MBD-isolated Genome Sequencing provides a high-throughput and comprehensive survey of DNA methylation in the human genome

Abstract

DNA methylation is an epigenetic modification involved in both normal developmental processes and disease states through the modulation of gene expression and the maintenance of genomic organization. Conventional methods of DNA methylation analysis, such as bisulfite sequencing, methylation sensitive restriction enzyme digestion and array-based detection techniques, have major limitations that impede high-throughput genome-wide analysis. We describe a novel technique, MBD-isolated Genome Sequencing (MiGS), which combines precipitation of methylated DNA by recombinant methyl-CpG binding domain of MBD2 protein and sequencing of the isolated DNA by a massively parallel sequencer. We utilized MiGS to study three isogenic cancer cell lines with varying degrees of DNA methylation. We successfully detected previously known methylated regions in these cells and identified hundreds of novel methylated regions. This technique is highly specific and sensitive and can be applied to any biological settings to identify differentially methylated regions at the genomic scale.

A methylome-wide study of aging using massively parallel sequencing of the methyl-CpG-enriched genomic fraction from blood in over 700 subjects

“…Our use of MBD protein, which only binds to methylated CpGs (30), led us to focus on the 27 million autosomal CpG sites in the human genome. Methylation measurements were obtained for each CpG by estimating the number of sequenced fragments covering it (37).…”

“…One such method is affinity-based capture. In two common variants of this approach, the methylated genomic fraction is captured using methyl-CpG binding domain (MBD) protein (30) or antibodies specific to 5 m C (31), followed by sequencing on an NGS instrument. The number of fragments mapping to a locus approximates the level of methylation.…”

Analysis of DNA modifications in aging research

“…This led initially to development of affinity-based methods in which modified DNA was Bpulled^out from unmethylated DNA by immunoprecipitation in a manner similar to chromatin immunoprecipitation sequencing (ChIP-Seq) (Furey 2012). Methylated DNA immunoprecipitation (meDIP-Seq) (Weber et al 2005;Jorgensen et al 2006), methyl-binding domain sequencing (MBD-Seq) (Rauch and Pfeifer 2005;Serre et al 2010), and a number of variations of these techniques (see Lister and Ecker 2009;Laird 2010) were developed. However, the limitation to any affinity-based approach is that the specific methylation sites (or hydroxymethylation; Tan et al 2013) cannot be determined, only a general genomic region of methylation.…”

Interplay of Binding Stoichiometry and Recognition Specificity for the Interaction of MBD2b Protein and Methylated DNA Revealed by Affinity Capillary Electrophoresis Coupled with Laser-Induced Fluorescence Analysis

“…Human gen promoters of about 60% are associated with CpG islands, which display high CpG density and are usually free of methylation in healthy cells. [2][3][4]6 Dynamic DNA methylation/demethylation regulates many cellular processes, including nuclear reprogramming, transcription, genomic imprinting, and X-chromosome inactivation. 4−9 Dysregulation of DNA methylation occurs in many cancer cells, in which the hypermethylation of promoter CpG islands may inactivate some tumor suppressor genes.…”

“…3,12,20 Among all known MBD proteins, MBD2 has the highest affinity for methylated DNA and the greatest capacity to differentiate between methylated and unmethylated DNA. 3 On the basis of the binding property of the MBD2 protein, a series of assays for detecting DNA methylation was established, e.g., methylated-CpG island recovery assay, 1 MBD-isolated genome sequencing, 4 and methylated DNA precipitation. 5 MBD proteins link DNA methylation with transcriptional repression via the recruitment of corepressors, such as HDAC, leading to chromatin condensation.…”