DNA methylation is an indispensible epigenetic modification of mammalian genomes. Consequently there is great interest in strategies for genome-wide/whole-genome DNA methylation analysis, and immunoprecipitation-based methods have proven to be a powerful option. Such methods are rapidly shifting the bottleneck from data generation to data analysis, necessitating the development of better analytical tools. Until now, a major analytical difficulty associated with immunoprecipitation-based DNA methylation profiling has been the inability to estimate absolute methylation levels. Here we report the development of a novel cross-platform algorithm – Bayesian Tool for Methylation Analysis (Batman) – for analyzing Methylated DNA Immunoprecipitation (MeDIP) profiles generated using arrays (MeDIP-chip) or next-generation sequencing (MeDIP-seq). The latter is an approach we have developed to elucidate the first high-resolution whole-genome DNA methylation profile (DNA methylome) of any mammalian genome. MeDIP-seq/MeDIP-chip combined with Batman represent robust, quantitative, and cost-effective functional genomic strategies for elucidating the function of DNA methylation.
Perturbation of DNA methylation is frequent in cancers and has emerged as an important mechanism involved in tumorigenesis. To determine how DNA methylation is modified in the genome of primary glioma, we used Methyl-DNA immunoprecipitation (MeDIP) and Nimblegen CpG promoter microarrays to identify differentially DNA methylation sequences between primary glioma and normal brain tissue samples.
MeDIP-chip technology was used to investigate the whole-genome differential methylation patterns in glioma and normal brain tissues. Subsequently, the promoter methylation status of eight candidate genes was validated in 40 glioma samples and 4 cell lines by Sequenom's MassARRAY system. Then, the epigenetically regulated expression of these genes and the potential mechanisms were examined by chromatin immunoprecipitation and quantitative real-time PCR.
A total of 524 hypermethylated and 104 hypomethylated regions were identified in glioma. Among them, 216 hypermethylated and 60 hypomethylated regions were mapped to the promoters of known genes related to a variety of important cellular processes. Eight promoter-hypermethylated genes (ANKDD1A, GAD1, HIST1H3E, PCDHA8, PCDHA13, PHOX2B, SIX3, and SST) were confirmed in primary glioma and cell lines. Aberrant promoter methylation and changed histone modifications were associated with their reduced expression in glioma. In addition, we found loss of heterozygosity (LOH) at the miR-185 locus located in the 22q11.2 in glioma and induction of miR-185 over-expression reduced global DNA methylation and induced the expression of the promoter-hypermethylated genes in glioma cells by directly targeting the DNA methyltransferases 1.
These comprehensive data may provide new insights into the epigenetic pathogenesis of human gliomas.
DNA methylation; MiR-185; Glioma; DNMT1
The methylated DNA immunoprecipitation microarray (MeDIP-chip) is a genome-wide, high-resolution approach to detect DNA methylation in whole genome or CpG (cytosine base followed by a guanine base) islands. The method utilizes anti-methylcytosine antibody to immunoprecipitate DNA that contains highly methylated CpG sites. Enriched methylated DNA can be interrogated using DNA microarrays or by massive parallel sequencing techniques. This combined approach allows researchers to rapidly identify methylated regions in a genome-wide manner, and compare DNA methylation patterns between two samples with diversely different DNA methylation status. MeDIP-chip has been applied successfully for analyses of methylated DNA in the different targets including animal and plant tissues (1, 2). Here we present a MeDIP-chip protocol that is routinely used in our laboratory, illustrated with specific examples from MeDIP-chip analysis of breast cancer cell lines. Potential technical pitfalls and solutions are also provided to serve as workflow guidelines.
DNA methylation; epigenetics; MeDIP-chip; microarray; cancer
Epigenetic mechanisms such as microRNA and histone modification are crucially responsible for dysregulated gene expression in heart failure. In contrast, the role of DNA methylation, another well-characterized epigenetic mark, is unknown. In order to examine whether human cardiomyopathy of different etiologies are connected by a unifying pattern of DNA methylation pattern, we undertook profiling with ischaemic and idiopathic end-stage cardiomyopathic left ventricular (LV) explants from patients who had undergone cardiac transplantation compared to normal control. We performed a preliminary analysis using methylated-DNA immunoprecipitation-chip (MeDIP-chip), validated differential methylation loci by bisulfite-(BS) PCR and high throughput sequencing, and identified 3 angiogenesis-related genetic loci that were differentially methylated. Using quantitative RT-PCR, we found that the expression of these genes differed significantly between CM hearts and normal control (p<0.01). Moreover, for each individual LV tissue, differential methylation showed a predicted correlation to differential expression of the corresponding gene. Thus, differential DNA methylation exists in human cardiomyopathy. In this series of heterogenous cardiomyopathic LV explants, differential DNA methylation was found in at least 3 angiogenesis-related genes. While in other systems, changes in DNA methylation at specific genomic loci usually precede changes in the expression of corresponding genes, our current findings in cardiomyopathy merit further investigation to determine whether DNA methylation changes play a causative role in the progression of heart failure.
Alcohol exposure during development can cause variable neurofacial deficit and growth retardation known as fetal alcohol spectrum disorders (FASD). The mechanism underlying FASD is not fully understood. However, alcohol, which is known to affect methyl donor metabolism, may induce aberrant epigenetic changes contributing to FASD. Using a tightly controlled whole-embryo culture, we investigated the effect of alcohol exposure (88 mM) at early embryonic neurulation on genome-wide DNA methylation and gene expression in the C57BL/6 mouse. The DNA methylation landscape around promoter CpG islands at early mouse development was analyzed using MeDIP (methylated DNA immunoprecipitation) coupled with microarray (MeDIP-chip). At early neurulation, genes associated with high CpG promoters (HCP) had a lower ratio of methylation but a greater ratio of expression. Alcohol-induced alterations in DNA methylation were observed, particularly in genes on chromosomes 7, 10 and X; remarkably, a >10 fold increase in the number of genes with increased methylation on chromosomes 10 and X was observed in alcohol-exposed embryos with a neural tube defect phenotype compared to embryos without a neural tube defect. Significant changes in methylation were seen in imprinted genes, genes known to play roles in cell cycle, growth, apoptosis, cancer, and in a large number of genes associated with olfaction. Altered methylation was associated with significant (p < 0.01) changes in expression for 84 genes. Sequenom EpiTYPER DNA methylation analysis was used for validation of the MeDIP-chip data. Increased methylation of genes known to play a role in metabolism (Cyp4f13) and decreased methylation of genes associated with development (Nlgn3, Elavl2, Sox21 and Sim1), imprinting (Igf2r) and chromatin (Hist1h3d) was confirmed. In a mouse model for FASD, we show for the first time that alcohol exposure during early neurulation can induce aberrant changes in DNA methylation patterns with associated changes in gene expression, which together may contribute to the observed abnormal fetal development.
fetal alcohol syndrome; epigenetics; MeDIP-chip; sequenom mass array; microarray; neural tube defect
DNA methylation plays critical roles in transcriptional regulation and chromatin remodeling. Differentially methylated regions (DMRs) have important implications for development, aging and diseases. Therefore, genome-wide mapping of DMRs across various temporal and spatial methylomes is important in revealing the impact of epigenetic modifications on heritable phenotypic variation. We present a quantitative approach, quantitative differentially methylated regions (QDMRs), to quantify methylation difference and identify DMRs from genome-wide methylation profiles by adapting Shannon entropy. QDMR was applied to synthetic methylation patterns and methylation profiles detected by methylated DNA immunoprecipitation microarray (MeDIP-chip) in human tissues/cells. This approach can give a reasonable quantitative measure of methylation difference across multiple samples. Then DMR threshold was determined from methylation probability model. Using this threshold, QDMR identified 10 651 tissue DMRs which are related to the genes enriched for cell differentiation, including 4740 DMRs not identified by the method developed by Rakyan et al. QDMR can also measure the sample specificity of each DMR. Finally, the application to methylation profiles detected by reduced representation bisulphite sequencing (RRBS) in mouse showed the platform-free and species-free nature of QDMR. This approach provides an effective tool for the high-throughput identification of potential functional regions involved in epigenetic regulation.
Methylated DNA immunoprecipitation followed by high-throughput sequencing (MeDIP-seq) has the potential to identify changes in DNA methylation important in cancer development. In order to understand the role of epigenetic modulation in the development of acute myeloid leukemia (AML) we have applied MeDIP-seq to the DNA of 12 AML patients and 4 normal bone marrows. This analysis revealed leukemia-associated differentially methylated regions that included gene promoters, gene bodies, CpG islands and CpG island shores. Two genes (SPHKAP and DPP6) with significantly methylated promoters were of interest and further analysis of their expression showed them to be repressed in AML. We also demonstrated considerable cytogenetic subtype specificity in the methylomes affecting different genomic features. Significantly distinct patterns of hypomethylation of certain interspersed repeat elements were associated with cytogenetic subtypes. The methylation patterns of members of the SINE family tightly clustered all leukemic patients with an enrichment of Alu repeats with a high CpG density (P<0.0001). We were able to demonstrate significant inverse correlation between intragenic interspersed repeat sequence methylation and gene expression with SINEs showing the strongest inverse correlation (R2 = 0.7). We conclude that the alterations in DNA methylation that accompany the development of AML affect not only the promoters, but also the non-promoter genomic features, with significant demethylation of certain interspersed repeat DNA elements being associated with AML cytogenetic subtypes. MeDIP-seq data were validated using bisulfite pyrosequencing and the Infinium array.
The parent-of-origin specific expression of imprinted genes relies on DNA methylation of CpG-dinucleotides at differentially methylated regions (DMRs) during gametogenesis. To date, four paternally methylated DMRs have been identified in screens based on conventional approaches. These DMRs are linked to the imprinted genes H19, Gtl2 (IG-DMR), Rasgrf1 and, most recently, Zdbf2 which encodes zinc finger, DBF-type containing 2. In this study, we applied a novel methylated-DNA immunoprecipitation-on-chip (meDIP-on-chip) method to genomic DNA from mouse parthenogenetic- and androgenetic-derived stem cells and sperm and identified 458 putative DMRs. This included the majority of known DMRs. We further characterized the paternally methylated Zdbf2/ZDBF2 DMR. In mice, this extensive germ line DMR spanned 16 kb and possessed an unusual tripartite structure. Methylation was dependent on DNA methyltransferase 3a (Dnmt3a), similar to H19 DMR and IG-DMR. In both humans and mice, the adjacent gene, Gpr1/GPR1, which encodes a G-protein-coupled receptor 1 protein with transmembrane domain, was also imprinted and paternally expressed. The Gpr1-Zdbf2 domain was most similar to the Rasgrf1 domain as both DNA methylation and the actively expressed allele were in cis on the paternal chromosome. This work demonstrates the effectiveness of meDIP-on-chip as a technique for identifying DMRs.
The FTO gene was recently identified as a susceptibility locus for both obesity and type 2 diabetes by whole-genome association analyses of several European populations. We tested for an association between FTO risk alleles and obesity and diabetes in a well-characterized multiethnic cohort of postmenopausal women in the United States. We genotyped two most significantly associated single-nucleotide polymorphisms (SNPs) (rs9939609 and rs8050136) in intron 1 of FTO gene in a nested case–control study of 1,517 diabetes cases and 2,123 controls from the Women’s Health Initiative–Observational Study (WHI-OS). The allelic frequencies of either rs9939609 or rs8050136 differed widely across four ethnic groups. The frequency of the rare allele A of rs9939609 among controls was much lower in Asians/Pacific Islanders (17%) than in blacks (45%), whites (40%), and Hispanics (31%). We found significant associations of rs9939609 with BMI and waist circumference in white and Hispanic women, but not among black and Asian/Pacific Islander women. On average, each copy of the risk-allele A at rs9939609 was significantly associated with 0.45 kg/m2 increase in BMI (95% confidence interval (CI): 0.16–0.74; P = 0.004) and 0.97 cm increase in waist circumference (95% CI: 0.21–0.65; P = 0.0002). Similar results were observed for rs8050136. However, we found no significant genetic associations with diabetes risk, either within the full study sample or in any ethnic group. In conclusion, common genetic variants in the intron 1 of FTO gene may confer a modest susceptibility to obesity in an ethnicity-specific manner, but may be unlikely to contribute to a clinically significant diabetes risk.
DNA methylation is an important epigenetic modification that regulates development and plays a role in the pathophysiology of many diseases. It is dynamically changed during germline development. Methylated DNA immunoprecipitation (MeDIP) is an efficient, cost-effective method for locus-specific and genome-wide analysis. Methylated DNA fragments are enriched by a 5-methylcytidine-recognizing antibody, therefore allowing the analysis of both CpG and non-CpG methylation. The enriched DNA fragments can be amplified and hybridized to tiling arrays covering CpG islands, promoters, or the entire genome. Comparison of different methylomes permits the discovery of differentially methylated regions that might be important in disease- or tissue-specific expression. Here, we describe an established MeDIP protocol and tiling array hybridization method for profiling methylation of testicular germ cells.
MeDIP; DNA methylation; Tiling arrays
It is evident that epigenetic factors, especially DNA methylation, play essential roles in obesity development. Using pig as a model, here we investigated the systematic association between DNA methylation and obesity. We sampled eight variant adipose and two distinct skeletal muscle tissues from three pig breeds living within comparable environments but displaying distinct fat level. We generated 1,381 gigabases (Gb) of sequence data from 180 methylated DNA immunoprecipitation (MeDIP) libraries, and provided a genome-wide DNA methylation map as well as a gene expression map for adipose and muscle studies. The analysis showed global similarity and difference among breeds, sexes and anatomic locations, and identified the differentially methylated regions (DMRs). The DMRs in promoters are highly associated with obesity development via expression repression of both known obesity-related genes and novel genes. This comprehensive map provides a solid basis for exploring epigenetic mechanisms of adipose deposition and muscle growth.
Growth traits are important in poultry production, however, little is known for its regulatory mechanism at epigenetic level. Therefore, in this study, we aim to compare DNA methylation profiles between fast- and slow-growing broilers in order to identify candidate genes for chicken growth. Methylated DNA immunoprecipitation-sequencing (MeDIP-seq) was used to investigate the genome-wide DNA methylation pattern in high and low tails of Recessive White Rock (WRRh; WRRl) and that of Xinhua Chickens (XHh; XHl) at 7 weeks of age. The results showed that the average methylation density was the lowest in CGIs followed by promoters. Within the gene body, the methylation density of introns was higher than that of UTRs and exons. Moreover, different methylation levels were observed in different repeat types with the highest in LINE/CR1. Methylated CGIs were prominently distributed in the intergenic regions and were enriched in the size ranging 200–300 bp. In total 13,294 methylated genes were found in four samples, including 4,085 differentially methylated genes of WRRh Vs. WRRl, 5,599 of XHh Vs. XHl, 4,204 of WRRh Vs. XHh, as well as 7,301 of WRRl Vs. XHl. Moreover, 132 differentially methylated genes related to growth and metabolism were observed in both inner contrasts (WRRh Vs. WRRl and XHh Vs. XHl), whereas 129 differentially methylated genes related to growth and metabolism were found in both across-breed contrasts (WRRh Vs. XHh and WRRl Vs. XHl). Further analysis showed that overall 75 genes exhibited altered DNA methylation in all four contrasts, which included some well-known growth factors of IGF1R, FGF12, FGF14, FGF18, FGFR2, and FGFR3. In addition, we validate the MeDIP-seq results by bisulfite sequencing in some regions.
This study revealed the global DNA methylation pattern of chicken muscle, and identified candidate genes that potentially regulate muscle development at 7 weeks of age at methylation level.
DNA methylation is an epigenetic mark linking DNA sequence and transcription regulation, and therefore plays an important role in phenotypic plasticity. The ideal whole genome methylation (methylome) assay should be accurate, affordable, high-throughput and agnostic with respect to genomic features. To this end, the methylated DNA immunoprecipitation (MeDIP) assay provides a good balance of these criteria. In this Methods paper, we present AutoMeDIP-seq, a technique that combines an automated MeDIP protocol with library preparation steps for subsequent second-generation sequencing. We assessed recovery of DNA sequences covering a range of CpG densities using in vitro methylated λ-DNA fragments (and their unmethylated counterparts) spiked-in against a background of human genomic DNA. We show that AutoMeDIP is more reliable than manual protocols, shows a linear recovery profile of fragments related to CpG density (R2 = 0.86), and that it is highly specific (>99%). AutoMeDIP-seq offers a competitive approach to high-throughput methylome analysis of medium to large cohorts.
DNA methylation; Automation; Whole genome; High-throughput sequencing; MeDIP
Previous observations have demonstrated that embryonic exposure to the endocrine disruptor vinclozolin during gonadal sex determination promotes transgenerational adult onset disease such as male infertility, kidney disease, prostate disease, immune abnormalities and tumor development. The current study investigates genome-wide promoter DNA methylation alterations in the sperm of F3 generation rats whose F0 generation mother was exposed to vinclozolin. A methylated DNA immunoprecipitation with methyl-cytosine antibody followed by a promoter tilling microarray (MeDIP-Chip) procedure was used to identify 52 different regions with statistically significant altered methylation in the sperm promoter epigenome. Mass spectrometry bisulfite analysis was used to map the CpG DNA methylation and 16 differential DNA methylation regions were confirmed, while the remainder could not be analyzed due to bisulfite technical limitations. Analysis of these validated regions identified a consensus DNA sequence (motif) that associated with 75% of the promoters. Interestingly, only 16.8% of a random set of 125 promoters contained this motif. One candidate promoter (Fam111a) was found to be due to a copy number variation (CNV) and not a methylation change, suggesting initial alterations in the germline epigenome may promote genetic abnormalities such as induced CNV in later generations. This study identifies differential DNA methylation sites in promoter regions three generations after the initial exposure and identifies common genome features present in these regions. In addition to primary epimutations, a potential indirect genetic abnormality was identified, and both are postulated to be involved in the epigenetic transgenerational inheritance observed. This study confirms that an environmental agent has the ability to induce epigenetic transgenerational changes in the sperm epigenome.
DNA methylation is one of the most studied epigenetic marks in the human genome, with the result that the desire to map the human methylome has driven the development of several methods to map DNA methylation on a genomic scale. Our study presents the first comparison of two of these techniques - the targeted approach of the Infinium HumanMethylation450 BeadChip® with the immunoprecipitation and sequencing-based method, MeDIP-seq. Both methods were initially validated with respect to bisulfite sequencing as the gold standard and then assessed in terms of coverage, resolution and accuracy. The regions of the methylome that can be assayed by both methods and those that can only be assayed by one method were determined and the discovery of differentially methylated regions (DMRs) by both techniques was examined. Our results show that the Infinium HumanMethylation450 BeadChip® and MeDIP-seq show a good positive correlation (Spearman correlation of 0.68) on a genome-wide scale and can both be used successfully to determine differentially methylated loci in RefSeq genes, CpG islands, shores and shelves. MeDIP-seq however, allows a wider interrogation of methylated regions of the human genome, including thousands of non-RefSeq genes and repetitive elements, all of which may be of importance in disease. In our study MeDIP-seq allowed the detection of 15,709 differentially methylated regions, nearly twice as many as the array-based method (8070), which may result in a more comprehensive study of the methylome.
Herpesvirus latency is generally thought to be governed by epigenetic modifications, but the dynamics of viral chromatin at early timepoints of latent infection are poorly understood. Here, we report a comprehensive spatial and temporal analysis of DNA methylation and histone modifications during latent infection with Kaposi Sarcoma-associated herpesvirus (KSHV), the etiologic agent of Kaposi Sarcoma and primary effusion lymphoma (PEL). By use of high resolution tiling microarrays in conjunction with immunoprecipitation of methylated DNA (MeDIP) or modified histones (chromatin IP, ChIP), our study revealed highly distinct landscapes of epigenetic modifications associated with latent KSHV infection in several tumor-derived cell lines as well as de novo infected endothelial cells. We find that KSHV genomes are subject to profound methylation at CpG dinucleotides, leading to the establishment of characteristic global DNA methylation patterns. However, such patterns evolve slowly and thus are unlikely to control early latency. In contrast, we observed that latency-specific histone modification patterns were rapidly established upon a de novo infection. Our analysis furthermore demonstrates that such patterns are not characterized by the absence of activating histone modifications, as H3K9/K14-ac and H3K4-me3 marks were prominently detected at several loci, including the promoter of the lytic cycle transactivator Rta. While these regions were furthermore largely devoid of the constitutive heterochromatin marker H3K9-me3, we observed rapid and widespread deposition of H3K27-me3 across latent KSHV genomes, a bivalent modification which is able to repress transcription in spite of the simultaneous presence of activating marks. Our findings suggest that the modification patterns identified here induce a poised state of repression during viral latency, which can be rapidly reversed once the lytic cycle is induced.
A characteristic feature of herpesviruses is their ability to establish a latent infection during which most of the viral genes are silenced. As a consequence, no viral progeny is produced and the host cell remains viable. While the viral genome may persist in the nucleus of such cells indefinitely, it retains the ability to re-enter the lytic cycle and produce new virions if conditions in the cell become unfavorable. The molecular requirements for the establishment of latency are poorly understood, but are thought to depend on epigenetic modifications of the viral episome. Here, we report a genome-wide screen to investigate DNA methylation and histone modification patterns associated with latent infection by Kaposi Sarcoma-associated herpesvirus (KSHV), a tumor virus linked to the development of several cancers. We find that latency is likely to be determined by modifications commonly associated with genes that are transcriptionally “poised”. The promoters of such genes harbor activating as well as repressive histone marks such that they are silenced, but they can be rapidly activated upon removal of the repressive marks. Our findings thus may explain how KSHV achieves efficient quiescence during latency, yet retains the potential to quickly revert to a fully active state upon induction of the lytic cycle.
DNA methylation contributes to the regulation of gene expression during development and cellular differentiation. The recently developed Methylated DNA ImmunoPrecipitation (MeDIP) assay allows a comprehensive analysis of this epigenetic mark at the genomic level in normal and disease-derived cells. However, estimating the efficiency of the MeDIP technique is difficult without previous knowledge of the methylation status of a given cell population. Attempts to circumvent this problem have involved the use of in vitro methylated DNA in parallel to the investigated samples. Taking advantage of this stratagem, we sought to improve the sensitivity of the approach and to assess potential biases resulting from DNA amplification and hybridization procedures using MeDIP samples.
We performed MeDIP assays using in vitro methylated DNA, with or without previous DNA amplification, and hybridization to a human promoter array. We observed that CpG content at gene promoters indeed correlates strongly with the MeDIP signal obtained using in vitro methylated DNA, even when lowering significantly the amount of starting material. In analyzing MeDIP products that were subjected to whole genome amplification (WGA), we also revealed a strong bias against CpG-rich promoters during this amplification procedure, which may potentially affect the significance of the resulting data.
We illustrate the use of in vitro methylated DNA to assess the efficiency and accuracy of MeDIP procedures. We report that efficient and reproducible genome-wide data can be obtained via MeDIP experiments using relatively low amount of starting genomic DNA; and emphasize for the precaution that must be taken in data analysis when an additional DNA amplification step is required.
AIM: To identify methylation profile and novel tumor marker of extrahepatic cholangiocarcinoma (CCA) with high throughout microarray.
METHODS: Differential methylation profile was compared between normal bile duct epithelial cell lines and CCA cell lines by methyl-DNA immunoprecipitation (MeDIP) microarray. Bisulfite-polymerase chain reaction (BSP) was performed to identify the methylated allels of target genes. Expression of target genes was investigated before and after the treatment with DNA demethylating agent. Expression of candidate genes was also evaluated by immunofluorescence in 30 specimens of CCA tissues and 9 normal bile duct tissues.
RESULTS: Methylation profile of CCA was identified with MeDIP microarray in the respects of different gene functions and signaling pathways. Interestingly, 97 genes with hypermethylated CpG islands in the promoter region were homeobox genes. The top 5 hypermethylated homeobox genes validated by BSP were HOXA2 (94.29%), HOXA5 (95.38%), HOXA11 (91.67%), HOXB4 (90.56%) and HOXD13 (94.38%). Expression of these genes was reactivated with 5’-aza-2’-deoxycytidine. Significant expression differences were found between normal bile duct and extrahepatic CCA tissues (66.67%-100% vs 3.33%-10%).
CONCLUSION: HOXA2, HOXA5, HOXA11, HOXB4 and HOXD13 may work as differential epigenetic biomarkers between malignant and benign biliary tissues.
DNA methylation; Epigenetic; Promoter microarray; Cholangiocarcinoma
The highly improved cognitive function is the most significant change in human evolutionary history. Recently, several large-scale studies reported the evolutionary roles of DNA methylation; however, the role of DNA methylation on brain evolution is largely unknown.
To test if DNA methylation has contributed to the evolution of human brain, with the use of MeDIP-Chip and SEQUENOM MassARRAY, we conducted a genome-wide analysis to identify differentially methylated regions (DMRs) in the brain between humans and rhesus macaques. We first identified a total of 150 candidate DMRs by the MeDIP-Chip method, among which 4 DMRs were confirmed by the MassARRAY analysis. All 4 DMRs are within or close to the CpG islands, and a MIR3 repeat element was identified in one DMR, but no repeat sequence was observed in the other 3 DMRs. For the 4 DMR genes, their proteins tend to be conserved and two genes have neural related functions. Bisulfite sequencing and phylogenetic comparison among human, chimpanzee, rhesus macaque and rat suggested several regions of lineage specific DNA methylation, including a human specific hypomethylated region in the promoter of K6IRS2 gene.
Our study provides a new angle of studying human brain evolution and understanding the evolutionary role of DNA methylation in the central nervous system. The results suggest that the patterns of DNA methylation in the brain are in general similar between humans and non-human primates, and only a few DMRs were identified.
DNA methylation; Brain evolution; Primates
DNA cytosine methylation is an epigenetic modification that has been implicated in many biological processes. However, large-scale epigenomic studies have been applied to very few plant species, and variability in methylation among specialized tissues and its relationship to gene expression is poorly understood.
We surveyed DNA methylation from seven distinct tissue types (vegetative bud, male inflorescence [catkin], female catkin, leaf, root, xylem, phloem) in the reference tree species black cottonwood (Populus trichocarpa). Using 5-methyl-cytosine DNA immunoprecipitation followed by Illumina sequencing (MeDIP-seq), we mapped a total of 129,360,151 36- or 32-mer reads to the P. trichocarpa reference genome. We validated MeDIP-seq results by bisulfite sequencing, and compared methylation and gene expression using published microarray data. Qualitative DNA methylation differences among tissues were obvious on a chromosome scale. Methylated genes had lower expression than unmethylated genes, but genes with methylation in transcribed regions ("gene body methylation") had even lower expression than genes with promoter methylation. Promoter methylation was more frequent than gene body methylation in all tissues except male catkins. Male catkins differed in demethylation of particular transposable element categories, in level of gene body methylation, and in expression range of genes with methylated transcribed regions. Tissue-specific gene expression patterns were correlated with both gene body and promoter methylation.
We found striking differences among tissues in methylation, which were apparent at the chromosomal scale and when genes and transposable elements were examined. In contrast to other studies in plants, gene body methylation had a more repressive effect on transcription than promoter methylation.
Epigenetics; epigenomics; DNA methylation; 5-methylcytosine; Populus
Inter-individual DNA methylation variations were frequently hypothesized to alter individual susceptibility to Type 2 Diabetes Mellitus (T2DM). Sequence-influenced methylations were described in T2DM-associated genomic regions, but evidence for direct, sequence-independent association with disease risk is missing. Here, we explore disease-contributing DNA methylation through a stepwise study design: first, a pool-based, genome-scale screen among 1169 case and control individuals revealed an excess of differentially methylated sites in genomic regions that were previously associated with T2DM through genetic studies. Next, in-depth analyses were performed at selected top-ranking regions. A CpG site in the first intron of the FTO gene showed small (3.35%) but significant (P = 0.000021) hypomethylation of cases relative to controls. The effect was independent of the sequence polymorphism in the region and persists among individuals carrying the sequence-risk alleles. The odds of belonging to the T2DM group increased by 6.1% for every 1% decrease in methylation (OR = 1.061, 95% CI: 1.032–1.090), the odds ratio for decrease of 1 standard deviation of methylation (adjusted to gender) was 1.5856 (95% CI: 1.2824–1.9606) and the sensitivity (area under the curve = 0.638, 95% CI: 0.586–0.690; males = 0.675, females = 0.609) was better than that of the strongest known sequence variant. Furthermore, a prospective study in an independent population cohort revealed significant hypomethylation of young individuals that later progressed to T2DM, relative to the individuals who stayed healthy. Further genomic analysis revealed co-localization with gene enhancers and with binding sites for methylation-sensitive transcriptional regulators. The data showed that low methylation level at the analyzed sites is an early marker of T2DM and suggests a novel mechanism by which early-onset, inter-individual methylation variation at isolated non-promoter genomic sites predisposes to T2DM.
Methylated DNA immunoprecipitation (MeDIP) is a popular enrichment based method and can be combined with sequencing (termed MeDIP-seq) to interrogate the methylation status of cytosines across entire genomes. However, quality control and analysis of MeDIP-seq data have remained to be a challenge.
We report genome-wide DNA methylation profiles of wild type (wt) and mutant mouse cells, comprising 3 biological replicates of Thymine DNA glycosylase (Tdg) knockout (KO) embryonic stem cells (ESCs), in vitro differentiated neural precursor cells (NPCs) and embryonic fibroblasts (MEFs). The resulting 18 methylomes were analysed with MeDUSA (Methylated DNA Utility for Sequence Analysis), a novel MeDIP-seq computational analysis pipeline for the identification of differentially methylated regions (DMRs). The observed increase of hypermethylation in MEF promoter-associated CpG islands supports a previously proposed role for Tdg in the protection of regulatory regions from epigenetic silencing. Further analysis of genes and regions associated with the DMRs by gene ontology, pathway, and ChIP analyses revealed further insights into Tdg function, including an association of TDG with low-methylated distal regulatory regions.
We demonstrate that MeDUSA is able to detect both large-scale changes between cells from different stages of differentiation and also small but significant changes between the methylomes of cells that only differ in the KO of a single gene. These changes were validated utilising publicly available datasets and confirm TDG's function in the protection of regulatory regions from epigenetic silencing.
Methylome; MeDIP-seq; Epigenetics; Epigenomics; DNA methylation; Computational pipeline; MeDUSA
Evidence suggests that epigenetic perturbations are involved in the adverse effects associated with some drugs and toxicants, including certain classes of non-genotoxic carcinogens. Such epigenetic changes (altered DNA methylation and covalent histone modifications) may take place at the earliest stages of carcinogenesis and their identification holds great promise for biomedical research. Here, we evaluate the sensitivity and specificity of genome-wide epigenomic and transcriptomic profiling in phenobarbital (PB)-treated B6C3F1 mice, a well-characterized rodent model of non-genotoxic liver carcinogenesis. Methylated DNA Immunoprecipitation (MeDIP)-coupled microarray profiling of 17,967 promoter regions and 4,566 intergenic CpG islands was combined with genome-wide mRNA expression profiling to identify liver tissue-specific PB-mediated DNA methylation and transcriptional alterations. Only a limited number of significant anti-correlations were observed between PB-induced transcriptional and promoter-based DNA methylation perturbations. However, the constitutive androstane receptor (CAR) target gene Cyp2b10 was found to be concomitantly hypomethylated and transcriptionally activated in a liver tissue-specific manner following PB treatment. Furthermore, analysis of active and repressive histone modifications using chromatin immunoprecipitation revealed a strong PB-mediated epigenetic switch at the Cyp2b10 promoter. Our data reveal that PB-induced transcriptional perturbations are not generally associated with broad changes in the DNA methylation status at proximal promoters and suggest that the drug-inducible CAR pathway regulates an epigenetic switch from repressive to active chromatin at the target gene Cyp2b10. This study demonstrates the utility of integrated epigenomic and transcriptomic profiling for elucidating early mechanisms and biomarkers of non-genotoxic carcinogenesis.
Noninvasive prenatal diagnosis of fetal aneuploidy by maternal plasma analysis is challenging owing to the low fractional and absolute concentrations of fetal DNA in maternal plasma. Previously, we demonstrated for the first time that fetal DNA in maternal plasma could be specifically targeted by epigenetic (DNA methylation) signatures in the placenta. By comparing one such methylated fetal epigenetic marker located on chromosome 21 with another fetal genetic marker located on a reference chromosome in maternal plasma, we could infer the relative dosage of fetal chromosome 21 and noninvasively detect fetal trisomy 21. Here we apply this epigenetic-genetic (EGG) chromosome dosage approach to detect Edwards syndrome (trisomy 18) in the fetus noninvasively.
We have systematically identified methylated fetal epigenetic markers on chromosome 18 by methylated DNA immunoprecipitation (MeDIP) and tiling array analysis with confirmation using quantitative DNA methylation assays. Methylated DNA sequences from an intergenic region between the VAPA and APCDD1 genes (the VAPA-APCDD1 DNA) were detected in pre-delivery, but not post-delivery, maternal plasma samples. The concentrations correlated positively with those of an established fetal genetic marker, ZFY, in pre-delivery maternal plasma. The ratios of methylated VAPA-APCDD1(chr18) to ZFY(chrY) were higher in maternal plasma samples of 9 male trisomy 18 fetuses than those of 27 male euploid fetuses (Mann-Whitney test, P = 0.029). We defined the cutoff value for detecting trisomy 18 fetuses as mean+1.96 SD of the EGG ratios of the euploid cases. Eight of 9 trisomy 18 and 1 of 27 euploid cases showed EGG ratios higher than the cutoff value, giving a sensitivity of 88.9% and a specificity of 96.3%.
Our data have shown that the methylated VAPA-APCDD1 DNA in maternal plasma is predominantly derived from the fetus. We have demonstrated that this novel fetal epigenetic marker in maternal plasma is useful for the noninvasive detection of fetal trisomy 18.
Next-generation sequencing is rapidly transforming our ability to profile the transcriptional, genetic, and epigenetic states of a cell. In particular, sequencing DNA from the immunoprecipitation of protein-DNA complexes (ChIP-seq) and methylated DNA (MeDIP-seq) can reveal the locations of protein binding sites and epigenetic modifications. These approaches contain numerous biases which may significantly influence the interpretation of the resulting data. Rigorous computational methods for detecting and removing such biases are still lacking. Also, multi-sample normalization still remains an important open problem. This theoretical paper systematically characterizes the biases and properties of ChIP-seq data by comparing 62 separate publicly available datasets, using rigorous statistical models and signal processing techniques. Statistical methods for separating ChIP-seq signal from background noise, as well as correcting enrichment test statistics for sequence-dependent and sonication biases, are presented. Our method effectively separates reads into signal and background components prior to normalization, improving the signal-to-noise ratio. Moreover, most peak callers currently use a generic null model which suffers from low specificity at the sensitivity level requisite for detecting subtle, but true, ChIP enrichment. The proposed method of determining a cell type-specific null model, which accounts for cell type-specific biases, is shown to be capable of achieving a lower false discovery rate at a given significance threshold than current methods.
ChIP-seq; wavelets; regression; normalization; order statistics