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PubMed2025

Single-cell multi-omic detection of DNA methylation and histone modifications reconstructs the dynamics of epigenomic maintenance.

DNA methylation and histone modifications encode epigenetic information. Recently, major progress was made to measure either mark at a single-cell resolution; however, a method for simultaneous detection is lacking, preventing study of their interactions. Here, to bridge this gap, we developed scEpi2-seq. Our technique provides a readout of histone modifications and DNA methylation at the single-cell and single-molecule level. Application in a cell line with the FUCCI cell cycle reporter system reveals how DNA methylation maintenance is influenced by the local chromatin context. In addition, profiling of H3K27me3 and DNA methylation in the mouse intestine yields insights into epigenetic interactions during cell type specification. Differentially methylated regions also demonstrated independent cell-type regulation in addition to H3K27me3 regulation, which reinforces that CpG methylation acts as an additional layer of control in facultative heterochromatin.

DNA Methylation↗
PubMed2005

Direct detection of methylation in genomic DNA.

The identification of methylated sites on bacterial genomic DNA would be a useful tool to study the major roles of DNA methylation in prokaryotes: distinction of self and nonself DNA, direction of post-replicative mismatch repair, control of DNA replication and cell cycle, and regulation of gene expression. Three types of methylated nucleobases are known: N6-methyladenine, 5-methylcytosine and N4-methylcytosine. The aim of this study was to develop a method to detect all three types of DNA methylation in complete genomic DNA. It was previously shown that N6-methyladenine and 5-methylcytosine in plasmid and viral DNA can be detected by intersequence trace comparison of methylated and unmethylated DNA. We extended this method to include N4-methylcytosine detection in both in vitro and in vivo methylated DNA. Furthermore, application of intersequence trace comparison was extended to bacterial genomic DNA. Finally, we present evidence that intrasequence comparison suffices to detect methylated sites in genomic DNA. In conclusion, we present a method to detect all three natural types of DNA methylation in bacterial genomic DNA. This provides the possibility to define the complete methylome of any prokaryote.

5-Methylcytosine↗
PubMed2004

Colorimetric silver detection of methylation using DNA microarray coupled with linker-PCR.

BACKGROUND: Aberrant DNA methylation of CpG site is among the earliest and most frequent alterations in cancer. Detection of promoter hypermethylation of cancer-related genes may be useful for cancer diagnosis or the detection of recurrence. Recently, several DNA microarray methods have been developed to detect the methylation status of the multiple genes. However, the microarrays are currently detected in fluorescence using a sophisticated laser-based scanner. These methods are limited by their lower sensitivity. METHODS: We present a sensitive colorimetric silver method coupled with linker-PCR to detect methylation status of four genes, including p16, E-cadherin, VHL, and hMLH1. The signal generated with this method results from the precipitation of silver onto nanogold particles bound to streptavidin used to detect biotinylated DNA. RESULTS: We show that p16, E-cadherin, VHL, and hMLH1 were all methylated in the positive control. However, no methylation was found in these genes for the negative control. P16 gene was only methylated in the sample, whereas others were not methylated. Furthermore, as little as 0.1 fmol of target DNA amplicons could be detected on the arrays. The results were further validated by methylation-specific PCR (MSP) and bisulfite DNA sequencing. CONCLUSIONS: The colorimetric silver detection of DNA microarray could be used as an inexpensive, useful, and sensitive tool to detect methylation status of the multiple genes in the future.

Adaptor Proteins, Signal Transducing↗
PubMedDate not supplied

Detection of DNA methylation. Potential applications to diagnostic cytopathology.

Inactivation of tumor suppressor genes is often the result of genetic mutations or deletions. However, another mechanism for silencing genes involves DNA methylation. In this setting, a methyl group is added to cytosine residues within the gene promoter region; that prevents transcription. Tumors usually contain multiple genes that have been silenced by methylation, unlike normal tissues, in which gene methylation events are less common. The list of genes methylated in a given tumor is often referred to as a gene methylation profile. Gene methylation profiles may be almost unique for each type of tumor. Methylation-specific polymerase chain reaction is a sensitive technique that can detect gene methylation in cytologic samples. Application of this technique to cytologic cancer screening tests may increase their sensitivity. Also, the introduction of novel chemotherapeutic drugs that target DNA methylation may utilize gene methylation assays on fine needle aspiration biopsies of tumors.

Biopsy, Needle↗
PubMed2003

Detection of methylation of human p16(Ink4a) gene 5'-CpG islands by electrochemical method coupled with linker-PCR.

Aberrant DNA methylation of the CpG site is among the earliest and most frequent alterations in cancer. Detection of promoter hypermethylation of cancer-related genes may be useful for cancer diagnosis or the detection of recurrence. p16, an inhibitor of the cyclin D-dependent protein kinases, is a classical tumor suppressor gene, and its inactivation is closely associated with carcinogenesis. p16 hypermethylation could be detected in each stage, which is consistent with the finding that aberrant methylation of p16 is a very early event in carcinogenesis. We have developed an electrochemical procedure for detecting DNA methylation of the human p16(Ink4a) gene. The procedure is based on the coupling of DNA electrochemical sensors with linker-PCR- amplified DNA from human gastric tumor tissue and whole blood cells of healthy human. The synthesized oligonucleotide was immobilized on the modified gold electrode to fabricate a DNA biosensor. The hybridization reaction on the electrode surface was monitored by cyclic voltammogram (CV) and square wave voltammogram (SWV), using [Co(phen)(3)](ClO(4))(3) as a redox indicator. Methylation status of human p16(Ink4a) gene was detected and the results were validated by bisulfite DNA sequencing. A good reproducibility was observed in several parallel experiments. The coupling of DNA electrochemical sensors with PCR allowed quick detection and have the potential of the quantitative evaluation of the methylation status of the human p16(Ink4a) gene.

Base Sequence↗
PubMed2003

Detection and analysis of enzymatic DNA methylation of oligonucleotide substrates by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) mass spectrometry was employed to analyze DNA methylation carried out by the Escherichia coli dam DNA methyltransferase using oligonucleotide substrates with molecular masses of 5000-10,000 Da per strand. The mass spectrometry assay offers several advantages: (i) it directly shows the methylation as the increase in the mass of the substrate DNA, (ii) it is nonradioactive, (iii) it is quantitative, and (iv) it can be automated for high-throughput applications. Since unmethylated and methylated DNA are detected, the ratio of methylation can be determined directly and accurately. Furthermore, the assay allows detection individually of the methylation of several substrates in competition, offering an ideal setup to analyze the specificity of DNA interacting with enzymes. We could not identify methylation at any noncanonical site, indicating that the dam MTase is a very specific enzyme. Finally, MALDI-TOF mass spectrometry permitted assessment of the number of methyl groups incorporated into each DNA strand, thereby, allowing study of mechanistic details such as the processivity of the methylation reaction. We provide evidence that the dam MTase modifies DNA in a processive reaction, confirming earlier findings.

DNA Methylation↗
PubMed2026

ReMeDy: A Flexible Statistical Framework for Region-Based Detection of DNA Methylation Dysregulation.

Region-based epigenome-wide association studies have demonstrated improved statistical power and biological interpretability compared with probe-wise analyses of DNA methylation data. However, most existing region-based methods characterize methylation dysregulation primarily through changes in mean methylation levels associated with a phenotype of interest. Substantial evidence indicates that phenotype-associated methylation alterations may also manifest through changes in methylation variability or through joint shifts in mean and variability. Despite this, no existing statistical framework jointly models mean-variance methylation changes in a region-based manner. We propose ReMeDy, a flexible statistical framework that uses a hierarchical likelihood approach within a generalized linear model setting to identify differentially methylated regions, variably methylated regions, and regions exhibiting joint differential and variable methylation at a genome-wide scale. Unlike existing models, ReMeDy operates directly on biologically defined co-methylated regions, allowing it to naturally capture spatial correlation inherent in DNA methylation array data, while avoiding reliance on heuristic, user-defined tuning parameters such as smoothing spans and kernel bandwidths that can substantially influence results and introduce subjectivity. Through extensive simulation studies and comprehensive benchmarking against popular models, we demonstrate that ReMeDy maintains false discovery and Type-I error rates at nominal levels while achieving consistently higher statistical power across a wide range of realistic scenarios. Application to population-level DNA methylation data further shows that ReMeDy identifies biologically meaningful regions and pathways implicated in complex human diseases that are not captured by conventional mean-based analyses alone. ReMeDy is implemented as an open-source R package and is freely available at https://github.com/SChatLab/ReMeDy.

DNA Methylation↗
PubMed2005

Methylation profiles of genes utilizing newly developed CpG island methylation microarray on colorectal cancer patients.

Aberrant methylation of DNA has been shown to play an important role in a variety of human cancers, developmental disorders and aging. Hence, aberrant methylation patterns in genes can be a molecular marker for such conditions. Therefore, a reliable but uncomplicated method to detect DNA methylation is preferred, not merely for research purposes but for daily clinical practice. To achieve these aims, we have established a precise system to identify DNA methylation patterns based on an oligonucleotide microarray technology. Our microarray method has an advantage over conventional methods and is unique because it allows the precise measurement of the methylation patterns within a target region. Our simple signal detection system depends on using an avidin-biotinylated peroxidase complex and does not require an expensive laser scanner or hazardous radioisotope. In this study, we applied our technique to detect promoter methylation status of O6-methylguanine-DNA methyltransferase (MGMT) gene. Our easy-handling technology provided reproducible and precise measurement of methylated CpGs in MGMT promoter and, thus, our method may bring about a potential evolution in the handling of a variety of high-throughput DNA methylation analyses for clinical purposes.

Avidin↗
PubMed2006

Bisulfite modification of immobilized DNAs for methylation detection.

We have developed a novel method for detecting DNA methylation status of multiple samples, in which the DNA samples were firstly immobilized on the slide and treated with bisulfite directly on the chip. In this experiment, DNAs of pUC19 plasmid were restricted by the enzymes, and ligated with a linker bearing 5'-terminal acrylamide group at the sticky ends. Using universal acrylamide gel polymerization technique, a large amount of DNAs could be immobilized on the slide. The immobilized DNAs were converted by soaking the chip in bisulfite reaction mixtures for 16 h. The probes for detection of the methylation patterns of CpG sites hybridized with the converted DNAs on the microarray, and non-specifically bound probes were cleaned by electrophoresis. We have optimized the experimental conditions of both bisulfite treatment and electrophoresis to increase sensitivity and specificity. The results were further validated by bisulfite DNA sequencing. The experiments show that the method can simplify the experimental processes and increase the efficiency of the bisulfite treatment. This novel method could be used as a convenient tool to detect the methylation status of the multiple genes for a large amount of samples in the future.

Adsorption↗
PubMed2006

Microarray-based DNA methylation profiling: technology and applications.

This work is dedicated to the development of a technology for unbiased, high-throughput DNA methylation profiling of large genomic regions. In this method, unmethylated and methylated DNA fractions are enriched using a series of treatments with methylation sensitive restriction enzymes, and interrogated on microarrays. We have investigated various aspects of the technology including its replicability, informativeness, sensitivity and optimal PCR conditions using microarrays containing oligonucleotides representing 100 kb of genomic DNA derived from the chromosome 22 COMT region in addition to 12 192 element CpG island microarrays. Several new aspects of methylation profiling are provided, including the parallel identification of confounding effects of DNA sequence variation, the description of the principles of microarray design for epigenomic studies and the optimal choice of methylation sensitive restriction enzymes. We also demonstrate the advantages of using the unmethylated DNA fraction versus the methylated one, which substantially improve the chances of detecting DNA methylation differences. We applied this methodology for fine-mapping of methylation patterns of chromosomes 21 and 22 in eight individuals using tiling microarrays consisting of over 340 000 oligonucleotide probe pairs. The principles developed in this work will help to make epigenetic profiling of the entire human genome a routine procedure.

Chromosome Mapping↗
PubMed2004

Circulating methylated DNA.

Early detection of cancer would facilitate better management of patients, because survival is greatly affected by stage of presentation. Circulating nucleic acids represent a biomarker that might be used in the early detection of cancer. In addition, such biomarkers could potentially be used to follow the progression of patients with cancer. DNA methylation of the promoter region CpG islands of genes represents one such nucleic acid-based marker, and possesses some advantages over other approaches. These include the stability of DNA as a molecule, the ability to detect such changes using simple polymerase chain reaction-based approaches, and the potential for methylated DNA to be preferentially present in nucleosomes in plasma or serum. These factors are presented and their impact on the detection of methylated DNA circulating in the bloodstream is demonstrated.

Biomarkers, Tumor↗
PubMed1996

Detection of cytosine methylation and mapping of a gene influencing cytosine methylation in the genome of Citrus.

A new method was developed to detect DNA methylation in the Citrus genome using random amplification coupled with restriction enzyme digestion. Genomic DNA from Citrus grandis (L.) Osb., Poncirus trifoliata (L.) Raf., and their F1 hybrid was amplified using 7 individual 10-mer random primers. Prior to amplification the DNA templates were digested with 2 pairs of restriction endonucleases (HpaII-MspI and (or) Sau3AI-NdeII) with different sensitivities to cytosine methylation and after PCR amplification their amplified products were further digested with the same enzymes. Using this method, it was possible to detect 28 methylation events involving 23 amplified bands with the 7 random primers and 2 pairs of enzymes. A methylation polymorphism was found at a Sau3AI site in a 1.2-kb band amplified with one primer. One locus influencing cytosine methylation at this restriction site was identified through genetic analysis of a BC1 population between C. grandis and P. trifoliata and was mapped to linkage group IV using an already developed core map. This technique for detecting methylation and methylation polymorphisms is simple and should be applicable to any eukaryotic species and to many situations where it is desirable to determine whether a sequence is methylated.

Base Sequence↗
PubMed1986

Isolation of mutants sensitive to 2-aminopurine and alkylating agents and evidence for the role of DNA methylation in Penicillium chrysogenum.

Using high performance liquid chromatography, the presence of N6-methyladenine has been found at a level of 0.1 mol percent in DNA extracted from Penicillium chrysogenum. No 5-methylcytosine was detected. A mutant strain HP547, which is sensitive to the lethal effects of N-methyl-N'-nitro-N-nitrosoguanidine, methylmethane sulphonate and the base analogue 2-aminopurine shows an increased spontaneous mutation rate and no detectable DNA methylation. Comparison of restriction enzyme digests of wild type and undermethylated strains indicated that methylation was occurring at a different sequence to that of the Dam methylase system of E. coli.

2-Aminopurine↗
PubMed2004

DNA methylation, cell proliferation, and histopathology in rats following repeated inhalation exposure to dimethyl sulfate.

Dimethyl sulfate (DMS) is an alkylating agent that is carcinogenic to the respiratory tract of rodents. DNA adducts, cell proliferation, and histopathology were assessed in rats to better understand the molecular dosimetry and tissue dynamics associated with repeated inhalation exposure to DMS. For DNA methylation, rats were exposed to DMS vapor 6 h/day for up to 10 days to 0.0, 0.1, 0.7 and 1.5 ppm. N7-Methylguanine and N3-methyladenine were detected in neutral thermal hydrolysates of DNA isolated from respiratory tract tissues by high-performance liquid chromatography (HPLC) using fluorescence and ultraviolet (UV) detection. DNA methylation was greatest in DNA isolated from nasal respiratory mucosa, less in olfactory, and little was found in lung. N7-Methylguanine levels in respiratory mucosa approached steady-state levels by day 5, and N7-methylguanine persistence following exposure for 5 consecutive days was also determined. Loss of N7-methylguanine from respiratory and olfactory mucosa appeared to follow first-order kinetics. N3-Methyladenine levels were at or below detection limits in all samples. The effect of DMS on histopathology and cell proliferation in the nasal epithelium was also investigated. Rats were exposed nose-only for 2 wk to DMS vapor at concentrations of 0, 0.1, 0.7, or 1.5 ppm. Inhalation exposure to DMS induced degenerative and inflammatory changes in nasal epithelium at >or=0.7 ppm. Cell proliferation evaluations showed a trend towards an increased response at 1.5 ppm. These experiments demonstrate that DMS can induce cytotoxic and proliferative effects and is a potent methylating agent of the nasal mucosa in vivo. These experiments will provide data for the development of dosimetry models useful for risk extrapolation.

Adenine↗
PubMed1993

Abnormal chromosome behavior in Neurospora mutants defective in DNA methylation.

The function and regulation of DNA methylation in eukaryotes remain unclear. Genes affecting methylation were identified in the fungus Neurospora crassa. A mutation in one gene, dim-2, resulted in the loss of all detectable DNA methylation. Abnormal segregation of the methylation defects in crosses led to the discovery that the methylation mutants frequently generate strains with extra chromosomes or chromosomal parts. Starvation for S-adenosylmethionine, the presumed methyl group donor for DNA methylation, also produced aneuploidy. These results suggest that DNA methylation plays a role in the normal control of chromosome behavior.

5-Methylcytosine↗
PubMed1999

A sensitive new method for rapid detection of abnormal methylation patterns in global DNA and within CpG islands.

To assess alterations in DNA methylation density in both global DNA and within CpG islands, we have developed a simple method based on the use of methylation-sensitive restriction endonucleases that leave a 5' guanine overhang after DNA cleavage, with subsequent single nucleotide extension with radiolabeled [(3)H]dCTP. The methylation-sensitive restriction enzymes HpaII and AciI have relatively frequent recognition sequences at CpG sites that occur randomly throughout the genome. BssHII is a methylation sensitive enzyme that similarly leaves a guanine overhang, but the recognition sequence is nonrandom and occurs predominantly at unmethylated CpG sites within CpG islands. The selective use of these enzymes can be used to screen for alterations in genome-wide methylation and CpG island methylation status, respectively. The extent of [(3)H]dCTP incorporation opposite the exposed guanine after restriction enzyme treatment is directly proportional to the number of unmethylated (cleaved) CpG sites. The "cytosine-extension assay" has several advantages over existing methods because (a) radiolabel incorporation is independent of the integrity of the DNA, (b) methylation detection does not require PCR amplification or DNA methylase reactions, and (c) it is applicable to ng quantities of DNA. Using DNA extracted from normal human liver and from human hepatocellular carcinoma, the applicability of the assay is demonstrated by the detection of an increase in genome-wide hypomethylation and CpG island hypermethylation in the tumor DNA.

Carcinoma, Hepatocellular↗