A disposable electrochemical magnetic immunosensor for the rapid and sensitive detection of 5-formylcytosine and 5-carboxylcytosine in DNA.

5-formylcytosine (5 fC) and 5-carboxylcytosine (5caC) serve as key intermediates in DNA demethylation process with significant implications for gene regulation and disease progression. In this study, we introduce a novel electrochemical sensing platform specifically designed for the sensitive and selective detection of 5 fC and 5caC in DNA. Protein A-modified magnetic beads (ProtA-MBs) coupled with specific antibodies facilitate the immunorecognition and enrichment of these modified bases. Signal amplification is achieved through several chemical reactions involving the interaction between N3-kethonaxl and guanine, copper-free click chemistry for the attachment of dibenzocyclooctyne (DBCO)-Biotin, and the subsequent recognition by streptavidin-conjugated horseradish peroxidase (SA-HRP). The assay's readout is performed on a disposable laser-induced graphene (LIG) electrode, modified with the bead-antibody-DNA complex in a magnetic field, and analyzed using differential pulse voltammetry in a system employing hydroquinone (HQ) as the redox mediator and H2O2 as the substrate. This immunosensor displayed excellent sensitivity, with detection limits of 14.8 fM for 5 fC across a 0.1-1000 pM linear range and 87.4 fM for 5caC across a 0.5-5000 pM linear range, and maintained high selectivity even in the presence of interferences from other DNA modifications. Successful application in quantifying 5 fC and 5caC in genomic DNA from cell extracts, with recovery rates between 97.7% to 102.9%, underscores its potential for clinical diagnostics. N3-kethoxal was used for the first time in an electrochemical sensor. This work not only broadens the toolkit for detecting DNA modifications but also provides a fresh impetus for the development of point-of-care testing (POCT) technologies.

Hepatic Global DNA Hypomethylation Phenotype in Rainbow Trout Fed Diets Varying in Carbohydrate to Protein Ratio.

BACKGROUND: A high carbohydrate-low protein diet can induce hepatic global DNA hypomethylation in trout. The mechanisms remain unclear. OBJECTIVES: We aimed to investigate whether an increase in dietary carbohydrates (dHCs) or a decrease in dietary proteins (dLPs) can cause hepatic global DNA hypomethylation, as well as explore the underlying mechanisms in trout. METHODS: Two feeding trials were conducted on juvenile males, both of which involved a 4-d fasting and 4-d refeeding protocol. In trial 1, trout were fed either a high protein-no carbohydrate [HP-NC, protein 60% dry matter (DM), carbohydrates 0% DM] or a moderate protein-high carbohydrate (MP-HC, protein 40% DM, carbohydrates 30% DM) diet. In trial 2, fish were fed either a moderate protein-no carbohydrate (MP-NC, protein 40% DM, carbohydrates 0% DM), an MP-HC (protein 40% DM, carbohydrates 30% DM), or a low protein-no carbohydrate (LP-NC, protein 20% DM, carbohydrates 0% DM) diet to separate the effects of dHCs and dLPs on the hepatic methylome. Global CmCGG methylation, DNA demethylation derivative concentrations, and mRNA expression of DNA (de)methylation-related genes were measured. Differences were tested by 1-factor ANOVA when data were normally distributed or by Kruskal-Wallis nonparametric test if not. RESULTS: In both trials, global CmCGG methylation concentrations remained unaffected, but the hepatic 5-mdC content decreased after refeeding (1-3%). The MP-HC group had 3.4-fold higher hepatic 5-hmdC and a similar 5-mdC concentration compared with the HP-NC group in trial 1. Both MP-HC and LP-NC diets lowered the hepatic 5-mdC content (1-2%), but only the LP-NC group had a significantly lower 5-hmdC concentration (P < 0.01) compared with MP-NC group in trial 2. CONCLUSIONS: dHC and dLP independently induced hepatic global DNA demethylation in trout. The alterations in other methylation derivative concentrations indicated the demethylation process was achieved through an active demethylation pathway and probably occurred at non-CmCGG sites.

Modified Forms of Cytosine in Eukaryotes: DNA (De)methylation and Beyond.

5-Methylcytosine (5mC) is an epigenetic mark known to contribute to the regulation of gene expression in a wide range of biological systems. Ten Eleven Translocation (TET) dioxygenases oxidize 5mC to 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine in metazoans and fungi. Moreover, two recent reports imply the existence of other species of modified cytosine in unicellular alga Chlamydomonas reinhardtii and malaria parasite Plasmodium falciparum. Here we provide an overview of the spectrum of cytosine modifications and their roles in demethylation of DNA and regulation of gene expression in different eukaryotic organisms.

Detection of Low-Abundance DNA Modifications Using Signal Amplification-Based Immunocytochemistry.

Immunocytochemistry can be instrumental in assessing the spatial distribution and relative levels of epigenetic modifications. Although conventional immunostaining has been utilized for the detection of 5-methylcytosine (5mC) in animal cells and tissues for several decades, the sensitivity of techniques based on the use of fluorophore-conjugated secondary antibodies is not always sufficient for studying DNA modifications that are less abundant in DNA compared with 5mC. Here we describe a protocol for sensitive immunocytochemistry that utilizes peroxidase-conjugated secondary antibodies coupled with catalyzed reporter deposition and allows for detection of low-abundance noncanonical bases (e.g., 5-carboxylcytosine, 5caC, 5-formylcytosine, 5fC, 5-hydroxymethyluracil, 5hmU) in mammalian DNA. This method can be employed for evaluation of the levels and nuclear distribution of DNA modifications and permits their colocalization with protein markers in animal cells.

Analysis of 5-Carboxylcytosine Distribution Using DNA Immunoprecipitation.

DNA methylation (5-methylcytosine, 5mC) is involved in regulation of a wide range of biological processes. TET proteins can oxidize 5mC to 5-hydroxymethylcytosine, 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Although both 5fC and 5caC serve as intermediates in active demethylation pathway, growing body of experimental evidence indicate that these DNA modifications may also interact with specific sets of reader proteins and therefore may represent bona fide epigenetic marks. Despite a number of single-base resolution techniques have recently been proposed for 5fC/5caC mapping, antibody-based approaches still represent a relatively simple and plausible alternative for the analysis of genomic distribution of these DNA modifications. Here, we describe a protocol for 5caC DNA immunoprecipitation (5caC DIP) that can be used for both locus-specific and genome-wide assessment of 5caC distribution. In combination with mass spectrometry-based techniques and single base resolution mapping methods, this approach may contribute to elucidating the role of 5caC in development, differentiation, and tumorigenesis.

A Bisulfite-free Approach for Base-Resolution Analysis of Genomic 5-Carboxylcytosine.

Due to an extreme rarity of 5-carboxylcytosine (5caC) in the mammalian genome, investigation of its role brings a considerable challenge. Methods based on bisulfite sequencing have been proposed for genome-wide 5caC analysis. However, bisulfite-based sequencing of scarcely abundant 5caC demands significant experimental and computational resources, increasing sequencing cost. Here, we present a bisulfite-free approach, caCLEAR, for high-resolution mapping of 5caCGs. The method uses an atypical activity of the methyltransferase eM.SssI to remove a carboxyl group from 5caC, generating unmodified CGs, which are localized by uTOP-seq sequencing. Validation of caCLEAR on model DNA systems and mouse ESCs supports the suitability of caCLEAR for analysis of 5caCGs. The 5caCG profiles of naive and primed pluripotent ESCs reflect their distinct demethylation dynamics and demonstrate an association of 5caC with gene expression. Generally, we demonstrate that caCLEAR is a robust economical approach that could help provide deeper insights into biological roles of 5caC.

Protein Interactions at Oxidized 5-Methylcytosine Bases.

5-Methylcytosine (5mC), the major modified DNA base in mammalian cells, can be oxidized enzymatically to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) by the Ten-Eleven-Translocation (TET) family of proteins. Whereas 5fC and 5caC are recognized and removed by base excision repair proteins, the 5hmC base accumulates to substantial levels in certain cell types such as brain-derived neurons and is viewed as a relatively stable DNA base. As such, the existence of "reader" proteins that recognize 5hmC would be a logical assumption, and various searches have been undertaken to identify proteins that specifically bind to 5hmC and the other oxidized 5mC bases. However, the existence of definitive 5hmC "readers" has remained unclear and proteins interacting specifically with 5fC or 5caC are also very few. On the other hand, 5hmC is incapable of interacting with a number of proteins that recognize 5mC at CpG sequences, suggesting that 5hmC is an anti-reader modification that may serve to displace 5mC readers from DNA. In this review article, we discuss candidate proteins that may interact with oxidized 5mC bases.

Enhanced immunological detection of epigenetic modifications of DNA in healthy and cancerous cells by fluorescence microscopy.

Epigenetic modifications of DNA, including methylation, hydroxymethylation, formylation, and carboxylation of cytosines, are proposed to function in gene regulation during reproduction and development. Changes in cytosine methylation are associated with a range of diseases, such as cancer. Immunofluorescence uses specific antibodies to quantitatively detect the global amount of cytosine modifications by fluorescence microscopy. The most critical stage of immunofluorescence is the antigen retrieval to remove the protein content around the DNA, allowing specific antibodies to bind to DNA epitopes. Acid treatments have commonly been used for antigen retrieval. Previously, trypsin was added after acid in the protocol, which increased the amount of detectable DNA methylation. In this study, the protocol was further enhanced by the addition of pepsin, which is able to target charged hydrophobic amino acids in proteins, unlike trypsin, which breaks positive hydrophilic amino acids. The global levels of cytosine modifications in CF-1, HeLa, and AR42J cells were compared using this protocol. In all cells, the sequential treatment of trypsin and pepsin increased the specificity of the staining. With the synergistic effect of the two enzymes, it is possible to target different protein groups packaging DNA molecules and removing them effectively. The findings suggest that this revised protocol can be conveniently used for each cytosine modification in the cells examined, and should be optimized for other cells. These new antigen retrieval conditions may more accurately detect the changes in cytosine modifications during development and in diseases.

Dysregulation and prognostic potential of 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) levels in prostate cancer.

BACKGROUND: Prognostic tools for prostate cancer (PC) are inadequate and new molecular biomarkers may improve risk stratification. The epigenetic mark 5-hydroxymethylcytosine (5hmC) has recently been proposed as a novel candidate prognostic biomarker in several malignancies including PC. 5hmC is an oxidized derivative of 5-methylcytosine (5mC) and can be further oxidized to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). The present study is the first to investigate the biomarker potential in PC for all four DNA methylation marks in parallel. Thus, we determined 5mC, 5hmC, 5fC, and 5caC levels in non-malignant (NM) and PC tissue samples from a large radical prostatectomy (RP) patient cohort (n = 546) by immunohistochemical (IHC) analysis of serial sections of a tissue microarray. Possible associations between methylation marks, routine clinicopathological parameters, ERG status, and biochemical recurrence (BCR) after RP were investigated. RESULTS: 5mC and 5hmC levels were significantly reduced in PC compared to NM prostate tissue samples (p ≤ 0.027) due to a global loss of both marks specifically in ERG- PCs. 5fC levels were significantly increased in ERG+ PCs (p = 0.004), whereas 5caC levels were elevated in both ERG- and ERG+ PCs compared with NM prostate tissue samples (p ≤ 0.019). Positive correlations were observed between 5mC, 5fC, and 5caC levels in both NM and PC tissues (p < 0.001), while 5hmC levels were only weakly positively correlated to 5mC in the PC subset (p = 0.030). There were no significant associations between 5mC, 5fC, or ERG status and time to BCR in this RP cohort. In contrast, high 5hmC levels were associated with BCR in ERG- PCs (p = 0.043), while high 5caC levels were associated with favorable prognosis in ERG+ PCs (p = 0.011) and were borderline significantly associated with worse prognosis in ERG- PCs (p = 0.058). Moreover, a combined high-5hmC/high-5caC score was a significant adverse predictor of post-operative BCR beyond routine clinicopathological variables in ERG- PCs (hazard ratio 3.18 (1.54-6.56), p = 0.002, multivariate Cox regression). CONCLUSIONS: This is the first comprehensive study of 5mC, 5hmC, 5fC, and 5caC levels in PC and the first report of a significant prognostic potential for 5caC in PC.

Epigenetic Modifications of Cytosine: Biophysical Properties, Regulation, and Function in Mammalian DNA.

To decode the function and molecular recognition of several recently discovered cytosine derivatives in the human genome - 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine - a detailed understanding of their effects on the structural, chemical, and biophysical properties of DNA is essential. Here, we review recent literature in this area, with particular emphasis on features that have been proposed to enable the specific recognition of modified cytosine bases by DNA-binding proteins. These include electronic factors, modulation of base-pair stability, flexibility, and radical changes in duplex conformation. We explore these proposals and assess whether or not they are supported by current biophysical data. This analysis is focused primarily on the properties of epigenetically modified DNA itself, which provides a basis for discussion of the mechanisms of recognition by different proteins.

Simultaneous mapping of active DNA demethylation and sister chromatid exchange in single cells.

To understand mammalian active DNA demethylation, various methods have been developed to map the genomic distribution of the demethylation intermediates 5-formylcysotine (5fC) and 5-carboxylcytosine (5caC). However, the majority of these methods requires a large number of cells to begin with. In this study, we describe low-input methylase-assisted bisulfite sequencing (liMAB-seq ) and single-cell MAB-seq (scMAB-seq), capable of profiling 5fC and 5caC at genome scale using ∼100 cells and single cells, respectively. liMAB-seq analysis of preimplantation embryos reveals the oxidation of 5mC to 5fC/5caC and the positive correlation between chromatin accessibility and processivity of ten-eleven translocation (TET) enzymes. scMAB-seq captures the cell-to-cell heterogeneity of 5fC and 5caC and reveals the strand-biased distribution of 5fC and 5caC. scMAB-seq also allows the simultaneous high-resolution mapping of sister chromatid exchange (SCE), facilitating the study of this type of genomic rearrangement. Therefore, our study not only establishes new methods for the genomic mapping of active DNA demethylation using limited numbers of cells or single cells but also demonstrates the utilities of the methods in different biological contexts.

Technical advances in global DNA methylation analysis in human cancers.

Prototypical abnormalities of genome-wide DNA methylation constitute the most widely investigated epigenetic mechanism in human cancers. Errors in the cellular machinery to faithfully replicate the global 5-methylcytosine (5mC) patterns, commonly observed during tumorigenesis, give rise to misregulated biological pathways beneficial to the rapidly propagating tumor mass but deleterious to the healthy tissues of the affected individual. A growing body of evidence suggests that the global DNA methylation levels could serve as utilitarian biomarkers in certain cancer types. Important breakthroughs in the recent years have uncovered further oxidized derivatives of 5mC - 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), thereby expanding our understanding of the DNA methylation dynamics. While the biological roles of these epigenetic derivatives are being extensively characterized, this review presents a perspective on the opportunity of innovation in the global methylation analysis platforms. While multiple methods for global analysis of 5mC in clinical samples exist and have been reviewed elsewhere, two of the established methods - Liquid Chromatography coupled with mass spectrometry (LC-MS/MS) and Immunoquantification have successfully evolved to include the quantitation of 5hmC, 5fC and 5caC. Although the analytical performance of LC-MS/MS is superior, the simplicity afforded by the experimental procedure of immunoquantitation ensures it's near ubiquity in clinical applications. Recent developments in spectroscopy, nanotechnology and sequencing also provide immense promise for future evaluations and are discussed briefly. Finally, we provide a perspective on the current scenario of global DNA methylation analysis tools and present suggestions to develop the next generation toolset.

Dynamics of 5-carboxylcytosine during hepatic differentiation: Potential general role for active demethylation by DNA repair in lineage specification.

Patterns of DNA methylation (5-methylcytosine, 5mC) are rearranged during differentiation contributing to the regulation of cell type-specific gene expression. TET proteins oxidize 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Both 5fC and 5caC can be recognized and excised from DNA by thymine-DNA glycosylase (TDG) followed by the subsequent incorporation of unmodified cytosine into the abasic site via the base excision repair (BER) pathway. We previously demonstrated that 5caC accumulates during lineage specification of neural stem cells (NSCs) suggesting that such active demethylation pathway is operational in this system; however, it is still unknown if TDG/BER-dependent demethylation is used during other types of cellular differentiation. Here we analyze dynamics of the global levels of 5hmC and 5caC during differentiation of human pluripotent stem cells toward hepatic endoderm. We show that, similar to differentiating NSCs, 5caC transiently accumulates during hepatic differentiation. The levels of 5caC increase during specification of foregut, peak at the stage of hepatic endoderm commitment, and drop in differentiating cells concurrently with the onset of expression of α fetoprotein, a marker of committed hepatic progenitors. Moreover, we show that 5caC accumulates at promoter regions of several genes expressed during hepatic specification at differentiation stages corresponding to the beginning of their expression. Our data indicate that transient 5caC accumulation is a common feature of 2 different types (neural/glial and endoderm/hepatic) of cellular differentiation. This suggests that oxidation of 5mC may represent a general mechanism of rearrangement of 5mC profiles during lineage specification of somatic cells in mammals.

Medulloblastoma and ependymoma cells display increased levels of 5-carboxylcytosine and elevated TET1 expression.

BACKGROUND: Alteration of DNA methylation (5-methylcytosine, 5mC) patterns represents one of the causes of tumorigenesis and cancer progression. Tet proteins can oxidise 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine and 5-carboxylcytosine (5caC). Although the roles of these oxidised forms of 5mC (oxi-mCs) in cancer pathogenesis are still largely unknown, there are indications that they may be involved in the mechanisms of malignant transformation. Thus, reduction of 5hmC content represents an epigenetic hallmark of human tumours, and according to our recent report, 5caC is enriched in a proportion of breast cancers and gliomas. Nevertheless, the distribution of oxi-mCs in paediatric brain tumours has not been assessed. FINDINGS: Here, we analyse the global levels and spatial distribution of 5hmC and 5caC in four brain tumour cell lines derived from paediatric sonic hedgehog (SHH) pathway-activated medulloblastomas (Daoy and UW228-3) and ependymomas (BXD-1425EPN and DKFZ-EP1NS). We show that, unlike HeLa cells, the paediatric tumour cell lines possess both 5hmC and 5caC at immunochemically detectable levels and demonstrate that both modifications display high degrees of spatial overlap in the nuclei of medulloblastomas and ependymomas. Moreover, although 5hmC levels are comparable in the four brain tumour cell lines, 5caC staining intensities differ dramatically between them with highest levels of this mark in a subpopulation of DKFZ-EP1NS cells. Remarkably, the 5caC enrichment does not correlate with 5hmC levels and is not associated with alterations in thymine DNA glycosylase (TDG) expression in SHH medulloblastoma and ependymoma cell lines but corresponds to elevated levels of TET1 transcript in UW228-3 and DKFZ-EP1NS cells. CONCLUSIONS: We demonstrate that both 5caC enrichment and elevated TET1 expression are observed in SHH medulloblastomas and ependymomas. Our results suggest that increased Tet-dependent 5mC oxidation may represent one of the epigenetic signatures of cancers with neural stem cell origin and, thus, may contribute to development of novel approaches for diagnosis and therapy of the brain tumours.

Comparative dynamics of 5-methylcytosine reprogramming and TET family expression during preimplantation mammalian development in mouse and sheep.

Despite previous assumption that paternal active DNA demethylation is an evolutionary conserved phenomenon in mammals, emerging studies in other species, particularly sheep, do not support this issue. Recently, ten eleven translocation (TET) enzymes have been suggested as intermediates in genome-wide DNA demethylation through the iterative conversion of five methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC)/5-formylcytosine/5-carboxylcytosine (5caC) derivatives. This study investigated whether TET enzymes and 5mC derivatives are also involved in dynamic reprogramming of early sheep embryos derived by fertilization. Mouse zygotes and developing embryos were considered as control. Obtained results reported substantial differences in dynamics of parent-of-origin-specific patterns of 5mC reprogramming and generation/dilution of 5mC derivatives (5hmC and 5caC) between mouse and sheep early zygotes. Sheep zygotes reported a gradual and insignificant decrease pattern of parental pronucleus 5mC, which was notably replication independent, coincided with gradual generation of 5hmC and 5caC. Although the expression profiles of TET family of enzymes (Tet1, Tet2, and Tet3), with the main exception being Tet2 at later developmental stages, were similar between mouse and sheep developing embryos. In addition, although the expression level of Tet3 was higher than Tet1 and Tet2 in MII oocytes and zygotes in both mouse and sheep, the expression of Tet3 in mouse was higher than sheep in both MII oocytes and zygotes. The contrasting dynamics of 5mC reprogramming between these two species may be associated with the particular evolutionary differences that exist between developmental program of rodents and ruminants, particularly during peri-implantation stages.

DNA repair and erasure of 5-methylcytosine in vertebrates.

DNA methylation plays important roles in development and disease. Yet, only recently has the dynamic nature of this epigenetic mark via oxidation and DNA repair-mediated demethylation been recognized. A major conceptual challenge to the model that DNA methylation is reversible is the risk of genomic instability, which may come with widespread DNA repair activity. Here, we focus on recent advances in mechanisms of TET-TDG mediated demethylation and cellular strategies that avoid genomic instability. We highlight the recently discovered involvement of NEIL DNA glycosylases, which cooperate with TDG in oxidative demethylation to accelerate substrate turnover and promote the organized handover of harmful repair intermediates to maintain genome stability.

DNA demethylation pathways: Additional players and regulators.

DNA demethylation can occur passively by "dilution" of methylation marks by DNA replication, or actively and independently of DNA replication. Direct conversion of 5-methylcytosine (5mC) to cytosine (C), as originally proposed, does not occur. Instead, active DNA methylation involves oxidation of the methylated base by ten-eleven translocations (TETs), or deamination of the methylated or a nearby base by activation induced deaminase (AID). The modified nucleotide, possibly together with surrounding nucleotides, is then replaced by the BER pathway. Recent data clarify the roles and the regulation of well-known enzymes in this process. They identify base excision repair (BER) glycosylases that may cooperate with or replace thymine DNA glycosylase (TDG) in the base excision step, and suggest possible involvement of DNA damage repair pathways other than BER in active DNA demethylation. Here, we review these new developments.

Progress in TET enzymes and their intermediate products / 中国病理生理杂志

DNA methylation is an important epigenetic modification mode , which plays a crucial role in gene expression , genome stability and development .DNA methylation is catalyzed and maintained in cell proliferation by the family of DNA methyltransferases.The ten-eleven translocation (TET) enzymes oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC).Here, we briefly describe the TET enzymes and their role in cancer , and the distribution , the role and detection method of those three oxidation products of cytosine in genome .