Nair SS, Coolen MW, Stirzaker C, Song JZ, Statham AL, Strbenac D et al. Comparison of methyl-DNA immunoprecipitation (MeDIP) and methyl-CpG binding domain (MBD) protein capture for genome-wide DNA methylation analysis reveal CpG sequence coverage bias. Epigenetics 6: 34-44

Epigenetics Laboratory, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia.
Epigenetics: official journal of the DNA Methylation Society (Impact Factor: 4.78). 01/2011; 6(1):34-44. DOI: 10.4161/epi.6.1.13313
Source: PubMed


DNA methylation primarily occurs at CpG dinucleotides in mammals and is a common epigenetic mark that plays a critical role in the regulation of gene expression. Profiling DNA methylation patterns across the genome is vital to understand DNA methylation changes that occur during development and in disease phenotype. In this study, we compared two commonly used approaches to enrich for methylated DNA regions of the genome, namely methyl-DNA immunoprecipitation (MeDIP) that is based on enrichment with antibodies specific for 5'-methylcytosine (5MeC), and capture of methylated DNA using a methyl-CpG binding domain-based (MBD) protein to discover differentially methylated regions (DMRs) in cancer. The enriched methylated DNA fractions were interrogated on Affymetrix promoter tiling arrays and differentially methylated regions were identified. A detailed validation study of 42 regions was performed using Sequenom MassCLEAVE technique. This detailed analysis revealed that both enrichment techniques are sensitive for detecting DMRs and preferentially identified different CpG rich regions of the prostate cancer genome, with MeDIP commonly enriching for methylated regions with a low CpG density, while MBD capture favors regions of higher CpG density and identifies the greatest proportion of CpG islands. This is the first detailed validation report comparing different methylated DNA enrichment techniques for identifying regions of differential DNA methylation. Our study highlights the importance of understanding the nuances of the methods used for DNA genome-wide methylation analyses so that accurate interpretation of the biology is not overlooked.

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Available from: Sue Clark, Jan 06, 2016
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    • "The advent of new DNA sequencing technology (Mardis, 2009), combined with enrichment of methylated DNA (Harris et al., 2010), has provided a genome-wide view of the epigenetic landscape in mammalian cells (Meissner et al., 2008; Ball et al., 2009; Lister et al., 2009; Hawkins et al., 2010; Serre et al., 2010; Nair et al., 2011). Here, we used that technology to examine the state of DNA methylation in mouse ESCs in which both Gsk-3α and Gsk-3β have been genetically deleted (Doble et al., 2007). "
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    ABSTRACT: Glycogen synthase kinase-3 (Gsk-3) is a key regulator of multiple signal transduction pathways. Recently, we described a novel role for Gsk-3 in the regulation of DNA methylation at imprinted loci in mouse embryonic stem cells (ESCs), suggesting that epigenetic changes regulated by Gsk-3 are likely an unrecognized facet of Gsk-3 signaling. Here, we extend our initial observation to the entire mouse genome by enriching for methylated DNA with the MethylMiner kit and performing next-generation sequencing (MBD-Seq) in wild-type and Gsk-3α(-/-);Gsk-3β(-/-) ESCs. Consistent with our previous data, we found that 77% of known imprinted loci have reduced DNA methylation in Gsk-3-deficient ESCs. More specifically, we unambiguously identified changes in DNA methylation within regions that have been confirmed to function as imprinting control regions (ICRs). In many cases, the reduced DNA methylation at imprinted loci in Gsk-3α(-/-);Gsk-3β(-/-) ESCs was accompanied by changes in gene expression as well. Furthermore, many of the Gsk-3-dependent differentially methylated regions (DMRs) are identical to the DMRs recently identified in uniparental ESCs. Our data demonstrate the importance of Gsk-3 activity in the maintenance of DNA methylation at a majority of the imprinted loci in ESCs, and emphasizes the importance for Gsk-3-mediated signal transduction on the epigenome. © 2015 by The American Society for Cell Biology.
    Full-text · Article · Apr 2015 · Molecular biology of the cell
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    • "Although widely used, these methods suffer from several limitations. For example, protein affinity-based enrichment of methylated cytosine has been reported to enrich for low CpG density regions and does not provide basepair resolution of methylated cytosines (Harris et al., 2010; Nair et al., 2011). In contrast, methods using methylated cytosinesensitive restriction enzymes can provide base-pair resolution, but enrich for CpG dense regions (Harris et al., 2010). "
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    ABSTRACT: Stress is a major contributor to anxiety and mood disorders. The recent discovery of epigenetic changes in the brain resulting from stress has enhanced our understanding of the mechanism by which stress is able to promote these disorders. Although epigeneticses encompasses chemical modifications that occur at both DNA and histones, much attention has been focused on stress-induced DNA methylation changes on behavior. Here, we review the effect of stress-induced DNA methylation changes on physiological mechanisms that govern behavior and cognition, dysregulation of which can be harmful to mental health. A literature review was performed in the areas of DNA methylation, stress, and their impact on the brain and psychiatric illness. Key findings center on genes involved in the hypothalamic-pituitary-adrenal axis, neurotransmission and neuroplasticity. Using animal models of different stress paradigms and clinical studies, we detail how DNA methylation changes to these genes can alter physiological mechanisms that influence behavior. Appropriate levels of gene expression in the brain play an important role in mental health. This dynamic control can be disrupted by stress-induced changes to DNA methylation patterns. Advancement in other areas of epigenetics, such as histone modifications and the discovery of the novel DNA epigenetic mark, 5-hydroxymethylcytosine, could provide additional avenues to consider when determining the epigenetic effects of stress on the brain. © 2014 Wiley Periodicals, Inc.
    Full-text · Article · Oct 2014 · American Journal of Medical Genetics Part B Neuropsychiatric Genetics
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    • "However, the MBD-seq analysis of Aid+/+ MEF-DMRs compared with Aid+/+ iPS cells did not include either of these promoters, although there were some mapped reads detected at the Nanog promoter in Aid+/+ MEFs (Fig. S13E). One possible explanation for this different result is that MBD-seq is a method based on immunoprecipitation, which can be affected by the density of CpGs [28]. "
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    ABSTRACT: It has been shown that DNA demethylation plays a pivotal role in the generation of induced pluripotent stem (iPS) cells. However, the underlying mechanism of this action is still unclear. Previous reports indicated that activation-induced cytidine deaminase (Aid, also known as Aicda) is involved in DNA demethylation in several developmental processes, as well as cell fusion-mediated reprogramming. Based on these reports, we hypothesized that Aid may be involved in the DNA demethylation that occurs during the generation of iPS cells. In this study, we examined the function of Aid in iPS cell generation using Aid knockout (Aid-/-) mice expressing a GFP reporter under the control of a pluripotent stem cell marker, Nanog. By introducing Oct3/4, Sox2, Klf4 and c-Myc, Nanog-GFP-positive iPS cells could be generated from the fibroblasts and primary B cells of Aid-/- mice. Their induction efficiency was similar to that of wild-type (Aid+/+) iPS cells. The Aid-/- iPS cells showed normal proliferation and gave rise to chimeras, indicating their capacity for self-renewal and pluripotency. A comprehensive DNA methylation analysis showed only a few differences between Aid+/+ and Aid-/- iPS cells. These data suggest that Aid does not have crucial functions in DNA demethylation during iPS cell generation.
    Full-text · Article · Apr 2014 · PLoS ONE
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