Cytosine Modifications Research Articles

Phage-encoded ten-eleven translocation dioxygenase (TET) is active in C5-cytosine hypermodification in DNA

TET/JBP (ten-eleven translocation/base J binding protein) enzymes are iron(II)- and 2-oxo-glutarate-dependent dioxygenases that are found in all kingdoms of life and oxidize 5-methylpyrimidines on the polynucleotide level. Despite their prevalence, few examples have been biochemically characterized. Among those studied are the metazoan TET enzymes that oxidize 5-methylcytosine in DNA to hydroxy, formyl, and carboxy forms and the euglenozoa JBP dioxygenases that oxidize thymine in the first step of base J biosynthesis. Both enzymes have roles in epigenetic regulation. It has been hypothesized that all TET/JBPs have their ancestral origins in bacteriophages, but only eukaryotic orthologs have been described. Here we demonstrate the 5mC-dioxygenase activity of several phage TETs encoded within viral metagenomes. The clustering of these TETs in a phylogenetic tree correlates with the sequence specificity of their genomically cooccurring cytosine C5-methyltransferases, which install the methyl groups upon which TETs operate. The phage TETs favor Gp5mC dinucleotides over the 5mCpG sites targeted by the eukaryotic TETs and are found within gene clusters specifying complex cytosine modifications that may be important for DNA packaging and evasion of host restriction.

Cytosine base modifications regulate DNA duplex stability and metabolism.

DNA base modifications diversify the genome and are essential players in development. Yet, their influence on DNA physical properties and the ensuing effects on genome metabolism are poorly understood. Here, we focus on the interplay of cytosine modifications and DNA processes. We show by a combination of in vitro reactions with well-defined protein compositions and conditions, and in vivo experiments within the complex networks of the cell that cytosine methylation stabilizes the DNA helix, increasing its melting temperature and reducing DNA helicase and RNA/DNA polymerase speed. Oxidation of methylated cytosine, however, reverts the duplex stabilizing and genome metabolic effects to the level of unmodified cytosine. We detect this effect with DNA replication and transcription proteins originating from different species, ranging from prokaryotic and viral to the eukaryotic yeast and mammalian proteins. Accordingly, lack of cytosine methylation increases replication fork speed by enhancing DNA helicase unwinding speed in cells. We further validate that this cannot simply be explained by altered global DNA decondensation, changes in histone marks or chromatin structure and accessibility. We propose that the variegated deposition of cytosine modifications along the genome regulates DNA helix stability, thereby providing an elementary mechanism for local fine-tuning of DNA metabolism.

Ten-eleven translocation protein 1 modulates medulloblastoma progression

BackgroundMedulloblastoma (MB) is the most common malignant pediatric brain tumor that originates in the cerebellum and brainstem. Frequent somatic mutations and deregulated expression of epigenetic regulators in MB highlight the substantial role of epigenetic alterations. 5-hydroxymethylcytosine (5hmC) is a highly abundant cytosine modification in the developing cerebellum and is regulated by ten-eleven translocation (TET) enzymes.ResultsWe investigate the alterations of 5hmC and TET enzymes in MB and their significance to cerebellar cancer formation. We show total abundance of 5hmC is reduced in MB, but identify significant enrichment of MB-specific 5hmC marks at regulatory regions of genes implicated in stem-like properties and Nanog-binding motifs. While TET1 and TET2 levels are high in MBs, only knockout of Tet1 in the smoothened (SmoA1) mouse model attenuates uncontrolled proliferation, leading to a favorable prognosis. The pharmacological Tet1 inhibition reduces cell viability and platelet-derived growth factor signaling pathway-associated genes.ConclusionsThese results together suggest a potential key role of 5hmC and indicate an oncogenic nature for TET1 in MB tumorigenesis, suggesting it as a potential therapeutic target for MBs.

Origins of human disease: the chrono-epigenetic perspective.

Epigenetics has enriched human disease studies by adding new interpretations to disease features that cannot be explained by genetic and environmental factors. However, identifying causal mechanisms of epigenetic origin has been challenging. New opportunities have risen from recent findings in intra-individual and cyclical epigenetic variation, which includes circadian epigenetic oscillations. Cytosine modifications display deterministic temporal rhythms, which may drive ageing and complex disease. Temporality in the epigenome, or the 'chrono' dimension, may help the integration of epigenetic, environmental and genetic disease studies, and reconcile several disparities stemming from the arbitrarily delimited research fields. The ultimate goal of chrono-epigenetics is to predict disease risk, age of onset and disease dynamics from within individual-specific temporal dynamics of epigenomes.

Distinct Epigenetic Reprogramming, Mitochondrial Patterns, Cellular Morphology, and Cytotoxicity after Bee Venom Treatment.

Bee venom is a promising agent for cancer treatment due to its selective cytotoxic potential for cancer cells through apoptotic pathways. However, there is no evidence for changes in the epigenome and mitochondrial DNA copy numbers after bee venom application. The purpose of this study was to determine the impact of bee venom on cytosine modifications and mitochondrial DNA copy number variation. A broad range of methods was applied to elucidate the impact of bee venom on neoplastic cells. These included MTT assay for detection of cytotoxicity, immunostaining of cytosine modifications and mitochondria, assessment of cellular morphology by flow cytometry, and quantification of mitochondrial DNA copy numbers using QPCR. Bee venom-induced cell death was selective for cancer cells, where it triggered a response characterized by alteration of cytosine modification. In contrast, normal cells were more resistant to DNA modifications. Furthermore, application of the venom resulted in variation of mitochondrial membrane permeability and mitochondrial DNA copy numbers, together with alterations in cell morphology, manifesting as reduced affected cell size. The study findings suggest that bee venom can be used as a selective DNA (de)methylating agent in cancer. Various agents (such as decitabine and 5-azacytidine) have been synthesized and developed for cancer treatment, and a range of syntheses and preparation and application methods have been described for these patented drugs. However, to the best of our knowledge, no previous research has investigated the use of bee venom or any component thereof for epigenetic therapy in cancer cells.

Subtraction-free and bisulfite-free specific sequencing of 5-methylcytosine and its oxidized derivatives at base resolution

Although various methods have been developed for sequencing cytosine modifications, it is still challenging for specific and quantitative sequencing of individual modification at base-resolution. For example, to obtain both true 5-methylcytosine (5mC) and true 5-hydroxymethylcytosine (5hmC) information, the two major epigenetic modifications, it usually requires subtraction of two methods, which increases noise and requires high sequencing depth. Recently, we developed TET-assisted pyridine borane sequencing (TAPS) for bisulfite-free direct sequencing of 5mC and 5hmC. Here we demonstrate that two sister methods, TAPSβ and chemical-assisted pyridine borane sequencing (CAPS), can be effectively used for subtraction-free and specific whole-genome sequencing of 5mC and 5hmC, respectively. We also demonstrate pyridine borane sequencing (PS) for whole-genome profiling of 5-formylcytosine and 5-carboxylcytosine, the further oxidized derivatives of 5mC and 5hmC. This work completes the set of versatile borane reduction chemistry-based methods as a comprehensive toolkit for direct and quantitative sequencing of all four cytosine epigenetic modifications.

Integrated analysis of the methylome and transcriptome of chickens with fatty liver hemorrhagic syndrome

BackgroundDNA methylation, a biochemical modification of cytosine, has an important role in lipid metabolism. Fatty liver hemorrhagic syndrome (FLHS) is a serious disease and is tightly linked to lipid homeostasis. Herein, we compared the methylome and transcriptome of chickens with and without FLHS.ResultsWe found genome-wide dysregulated DNA methylation pattern in which regions up- and down-stream of gene body were hypo-methylated in chickens with FLHS. A total of 4155 differentially methylated genes and 1389 differentially expressed genes were identified. Genes were focused when a negative relationship between mRNA expression and DNA methylation in promoter and gene body were detected. Based on pathway enrichment analysis, we found expression of genes related to lipogenesis and oxygenolysis (e.g., PPAR signaling pathway, fatty acid biosynthesis, and fatty acid elongation) to be up-regulated with associated down-regulated DNA methylation. In contrast, genes related to cellular junction and communication pathways (e.g., vascular smooth muscle contraction, phosphatidylinositol signaling system, and gap junction) were inhibited and with associated up-regulation of DNA methylation.ConclusionsIn the current study, we provide a genome-wide scale landscape of DNA methylation and gene expression. The hepatic hypo-methylation feature has been identified with FLHS chickens. By integrated analysis, the results strongly suggest that increased lipid accumulation and hepatocyte rupture are central pathways that are regulated by DNA methylation in chickens with FLHS.

Ten-eleven translocation proteins (TETs): tumor suppressors or tumor enhancers?

The epigenetic memory stored in the dynamic modifications, such as base modifications of cytosine (C) in DNA, including methylation/hydroxymethylation/demethylation, causes heritable phenotypes via regulating gene expression without alteration of DNA sequence. The process from cytosine modification to the epigenetic effect is orchestrated by complicated machinery consisting of writers, erasers, readers, and other factors. The two major forms of cytosine modification include methylcytosine (5-mC) and hydroxymethylcytosine (5-hmC). DNA methyltransferases (DNMTs) including DNMT1, DNMT3A, and DNMT3B function as writers for 5-mC. The ten-eleven translocation proteins (TET) including TET1, TET2, and TET3 in the mammalian genome are responsible for hydroxymethylation of 5-mC to generate 5-hmC, 5-formylcytosine (5-fC), and 5-carboxylcytosine (5-caC). The 5-mC and 5-hmC have become the two most extensively investigated epigenetic markers, and the dynamic balance of these two markers shape the landscape of the epigenome, functioning as a platform to regulate gene expression epigenetically. The landscape of the 5-hmC in epigenome is precisely and tightly regulated during the development. Aberrant alterations of the epigenetic regulation may cause severe consequences such as phenotype change as well as initiation of disease. Progressively, significant achievements have been made in characterization of writers, erasers, and readers of 5-mC and 5-hmC, as well as the contribution of aberrant alteration of 5-hmC/5-mC landscape to the pathogenesis of human diseases, such as cancers and neurological disorders. This article will highlight the research advances in the distinct contribution of TET proteins as suppressors or promoters to the pathogenesis of tumorigenesis and progression. Furthermore, this article also discusses the challenges and the directions for research in the future.

A comprehensive mechanism for 5-carboxylcytosine-induced transcriptional pausing revealed by Markov state models

RNA polymerase II (Pol II) surveils the genome, pausing as it encounters DNA lesions and base modifications and initiating signals for DNA repair among other important regulatory events. Recent work suggests that Pol II pauses at 5-carboxycytosine (5caC), an epigenetic modification of cytosine, because of a specific hydrogen bond between the carboxyl group of 5caC and a specific residue in fork loop 3 of Pol II. This hydrogen bond compromises productive NTP binding and slows down elongation. Apart from this specific interaction, the carboxyl group of 5caC can potentially interact with numerous charged residues in the cleft of Pol II. However, it is not clear how other interactions between Pol II and 5caC contribute to pausing. In this study, we use Markov state models (a type of kinetic network models) built from extensive molecular dynamics simulations to comprehensively study the impact of 5caC on Pol II translocation. We describe two translocation intermediates with specific interactions that prevent the template base from loading into the Pol II active site. In addition to the previously observed state with 5caC constrained by fork loop 3, we discovered a new intermediate state with a hydrogen bond between 5caC and fork loop 2. Surprisingly, we find that 5caC may curb translocation by suppressing kinking of the helix bordering the active site (the bridge helix) because its high flexibility is critical to translocation. Our work provides new insights into how epigenetic modifications of genomic DNA can modulate Pol II translocation, inducing pauses in transcription.

TBIO-05. GENOME-SCALE NUCLEOTIDE-SPECIFIC CHARACTERIZATION OF 5-HYDROXYMETHYLCYTOSINE IN PEDIATRIC CENTRAL NERVOUS SYSTEM TUMORS

Though aberrant cytosine modifications are prevalent in cancer, nucleotide-specific 5-hydroxymethylcytosine (5hmC) modifications remain understudied, including in pediatric CNS tumors. Brain 5-hydroxymethylation is linked with development and differentiation. We measured genome-scale nucleotide-specific 5hmC in patients with diagnoses of glioma, ependymoma, and embryonal tumors under age 18 (n=36), and in non-tumor pediatric brain tissues (n=3). DNA was processed with tandem oxidative (OxBS) and bisulfite (BS) treatments followed by hybridization to the Illumina Methylation EPIC Array that interrogates over 860,000 CpG sites. We used the OxyBS R package to determine levels of 5hmC and 5mC. Mean 5hmC levels were lower in tumors (gliomas 4.1%, ependymomas 3.9%, and embryonal tumors 3.4%) compared to nontumor tissues (5.3%). We subset to the CpGs with the 5% highest 5hmC content for downstream analyses (37,173 CpGs). These sites were enriched among regulatory elements, including TFBS (Odds Ratio 1.14 p-value 3.57E-20) and super-enhancers (OR 1.93, p-value 1.14E-126). Linear mixed-effects models adjusted for age, sex, and cell type proportions tested the CpG-specific differences in 5hmC between tumor and nontumor samples, as well as between tumor subtypes. 5hmC levels were depleted across tumors compared with nontumor brain tissues, including at CpG islands. Model-based clustering (RPMM) results indicated that patients with low 5hmC patterns have poorer overall survival and increased risk of recurrence. Our results indicate that 5hmC localizes to sites in the DNA critical to gene regulation and is associated with patient outcomes. This study offers an opportunity to potentially contribute to classification markers for childhood brain tumors.

Active turnover of DNA methylation during cell fate decisions.

DNA methylation is a key layer of epigenetic regulation. The deposition of methylation marks relies on the catalytic activity of DNA methyltransferases (DNMTs), and their active removal relies on the activity of ten-eleven translocation (TET) enzymes. Paradoxically, in important biological contexts these antagonistic factors are co-expressed and target overlapping genomic regions. The ensuing cyclic biochemistry of cytosine modifications gives rise to a continuous, out-of-thermal equilibrium transition through different methylation states. But what is the purpose of this intriguing turnover of DNA methylation? Recent evidence demonstrates that methylation turnover is enriched at gene distal regulatory elements, including enhancers, and can give rise to large-scale oscillatory dynamics. We discuss this phenomenon and propose that DNA methylation turnover might facilitate key lineage decisions.

Mapping 5-Hydroxymethylcytosine (5hmC) Modifications in Skeletal Tissues Using High-Throughput Sequencing.

Cytosine modifications can alter the epigenetic landscape of a cell, affecting the binding of transcription factors, chromatin organizing complexes, and ultimately affecting gene expression and cell fate. 5-Hydroxymethylcytosine (5hmC) modifications are generated by the Ten-eleven-translocation (TET) family of enzymes, TET 1, 2, and 3, through the oxidation of methylated cytosines (5mC). The TET family is capable of further oxidizing 5hmC to 5fC and 5caC, leading to eventual DNA demethylation. However, 5hmC marks can also exist stably in DNA. Stable 5hmC is enriched in the gene bodies of activated genes in multiple tissues, as well as associated with regulatory regions such as enhancers. Alterations to 5hmC patterns have now been found in multiple diseases including osteoarthritis. Here, we describe a method to map 5hmC modifications by next-generation sequencing using a technique based on the selective modification and enrichment of the 5hmC mark. We additionally provide a bioinformatic analysis pipeline to interpret the resulting data.

BZIP Dimers CREB1, ATF2, Zta, ATF3|cJun, and cFos|cJun Prefer to Bind to Some Double-Stranded DNA Sequences Containing 5-Formylcytosine and 5-Carboxylcytosine.

In mammalian cells, 5-methylcytosine (5mC) occurs in genomic double-stranded DNA (dsDNA) and is enzymatically oxidized to 5-hydroxymethylcytosine (5hmC), then to 5-formylcytosine (5fC), and finally to 5-carboxylcytosine (5caC). These cytosine modifications are enriched in regulatory regions of the genome. The effect of these oxidative products on five bZIP dimers (CREB1, ATF2, Zta, ATF3|cJun, and cFos|cJun) binding to five types of dsDNA was measured using protein binding microarrays. The five dsDNAs contain either cytosine in both DNA strands or cytosine in one strand and either 5mC, 5hmC, 5fC, or 5caC in the second strand. Some sequences containing the CEBP half-site GCAA are bound more strongly by all five bZIP domains when dsDNA contains 5mC, 5hmC, or 5fC. dsDNA containing 5caC in some TRE (AP-1)-like sequences, e.g., TGACTAA, is better bound by Zta, ATF3|cJun, and cFos|cJun.

In silico structural analysis of sequences containing 5-hydroxymethylcytosine reveals its potential as binding regulator for development, ageing and cancer-related transcription factors

ABSTRACT The presence of 5-hydroxymethyl cytosine in DNA has been previously associated with ageing. Using in silico analysis of normal liver samples we presently observed that in 5-hydroxymethyl cytosine sequences, DNA methylation is dependent on the co-presence of G-quadruplexes and palindromes. This association exhibits discrete patterns depending on G-quadruplex and palindrome densities. DNase-Seq data show that 5-hydroxymethyl cytosine sequences are common among liver nucleosomes (p < 2.2x10−16) and threefold more frequent than nucleosome sequences. Nucleosomes lacking palindromes and potential G-quadruplexes are rare in vivo (1%) and nucleosome occupancy potential decreases with increasing G-quadruplexes. Palindrome distribution is similar to that previously reported in nucleosomes. In low and mixed complexity sequences 5-hydroxymethyl cytosine is frequently located next to three elements: G-quadruplexes or imperfect G-quadruplexes with CpGs, or unstable hairpin loops (TCCCAY6TGGGA) mostly located in antisense strands or finally A-/T-rich segments near these motifs. The high frequencies and selective distribution of pentamer sequences (including TCCCA, TGGGA) probably indicate the positive contribution of 5-hydroxymethyl cytosine to stabilize the formation of structures unstable in the absence of this cytosine modification. Common motifs identified in all total 5-hydroxymethyl cytosine-containing sequences exhibit high homology to recognition sites of several transcription factor families: homeobox, factors involved in growth, mortality/ageing, cancer, neuronal function, vision, and reproduction. We conclude that cytosine hydroxymethylation could play a role in the recognition of sequences with G-quadruplexes/palindromes by forming epigenetically regulated DNA ‘springs’ and governing expansions or compressions recognized by different transcription factors or stabilizing nucleosomes. The balance of these epigenetic elements is lost in hepatocellular carcinoma.

Antigen Retrieval for Immunostaining of Modified Cytosine Species.

Immunostaining (also called as immunofluorescence) is a fluorescence labeling method to stain one or more epitopes of interest on DNA and/or protein using specific antibodies. Cytosine modifications can be detected quantitatively by immunostaining. The protocol commonly includes sequential steps. These include fixation, permeabilization, antigen retrieval, blocking, incubation with primary and secondary antibodies, and visualization under the microscope followed by image-based intensity analysis of staining. Each step is important, but antigen retrieval is especially necessary for DNA epitopes such as cytosine modifications as antibodies can access cytosines in DNA only once the DNA double-strand is denatured and DNA-packaging proteins have been removed. Hydrochloric acid is commonly used for this purpose. However, there are additional treatments with enzymes to enhance antigen retrieval and improve the detection by increasing staining intensity. This chapter describes current methodology for improving antigen retrieval for the staining of the cytosine modifications 5'-methylcytosine (5meC), 5'-hydroxymethylcytosine (5hmC), 5'-formylcytosine (5fC), and 5'-carboxycytosine (5caC).

Immunochemical Detection of Modified Species of Cytosine in Plant Tissues.

Methylated cytosine (5-methylcytosine) is the most studied epigenetic mark involved in the regulation of gene expression. Although it displays highly variable dynamics during plant ontogenesis, it is possible to gain a fine spatial perspective with immunohistochemistry techniques that use specific antibodies and fluorochromes. Besides, there are other cytosine modifications described in plants, although their biological significance is still unknown (i.e., 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine). Here we present a standardized protocol to detect cytosine modifications in plant tissues.

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.

High-Resolution Analysis of 5-Hydroxymethylcytosine by TET-Assisted Bisulfite Sequencing.

DNA cytosine modification is an important epigenetic mechanism that serves critical functions in a variety of biological processes in development and disease. 5-Methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are the two most common epigenetic marks found in the mammalian genome. 5hmC is generated from 5mC by the ten-eleven translocation (TET) family of dioxygenase enzymes. This modification can reach substantial levels in certain cell types such as embryonic stem cells and neurons. Standard bisulfite sequencing techniques cannot distinguish between 5mC and 5hmC. Therefore, the method of TET-assisted bisulfite sequencing has been developed for detecting 5hmC specifically. The method is based on protection of 5hmC by glycosylation followed by complete oxidation of both 5mC and 5fC to 5caC, which converts to uracil after bisulfite treatment leaving only 5hmC remaining as a cytosine signal after PCR and sequencing. The method requires a highly active TET protein for the conversion steps. Here, we present an efficient TET protein purification method and a streamlined TAB-sequencing protocol for 5hmC analysis at single base resolution.

Mapping DNA Methylation in Mammals: The State of the Art.

A complete understanding of the dynamics and function of cytosine modifications in mammalian biology is lacking. Central to achieving this understanding is the availability of techniques that permit sensitive and specific genome-wide mapping of DNA modifications in mammalian DNA. The last decade has seen the development of a vast arsenal of novel profiling approaches enabling epigeneticists to tackle research questions that were previously out of reach. Here, we review the techniques currently available for profiling DNA modifications in mammals, discuss their strengths and weaknesses, and speculate on the future direction of DNA modification profiling technologies.

Liquid Chromatography-Mass Spectrometry Analysis of Cytosine Modifications.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a widely used technique in the global analysis of epigenetic DNA modifications. The high-resolution chromatographic separation along with sensitive MS detection permits the identification and quantification of deoxyribonucleosides with precision and reliability. Although there have been tremendous advances in LC and MS instrumentation in recent years, sample preparation has not experienced a similar rate of development and is often a bottleneck to chemical analysis. Here we present a protocol for identification and quantification of cytosine modifications that combines a robust and efficient method to generate single nucleosides from genomic DNA samples followed by direct LC-MS/MS analysis.