Epigenetic Quantification of DNA 5-Hydroxymethylcytosine Using DNA Hybridization-Based Single-Molecule Immunofluorescent Imaging.

5-Hydroxymethylcytosine (5hmC) is a deoxyribonucleic acid (DNA) epigenetic modification that has an important function in embryonic development and human diseases. However, the numerous methods that have been developed to detect and quantify 5hmC, require large amounts of DNA sample to be modified via chemical reactions, which considerably limits their application with cell-free DNA (cfDNA). Meanwhile, other antibody-based methods of detecting 5hmC do not offer information about the DNA sequence. Here, in this article DNA hybridization-based single-molecule immunofluorescent imaging is presented, an ultrasensitive method of detecting 5hmC modification in DNA. Via using the probe DNA to capture the DNA fragment of interest and the 5hmC antibody to detect the 5hmC modification in DNA, the fluorescent response signal of the 5hmC modification from the secondary antibody at the single-molecule level is successfully detected. Using the method, one could determine the quantity of 5hmC in the gene of interest within 6 h. In addition, it requires only 3 pg of the DNA sample and minimal experience and training for operation and analysis.

ELISA-Based Quantitation of Global 5hmC Levels.

5-Hydroxymethylcytosine (5hmC) is an abundant DNA modification in human and mouse brain, as well as in embryonic stem cells, while severely depleted in multiple types of cancer. Assays for 5hmC detection and quantification, both on a locus-specific and global level, are limited in number and often resource-intensive. Immunodetection of 5hmC through antibodies remains a cost-effective and widely accessible approach. This chapter describes an ELISA-based protocol for 5hmC detection and quantification in genomic or in vitro modified DNA. It is based on the passive adsorption of DNA onto a solid polystyrene surface and the specific detection of 5hmC, which generates a measurable chemiluminescent signal, proportional to the amount of immobilized 5hmC. The assay utilizes a standard curve for interpolation of 5hmC percentage and a loading standard for monitoring loading precision.

Avidin-Biotin ELISA-Based Detection of 5hmC.

The enzyme-linked immunosorbent assay (ELISA) technique has been developed half a century ago, and yet its role in molecular biology remains significant. Among the most sensitive of immunoassays, it offers high throughput, combined with affordability and ease of use. This chapter provides the procedure of a highly reproducible indirect sandwich ELISA protocol, which can be applied to a variety of semi-quantitative assays for the investigation of the molecular biology of 5-hydroxymethylcytosine (5hmC) or TET enzymes. Three variations of this protocol are described: assessment and validation of 5hmC-binding proteins, screening and validation of anti-5hmC antibodies, or a readout of TET catalytic activity in in vitro experiments. The assay principle is based on the use of a high affinity avidin-biotin system for efficient immobilization of DNA fragments for further detection by high specificity antibodies. A colorimetric enzymatic reaction is ultimately developed with intensity correlating with the amount of attached antigen.

The function and regulation of TET2 in innate immunity and inflammation.

TET2, a member of ten-eleven translocation (TET) family as α-ketoglutarate- and Fe2+-dependent dioxygenase catalyzing the iterative oxidation of 5-methylcytosine (5mC), has been widely recognized to be an important regulator for normal hematopoiesis especially myelopoiesis. Mutation and dysregulation of TET2 contribute to the development of multiple hematological malignancies. Recent studies reveal that TET2 also plays an important role in innate immune homeostasis by promoting DNA demethylation or independent of its enzymatic activity. Here, we focus on the functions of TET2 in the initiation and resolution of inflammation through epigenetic regulation and signaling network. In addition, we highlight regulation of TET2 at various molecular levels as well as the correlated inflammatory diseases, which will provide the insight to intervene in the pathological process caused by TET2 dysregulation.

Identification of cross-talk between m6A and 5mC regulators associated with onco-immunogenic features and prognosis across 33 cancer types.

Methylation of RNA and DNA, notably in the forms of N6-methyladenosine (m6A) and 5-methylcytosine (5mC) respectively, plays crucial roles in diverse biological processes. Currently, there is a lack of knowledge regarding the cross-talk between m6A and 5mC regulators. Thus, we systematically performed a pan-cancer genomic analysis by depicting the molecular correlations between m6A and 5mC regulators across ~ 11,000 subjects representing 33 cancer types. For the first time, we identified cross-talk between m6A and 5mC methylation at the multiomic level. Then, we further established m6A/5mC epigenetic module eigengenes by combining hub m6A/5mC regulators and informed a comprehensive epigenetic state. The model reflected status of the tumor-immune-stromal microenvironment and was able to predict patient survival in the majority of cancer types. Our results lay a solid foundation for epigenetic regulation in human cancer and pave a new road for related therapeutic targets.

Ten-Eleven Translocation (TET) Enzymes Modulate the Activation of Dendritic Cells in Allergic Rhinitis.

Background: The prevalence of allergic rhinitis (AR) has increased in recent decades. Accumulating evidence indicates that aberrant DNA demethylation modulated by enzymes of ten-eleven translocation (TET) promotes an imbalanced immune response. Objective: This study aimed to explore TETs on the activation of dendritic cells (DCs) in AR. Methods: The levels of TETs in peripheral blood mononuclear cells (PBMCs), peripheral myeloid DCs (mDCs), and plasmacytoid DCs (pDCs) from house dust mite (HDM)-sensitive AR patients and healthy volunteers (HC) were evaluated by qPCR and flow cytometry. The levels of 5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) in PBMCs were determined by DNA-5hmC and DNA-5mC ELISA. The major HDM allergen, Dermatophagoides pteronyssinus (Der p 1), was used to stimulate atopic monocyte-derived DCs (moDCs) to assess its effect on the TETs. TET1 knockdown effect on the activation of non-atopic and atopic moDCs was investigated. Results: TETs and global 5hmC were higher in PBMCs of AR than HC. So was TET1 in peripheral mDCs and pDCs of AR. In vitro, TET1 in atopic moDCs was significantly decreased by allergen challenge. Knockdown of TET1 in moDCs tended to induce CD86, CD80, and CD40 in AR but not in HC. TET1-knockdown moDCs significantly decreased the differentiation of activated regulatory T cells in AR. Conclusion: DCs from AR patients express higher TET1 and are susceptible to be activated by TET1 decrease, which can be triggered by allergen challenge. Collectively, this suggests a role for TET in the pathogenesis of AR and potential for novel TET1-related, preventive, and therapeutic targets.

The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice.

Multiple DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic mechanisms have been intensively studied. 5-hydroxymethylcytosine displays dynamic features during embryonic and postnatal development of the brain, plays a regulatory function in gene expression, and is involved in multiple neurological disorders. Here, we describe the detailed methods including immunofluorescence staining and DNA dot-blot to detect 5-hydroxymethylcytosine in cultured cells and brain tissues of mouse.

A Practical Guide to the Measurement and Analysis of DNA Methylation.

DNA methylation represents a fundamental epigenetic mark that is associated with transcriptional repression during development, maintenance of homeostasis, and disease. In addition to methylation-sensitive PCR and targeted deep-amplicon bisulfite sequencing to measure DNA methylation at defined genomic loci, numerous unsupervised techniques exist to quantify DNA methylation on a genome-wide scale, including affinity enrichment strategies and methods involving bisulfite conversion. Both affinity-enriched and bisulfite-converted DNA can serve as input material for array hybridization or sequencing using next-generation technologies. In this practical guide to the measurement and analysis of DNA methylation, the goal is to convey basic concepts in DNA methylation biology and explore genome-scale bisulfite sequencing as the current gold standard for assessment of DNA methylation. Bisulfite conversion chemistry and library preparation are discussed in addition to a bioinformatics approach to quality assessment, trimming, alignment, and methylation calling of individual cytosine residues. Bisulfite-converted DNA presents challenges for standard next-generation sequencing library preparation protocols and data-processing pipelines, but these challenges can be met with elegant solutions that leverage the power of high-performance computing systems. Quantification of DNA methylation, data visualization, statistical approaches to compare DNA methylation between sample groups, and examples of integrating DNA methylation data with other -omics data sets are also discussed. The reader is encouraged to use this article as a foundation to pursue advanced topics in DNA methylation measurement and data analysis, particularly the application of bioinformatics and computational biology principles to generate a deeper understanding of mechanisms linking DNA methylation to cellular function.

5-Methylcytosine-Rich Heterochromatin in Reptiles.

An experimental approach using monoclonal anti-5-methylcytosine antibodies and indirect immunofluorescence was elaborated for detecting 5-methylcytosine-rich chromosome regions in reptilian chromosomes. This technique was applied to conventionally prepared mitotic metaphases of 2 turtle species and 12 squamate species from 8 families. The hypermethylation patterns were compared with C-banding patterns obtained by conventional banding techniques. The hypermethylated DNA sequences are species-specific and are located in constitutive heterochromatin. They are highly reproducible and often found in centromeric, pericentromeric, and interstitial positions of the chromosomes. Heterochromatic regions in differentiated sex chromosomes are particularly hypermethylated.

Versatile Electroanalytical Bioplatforms for Simultaneous Determination of Cancer-Related DNA 5-Methyl- and 5-Hydroxymethyl-Cytosines at Global and Gene-Specific Levels in Human Serum and Tissues.

This paper reports the preparation of versatile electrochemical biosensing platforms for the simple, rapid, and PCR-independent detection of the most frequent DNA methylation marks (5-methylcytosine, 5-mC, and/or 5-hydroxymethylcytosine, 5-hmC) both at global and gene-specific levels. The implemented strategies, relying on the smart coupling of immuno-magnetic beads (MBs), specific DNA probes and amperometric detection at screen-printed carbon electrodes (SPCEs), provided sensitive and selective determination of the target methylated DNAs in less than 90 min with a great reproducibility and demonstrated feasibility for the simultaneous detection of the same or different cytosine epimarks both at global level and in different loci of the same gene or in different genes. The bioplatforms were applied to determine global methylation events in paraffin-embedded colorectal tissues and specific methylation at promoters of tumor suppressor genes in genomic DNA extracted from cancer cells and paraffin-embedded colorectal tissues, and in serum without previous DNA extraction from cancer patients.

Immobilization of DNA with nitrogen mustard-biotin conjugate for global epigenetic analysis.

We report the quantitative analysis of 5-methylcytosine, a representative epigenetic modification in genomic DNA, with an enzyme-linked immunosorbent assay (ELISA). We synthesized a novel hetero-bifunctional linker molecule consisting of nitrogen mustard and biotin to capture DNA on the surface of biosensing devices. The molecule can successfully immobilize genomic DNA on a streptavidin coated 96-well microplate, which was then employed for immunochemical epigenetic assessment. We achieved the sensitive and quantitative detection of 5-mC in genomic DNA samples. The CpG methylation ratios obtained from our system for mouse brain and mouse small intestine genomes were 79% and 82%, respectively. These numbers are in good agreement with the previously reported methylation ratio of 75-85%, which was identified by whole genome bisulfite sequencing. Accordingly, the present technology using our novel bifunctional linker molecule provides a fast, easy, and inexpensive method for epigenetic assessment, without the need for any conventional bisulfite treatment, polymerase chain reaction (PCR), or sequencing.

Rapid Electrochemical Assessment of Tumor Suppressor Gene Methylations in Raw Human Serum and Tumor Cells and Tissues Using Immunomagnetic Beads and Selective DNA Hybridization.

We report a rapid and sensitive electrochemical strategy for the detection of gene-specific 5-methylcytosine DNA methylation. Magnetic beads (MBs) modified with an antibody for 5-methylcytosines (5-mC) are used for the capture of any 5-mC methylated single-stranded (ss)DNA sequence. A flanking region next to the 5-mCs of the captured methylated ssDNA is recognized by hybridization with a synthetic biotinylated DNA sequence. Amperometric transduction at disposable screen-printed carbon electrodes (SPCEs) is employed. The developed biosensor has a dynamic range from 3.9 to 500 pm and a limit of detection of 1.2 pm for the methylated synthetic sequence of the tumor suppressor gene O-6-methylguanine-DNA methyltransferase (MGMT) promoter region. The method is applied in the 45-min analysis of specific methylation in the MGMT promoter region directly in raw spiked human serum samples and in genomic DNA extracted from U-87 glioblastoma cells and paraffin-embedded brain tumor tissues without any amplification and pretreatment step.

Antibody-Based Detection of Global Nuclear DNA Methylation in Cells, Tissue Sections, and Mammalian Embryos.

Immunostaining is widely used in cell biology for the in situ detection of proteins in fixed cells. The method is based on the specificity of antibodies for recognizing and binding to a selected target, combined with immunolabeling techniques for microscopic imaging. Antibodies with high specificities for modified nucleotides have also been widely developed, and among those, antibodies that recognize modified cytosine: 5-methylcytosine (5mC), and more recently, its derivates 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). To allow for their detection, primary antibody signals can be amplified using secondary antibodies coupled to fluorophores for immunofluorescence, or other molecules for immunocytochemistry.Immunostaining can be used to gain information on the spatial distribution and levels of DNA methylation states within the nucleus. Although the resolution remains quite low in genomic terms, advanced microscopy techniques and image analysis can obtain detailed spatial information content from immunostained sites. The technique complements genomic approaches that permit the assessment of DNA methylation on specific sequences, but that cannot provide global nuclear spatial context. Immunostaining is an accessible method of great benefit in several cases: when working with limited material (such as embryos or primary cells), to quickly assess at the level of individual cells the effect of siRNA, drugs, or biological processes that promote or inhibit DNA methylation or demethylation, or to study the 3D nuclear organization of regions with high DNA methylation, such as constitutive heterochromatin.Here, we review and outline protocols for the fluorescent and enzymatic immunodetection of DNA methylation in the nuclei of cells, tissue sections, and mammalian embryos.

Immunofluorescence Imaging Strategy for Evaluation of the Accessibility of DNA 5-Hydroxymethylcytosine in Chromatins.

DNA 5-hydroxymethylcytosine (5hmC) is an important epigenetic modification found in various mammalian cells. Immunofluorescence imaging analysis essentially provides visual pictures for the abundance and distribution of DNA 5hmC in single cells. However, nuclear DNA is usually wrapped around nucleosomes, packaged into chromatins, and further bound with many functional proteins. These physiologically relevant events would generate barriers to the anti-5hmC antibody to selectively recognize 5hmC in DNA. By taking advantage of these naturally generated barriers, here, we present a strategy to evaluate the accessibility of DNA 5hmC in chromatins in situ. We demonstrate that a few of the 5hmC sites in DNA are exposed or accessible to anti-5hmC antibody under nondenaturing conditions, suggesting that these 5hmC sites are not covered by functional DNA-binding proteins in mouse embryonic stem cells. Consistently, these 5hmC foci were distributed in open euchromatin regions as revealed by the 4',6-diamidino-2-phenylindole (DAPI) staining. By overexpressing TET1 catalytic domain (responsible for oxidation 5mC to produce 5hmC) in human MCF-7 cells, we observed a significant increase in accessible 5hmC along with an increase in total 5hmC sites. Collectively, by the use of the nondenaturing immunofluorescence imaging approach, we could obtain a visual landscape on the accessibility of DNA 5hmC in chromatins.

Quantitation and mapping of the epigenetic marker 5-hydroxymethylcytosine.

We here review primary methods used in quantifying and mapping 5-hydroxymethylcytosine (5hmC), including global quantification, restriction enzyme-based detection, and methods involving DNA-enrichment strategies and the genome-wide sequencing of 5hmC. As discovered in the mammalian genome in 2009, 5hmC, oxidized from 5-methylcytosine (5mC) by ten-eleven translocation (TET) dioxygenases, is increasingly being recognized as a biomarker in biological processes from development to pathogenesis, as its various detection methods have shown. We focus in particular on an ultrasensitive single-molecule imaging technique that can detect and quantify 5hmC from trace samples and thus offer information regarding the distance-based relationship between 5hmC and 5mC when used in combination with fluorescence resonance energy transfer.

Chromosome Banding in Amphibia. XXXIII. Demonstration of 5-Methylcytosine-Rich Heterochromatin in Anura.

An experimental approach using monoclonal anti-5-methylcytosine (5-MeC) antibodies and indirect immunofluorescence was elaborated for detecting 5-MeC-rich chromosome regions in anuran chromosomes. This technique was applied to mitotic metaphases of 6 neotropical frog species belonging to 6 genera and 4 families. The hypermethylation patterns were compared with a variety of banding patterns obtained by conventional banding techniques. The hypermethylated DNA sequences are species-specific and located exclusively in constitutive heterochromatin. They are found in centromeric, pericentromeric, telomeric, and interstitial positions of the chromosomes and adjacent to nucleolus organizer regions. 5-MeC-rich DNA sequences can be embedded both in AT- and GC-rich repetitive DNA. The experimental parameters that have major influence on the reproducibility and quality of the anti-5-MeC antibody labeling are discussed.

Presence of DNA methyltransferase activity and CpC methylation in Drosophila melanogaster.

Drosophila melanogaster lacks DNMT1/DNMT3 based methylation machinery. Despite recent reports confirming the presence of low DNA methylation in Drosophila; little is known about the methyltransferase. Therefore, in this study, we have aimed to investigate the possible functioning of DNA methyltransferase in Drosophila. The 14 K oligo microarray slide was incubated with native cell extract from adult Drosophila to check the presence of the methyltransferase activity. After incubation under appropriate conditions, the methylated oligo sequences were identified by the binding of anti 5-methylcytosine monoclonal antibody. The antibody bound to the methylated oligos was detected using Cy3 labeled secondary antibody. Methylation sensitive restriction enzyme mediated PCR was used to assess the methylation at a few selected loci identified on the array. It could be seen that a few of the total oligos got methylated under the assay conditions. Analysis of methylated oligo sequences provides evidence for the presence of de novo methyltransferase activity and allows identification of its sequence specificity in adult Drosophila. With the help of methylation sensitive enzymes we could detect presence of CpC methylation in the selected genomic regions. This study reports presence of an active DNA methyltransferase in adult Drosophila, which exhibits sequence specificity confirmed by presence of asymmetric methylation at corresponding sites in the genomic DNA. It also provides an innovative approach to investigate methylation specificity of a native methyltransferase.

Understanding the complexity of antigen retrieval of DNA methylation for immunofluorescence-based measurement and an approach to challenge.

Cytosine methylation (5-methylcytosine, 5meC) in the CpG-rich regions of the mammalian genome is an important epigenetic mechanism playing roles in transcription regulation and genomic stability. The abnormalities in DNA methylation can occur in various types of cancer and some genetic diseases. The measurement of DNA methylation is therefore important and there is a range of methodologies used to detect DNA methylation. Many methods based on bisulfite treatment appeared with a lack of specificity after recent discoveries of various modifications of methylated cytosine, however there are new treatments developed to overcome this limitation. Immunofluorescence is currently known to be able to specifically detect DNA methylation as it uses different antibodies against 5meC and its derivatives, but it is a semi-quantitative method. Immunofluorescence protocols commonly include fixation of cells followed by permeabilisation, antigen retrieval, and treatments with antibodies. Establishing the strategy for antigen retrieval of immunofluorescence is important to unmask epitopes (i.e. 5meC) from other proteins, and therefore to access the antigen of interest. There are many approaches used for antigen retrieval induced by acid, enzyme and/or heat. The selection of antigen retrieval method can depend on a variety of such antigen-based or cell-based conditions, since the dynamic structure of DNA and chromatin accounts for the complexity of involved proteins to mask the epitope. This review aims to specifically focus on the complexity of in situ detection of DNA methylation by immunofluorescence-based methods using antigen retrieval with the current understanding of DNA methylation mechanism, and suggests conditions for antigenic retrieval of 5meC epitope.

Prenatal exposure to BPA alters the epigenome of the rat mammary gland and increases the propensity to neoplastic development.

Exposure to environmental estrogens (xenoestrogens) may play a causal role in the increased breast cancer incidence which has been observed in Europe and the US over the last 50 years. The xenoestrogen bisphenol A (BPA) leaches from plastic food/beverage containers and dental materials. Fetal exposure to BPA induces preneoplastic and neoplastic lesions in the adult rat mammary gland. Previous results suggest that BPA acts through the estrogen receptors which are detected exclusively in the mesenchyme during the exposure period by directly altering gene expression, leading to alterations of the reciprocal interactions between mesenchyme and epithelium. This initiates a long sequence of altered morphogenetic events leading to neoplastic transformation. Additionally, BPA induces epigenetic changes in some tissues. To explore this mechanism in the mammary gland, Wistar-Furth rats were exposed subcutaneously via osmotic pumps to vehicle or 250 µg BPA/kg BW/day, a dose that induced ductal carcinomas in situ. Females exposed from gestational day 9 to postnatal day (PND) 1 were sacrificed at PND4, PND21 and at first estrus after PND50. Genomic DNA (gDNA) was isolated from the mammary tissue and immuno-precipitated using anti-5-methylcytosine antibodies. Detection and quantification of gDNA methylation status using the Nimblegen ChIP array revealed 7412 differentially methylated gDNA segments (out of 58207 segments), with the majority of changes occurring at PND21. Transcriptomal analysis revealed that the majority of gene expression differences between BPA- and vehicle-treated animals were observed later (PND50). BPA exposure resulted in higher levels of pro-activation histone H3K4 trimethylation at the transcriptional initiation site of the alpha-lactalbumin gene at PND4, concomitantly enhancing mRNA expression of this gene. These results show that fetal BPA exposure triggers changes in the postnatal and adult mammary gland epigenome and alters gene expression patterns. These events may contribute to the development of pre-neoplastic and neoplastic lesions that manifest during adulthood.

The exit of mouse embryonic fibroblasts from the cell-cycle changes the nature of solvent exposure of the 5'-methylcytosine epitope within chromatin.

The methylation of CpG dinucleotides is a pervasive epigenetic signature with critical roles governing genomic stability and lineage-specific patterns of gene expression. Reprogramming the patterns of CpG methylation accompanies key developmental transitions and the onset of some pathologies, such as cancer. In this study we show that levels of immuno-detectable 5meC decreased as mouse embryonic fibroblasts withdraw from the cell-cycle (became mitotically quiescent), but increased as they aged in culture. Two pools of 5meC epitope were found to exist, one solvent exposed after acid-induced denaturation of chromatin and another that required the additional step of tryptic digestion for detection. Proliferative cells displayed a relatively greater accumulation of detectable 5meC within the trypsin-sensitive pool than did quiescent cells. A substantial proportion of the 5meC was associated with a large number of heterochromatic foci scattered throughout nuclei, yet much of this was masked in a trypsin-sensitive manner, particularly in young proliferative cells. This study showed that the growth status of cells changed the level of solvent exposure of 5meC in fibroblasts and the long-accepted conventional methods of immunolocalization underestimate the level of 5meC in cells. This resulted in an artefactual assessment of the levels and patterns of nuclear localization of the antigen. The use of an additional tryptic digestion step improved antigen retrieval and revealed a more dynamic response of 5meC levels and distribution patterns to changes in the cell's growth state. This discovery will provide a basis for investigating the role of changes in chromatin structure that underlie this dynamism.