A nuclear protein that binds preferentially to methylated DNA in vitro may play a role in the inaccessibility of methylated CpGs in mammalian nuclei

The effects of DNA methylation on gene expression and chromatin structure suggest the existence of a mechanism in the nucleus capable of distinguishing methylated and non-methylated sequences. We report the finding of a nuclear protein in several vertebrate tissues and cell lines that binds preferentially to methylated DNA in vitro. Its lack of sequence-specific requirements makes it potentially capable of binding to any methylated sequence in mammalian nuclei. An in vivo counterpart of these results is that methylated CpGs are inaccessible to nucleases within nuclei. In contrast, non-methylated CpG sites, located mainly at CpG islands, and restriction sites not containing this dinucleotide, are relatively accessible. The possibility that DNA methylation acts through binding to specific proteins that could alter chromatin structure is discussed.     

Methylated DNA Immunoprecipitation

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on gene expression, and is involved with normal development as well as disease ^1-4. Thus, the ability to discriminate between methylated DNA and non-methylated DNA is essential for generating methylation profiles for such studies. Methylated DNA immunoprecipitation (MeDIP) is an efficient technique for the extraction of methylated DNA from a sample of interest ^5-7. A sample of as little as 200 ng of DNA is sufficient for the antibody, or immunoprecipitation (IP), reaction. DNA is sonicated into fragments ranging in size from 300-1000 bp, and is divided into immunoprecipitated (IP) and input (IN) portions. IP DNA is subsequently heat denatured and then incubated with anti-5''mC, allowing the monoclonal antibody to bind methylated DNA. After this, magnetic beads containing a secondary antibody with affinity for the primary antibody are added, and incubated. These bead-linked antibodies will bind the monoclonal antibody used in the first step. DNA bound to the antibody complex (methylated DNA) is separated from the rest of the DNA by using a magnet to pull the complexes out of solution. Several washes using IP buffer are then performed to remove the unbound, non-methylated DNA. The methylated DNA/antibody complexes are then digested with Proteinase K to digest the antibodies leaving only the methylated DNA intact. The enriched DNA is purified by phenol:chloroform extraction to remove the protein matter and then precipitated and resuspended in water for later use. PCR techniques can be used to validate the efficiency of the MeDIP procedure by analyzing the amplification products of IP and IN DNA for regions known to lack and known to contain methylated sequences. The purified methylated DNA can then be used for locus-specific (PCR) or genome-wide (microarray and sequencing) methylation studies, and is particularly useful when applied in conjunction with other research tools such as gene expression profiling and array comparative genome hybridization (CGH) ⁸. Further investigation into DNA methylation will lead to the discovery of new epigenetic targets, which in turn, may be useful in developing new therapeutic or prognostic research tools for diseases such as cancer that are characterized by aberrantly methylated DNA ^{2, 4, 9-11}.     

Unmethylated CpG islands associated with genes in higher plant DNA

The genomes of many higher plant species are the most highly methylated among eukaryotes. We report here that in spite of their heavy methylation, genomic DNAs from four plant species contain a fraction that is very rich in non-methylated sites. The fraction was characterized in maize where it represents about 2.5% of the total nuclear genome. In order to establish the genomic origin of the fraction, three maize genes containing clustered CpG were tested for methylation and were found to be non-methylated in the CpG-rich regions. By contrast, tested CpGs were methylated in a gene whose sequence showed no clustering of CpG. These observations suggest that the CpG-rich fraction of plants is at least partially derived from non-methylated regions that are associated with genes. A similar phenomenon has been described in vertebrate genomes. We discuss the evolution of CpG islands in both groups of organisms, and their possible uses in mapping and gene isolation in plants.     

Non-methylated Genomic Sites Coincidence Cloning (NGSCC): an approach to large scale analysis of hypomethylated CpG patterns at predetermined genomic loci

We have developed a new approach to the analysis of hypomethylated CpG patterns within predetermined, megabase long, genome regions. The approach, which we term Non-methylated Genomic Sites Coincidence Cloning (NGSCC), includes three main steps. First, total genomic DNA is digested with a methylation sensitive restriction endonuclease, such as Hpa II or Hha I. Then the fragments corresponding to the genomic area of interest are selected. To this end the fragmented genome DNA is hybridized with a mixture of clones (BACs, cosmids etc.) representing a given region and digested with the same restriction enzyme(s). A special version of the coincidence cloning procedure was developed to make this hybridization selection highly efficient and specific. Finally, fragments of the locus under study are mapped and sequenced. The technique proved to be efficient and specific. As a test, it was applied to the analysis of hypomethylated CpG patterns along the 1-Mb D19S208-COX7A1 (Chr 19q13.12) locus, on human chromosome 19, in normal testis and in seminoma tissues. Some differences in the distribution of hypomethylated CpGs between the two tissues were demonstrated. The methylation profiles in both tissues revealed a clear trend to clustering of non-methylated sites. We also analyzed the expression of genes located within hypomethylated clusters in both tissues. It was shown that, whereas the expression of some of the genes investigated was correlated with hypomethylation of the region, other genes were expressed regardless of their methylation status. NGSCC thus promises to be a useful approach for the analysis of the role of dynamic epigenetic factors in genome function.Communicated by G. P. Georgiev     

Semiconductor-based sequencing of genome-wide DNA methylation states

Methylated DNA immunoprecipitation sequencing (MeDIP-Seq) is a widely used approach to study DNA methylation genome-wide. Here, we developed a MeDIP-Seq protocol compatible with the Ion Torrent semiconductor-based sequencing platform that is low cost, rapid, and scalable. We applied this protocol to demonstrate MeDIP-Seq on the Ion Torrent platform provides adequate coverage of CpG cytosines, the methylation states of which we validated at single-base resolution on the Infinium HumanMethylation450 BeadChip array, and accurately identifies sites of differential DNA methylation. Furthermore, we applied an integrative approach to further investigate and confirm the role of DNA methylation in alternative splicing and to profile 5mC and 5hmC variants of DNA methylation in normal human brain tissue that is localized over distinct genomic regions. These applications of MeDIP-Seq on the Ion Torrent platform have broad utility and add to the current methodologies for profiling genome-wide DNA methylation states in normal and disease conditions.     

DNA methylation analysis by pyrosequencing

Pyrosequencing is a sequencing-by-synthesis method that quantitatively monitors the real-time incorporation of nucleotides through the enzymatic conversion of released pyrophosphate into a proportional light signal. Quantitative measures are of special importance for DNA methylation analysis in various developmental and pathological situations. Analysis of DNA methylation patterns by pyrosequencing combines a simple reaction protocol with reproducible and accurate measures of the degree of methylation at several CpGs in close proximity with high quantitative resolution. After bisulfite treatment and PCR, the degree of each methylation at each CpG position in a sequence is determined from the ratio of T and C. The process of purification and sequencing can be repeated for the same template to analyze other CpGs in the same amplification product. Quantitative epigenotypes are obtained using this protocol in approximately 4 h for up to 96 DNA samples when bisulfite-treated DNA is already available as the starting material.     

Densely methylated sequences that are preferentially localized at telomere-proximal regions of human chromosomes

We have constructed a library of densely methylated DNA sequences from human blood DNA by selecting fragments with a high affinity for a methyl-CpG binding domain (MBD) column. PCR analysis of the library confirmed the presence of known densely methylated CpG island sequences. Analysis of random clones, however, showed that the library was dominated by sequences whose G+C content and CpG frequency were intermediate between those of bulk genomic DNA and bona fide CpG islands. When human chromosomes were probed with the library by fluorescent in situ hybridisation (FISH), the predominant sites of labelling were at terminal regions of many chromosomes, approximately corresponding to T-bands. Analysis of the methylation status of random clones indicated that all were heavily methylated at CpGs in blood DNA, but many were under-methylated in sperm DNA. Lack of methylation in germ cells may reduce CpG depletion at some sub-terminal sequences and result in a high density of methyl-CpG when these regions become methylated in somatic cells.     

De novo quantitative bisulfite sequencing using the pyrosequencing technology

Current protocols for DNA methylation analysis are either labor intensive or limited to the measurement of only one or two CpG positions. Pyrosequencing is a real-time sequencing technology that can overcome these limitations and be used as an epigenotype-mapping tool. Initial experiments demonstrated reliable quantification of the degree of DNA methylation when 2-6 CpGs were analyzed. We sought to improve the sequencing protocol so as to analyze as many CpGs as possible in a single sequencing run. By using an improved enzyme mix and adding single-stranded DNA-binding protein to the reaction, we obtained reproducible results for as many as 10 successive CpGs in a single sequencing reaction spanning up to 75 nucleotides. A minimum amount of 10 ng of bisulfite-treated DNA is necessary to obtain good reproducibility and avoid preferential amplification. We applied the assay to the analysis of DNA methylation patterns in four CpG islands in the vicinity of IGF2 and H19 genes. This allowed accurate and quantitative de novo sequencing of the methylation state of each CpG, showing reproducible variations of methylation state in contiguous CpGs, and proved to be a useful adjunct to current technologies.     

Early- and late-onset preeclampsia and the DNA methylation of circadian clock and clock-controlled genes in placental and newborn tissues

The placenta is important in providing a healthy environment for the fetus and plays a central role in the pathophysiology of preeclampsia (PE). Fetal and placental developments are influenced by epigenetic programming. There is some evidence that PE is controlled to an altered circadian homeostasis. In a nested case–control study embedded in the Rotterdam Periconceptional Cohort, we obtained placental tissue, umbilical cord leukocytes (UCL), and human umbilical venous endothelial cells of 13 early-onset PE, 16 late-onset PE and 83 controls comprising 36 uncomplicated and 47 complicated pregnancies, i.e. 27 fetal growth restricted and 20 spontaneous preterm birth. To investigate the associations between PE and the epigenetics of circadian clock and clock-controlled genes in placental and newborn tissues, genome-wide DNA methylation analysis was performed using the Illumina HumanMethylation450K BeadChip and a candidate-gene approach using ANCOVA was applied on 939 CpGs of 39 circadian clock and clock-controlled genes. DNA methylation significantly differed in early-onset PE compared with spontaneous preterm birth at 6 CpGs in placental tissue (3.73^E-5 ≤ p ≤ 0.016) and at 21 CpGs in UCL (1.09^E-5≤ p ≤ 0.024). In early-onset PE compared with fetal growth restriction 2 CpGs in placental tissue (p < 0.05) and 8 CpGs in uncomplicated controls (4.78^E-5≤ p ≤ 0.049) were significantly different. Moreover, significantly different DNA methylation in early-onset PE compared with uncomplicated controls was shown at 6 CpGs in placental tissue (1.36^E-4≤ p ≤ 0.045) and 11 CpGs in uncomplicated controls (2.52^E-6≤ p ≤ 0.009). No significant associations were shown with late-onset PE between study groups or tissues. The most differentially methylated CpGs showed hypomethylation in placental tissue and hypermethylation in uncomplicated controls. In conclusion, DNA methylation of circadian clock and clock-controlled genes demonstrated most differences in UCL of early-onset PE compared with spontaneous preterm birth. Implications of the tissue-specific variations in epigenetic programming for circadian performance and long-term health need further investigation.     

Racial differences in genome-wide methylation profiling and gene expression in breast tissues from healthy women

Breast cancer is more common in European Americans (EAs) than in African Americans (AAs) but mortality from breast cancer is higher among AAs. While there are racial differences in DNA methylation and gene expression in breast tumors, little is known whether such racial differences exist in breast tissues of healthy women. Genome-wide DNA methylation and gene expression profiling was performed in histologically normal breast tissues of healthy women. Linear regression models were used to identify differentially-methylated CpG sites (CpGs) between EAs (n = 61) and AAs (n = 22). Correlations for methylation and expression were assessed. Biological functions of the differentially-methylated genes were assigned using the Ingenuity Pathway Analysis. Among 485 differentially-methylated CpGs by race, 203 were hypermethylated in EAs, and 282 were hypermethylated in AAs. Promoter-related differentially-methylated CpGs were more frequently hypermethylated in EAs (52%) than AAs (27%) while gene body and intergenic CpGs were more frequently hypermethylated in AAs. The differentially-methylated CpGs were enriched for cancer-associated genes with roles in cell death and survival, cellular development, and cell-to-cell signaling. In a separate analysis for correlation in EAs and AAs, different patterns of correlation were found between EAs and AAs. The correlated genes showed different biological networks between EAs and AAs; networks were connected by Ubiquitin C. To our knowledge, this is the first comprehensive genome-wide study to identify differences in methylation and gene expression between EAs and AAs in breast tissues from healthy women. These findings may provide further insights regarding the contribution of epigenetic differences to racial disparities in breast cancer.     

High-throughput assay of DNA methylation based on methylation-specific primer and SAGE

Mapping of genomic DNA methylation is a dispensable part of functional genome. We have developed a novel method based on methylation-specific primer and serial analysis of gene expression, called MSP-SAGE, with potential of high-throughput quantification of genomic DNA methylation. We used a 6-mer methylation-specific primer to extend the methylated CpG sequences other than non-methylated CpG sequences. The 17 bp tags contained methylated CpG sequence, which were obtained from extended methylation sequence by digestion of restriction endonuclease, and then the tags were concatenated and cloned for sequencing. We can identify the locations of methylation according to the sequences of tags and quantify the methylation status from the frequency of the tags. MSP-SAGE has a good linearity in a broad methylation range from 5% to 100% with good accuracy and high precision. The proof-of-principle study shows that MSP-SAGE is a reliable high-throughput assay for quantification of DNA methylation.     

Existence and possible roles of independent non-CpG methylation in the mammalian brain

Methylated non-CpGs (mCpHs) in mammalian cells yield weak enrichment signals and colocalize with methylated CpGs (mCpGs), thus have been considered byproducts of hyperactive methyltransferases. However, mCpHs are cell type-specific and associated with epigenetic regulation, although their dependency on mCpGs remains to be elucidated. In this study, we demonstrated that mCpHs colocalize with mCpGs in pluripotent stem cells, but not in brain cells. In addition, profiling genome-wide methylation patterns using a hidden Markov model revealed abundant genomic regions in which CpGs and CpHs are differentially methylated in brain. These regions were frequently located in putative enhancers, and mCpHs within the enhancers increased in correlation with brain age. The enhancers with hypermethylated CpHs were associated with genes functionally enriched in immune responses, and some of the genes were related to neuroinflammation and degeneration. This study provides insight into the roles of non-CpG methylation as an epigenetic code in the mammalian brain genome.     

Nucleosome-interacting proteins regulated by DNA and histone methylation

Modifications on histones or on DNA recruit proteins that regulate chromatin function. Here, we use nucleosomes methylated on DNA and on histone H3 in an affinity assay, in conjunction with a SILAC-based proteomic analysis, to identify "crosstalk" between these two distinct classes of modification. Our analysis reveals proteins whose binding to nucleosomes is regulated by methylation of CpGs, H3K4, H3K9, and H3K27 or a combination thereof. We identify the origin recognition complex (ORC), including LRWD1 as a subunit, to be a methylation-sensitive nucleosome interactor that is recruited cooperatively by DNA and histone methylation. Other interactors, such as the lysine demethylase Fbxl11/KDM2A, recognize nucleosomes methylated on histones, but their recruitment is disrupted by DNA methylation. These data establish SILAC nucleosome affinity purifications (SNAP) as a tool for studying the dynamics between different chromatin modifications and provide a modification binding "profile" for proteins regulated by DNA and histone methylation.