Marcel W. Coolen

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

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.

Bisulfite sequencing of chromatin immunoprecipitated DNA (BisChIP-seq) directly informs methylation status of histone-modified DNA

The complex relationship between DNA methylation, chromatin modification, and underlying DNA sequence is often difficult to unravel with existing technologies. Here, we describe a novel technique based on high-throughput sequencing of bisulfite-treated chromatin immunoprecipitated DNA (BisChIP-seq), which can directly interrogate genetic and epigenetic processes that occur in normal and diseased cells. Unlike most previous reports based on correlative techniques, we found using direct bisulfite sequencing of Polycomb H3K27me3-enriched DNA from normal and prostate cancer cells that DNA methylation and H3K27me3-marked histones are not always mutually exclusive, but can co-occur in a genomic region-dependent manner. Notably, in cancer, the co-dependency of marks is largely redistributed with an increase of the dual repressive marks at CpG islands and transcription start sites of silent genes. In contrast, there is a loss of DNA methylation in intergenic H3K27me3-marked regions. Allele-specific methylation status derived from the BisChIP-seq data clearly showed that both methylated and unmethylated alleles can simultaneously be associated with H3K27me3 histones, highlighting that DNA methylation status in these regions is not dependent on Polycomb chromatin status. BisChIP-seq is a novel approach that can be widely applied to directly interrogate the genomic relationship between allele-specific DNA methylation, histone modification, or other important epigenetic regulators.

Evaluation of affinity-based genome-wide DNA methylation data: Effects of CpG density, amplification bias, and copy number variation

DNA methylation is an essential epigenetic modification that plays a key role associated with the regulation of gene expression during differentiation, but in disease states such as cancer, the DNA methylation landscape is often deregulated. There are now numerous technologies available to interrogate the DNA methylation status of CpG sites in a targeted or genome-wide fashion, but each method, due to intrinsic biases, potentially interrogates different fractions of the genome. In this study, we compare the affinity-purification of methylated DNA between two popular genome-wide techniques, methylated DNA immunoprecipitation (MeDIP) and methyl-CpG binding domain-based capture (MBDCap), and show that each technique operates in a different domain of the CpG density landscape. We explored the effect of whole-genome amplification and illustrate that it can reduce sensitivity for detecting DNA methylation in GC-rich regions of the genome. By using MBDCap, we compare and contrast microarray- and sequencing-based readouts and highlight the impact that copy number variation (CNV) can make in differential comparisons of methylomes. These studies reveal that the analysis of DNA methylation data and genome coverage is highly dependent on the method employed, and consideration must be made in light of the GC content, the extent of DNA amplification, and the copy number.

Impact of the Genome on the Epigenome Is Manifested in DNA Methylation Patterns of Imprinted Regions in Monozygotic and Dizygotic Twins

One of the best studied read-outs of epigenetic change is the differential expression of imprinted genes, controlled by differential methylation of imprinted control regions (ICRs). To address the impact of genotype on the epigenome, we performed a detailed study in 128 pairs of monozygotic (MZ) and 128 pairs of dizygotic (DZ) twins, interrogating the DNA methylation status of the ICRs of IGF2, H19, KCNQ1, GNAS and the non-imprinted gene RUNX1. While we found a similar overall pattern of methylation between MZ and DZ twins, we also observed a high degree of variability in individual CpG methylation levels, notably at the H19/IGF2 loci. A degree of methylation plasticity independent of the genome sequence was observed, with both local and regional CpG methylation changes, discordant between MZ and DZ individual pairs. However, concordant gains or losses of methylation, within individual twin pairs were more common in MZ than DZ twin pairs, indicating that de novo and/or maintenance methylation is influenced by the underlying DNA sequence. Specifically, for the first time we showed that the rs10732516 [A] polymorphism, located in a critical CTCF binding site in the H19 ICR locus, is strongly associated with increased hypermethylation of specific CpG sites in the maternal H19 allele. Together, our results highlight the impact of the genome on the epigenome and demonstrate that while DNA methylation states are tightly maintained between genetically identical and related individuals, there remains considerable epigenetic variation that may contribute to disease susceptibility.

Repitools: an R package for the analysis of enrichment-based epigenomic data

Summary: Epigenetics, the study of heritable somatic phenotypic changes not related to DNA sequence, has emerged as a critical component of the landscape of gene regulation. The epigenetic layers, such as DNA methylation, histone modifications and nuclear architecture are now being extensively studied in many cell types and disease settings. Few software tools exist to summarize and interpret these datasets. We have created a toolbox of procedures to interrogate and visualize epigenomic data (both array- and sequencing-based) and make available a software package for the cross-platform R language. Availability: The package is freely available under LGPL from the R-Forge web site (http://repitools.r-forge.r-project.org/) Contact: mrobinson@wehi.edu.au

Dietary methyl donor deficiency during pregnancy in rats shapes learning and anxiety in offspring

Two important lines of research have enhanced our understanding of the molecular role of nutrition in influencing behavior. First, exposure to an adverse environment during early life can influence the long-term behavior of the offspring. Second, regulation of the nervous system development and functioning appears to involve epigenetic mechanisms that require a continuous supply of methyl group donors in food. We hypothesized that a maternal diet during pregnancy deficient in methyl donors (MDD) may lead to altered behavior in offspring through permanent changes in hippocampal DNA methylation. We used a rat model of prenatal dietary MDD to test this hypothesis in female offspring as they aged. Prenatal MDD reduced birth weight, litter size, and newborn viability. Aged female offspring of MDD mothers showed increased anxiety and increased learning ability in comparison with control diet group offspring. To explore the role of MDD on epigenetic mechanisms in the brain of adult offspring, we studied expression and methylation of 4 selected genes coding for glucocorticoid receptor, hydroxysteroid dehydrogenase 11 type 2, neuronatin, and reelin proteins in the hippocampus. No major group differences in methylation or expression of the studied genes were detected, except for a significant down-regulation of the reelin gene in the MDD female offspring. The prenatal MDD diet caused intrauterine growth restriction, associated with long-term effects on the behavior of the offspring. However, the observed behavioral differences between the MDD and control diet offspring cannot be explained by epigenetic regulation of the specific genes investigated in this study.

Regulatory Roles for Long ncRNA and mRNA

Recent advances in high-throughput sequencing technology have identified the transcription of a much larger portion of the genome than previously anticipated. Especially in the context of cancer it has become clear that aberrant transcription of both protein-coding and long non-coding RNAs (lncRNAs) are frequent events. The current dogma of RNA function describes mRNA to be responsible for the synthesis of proteins, whereas non-coding RNA can have regulatory or epigenetic functions. However, this distinction between protein coding and regulatory ability of transcripts may not be that strict. Here, we review the increasing body of evidence for the existence of multifunctional RNAs that have both protein-coding and trans-regulatory roles. Moreover, we demonstrate that coding transcripts bind to components of the Polycomb Repressor Complex 2 (PRC2) with similar affinities as non-coding transcripts, revealing potential epigenetic regulation by mRNAs. We hypothesize that studies on the regulatory ability of disease-associated mRNAs will form an important new field of research.

Epigenetic Deregulation Across Chromosome 2q14.2 Differentiates Normal from Prostate Cancer and Provides a Regional Panel of Novel DNA Methylation Cancer Biomarkers

Background: Previously, we showed that gene suppression commonly occurs across chromosome 2q14.2 in colorectal cancer, through a process of long-range epigenetic silencing (LRES), involving a combination of DNA methylation and repressive histone modifications. We now investigate whether LRES also occurs in prostate cancer across this 4-Mb region and whether differential DNA methylation of 2q14.2 genes could provide a regional panel of prostate cancer biomarkers. Methods: We used highly sensitive DNA methylation headloop PCR assays that can detect 10 to 25 pg of methylated DNA with a specificity of at least 1:1,000, and chromatin immunoprecipitation assays to investigate regional epigenetic remodeling across 2q14.2 in prostate cancer, in a cohort of 195 primary prostate tumors and 90 matched normal controls. Results: Prostate cancer cells exhibit concordant deacetylation and methylation of histone H3 Lysine 9 (H3K9Ac and H3K9me2, respectively), and localized DNA hypermethylation of EN1, SCTR, and INHBB and corresponding loss of H3K27me3. EN1 and SCTR were frequently methylated (65% and 53%, respectively), whereas INHBB was less frequently methylated. Conclusions: Consistent with LRES in colorectal cancer, we found regional epigenetic remodeling across 2q14.2 in prostate cancer. Concordant methylation of EN1 and SCTR was able to differentiate cancer from normal (P < 0.0001) and improved the diagnostic specificity of GSTP1 methylation for prostate cancer detection by 26%. Impact: For the first time we show that DNA methylation of EN1 and SCTR promoters provide potential novel biomarkers for prostate cancer detection and in combination with GSTP1 methylation can add increased specificity and sensitivity to improve diagnostic potential. Cancer Epidemiol Biomarkers Prev; 20(1); 148–59. ©2011 AACR.

GWAS of DNA methylation variation within imprinting control regions suggests parent-of-origin association.

Imprinting control regions (ICRs) play a fundamental role in establishing and maintaining the non-random monoallelic expression of certain genes, via common regulatory elements such as non-coding RNAs and differentially methylated regions (DMRs) of DNA. We recently surveyed DNA methylation levels within four ICRs (H19-ICR, IGF2-DMR, KvDMR, and NESPAS-ICR) in whole-blood genomic DNA from 128 monozygotic (MZ) and 128 dizygotic (DZ) human twin pairs. Our analyses revealed high individual variation and intra-domain covariation in methylation levels across CpGs and emphasized the interaction between epigenetic variation and the underlying genetic sequence in a parent-of-origin fashion. Here, we extend our analysis to conduct two genome-wide screenings of single nucleotide polymorphisms (SNPs) underlying either intra-domain covariation or parent-of-origin-dependent association with methylation status at individual CpG sites located within ICRs. Although genome-wide significance was not surpassed due to sample size limitations, the most significantly associated SNPs found through multiple-trait genome-wide association (MQFAM) included the previously described rs10732516, which is located in the vicinity of the H19-ICR. Similarly, we identified an association between rs965808 and methylation status within the NESPAS-ICR. This SNP is positioned within an intronic region of the overlapping genes GNAS and GNAS-AS1, which are imprinted genes regulated by the NESPAS-ICR. Sixteen other SNPs located in regions apart from the analyzed regions displayed suggestive association with intra-domain methylation. Additionally, we identified 13 SNPs displaying parent-of-origin association with individual methylation sites through family-based association testing. In this exploratory study, we show the value and feasibility of using alternative GWAS approaches in the study of the interaction between epigenetic state and genetic sequence within imprinting regulatory domains. Despite the relatively small sample size, we identified a number of SNPs displaying suggestive association either in a domain-wide or in a parent-of-origin fashion. Nevertheless, these associations will require future experimental validation or replication in larger and independent samples.

Dynamics of bivalent chromatin domains upon drug induced reactivation and resilencing in cancer cells

Epigenetic deregulation revealed by altered profiles of DNA methylation and histone modifications is a frequent event in cancer cells and results in abnormal patterns of gene expression. Cancer silenced genes constitute prime therapeutic targets and considerable progress has been made in the epigenetic characterization of the chromatin scenarios associated with their inactivation and drug induced reactivation. Despite these advances, the mechanisms involved in the maintenance or resetting of epigenetic states in both physiological and pharmacological situations are poorly known. To get insights into the dynamics of chromatin regulation upon drug-induced reactivation, we have investigated the epigenetic profiles of two chromosomal regions undergoing long range epigenetic silencing in colon cancer cells in time-course settings after exposure of cells to chromatin reactivating agents. The DNA methylation states and the balance between histone H3K4 methylation and H3K27 methylation marks clearly define groups of genes with alternative responses to therapy. We show that the expected epigenetic remodeling induced by the reactivating drugs, just achieves a transient disruption of the bivalent states, which overcome the treatment and restore the transcriptional silencing approximately four weeks after drug exposure. The interplay between DNA methylation and bivalent histone marks appears to configure a plastic but stable chromatin scenario that is fully restored in silenced genes after drug withdrawal. These data suggest that improvement of epigenetic therapies may be achieved by designing strategies with long lasting effects.