNA with a wide range of observed-to-expected CpG ratios due to the binding properties of the MBD2b/MBD3L1 protein complex. This methodology enabled the first analysis of genome-wide methylation in the mouse retina, characterizing novel T-DMRs.

ORGANISM(S): Mus musculus

PROVIDER: GSE46683 | GEO | 2013/06/10

SECONDARY ACCESSION(S): PRJNA201495

REPOSITORIES: GEO

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Project description:To further our understanding of the role of DNA methylation in development, Methylated DNA Immunoprecipitation (MeDIP) was used in conjunction with a NimbleGen promoter plus CpG island array to identify Tissue and Developmental Stage specific Differentially Methylated DNA Regions (T-DMRs and DS-DMRs) on a genome-wide basis. Four tissues (brain, heart, liver, and testis) from C57BL/6J mice were analyzed at three developmental stages (15 day embryo, E15; new born, NB; 12 week adult, AD). Almost 5,000 adult T-DMRs and 10,000 DS-DMRs were identified. Surprisingly, almost all DS-DMRs were tissue specific (i.e., methylated and ummenthylated in one or more non-overlapping tissues), indicating that the vast majority of unique sequence DNA methylation has tissue specificity. Also, many DS-DMRs were methylated at early development stages (E15 and NB) but unmethylated in adult, indicating “demethylation” has a prominent role in tissue differentiation. The pattern of DNA methylation in adult testis was dramatically different from somatic tissues in many aspects, mostly notably with a very strong bias of methylation in non-CpGi (CpG island) promoter regions (94%). Although the majority of T-DMRs and DS-DMRs tended to be in non-CpGi promoter regions, a relatively large number were also located in CpGi in promoter, intra-genic and inter-genic regions (>15% of all CpGi). Gene Ontology analysis of genes with methylation in non-CpGi promoters indicates enrichment of genes related to membrane proteins and G-protein coupled receptors. Our data also suggest regulatory roles of DNA methylation outside of promoter regions and in alternative promoter selection. Overall, our studies indicate that change in DNA methylation during development is a dynamic, widespread and tissue-specific process involving both DNA methylation and demethylation. Comparison of DNA methylation across 3 developmental stages (15 day embryo, newborn, and adult) for four tissues (brain, heart, liver and testis)

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A novel methyl-binding domain protein enrichment method for identifying genome-wide tissue-specific DNA methylation from nanogram DNA samples

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