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

Verity F Oliver,Saurabh Agarwal,Jun Wan,Shannath L Merbs,Jiang Qian,Donald J Zack

doi:10.1186/1756-8935-6-17

Abstract

BackgroundGrowing evidence suggests that DNA methylation plays a role in tissue-specific differentiation. Current approaches to methylome analysis using enrichment with the methyl-binding domain protein (MBD) are restricted to large (≥1 μg) DNA samples, limiting the analysis of small tissue samples. Here we present a technique that enables characterization of genome-wide tissue-specific methylation patterns from nanogram quantities of DNA.ResultsWe 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 tiling arrays, which we termed MeKL-chip. Kinase modification in combination with the addition of PEG has increased 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 of DNA, enabling comprehensive analysis of small DNA samples. Applying MeKL-chip to the mouse retina (a limited tissue source) and brain, 2,498 tissue-specific differentially methylated regions (T-DMRs) were characterized. The top five T-DMRs (Rgs20, Hes2, Nfic, Cckbr and Six3os1) were validated by pyrosequencing.ConclusionsMeKL-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.

Highlights

Growing evidence suggests that DNA methylation plays a role in tissue-specific differentiation
With the goal of establishing a user-friendly method for assessing genome-wide DNA methylation in small DNA samples, we developed a new protocol based upon MBD2b/MBD3L1 enrichment [5] followed by amplification using modified Ligation-mediated Polymerase chain reaction (PCR) (LM-PCR), which we call MBD2b/MBD3L1 enrichment of DNA followed by KLM-PCR (MeKL)-chip
We increased the efficiency by adding a pre-ligation kinase treatment to repair DNA damage induced during fragmentation

Summary

Introduction

Growing evidence suggests that DNA methylation plays a role in tissue-specific differentiation. Current approaches to methylome analysis using enrichment with the methyl-binding domain protein (MBD) are restricted to large (≥1 μg) DNA samples, limiting the analysis of small tissue samples. We present a technique that enables characterization of genome-wide tissue-specific methylation patterns from nanogram quantities of DNA. DNA methylation is an epigenetic modification known to be important in many cellular processes, including tissue-specific gene expression. There is no standardized method for DNA methylation analysis and most arraybased methods for genome-wide profiling require input DNA in microgram quantities, making them impractical for small tissue samples (Table 1). Sodium bisulfite modification is traditionally considered the gold-standard technique for assessing methylation [1]. Alternatives to bisulfite conversion include enrichment of methylated/

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Discussion

Conclusion