Determination Of Global DNA Methylation Research Articles

Gene body DNA methylation in seagrasses: inter- and intraspecific differences and interaction with transcriptome plasticity under heat stress

The role of DNA methylation and its interaction with gene expression and transcriptome plasticity is poorly understood, and current insight comes mainly from studies in very few model plant species. Here, we study gene body DNA methylation (gbM) and gene expression patterns in ecotypes from contrasting thermal environments of two marine plants with contrasting life history strategies in order to explore the potential role epigenetic mechanisms could play in gene plasticity and responsiveness to heat stress. In silico transcriptome analysis of CpGO/E ratios suggested that the bulk of Posidonia oceanica and Cymodocea nodosa genes possess high levels of intragenic methylation. We also observed a correlation between gbM and gene expression flexibility: genes with low DNA methylation tend to show flexible gene expression and plasticity under changing conditions. Furthermore, the empirical determination of global DNA methylation (5-mC) showed patterns of intra and inter-specific divergence that suggests a link between methylation level and the plants’ latitude of origin and life history. Although we cannot discern whether gbM regulates gene expression or vice versa, or if other molecular mechanisms play a role in facilitating transcriptome responsiveness, our findings point to the existence of a relationship between gene responsiveness and gbM patterns in marine plants.

The effect of FOXO gene family variants and global DNA metylation on RRMS disease

Multiple sclerosis is a chronic disease that usually occurs with exacerbations and remissions in young adults, affects the central nervous system white matter in multiple localization, and is thought to be the result of complex interactions of genetic and environmental factors, the most common form is relapsing-remitting MS. Forkhead transcription factors O class (FOXO) are responsible for the regulation of various cellular processes including cell cycle, apoptosis, DNA repair, cellular resistance and metabolism. DNA methylation is such an epigenetic change and has been shown to be associated with almost any biological process. The aim of our study to show the relation between the genetic variants of FOXO3a (rs2253310 rs4966936) and FOXO1 (rs3900833, rs4581585) and global DNA methylation in RRMS. We analyzed DNA obtained from 79 RRMS patients and 104 healthy individuals by PCR-RFLP method for the detection of genetic variants. For the determination of global DNA methylation, results were obtained using ELISA method. The data were evaluated statistically. As a result of our analysis; global DNA methylation is higher in RRMS patients compared to control individuals and it can be effective on the disease. In addition, it has been determined that variants of FOXO3a (rs2253310, rs4966936) and FOXO1 (rs3900833), which have been genotyped, may be effective in disease pathogenesis. These results suggest that DNAmethylation and FOXO gene variants may be effective in neuronal loss in RRMS.

Two-color fluorescent cytosine extension assay for the determination of global DNA methylation.

Here, we present a DNA restriction enzyme-based, fluorescent cytosine extension assay (CEA) to improve normalization and technical variation among sample-to-sample measurements. The assay includes end-labeling of parallel methylation-sensitive and methylation-insensitive DNA restriction enzyme digests along with co-purification and subsequent co-measurement of incorporated fluorescence. This non-radioactive, two-color fluorescent CEA (TCF-CEA) was shown to be a relatively rapid and accurate, with 3-fold greater precision than the one-color CEA. In addition, TCF-CEA provided an index of global DNA methylation that was sensitive to differences >5%. TCF-CEA results were highly correlated with LUminometric Methylation Assay (LUMA) results using human liver cell lines (HepG2, HepaRG, HC-04) as well as a human liver primary cell culture. Hypomethylation was observed in cells treated with the de-methylating agent 5-aza-2'-deoxycytidine. These results demonstrate that TCF-CEA provides a simple method for measuring relative degrees of global DNA methylation that could potentially be scaled up to higher-throughput formats.

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BioTechniquesVol. 62, No. 4 BioSpotlight / CitationsOpen AccessBioSpotlight / CitationsPatrick C.H. Lo & Nathan S. BlowPatrick C.H. LoSearch for more papers by this author & Nathan S. BlowSearch for more papers by this authorPublished Online:16 Mar 2018https://doi.org/10.2144/000114530AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInReddit Stabler expression in stable cell linesA major issue with stable cell lines generated by standard methods is the heterogeneous levels of recombinant protein expression in different cell clones due to the integration of variable numbers of the transgene and/or position effects caused by integration of the transgene into random locations in the genome. In addition, the use of strong heterologous promoters in transgenic vectors can drive expression to very high, non-physiological levels that harm the cells. In this month's issue, researchers led by Brian Chen at McGill University describe an efficient method for stable cell line generation that achieves controlled, physiological expression of a transgene. Using CRISPR/Cas9 gene editing, the team inserted a single copy of a transgene at a specific gene locus, placing it under the control of an endogenous promoter with the desired level of gene expression. Their transgenic vector also includes a protein quantitation reporter gene to produce an equimolar amount of a fluorescent protein, allowing easy quantitation of the recombinant protein.See “Generating stable cell lines with quantifiable protein production using CRISPR/Cas9 mediated knock-in” on page 165.Two colors are better than one for dna methylation assayThe cytosine extension assay (CEA) assesses global DNA methylation levels by tracking the methylation of a CpG-containing restriction enzyme site, for example CCGG, which when methylated cannot be digested by HpaII but can be digested by its isoschizomer, MspI. The relative digestion of CCGG sites in a DNA sample by HpaII compared to MspI reflects the extent of DNA methylation and is assessed in CEA by the incorporation of a fluorescently labeled cytosine through end-filling by DNA polymerase of the 5′ guanosine overhang at the HpaII/MspI cut site. In this issue of BioTechniques, Craig Parfett and his colleagues at Health Canada present an improved version of CEA. In standard CEA, the same fluorescently colored cytosine is used in the extension reactions following HpaII and MspI digestion. In their version, Parfett's team used a different fluorescently colored cytosine for the extension reaction of each digest, after which the reactions were combined for downstream purification and signal detection. This two-color CEA showed greater precision compared to the one-color CEA and is well-suited to high-throughput screening.See “Two-color fluorescent cytosine extension assay for the determination of global DNA methylation” on page 157.Root problemsPlant biologists have long been interested in understanding the interactions that occur in nature between microorganisms and plant roots. Plants both obtain information about their environment and send out signals to microbes through their roots. However, deciphering the interplay between plant roots and the microbial communities surrounding them has proven difficult. Now, researchers report in PNAS a new microfluidic-based device designed specifically for monitoring root–bacteria interactions over time. The device, which the authors named the tracking root interaction system (TRIS), is composed of 9 independent microfluidic chambers where roots can be established and into which bacteria are introduced for monitoring. The chambers are coupled to a microscope assembly that makes it possible to obtain both spatial and temporal information on root–bacteria interactions. In proof-of-concept experiments, the authors fluorescently labeled bacteria and were able to observe root colonization in real time over extended periods using their TRIS device.Massalha, H. et al. 2017. Live imaging of root-bacteria interactions in a microfluidics setup. Proc Natl Acad Sci USA. doi:10.1073/pnas.1618584114.Western blotting goes smallWestern blotting is a commonly used technique for studying cellular proteins. While effective when using large numbers of cells as starting material, western blot analysis has proven more difficult when scaled down for studies of single cells with their much lower protein concentrations. Seeking to overcome this limitation and extend the range of protein analysis, researchers describe a new rare-cell, single-cell western blotting procedure in Nature Communications. Building on previous work that established the feasibility of single-cell resolution western blotting, the authors sought to measure panels of proteins from extremely rare circulating tumor cell (CTC) isolates. For protein analysis, each CTC was placed into an individual microwell, where the cell was first lysed and then immediately electrophoresed into a polyacrylamide matrix. Following a miniaturized version of polyacrylamide gel electrophoresis (PAGE), in-gel immunoprobing was done on the size-separated CTC proteins. Using this method, the authors were able to successfully perform multiplexed protein analyses and report protein expression on a per-cell basis.Sinkala, E. et al. 2017. Profiling protein expression in circulating tumor cells using microfluidic western blotting. Nat Commun. 8:14622.FiguresReferencesRelatedDetails Vol. 62, No. 4 Follow us on social media for the latest updates Metrics History Published online 16 March 2018 Published in print April 2017 Information© 2017 Author(s)PDF download

Determination of Global DNA Methylation in Biological Samples by Liquid Chromatography-Tandem Mass Spectrometry

Determination of Global DNA Methylation in Biological Samples by Liquid Chromatography-Tandem Mass Spectrometry

Simultaneous Determination of Global DNA Methylation and Hydroxymethylation Levels by Hydrophilic Interaction Liquid Chromatography–Tandem Mass Spectrometry

Methylation of DNA at the 5-position of cytosine (Cyt) is a well-studied epigenetic pathway implicated in gene silencing and embryogenesis. Recently, in addition to 5-methylcytosine (5mC), substantial amounts of 5-hydroxymethylcytosine (5hmC) have been detected in certain mammalian tissues. Here, we developed and validated a hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC-MS/MS) method for the simultaneous determination of Cyt, 5mC, and 5hmC levels in biological samples. DNA was extracted with phenol-chloroform, hydrolyzed using 88% formic acid at 140 °C, separated using a bridged ethylene hybrid HILIC column, and analyzed by tandem MS. The linearity was established over the concentration range of 1 to 500 ng/mL for Cyt, 0.2 to 100 ng/mL for 5mC, and 0.1 to 50 ng/mL for 5hmC, and the correlation coefficients were all >0.99. Limits of detection were 1 pg/mL for Cyt, 45 pg/mL for 5mC, and 57 pg/mL for 5hmC, and the limit of quantification values for Cyt, 5mC, and 5hmC were 2 pg/mL, 90 pg/mL, and 100 pg/mL, respectively. The relative standard deviation (RSD) of the intraday precision ranged from 1.87% to 4.84% and the interday precision from 2.69% to 4.98%. The recovery of the method varied from 88.25% to 104.39%. The method was then applied to the analysis of DNA from biological samples, establishing its potential for helping researchers understand the roles of modified nucleobases in DNA.

Determination of Global DNA Methylation in Biological Samples by Liquid Chromatography-Tandem Mass Spectrometry

Abstract DNA methylation is one of the most common epigenetic modifications. Global genomic DNA hypomethylation is considered to induce chromosome instability and high incidence of genetic mutation, which is closely related to the emergence and development of cancer. In this study, a method based on liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) was developed to determine global DNA methylation ratios in various biological samples. DNA was extracted from the samples and hydrolyzed by three specific enzymes into single nucleosides. Liquid chromatography coupled to tandem mass spectrometry was used to measure the concentrations of 2'-deoxycytidine and 2'-deoxy-5-methylcytidine respectively, so as to calculate the global DNA methylation ratios. The developed method was further used to explore the global DNA methylation ratios in normal human liver cell L-02 exposed to perfluorooctane sulfonate (PFOS) and plasma samples of 10 hepatocellular carcinoma patients and 10 healthy participants as controls. This approach has high sensitivity and stability, and is easy to be operated, enabling us to analyze the global DNA methylation ratios in various biological samples, especially those valuable samples (such as serum, plasma et al.) with extremely low concentration of DNA.

Selective Derivatization of Cytosine and Methylcytosine Moieties with 2-Bromoacetophenone for Submicrogram DNA Methylation Analysis by Reversed Phase HPLC with Spectrofluorimetric Detection

In eukaryotes, actual DNA methylation patterns provide biologically important information, for which both, genome-wide and locus-specific methylation at cytosine residues have been extensively studied. The original contribution of this work relies on the selective derivatization of cytosine moieties with 2-bromoacetophenone for the determination of global DNA methylation by reversed phase high performance liquid chromatography with spectrofluorimetric detection. The important features of the proposed procedure are as follows: (1) no need for the elimination of RNA, (2) detection limits for cytidine, 2'-deoxycytidine, 5-methylcytidine, and 5-methyl-2'-deoxycytidine in the range of 14.4-22.7 fmol, (3) feasibility for the detection of 0.06% of methylation in a low amount of DNA (80 ng), (4) potential viability for the evaluation of RNA methylation, and (5) relative simplicity in terms of analytical instrumentation and personnel training. The results obtained in the analysis of salmon testes DNA and nucleic acids from plant, human blood, and earthworms demonstrate the utility of the proposed procedure in biological studies and, in particular, for evaluation of the potential effect of environmental factors on actual DNA methylation in different types of living organisms.

Determination of global DNA methylation in tissues by hydrophilic interaction liquid chromatography

A hydrophilic interaction liquid chromatographic (HILIC) method has been developed for the determination of global DNA methylation in tissues. The DNA was extracted by phenol-chloroform, hydrolyzed with 88% formic acid at 140 degrees C, evaporated under nitrogen at 60 degrees C, and reconstituted in a mixture of acetonitrile/water (90:10, v/v); the separation was achieved on a Waters bridged ethylene hybrid (BEH) HILIC column (100 mm x 2.1 mm, 1.7 microm). The cytosine (Cyt) and 5-methylcytosine (5-mCyt) were separated in a fairly short time (3.5 min) by isocratic elution with a mixture of acetonitrile/10 mmol/L ammonium formate (94:6, v/v) as the mobile phase. Under the optimized conditions, calibration standard curve showed a good linearity in the range 1-900 micromol/L for Cyt and in the range 1-64 micromol/L for 5-mCyt with correlation coefficients of 0.9999 and 0.9998, respectively. The limit of detection (LOD) was 54 nmol/L (0.54 pmol on-column) both for Cyt and 5-mCyt, and the limit of quantification was 250 nmol/L (2.5 pmol on-column) both for Cyt and 5-mCyt. The recoveries of Cyt at the spiked levels of 90, 450, 900 micromol/L and 5-mCyt at the spiked levels of 5, 16, 64 micromol/L all ranged from 94.7% to 100.5% with a relative standard deviations less than 1.48%. The method was applied to the analysis of DNA from colon cancer tissue, and the average degree of methylation was 4.0%. The method is rapid, simple, sensitive, reliable, and suitable for the determination of global DNA methylation.

Analysis of global DNA methylation by hydrophilic interaction ultra high-pressure liquid chromatography tandem mass spectrometry

We developed and validated a rapid, sensitive, and specific liquid chromatography tandem mass spectrometry (LC–MS/MS) method for determination of global DNA methylation in tissue. DNA was extracted by phenol–chloroform, hydrolyzed using 88% formic acid at 140 °C, spiked with cytosine-2,4- 13C 15N 2 as internal standard, evaporated under nitrogen, reconstituted in methanol, and analyzed by LC–MS/MS in multiple reaction monitoring mode to reflect the global DNA methylation of the tissue. The method was linear throughout the range of clinical interest and had good sensitivity, with a limit of quantification of 0.5 pg for both cytosine (Cyt) and 5-methylcytosine (5mCyt). The linear range of calibration curve was 1–50 and 1–100 ng/ml for 5mCyt and Cyt, respectively, with a correlation coefficient higher than 0.99. The relative standard deviation (RSD) was 0.70–4.09% and 0.60–4.81% for Cyt and 5mCyt, respectively. The intraday precision expressed as RSD ranged from 1.86% to 4.67%, whereas the interday values ranged from 3.72% to 4.68%. The recovery of the method varied from 86.52% to 105.14%. This yielded a simple and reliable LC–MS/MS assay for detection of Cyt and 5mCyt, thereby enabling the evaluation of global DNA methylation.

Phosphorus and osmium as elemental tags for the determination of global DNA methylation—A novel application of high performance liquid chromatography inductively coupled plasma mass spectrometry in epigenetic studies

The hyphenation of high performance liquid chromatography with inductively coupled plasma mass spectrometry (HPLC–ICP-MS) is proposed in this work as a novel approach for the evaluation of DNA methylation, defined as the ratio between methylated cytosine and total cytosine bases in DNA. In the first part, reversed phase separation of 5-methyl-2′-deoxycytidine monophosphate (5mdCMP) and four deoxynucleotides with specific ICP-MS detection on 31P had been explored. In further development, selective labeling of 5-methylcytosine in ssDNA was carried out using potassium osmate (K 2OsO 4) in the presence of strong oxidant (K 3Fe(CN) 6) and N, N, N′, N′-tetramethylethylenediamine (TEMED). The sample was then cleaned up and introduced to size exclusion chromatography–ICP-MS for specific detection at 31P and 189Os and for evaluation of the molar ratio between Os and P eluted in DNA molecular mass fraction. The quantification of the two elemental tags was achieved by external calibration with phosphoric acid and Os(VI)-TEMED, respectively. The amount of Os in DNA fraction corresponded to methylated cytosines, while P signal was directly proportional to the total amount of DNA and could be recalculated to the amount of cytosine bases. The two procedures were tested by analyzing salmon testes DNA and a commercial oligonucleotide of known composition. For comparative purposes, these same samples were digested to deoxynucleosides and analyzed by reversed phase HPLC with spectrophotometric detection (DAD) at 280 nm. The results obtained using two procedures based on ICP-MS detection were in good agreement and also in agreement with the results obtained by HPLC–DAD procedure. In conclusion, ICP-MS specific detection at internal or external element tags seems to be an interesting alternative for the evaluation of global DNA in epigenetic studies.

High-performance capillary electrophoretic method for the quantification of global DNA methylation: Application to methotrexate-resistant cells

Global DNA hypomethylation in tumor tissue is a common characteristic in a variety of malignancies such as breast, colon, oral, lung, and blood cancers. A rapid and sensitive method has been developed for the determination of global DNA methylation in cells. Five substances—2′-deoxycytidine (dC), 5-methyl 2′-deoxycytidine (mdC), 2′-deoxyadenosine (dA), 2′-deoxythymidine (dT), and 2′-deoxyguanosine (dG)—were completely separated by high-performance capillary electrophoresis in 10 min. Intraday coefficient of variation was less than 1%, and interday coefficient of variation was less than 2%. The minimal detection limit was 1 μM. Acquired drug resistance to methotrexate (MTX) is one of the most serious problems in cancer chemotherapy. Under optimal conditions, we analyzed global DNA methylation levels in A549 and A549/MTX cells, and only 10 5 cells are needed to obtain reliable results. The percentage of 5-methyl-2′-deoxycytidine (5-mC) was 4.80 ± 0.52% in A549 cells, and this decreased to 4.20 ± 0.44% in A549/MTX cells. It was considered as statistically significant. This demonstrated that the mechanisms of acquired drug resistance to MTX might be concerned with DNA methylation.

Characterization of in vitro and in vivo hypomethylating effects of decitabine in acute myeloid leukemia by a rapid, specific and sensitive LC-MS/MS method.

DNA hypermethylation is a common finding in malignant cells and has been explored as a therapeutic target for hypomethylating agents (e.g., decitabine). Detection of changes in DNA methylation might serve as a pharmacodynamic endpoint to establish the biological activity of these agents and predict clinical response. We developed and validated a rapid, sensitive and specific LC-MS/MS method for determination of global DNA methylation (GDM) in vitro and in vivo. Ratios of 5-methyl-2′-deoxycytidine (5mdC) to the internal standard 2-deoxyguanosine (2dG) in mass signal were used to quantify GDM levels. The assay was validated in a linear range from 40 fmol to 200 pmol 5mdC. The intra-day precision values ranged from 2.8 to 9.9% and the inter-day values from 1.1 to 15.0%. The accuracy of the assay varied between 96.7 and 109.5%. This method was initially applied for characterization of decitabine-induced GDM changes in in-vitro-treated leukemia cells. Following exposure to 2.5 μM decitabine, GDM decreased to ∼50% of the baseline value. The clinical applicability of this method was then demonstrated in bone marrow samples from patients with acute myeloid leukemia treated with decitabine. Our data support the use of our LC-MS/MS method for clinical pharmacodynamic determination of changes in GDM in vivo.