Genome-Wide Analysis of DNA Methylation before-and after Exercise in the Thoroughbred Horse with MeDIP-Seq

Athletic performance is an important criteria used for the selection of superior horses. However, little is known about exercise-related epigenetic processes in the horse. DNA methylation is a key mechanism for regulating gene expression in response to environmental changes. We carried out comparative genomic analysis of genome-wide DNA methylation profiles in the blood samples of two different thoroughbred horses before and after exercise by methylated-DNA immunoprecipitation sequencing (MeDIP-Seq). Differentially methylated regions (DMRs) in the pre-and post-exercise blood samples of superior and inferior horses were identified. Exercise altered the methylation patterns. After 30 min of exercise, 596 genes were hypomethylated and 715 genes were hypermethylated in the superior horse, whereas in the inferior horse, 868 genes were hypomethylated and 794 genes were hypermethylated. These genes were analyzed based on gene ontology (GO) annotations and the exercise-related pathway patterns in the two horses were compared. After exercise, gene regions related to cell division and adhesion were hypermethylated in the superior horse, whereas regions related to cell signaling and transport were hypermethylated in the inferior horse. Analysis of the distribution of methylated CpG islands confirmed the hypomethylation in the gene-body methylation regions after exercise. The methylation patterns of transposable elements also changed after exercise. Long interspersed nuclear elements (LINEs) showed abundance of DMRs. Collectively, our results serve as a basis to study exercise-based reprogramming of epigenetic traits.     

Comparative DNA methylome analysis of endometrial carcinoma reveals complex and distinct deregulation of cancer promoters and enhancers

Background

Aberrant DNA methylation is a hallmark of many cancers. Classically there are two types of endometrial cancer, endometrioid adenocarcinoma (EAC), or Type I, and uterine papillary serous carcinoma (UPSC), or Type II. However, the whole genome DNA methylation changes in these two classical types of endometrial cancer is still unknown.

Results

Here we described complete genome-wide DNA methylome maps of EAC, UPSC, and normal endometrium by applying a combined strategy of methylated DNA immunoprecipitation sequencing (MeDIP-seq) and methylation-sensitive restriction enzyme digestion sequencing (MRE-seq). We discovered distinct genome-wide DNA methylation patterns in EAC and UPSC: 27,009 and 15,676 recurrent differentially methylated regions (DMRs) were identified respectively, compared with normal endometrium. Over 80% of DMRs were in intergenic and intronic regions. The majority of these DMRs were not interrogated on the commonly used Infinium 450K array platform. Large-scale demethylation of chromosome X was detected in UPSC, accompanied by decreased XIST expression. Importantly, we discovered that the majority of the DMRs harbored promoter or enhancer functions and are specifically associated with genes related to uterine development and disease. Among these, abnormal methylation of transposable elements (TEs) may provide a novel mechanism to deregulate normal endometrium-specific enhancers derived from specific TEs.

Conclusions

DNA methylation changes are an important signature of endometrial cancer and regulate gene expression by affecting not only proximal promoters but also distal enhancers.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-868) contains supplementary material, which is available to authorized users.     

Methylome analysis identifies a Wilms tumor epigenetic biomarker detectable in blood

Background

Wilms tumor is the most common pediatric renal malignancy and there is a clinical need for a molecular biomarker to assess treatment response and predict relapse. The known mutated genes in this tumor type show low mutation frequencies, whereas aberrant methylation at 11p15 is by far the most common aberration. We therefore analyzed the epigenome, rather than the genome, to identify ubiquitous tumor-specific biomarkers.

Results

Methylome analysis of matched normal kidney and Wilms tumor identifies 309 preliminary methylation variable positions which we translate into three differentially methylated regions (DMRs) for use as tumor-specific biomarkers. Using two novel algorithms we show that these three DMRs are not confounded by cell type composition. We further show that these DMRs are not methylated in embryonic blastema but are intermediately methylated in Wilms tumor precursor lesions. We validate the biomarker DMRs using two independent sample sets of normal kidney and Wilms tumor and seven Wilms tumor histological subtypes, achieving 100% and 98% correct classification, respectively. As proof-of-principle for clinical utility, we successfully use biomarker DMR-2 in a pilot analysis of cell-free circulating DNA to monitor tumor response during treatment in ten patients.

Conclusions

These findings define the most common methylated regions in Wilms tumor known to date which are not associated with their embryonic origin or precursor stage. We show that this tumor-specific methylated DNA is released into the blood circulation where it can be detected non-invasively showing potential for clinical utility.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0434-y) contains supplementary material, which is available to authorized users.     

Bisulfite Conversion of DNA: Performance Comparison of Different Kits and Methylation Quantitation of Epigenetic Biomarkers that Have the Potential to Be Used in Non-Invasive Prenatal Testing

Introduction

Epigenetic alterations, including DNA methylation, play an important role in the regulation of gene expression. Several methods exist for evaluating DNA methylation, but bisulfite sequencing remains the gold standard by which base-pair resolution of CpG methylation is achieved. The challenge of the method is that the desired outcome (conversion of unmethylated cytosines) positively correlates with the undesired side effects (DNA degradation and inappropriate conversion), thus several commercial kits try to adjust a balance between the two. The aim of this study was to compare the performance of four bisulfite conversion kits [Premium Bisulfite kit (Diagenode), EpiTect Bisulfite kit (Qiagen), MethylEdge Bisulfite Conversion System (Promega) and BisulFlash DNA Modification kit (Epigentek)] regarding conversion efficiency, DNA degradation and conversion specificity.

Methods

Performance was tested by combining fully methylated and fully unmethylated λ-DNA controls in a series of spikes by means of Sanger sequencing (0%, 25%, 50% and 100% methylated spikes) and Next-Generation Sequencing (0%, 3%, 5%, 7%, 10%, 25%, 50% and 100% methylated spikes). We also studied the methylation status of two of our previously published differentially methylated regions (DMRs) at base resolution by using spikes of chorionic villus sample in whole blood.

Results

The kits studied showed different but comparable results regarding DNA degradation, conversion efficiency and conversion specificity. However, the best performance was observed with the MethylEdge Bisulfite Conversion System (Promega) followed by the Premium Bisulfite kit (Diagenode). The DMRs, EP6 and EP10, were confirmed to be hypermethylated in the CVS and hypomethylated in whole blood.

Conclusion

Our findings indicate that the MethylEdge Bisulfite Conversion System (Promega) was shown to have the best performance among the kits. In addition, the methylation level of two of our DMRs, EP6 and EP10, was confirmed. Finally, we showed that bisulfite amplicon sequencing is a suitable approach for methylation analysis of targeted regions.     

Role of CpG deserts in the epigenetic transgenerational inheritance of differential DNA methylation regions

Background

Previously a variety of environmental toxicants were found to promote the epigenetic transgenerational inheritance of disease through differential DNA methylation regions (DMRs), termed epimutations, present in sperm. The transgenerational epimutations in sperm and somatic cells identified in a number of previous studies were further investigated.

Results

The epimutations from six different environmental exposures were found to be predominantly exposure specific with negligible overlap. The current report describes a major genomic feature of all the unique epimutations identified (535) as a very low (<10 CpG/100 bp) CpG density in sperm and somatic cells associated with transgenerational disease. The genomic locations of these epimutations were found to contain DMRs with small clusters of CpG within a general region of very low density CpG. The potential role of these epimutations on gene expression is suggested to be important.

Conclusions

Observations suggest a potential regulatory role for lower density CpG regions termed “CpG deserts”. The potential evolutionary origins of these regions is also discussed.     

Deciphering the heterogeneity in DNA methylation patterns during stem cell differentiation and reprogramming

Background

Human induced pluripotent stem cells (iPSCs) have a wide range of applications throughout the fields of basic research, disease modeling and drug screening. Epigenetic instable iPSCs with aberrant DNA methylation may divide and differentiate into cancer cells. Unfortunately, little effort has been taken to compare the epigenetic variation in iPSCs with that in differentiated cells. Here, we developed an analytical procedure to decipher the DNA methylation heterogeneity of mixed cells and further exploited it to quantitatively assess the DNA methylation variation in the methylomes of adipose-derived stem cells (ADS), mature adipocytes differentiated from ADS cells (ADS-adipose) and iPSCs reprogrammed from ADS cells (ADS-iPSCs).

Results

We observed that the degree of DNA methylation variation varies across distinct genomic regions with promoter and 5’UTR regions exhibiting low methylation variation and Satellite showing high methylation variation. Compared with differentiated cells, ADS-iPSCs possess globally decreased methylation variation, in particular in repetitive elements. Interestingly, DNA methylation variation decreases in promoter regions during differentiation but increases during reprogramming. Methylation variation in promoter regions is negatively correlated with gene expression. In addition, genes showing a bipolar methylation pattern, with both completely methylated and completely unmethylated reads, are related to the carbohydrate metabolic process, cellular development, cellular growth, proliferation, etc.

Conclusions

This study delivers a way to detect cell-subset specific methylation genes in a mixed cell population and provides a better understanding of methylation dynamics during stem cell differentiation and reprogramming.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-978) contains supplementary material, which is available to authorized users.     

DNA methylation profiles of primary colorectal carcinoma and matched liver metastasis

Background

The contribution of DNA methylation to the metastatic process in colorectal cancers (CRCs) is unclear.

Methods

We evaluated the methylation status of 13 genes (MINT1, MINT2, MINT31, MLH1, p16, p14, TIMP3, CDH1, CDH13, THBS1, MGMT, HPP1 and ERα) by bisulfite-pyrosequencing in 79 CRCs comprising 36 CRCs without liver metastasis and 43 CRCs with liver metastasis, including 16 paired primary CRCs and liver metastasis. We also performed methylated CpG island amplification microarrays (MCAM) in three paired primary and metastatic cancers.

Results

Methylation of p14, TIMP3 and HPP1 in primary CRCs progressively decreased from absence to presence of liver metastasis (13.1% vs. 4.3%; 14.8% vs. 3.7%; 43.9% vs. 35.8%, respectively) (P<.05). When paired primary and metastatic tumors were compared, only MGMT methylation was significantly higher in metastatic cancers (27.4% vs. 13.4%, P = .013), and this difference was due to an increase in methylation density rather than frequency in the majority of cases. MCAM showed an average 7.4% increase in DNA methylated genes in the metastatic samples. The numbers of differentially hypermethylated genes in the liver metastases increased with increasing time between resection of the primary and resection of the liver metastasis. Bisulfite-pyrosequencing validation in 12 paired samples showed that most of these increases were not conserved, and could be explained by differences in methylation density rather than frequency.

Conclusions

Most DNA methylation differences between primary CRCs and matched liver metastasis are due to random variation and an increase in DNA methylation density rather than de-novo inactivation and silencing. Thus, DNA methylation changes occur for the most part before progression to liver metastasis.     

Bis-class: a new classification tool of methylation status using bayes classifier and local methylation information

Background

Whole genome sequencing of bisulfite converted DNA (‘methylC-seq’) method provides comprehensive information of DNA methylation. An important application of these whole genome methylation maps is classifying each position as a methylated versus non-methylated nucleotide. A widely used current method for this purpose, the so-called binomial method, is intuitive and straightforward, but lacks power when the sequence coverage and the genome-wide methylation level are low. These problems present a particular challenge when analyzing sparsely methylated genomes, such as those of many invertebrates and plants.

Results

We demonstrate that the number of sequence reads per position from methylC-seq data displays a large variance and can be modeled as a shifted negative binomial distribution. We also show that DNA methylation levels of adjacent CpG sites are correlated, and this similarity in local DNA methylation levels extends several kilobases. Taking these observations into account, we propose a new method based on Bayesian classification to infer DNA methylation status while considering the neighborhood DNA methylation levels of a specific site. We show that our approach has higher sensitivity and better classification performance than the binomial method via multiple analyses, including computational simulations, Area Under Curve (AUC) analyses, and improved consistencies across biological replicates. This method is especially advantageous in the analyses of sparsely methylated genomes with low coverage.

Conclusions

Our method improves the existing binomial method for binary methylation calls by utilizing a posterior odds framework and incorporating local methylation information. This method should be widely applicable to the analyses of methylC-seq data from diverse sparsely methylated genomes. Bis-Class and example data are provided at a dedicated website (http://bibs.snu.ac.kr/software/Bisclass).

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-608) contains supplementary material, which is available to authorized users.     

Adult monozygotic twins discordant for intra-uterine growth have indistinguishable genome-wide DNA methylation profiles

Background

Low birth weight is associated with an increased adult metabolic disease risk. It is widely discussed that poor intra-uterine conditions could induce long-lasting epigenetic modifications, leading to systemic changes in regulation of metabolic genes. To address this, we acquire genome-wide DNA methylation profiles from saliva DNA in a unique cohort of 17 monozygotic monochorionic female twins very discordant for birth weight. We examine if adverse prenatal growth conditions experienced by the smaller co-twins lead to long-lasting DNA methylation changes.

Results

Overall, co-twins show very similar genome-wide DNA methylation profiles. Since observed differences are almost exclusively caused by variable cellular composition, an original marker-based adjustment strategy was developed to eliminate such variation at affected CpGs. Among adjusted and unchanged CpGs 3,153 are differentially methylated between the heavy and light co-twins at nominal significance, of which 45 show sensible absolute mean β-value differences. Deep bisulfite sequencing of eight such loci reveals that differences remain in the range of technical variation, arguing against a reproducible biological effect. Analysis of methylation in repetitive elements using methylation-dependent primer extension assays also indicates no significant intra-pair differences.

Conclusions

Severe intra-uterine growth differences observed within these monozygotic twins are not associated with long-lasting DNA methylation differences in cells composing saliva, detectable with up-to-date technologies. Additionally, our results indicate that uneven cell type composition can lead to spurious results and should be addressed in epigenomic studies.     

Elucidating the Landscape of Aberrant DNA Methylation in Hepatocellular Carcinoma

Background

Hepatocellular carcinoma (HCC) is one of the most common cancers and frequently presents with an advanced disease at diagnosis. There is only limited knowledge of genome-scale methylation changes in HCC.

Methods and Findings

We performed genome-wide methylation profiling in a total of 47 samples including 27 HCC and 20 adjacent normal liver tissues using the Illumina HumanMethylation450 BeadChip. We focused on differential methylation patterns in the promoter CpG islands as well as in various less studied genomic regions such as those surrounding the CpG islands, i.e. shores and shelves. Of the 485,577 loci studied, significant differential methylation (DM) was observed between HCC and adjacent normal tissues at 62,692 loci or 13% (p<1.03e-07). Of them, 61,058 loci (97%) were hypomethylated and most of these loci were located in the intergenic regions (43%) or gene bodies (33%). Our analysis also identified 10,775 differentially methylated (DM) loci (17% out of 62,692 loci) located in or surrounding the gene promoters, 4% of which reside in known Differentially Methylated Regions (DMRs) including reprogramming specific DMRs and cancer specific DMRs, while the rest (10,315) involving 4,106 genes could be potential new HCC DMR loci. Interestingly, the promoter-related DM loci occurred twice as frequently in the shores than in the actual CpG islands. We further characterized 982 DM loci in the promoter CpG islands to evaluate their potential biological function and found that the methylation changes could have effect on the signaling networks of Cellular development, Gene expression and Cell death (p = 1.0e-38), with BMP4, CDKN2A, GSTP1, and NFATC1 on the top of the gene list.

Conclusion

Substantial changes of DNA methylation at a genome-wide level were observed in HCC. Understanding epigenetic changes in HCC will help to elucidate the pathogenesis and may eventually lead to identification of molecular markers for liver cancer diagnosis, treatment and prognosis.     

Association of Childhood Chronic Physical Aggression with a DNA Methylation Signature in Adult Human T Cells

Background

Chronic physical aggression (CPA) is characterized by frequent use of physical aggression from early childhood to adolescence. Observed in approximately 5% of males, CPA is associated with early childhood adverse environments and long-term negative consequences. Alterations in DNA methylation, a covalent modification of DNA that regulates genome function, have been associated with early childhood adversity.

Aims

To test the hypothesis that a trajectory of chronic physical aggression during childhood is associated with a distinct DNA methylation profile during adulthood.

Methods

We analyzed genome-wide promoter DNA methylation profiles of T cells from two groups of adult males assessed annually for frequency of physical aggression between 6 and 15 years of age: a group with CPA and a control group. Methylation profiles covering the promoter regions of 20 000 genes and 400 microRNAs were generated using MeDIP followed by hybridization to microarrays.

Results

In total, 448 distinct gene promoters were differentially methylated in CPA. Functionally, many of these genes have previously been shown to play a role in aggression and were enriched in biological pathways affected by behavior. Their locations in the genome tended to form clusters spanning millions of bases in the genome.

Conclusions

This study provides evidence of clustered and genome-wide variation in promoter DNA methylation in young adults that associates with a history of chronic physical aggression from 6 to 15 years of age. However, longitudinal studies of methylation during early childhood will be necessary to determine if and how this methylation variation in T cells DNA plays a role in early development of chronic physical aggression.     

Differential DNA methylation with age displays both common and dynamic features across human tissues that are influenced by CpG landscape

Background

DNA methylation is an epigenetic modification that changes with age in human tissues, although the mechanisms and specificity of this process are still poorly understood. We compared CpG methylation changes with age across 283 human blood, brain, kidney, and skeletal muscle samples using methylation arrays to identify tissue-specific age effects.

Results

We found age-associated CpGs (ageCGs) that are both tissue-specific and common across tissues. Tissue-specific ageCGs are frequently located outside CpG islands with decreased methylation, and common ageCGs show the opposite trend. AgeCGs are significantly associated with poorly expressed genes, but those with decreasing methylation are linked with higher tissue-specific expression levels compared with increasing methylation. Therefore, tissue-specific gene expression may protect against common age-dependent methylation. Distinguished from other tissues, skeletal muscle ageCGs are more associated with expression, enriched near genes related to myofiber contraction, and closer to muscle-specific CTCF binding sites. Kidney-specific ageCGs are more increasingly methylated compared to other tissues as measured by affiliation with kidney-specific expressed genes. Underlying chromatin features also mark common and tissue-specific age effects reflective of poised and active chromatin states, respectively. In contrast with decreasingly methylated ageCGs, increasingly methylated ageCGs are also generally further from CTCF binding sites and enriched within lamina associated domains.

Conclusions

Our data identified common and tissue-specific DNA methylation changes with age that are reflective of CpG landscape and suggests both common and unique alterations within human tissues. Our findings also indicate that a simple epigenetic drift model is insufficient to explain all age-related changes in DNA methylation.     

DNA Methylation Profiles of Airway Epithelial Cells and PBMCs from Healthy, Atopic and Asthmatic Children

Background

Allergic inflammation is commonly observed in a number of conditions that are associated with atopy including asthma, eczema and rhinitis. However, the genetic, environmental or epigenetic factors involved in these conditions are likely to be different. Epigenetic modifications, such as DNA methylation, can be influenced by the environment and result in changes to gene expression.

Objectives

To characterize the DNA methylation pattern of airway epithelial cells (AECs) compared to peripheral blood mononuclear cells (PBMCs) and to discern differences in methylation within each cell type amongst healthy, atopic and asthmatic subjects.

Methods

PBMCs and AECs from bronchial brushings were obtained from children undergoing elective surgery for non-respiratory conditions. The children were categorized as atopic, atopic asthmatic, non-atopic asthmatic or healthy controls. Extracted DNA was bisulfite treated and 1505 CpG loci across 807 genes were analyzed using the Illumina GoldenGate Methylation Cancer Panel I. Gene expression for a subset of genes was performed using RT-PCR.

Results

We demonstrate a signature set of CpG sites that are differentially methylated in AECs as compared to PBMCs regardless of disease phenotype. Of these, 13 CpG sites were specific to healthy controls, 8 sites were only found in atopics, and 6 CpGs were unique to asthmatics. We found no differences in the methylation status of PBMCs between disease phenotypes. In AECs derived from asthmatics compared to atopics, 8 differentially methylated sites were identified including CpGs in STAT5A and CRIP1. We demonstrate STAT5A gene expression is decreased whereas CRIP1 gene expression is elevated in the AECs from asthmatic compared to both healthy and atopic subjects.

Discussion

We characterized a cell specific DNA methylation signature for AECs compared to PBMCs regardless of asthmatic or atopic status. Our data highlight the importance of understanding DNA methylation in the epithelium when studying the epithelial contribution to asthma.     

The Diagnostic Value of DNA Methylation in Leukemia: A Systematic Review and Meta-Analysis

Background

Accumulating evidence supports a role of DNA methylation in the pathogenesis of leukemia. The aim of our study was to evaluate the potential genes with aberrant DNA methylation in the prediction of leukemia risk by a comprehensive meta-analysis of the published data.

Methods

A series of meta-analyses were done among the eligible studies that were harvested after a careful filtration of the searching results from PubMed literature database. Mantel-Haenszel odds ratios and 95% confidence intervals were computed for each methylation event assuming the appropriate model.

Results

A total of 535 publications were initially retrieved from PubMed literature database. After a three-step filtration, we harvested 41 case-control articles that studied the role of gene methylation in the prediction of leukemia risk. Among the involving 30 genes, 20 genes were shown to be aberrantly methylated in the leukemia patients. A further subgroup meta-analysis by subtype of leukemia showed that CDKN2A, CDKN2B, ID4 genes were significantly hypermethylated in acute myeloid leukemia.

Conclusions

Our meta-analyses identified strong associations between a number of genes with aberrant DNA methylation and leukemia. Further studies should be required to confirm the results in the future.