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
ABSTRACT 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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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.Molecular biology of the cell 04/2015; DOI:10.1091/mbc.E15-01-0013 · 5.98 Impact Factor
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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.American Journal of Medical Genetics Part B Neuropsychiatric Genetics 10/2014; 165(7). DOI:10.1002/ajmg.b.32265 · 3.27 Impact Factor
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ABSTRACT: Recent advancements in sequencing-based DNA methylation profiling methods provide an unprecedented opportunity to map complete DNA methylomes. These include whole genome bisulfite sequencing (WGBS, MethylC-seq or BS-seq), Reduced-Representation Bisulfite-Sequencing (RRBS), and enrichment-based methods such as MeDIP-seq, MBD-seq and MRE-seq. These methods yield largely comparable results, but differ significantly in extent of genomic CpG coverage, resolution, quantitative accuracy, and cost, at least while using current algorithms to interrogate the data. None of these existing methods provides single-CpG resolution, comprehensive genome-wide coverage, and cost feasibility for a typical laboratory. We introduce methylCRF, a novel Conditional Random Fields-based algorithm that integrates methylated DNA immunoprecipitation (MeDIP-seq) and methylation-sensitive restriction enzyme (MRE-seq) sequencing data to predict DNA methylation levels at single CpG resolution. Our method is a combined computational and experimental strategy to produce DNA methylomes of all 28 million CpGs in the human genome for a fraction (<10%) of the cost of whole genome bisulfite sequencing methods. MethylCRF was benchmarked for accuracy against Infinium arrays, RRBS, WGBS sequencing and locus specific-bisulfite sequencing performed on the same embryonic stem cell line. MethylCRF transformation of MeDIP-seq/MRE-seq was equivalent to a biological replicate of WGBS in quantification, coverage and resolution. We used conventional bisulfite conversion, PCR, cloning and sequencing to validate loci where our predictions do not agree with whole genome bisulfite data, and in 11 out of 12 cases methylCRF predictions of methylation level agree better with validated results than does whole genome bisulfite sequencing. Therefore, methylCRF transformation of MeDIP-seq/MRE-seq data provides an accurate, inexpensive and widely accessible strategy to create full DNA methylomes.Genome Research 06/2013; DOI:10.1101/gr.152231.112 · 13.85 Impact Factor