5-formylcytosine and hydrogen-sulfite

Epigenetics has undergone an explosion in the past decade. DNA methylation, consisting of the addition of a methyl group at the fifth position of cytosine (5-methylcytosine, 5-mC) in a CpG dinucleotide, is a well-recognized epigenetic mark with important functions in cellular development and pathogenesis. Numerous studies have focused on the characterization of DNA methylation marks associated with disease development as they may serve as useful biomarkers for diagnosis, prognosis, and prediction of response to therapy. Recently, novel cytosine modifications with potential regulatory roles such as 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-foC), and 5-carboxylcytosine (5-caC) have been discovered. Study of the functions of 5-mC and its oxidation derivatives promotes the understanding of the mechanism underlying association of epigenetic modifications with disease biology. In this respect, much has been accomplished in the development of methods for the discovery, detection, and location analysis of 5-mC and its oxidation derivatives. In this review, we focus on the recent advances for the global detection and location study of 5-mC and its oxidation derivatives 5-hmC, 5-foC, and 5-caC.

Topics: 5-Methylcytosine; Chromatography, Thin Layer; Cytosine; DNA Methylation; Electrophoresis, Capillary; Endonucleases; Epigenesis, Genetic; Gas Chromatography-Mass Spectrometry; Humans; Microarray Analysis; Oxidation-Reduction; Polymerase Chain Reaction; Sulfites

Posttranscriptional RNA modifications have recently emerged as essential posttranscriptional regulators of gene expression. Here we present two methods for single nucleotide resolution detection of 5-formylcytosine (f

Topics: Cytosine; DNA, Mitochondrial; Gene Expression Profiling; Mitochondria; Nucleotides; RNA Processing, Post-Transcriptional; RNA-Seq; RNA, Mitochondrial; Sulfites; Transcriptome

The generation of tools to study mammalian epigenetics is vital to understanding normal biological function and to identify how it is dysregulated in disease. The well-studied epigenetic DNA modification 5-methylcytosine can be enzymatically oxidized to 5-formylcytosine (5fC) in vivo. 5fC has been demonstrated to be an intermediate in demethylation, but recent evidence suggests that 5fC may have an epigenetic function of its own. We have developed reduced bisulfite sequencing (redBS-seq), which can quantitatively locate 5fC bases at single-base resolution in genomic DNA. In bisulfite sequencing (BS-seq), 5fC is converted to uracil, as happens to unmodified cytosine (C), and thus cannot be discriminated from C. However, in redBS-seq, a specific reduction of 5fC to 5-hydroxymethylcytosine (5hmC) stops this conversion, allowing its discrimination from C. 5fC levels are inferred by comparison of a redBS-Seq run with a BS-seq run.

Topics: Animals; Cytosine; DNA; DNA Methylation; Epigenomics; Humans; Sequence Analysis, DNA; Sulfites

The 5-carbon positions on cytosine nucleotides preceding guanines in genomic DNA (CpG) are common targets for DNA methylation (5mC). DNA methylation removal can occur through both active and passive mechanisms. Ten-eleven translocation enzymes (TETs) oxidize 5mC in a stepwise manner to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5mC can also be removed passively through sequential cell divisions in the absence of DNA methylation maintenance. In this chapter, we describe approaches that couple TET-assisted bisulfite (TAB) and oxidative bisulfite (OxBS) conversion to the Illumina MethylationEPIC BeadChIP (EPIC array) and show how these technologies can be used to distinguish active versus passive DNA demethylation. We also describe integrative bioinformatics pipelines to facilitate this analysis.

Topics: 5-Methylcytosine; Computational Biology; Cytosine; DNA; DNA Demethylation; DNA Methylation; Epigenesis, Genetic; High-Throughput Nucleotide Sequencing; Humans; Microarray Analysis; Oxidation-Reduction; Sulfites

The controlled and stepwise oxidation of 5mC to 5hmC, 5fC and 5caC by Tet enzymes is influencing the chemical and biological properties of cytosine. Besides direct effects on gene regulation, oxidised forms influence the dynamics of demethylation and re-methylation processes. So far, no combined methods exist which allow to precisely determine the strand specific localisation of cytosine modifications along with their CpG symmetric distribution. Here we describe a comprehensive protocol combining conventional hairpin bisulfite with oxidative bisulfite sequencing (HPoxBS) to determine the strand specific distribution of 5mC and 5hmC at base resolution. We apply this method to analyse the contribution of local oxidative effects on DNA demethylation in mouse ES cells. Our method includes the HPoxBS workflow and subsequent data analysis using our developed software tools. Besides a precise estimation and display of strand specific 5mC and 5hmC levels at base resolution we apply the data to predict region specific activities of Dnmt and Tet enzymes. Our experimental and computational workflow provides a precise double strand display of 5mC and 5hmC modifications at single base resolution. Based on our data we predict region specific Tet and Dnmt enzyme efficiencies shaping the distinct locus levels and patterns of 5hmC and 5mC.

Topics: 5-Methylcytosine; Animals; Cytosine; DNA; DNA (Cytosine-5-)-Methyltransferase 1; DNA Methylation; DNA-Binding Proteins; Embryonic Stem Cells; Gene Expression Regulation; High-Throughput Nucleotide Sequencing; Mice; Oxidation-Reduction; Proto-Oncogene Proteins; Sulfites

A complete understanding of the function of the ten-eleven translocation (TET) family of dioxygenase-mediated DNA demethylation requires new methods to quantitatively map oxidized 5-methylcytosine (5mC) bases at high resolution. We have recently developed a methylase-assisted bisulfite sequencing (MAB-seq) method that allows base-resolution mapping of 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), two oxidized 5mC bases indicative of active DNA demethylation events. In standard bisulfite sequencing (BS-seq), unmodified C, 5fC and 5caC are read as thymine; thus 5fC and 5caC cannot be distinguished from C. In MAB-seq, unmodified C is enzymatically converted to 5mC, allowing direct mapping of rare modifications such as 5fC and 5caC. By combining MAB-seq with chemical reduction of 5fC to 5hmC, we also developed caMAB-seq, a method for direct 5caC mapping. Compared with subtraction-based mapping methods, MAB-seq and caMAB-seq require less sequencing effort and enable robust statistical calling of 5fC and/or 5caC. MAB-seq and caMAB-seq can be adapted to map 5fC/5caC at the whole-genome scale (WG-MAB-seq), within specific genomic regions enriched for enhancer-marking histone modifications (chromatin immunoprecipitation (ChIP)-MAB-seq), or at CpG-rich sequences (reduced-representation (RR)-MAB-seq) such as gene promoters. The full protocol, including DNA preparation, enzymatic treatment, library preparation and sequencing, can be completed within 6-8 d.

Topics: 5-Methylcytosine; Animals; Cell Line; Cytosine; DNA; DNA Methylation; DNA Modification Methylases; Mice; Sequence Analysis, DNA; Sulfites

Active DNA demethylation is mediated by ten-eleven translocation (TET) proteins that progressively oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). We have developed a methylation-assisted bisulfite sequencing (MAB-seq) method that enables direct genome-scale mapping and quantification of 5fC and 5caC marks together at single-base resolution. In bisulfite sequencing (BS), unmethylated cytosine residues (Cs), 5fCs and 5caCs, are converted to uracil and cannot be discriminated from each other. The pretreatment of the DNA with the CpG methylation enzyme M.SssI, which converts only the Cs to 5mCs, protects Cs but not 5fCs and 5caCs, which enables direct detection of 5fCs and 5caCs as uracils. Here we also describe an adapted version of the protocol to perform reduced-representation MAB-seq (RRMAB-seq) that provides increased coverage on CpG-rich regions, thus reducing the execution costs and increasing the feasibility of the technique. The main advantage of MAB-seq is to reduce the number of chemical/enzymatic DNA treatments required before bisulfite treatment and to avoid the need for prohibitive sequencing coverage, thus making it more reliable and affordable than subtractive approaches. The method presented here is the ideal tool for studying DNA demethylation dynamics in any biological system. Overall timing is ∼3 d for library preparation.

Topics: 5-Methylcytosine; Animals; Base Sequence; Cell Line; CpG Islands; Cytosine; DNA; DNA Methylation; Epigenesis, Genetic; Humans; Mice; Sequence Analysis, DNA; Sulfites

Oxidation of 5-methylcytosine (5mC) is catalyzed by ten-eleven translocation (TET) enzymes to produce 5-hydroxymethylcytosine (5hmC) and following oxidative products. The oxidized nucleotides were shown to be the intermediates for DNA demethylation, as the nucleotides are removed by base excision repair system initiated by thymine DNA glycosylase. A simple and accurate method to determine initial oxidation product 5hmC at single base resolution in genomic DNA is necessary to understand demethylation mechanism. Recently, we have developed a new catalytic oxidation reaction using micelle-incarcerated oxidants to oxidize 5hmC to form 5-formylcytosine (5fC), and subsequent bisulfite sequencing can determine the positions of 5hmC in DNA. In the present study, we described the optimization of the catalytic oxidative bisulfite sequencing (coBS-seq), and its application to the analysis of 5hmC in genomic DNA at single base resolution in a quantitative manner. As the oxidation step showed quite low damage on genomic DNA, the method allows us to down scale the sample to be analyzed.

Topics: 5-Methylcytosine; Adamantane; Animals; Cyclic N-Oxides; Cytosine; DNA, Single-Stranded; Embryonic Stem Cells; Iodobenzenes; Mice; Micelles; Onium Compounds; Oxidants; Oxidation-Reduction; Sequence Analysis, DNA; Sodium Dodecyl Sulfate; Sulfites; Temperature

Active DNA demethylation in mammals involves oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). However, genome-wide detection of 5fC at single-base resolution remains challenging. Here we present fC-CET, a bisulfite-free method for whole-genome analysis of 5fC based on selective chemical labeling of 5fC and subsequent C-to-T transition during PCR. Base-resolution 5fC maps showed limited overlap with 5hmC, with 5fC-marked regions more active than 5hmC-marked ones.

Topics: 5-Methylcytosine; Animals; Cell Line; CpG Islands; Cytosine; DNA Methylation; DNA Primers; Epigenomics; Gene Expression Regulation; Genome; Mice; Mice, Transgenic; Oligonucleotides; Oxygen; Polymerase Chain Reaction; Sequence Analysis, DNA; Stem Cells; Sulfites

5-Methylcytosine (5-MeCyt) can be converted to 5-hydroxymethylcytosine (5-hmCyt) in mammalian DNA by the ten-eleven translocation enzymes. The conventional bisulfite sequencing cannot discriminate 5-hmCyt from 5-MeCyt, whereas the oxidation products of 5-hmCyt, 5-carboxycytosine (5-caCyt) and 5-formylcytosine (5-fCyt) enable them to be identified in bisulfite sequencing. This mechanism likely involves the decarboxylation of 5-caCyt and deformylation of 5-fCyt to cytosine (Cyt) before deamination. Another possibility could be a direct bisulfite-induced deamination reaction followed by decarboxylation and deformylation. Here the HSO3(-)-induced direct hydrolytic deamination of 5-caCytN3(+)-SO3(-) (paths A and B) and 5-O(+)fCytN3(+)-SO3(-) (paths C and D) has been explored at the MP2/6-311++G(3df,3pd)//B3LYP/6-311++G(d,p) level. The activation free energy (ΔG(s≠) = 54.16 kJ mol(-1)) of the direct hydrolytic deamination of 5-caCytN3(+)-SO3(-) path A is much lower than the ΔG(s≠) of CytN3(+)-SO3(-) (100.91 kJ mol(-1)) under bisulfite conditions, implying that 5-caCyt may firstly involve a process of deamination. Meanwhile, the ΔG(s≠) (103.84 kJ mol(-1)) of the HSO3(-)-induced direct hydrolytic deamination of 5-O(+)fCytN3(+)-SO3(-) path C is in close proximity to our previous theoretical data for CytN3(+)-SO3(-), indicating that the deamination of 5-fCyt is also likely to occur in the presence of bisulfite. Meanwhile, the HSO3(-)-induced direct hydrolytic deamination of 5-caCytN3(+)-SO3(-) path A and 5-O(+)fCytN3(+)-SO3(-) path C is represented and has been further explored in the presence of one and two water molecules. The results show that both in the gas and aqueous phases, the participation of one and two water molecules makes the HSO3(-)-induced direct hydrolytic deamination of 5-caCytN3(+)-SO3(-) path A unfavorable, whereas the contribution of one and two water molecules facilitates the HSO3(-)-induced direct hydrolytic deamination of 5-O(+)fCytN3(+)-SO3(-) path C.

Topics: Cytosine; Deamination; Gases; Hydrogen Bonding; Isomerism; Models, Molecular; Solvents; Sulfites; Thermodynamics; Water

Recently, the cytosine modifications 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) were found to exist in the genomic deoxyribonucleic acid (DNA) of a wide range of mammalian cell types. It is now important to understand their role in normal biological function and disease. Here we introduce reduced bisulfite sequencing (redBS-Seq), a quantitative method to decode 5fC in DNA at single-base resolution, based on a selective chemical reduction of 5fC to 5hmC followed by bisulfite treatment. After extensive validation on synthetic and genomic DNA, we combined redBS-Seq and oxidative bisulfite sequencing (oxBS-Seq) to generate the first combined genomic map of 5-methylcytosine, 5hmC and 5fC in mouse embryonic stem cells. Our experiments revealed that in certain genomic locations 5fC is present at comparable levels to 5hmC and 5mC. The combination of these chemical methods can quantify and precisely map these three cytosine derivatives in the genome and will help provide insights into their function.

Topics: Animals; Cytosine; DNA; Embryonic Stem Cells; Mice; Sulfites

5-Methylcytosine can be converted to 5-hydroxymethylcytosine (5hmC) in mammalian DNA by the ten-eleven translocation (TET) enzymes. We introduce oxidative bisulfite sequencing (oxBS-Seq), the first method for quantitative mapping of 5hmC in genomic DNA at single-nucleotide resolution. Selective chemical oxidation of 5hmC to 5-formylcytosine (5fC) enables bisulfite conversion of 5fC to uracil. We demonstrate the utility of oxBS-Seq to map and quantify 5hmC at CpG islands (CGIs) in mouse embryonic stem (ES) cells and identify 800 5hmC-containing CGIs that have on average 3.3% hydroxymethylation. High levels of 5hmC were found in CGIs associated with transcriptional regulators and in long interspersed nuclear elements, suggesting that these regions might undergo epigenetic reprogramming in ES cells. Our results open new questions on 5hmC dynamics and sequence-specific targeting by TETs.

Topics: 5-Methylcytosine; Animals; CpG Islands; Cytosine; DNA; DNA Methylation; Embryonic Stem Cells; Epigenesis, Genetic; Genes, Intracisternal A-Particle; High-Throughput Nucleotide Sequencing; Long Interspersed Nucleotide Elements; Mice; Oxidation-Reduction; Rhenium; Sequence Analysis, DNA; Sulfites; Transcription, Genetic; Uracil