Site-specific quantification of 5-carboxylcytosine in DNA by chemical conversion coupled with ligation-based PCR

5-Methylcytosine (5mC) is the most important epigenetic modification in mammals. The active DNA demethylation could be achieved through the ten-eleven translocation (TET) protein-mediated oxidization of 5mC with the generation of 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). It has been known that 5mC, 5hmC and 5fC play critical roles in modulating gene expression. However, unlike the 5mC, 5hmC, and 5fC, the functions of 5caC are still underexplored. Investigation of the functions of 5caC relies on the accurate quantification and localization analysis of 5caC in DNA. In the current study, we developed a method by chemical conversion in conjugation with ligation-based real-time quantitative PCR (qPCR) for the site-specific quantification of 5caC in DNA. This method depends on the selective conversion of 5caC to form dihydrouracil (DHU) by pyridine borane treatment. DHU behaves like thymine and pairs with adenine (DHU-A). Thus, the chemical conversion by pyridine borane leads to the transformation of base paring from 5caC-G to DHU-A, which is utilized to achieve the site-specific detection and quantification of 5caC in DNA. As a proof-of-concept, the developed method was successfully applied in the site-specific quantification of 5caC in synthesized DNA spiked in complex biological samples. The method is rapid, straightforward and cost-effective, and shows promising in promoting the investigation of the functional roles of 5caC in future study.     

Direct decarboxylation of ten-eleven translocation-produced 5-carboxylcytosine in mammalian genomes forms a new mechanism for active DNA demethylation

DNA cytosine methylation (5-methylcytosine, 5mC) is the most important epigenetic mark in higher eukaryotes. 5mC in genomes is dynamically controlled by writers and erasers. DNA (cytosine-5)-methyltransferases (DNMTs) are responsible for the generation and maintenance of 5mC in genomes. Active demethylation of 5-methylcytosine (5mC) is achieved by ten-eleven translocation (TET) dioxygenase-mediated oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5fC and 5caC are further processed by thymine DNA glycosylase (TDG)-initiated base excision repair (BER) to restore unmodified cytosines. The TET-TDG-BER pathway could cause the production of DNA strand breaks and therefore jeopardize the integrity of genomes. Here, we investigated the direct decarboxylation of 5caC in mammalian genomes by using metabolic labeling with 2′-fluorinated 5caC (F-5caC) and mass spectrometry analysis. Our results clearly demonstrated the decarboxylation of 5caC occurring in mammalian genomes, which unveiled that, in addition to the TET-TDG-BER pathway, the direct decarboxylation of TET-produced 5caC constituted a new pathway for active demethylation of 5mC in mammalian genomes.

We demonstrated that the ten-eleven translocation (TET) dioxygenase-mediated oxidation of 5-methylcytosine followed by direct decarboxylation of 5-carboxylcytosine constitutes a novel pathway for active DNA demethylation in mammalian genomes.     

Preferential 5‐Methylcytosine Oxidation in the Linker Region of Reconstituted Positioned Nucleosomes by Tet1 Protein

Tet (ten–eleven translocation) family proteins oxidize 5‐methylcytosine (mC) to 5‐hydroxymethylcytosine (hmC), 5‐formylcytosine (fC), and 5‐carboxycytosine (caC), and are suggested to be involved in the active DNA demethylation pathway. In this study, we reconstituted positioned mononucleosomes using CpG‐methylated 382 bp DNA containing the Widom 601 sequence and recombinant histone octamer, and subjected the nucleosome to treatment with Tet1 protein. The sites of oxidized methylcytosine were identified by bisulfite sequencing. We found that, for the oxidation reaction, Tet1 protein prefers mCs located in the linker region of the nucleosome compared with those located in the core region.     

Synthesis of a DNA Promoter Segment Containing All Four Epigenetic Nucleosides: 5‐Methyl‐, 5‐Hydroxymethyl‐, 5‐Formyl‐, and 5‐Carboxy‐2′‐Deoxycytidine

A 5‐formyl‐2′‐deoxycytidine (fdC) phosphoramidite building block that enables the synthesis of fdC‐containing DNA with excellent purity and yield has been developed. In combination with phosphoramidites for 5‐methyl‐dC, 5‐hydroxymethyl‐dC, and carboxy‐dC, it was possible to prepare a segment of the OCT‐4 promoter that contains all four epigenetic bases. Because of the enormous interest in these new epigenetic bases, the ability to insert all four of them into DNA should be of great value for the scientific community.     

Synthesis of DNA oligos containing 2'-deoxy-2'-fluoro-D-arabinofuranosyl-5-carboxylcytosine as hTDG inhibitor

As an important step of the active demethylation of 5-methylcytosine (5mC), human thymine DNA glycosylase (hTDG) efficiently excises 5-carboxylcytosine (5caC) from double-stranded DNA (dsDNA). Here, we present synthesis of DNA oligos containing a 2'-deoxy-2'-fluoro-D-arabinofuranosyl-5-carboxylcytidine (F-5caC) modification that act as hTDG inhibitors. The glycosylase activity assay showed that F-5caC oligos were resistant to excision by the hTDG catalytic domain (hTDG(cat), residues 111-308) and they could inhibit the excision of DNA oligos containing 5caC. The electrophoretic mobility shift assay confirmed that DNA oligos containing F-5caC could bind well with unmodified hTDG(cat) to form a stable complex, which makes it possible to obtain the crystal structure of the complex to reveal details on how hTDG(cat) recognizes the DNA substrate.     

Pyrene‐Based Quantitative Detection of the 5‐Formylcytosine Loci Symmetry in the CpG Duplex Content during TET‐Dependent Demethylation

Methylcytosine (5mC) is mostly symmetrically distributed in CpG sites. Ten‐eleven‐translocation (TET) proteins are the key enzymes involved in active DNA demethylation through stepwise oxidation of 5mC. However, oxidation pathways of TET enzymes in the symmetrically methylated CpG context are still elusive. Employing the unique fluorescence properties of pyrene group, we designed and synthesized a sensitive fluorescence‐based probe not only to target 5‐formylcytosine (5fC) sites, but also to distinguish symmetric from asymmetric 5fC sites in the double stranded DNA context during TET‐dependent 5mC oxidation process. Using this novel probe, we revealed dominant levels of symmetric 5fC among total 5fC sites during in vitro TET‐dependent 5mC oxidation and novel mechanistic insights into the TET‐dependent 5mC oxidation in the mCpG context.     

Analysis of an Active Deformylation Mechanism of 5‐Formyl‐deoxycytidine (fdC) in Stem Cells

The removal of 5‐methyl‐deoxycytidine (mdC) from promoter elements is associated with reactivation of the silenced corresponding genes. It takes place through an active demethylation process involving the oxidation of mdC to 5‐hydroxymethyl‐deoxycytidine (hmdC) and further on to 5‐formyl‐deoxycytidine (fdC) and 5‐carboxy‐deoxycytidine (cadC) with the help of α‐ketoglutarate‐dependent Tet oxygenases. The next step can occur through the action of a glycosylase (TDG), which cleaves fdC out of the genome for replacement by dC. A second pathway is proposed to involve C?C bond cleavage that converts fdC directly into dC. A 6‐aza‐5‐formyl‐deoxycytidine (a‐fdC) probe molecule was synthesized and fed to various somatic cell lines and induced mouse embryonic stem cells, together with a 2′‐fluorinated fdC analogue (F‐fdC). While deformylation of F‐fdC was clearly observed in vivo, it did not occur with a‐fdC, thus suggesting that the C?C bond‐cleaving deformylation is initiated by nucleophilic activation.     

Ultrafast proton-coupled electron-transfer dynamics in pyrene-modified pyrimidine nucleosides: model studies towards an understanding of reductive electron transport in DNA.

5-(Pyren-1-yl)-2'-deoxyuridine (PydU) and 5-(Pyren-1-yl)-2'-deoxycytidine (PydC) were used as model nucleosides for DNA-mediated reductive electron transport (ET) in steady-state fluorescence and femtosecond time-resolved transient absorption spectroscopy studies. Excitation of the pyrene moiety in PydU and PydC leads to an intramolecular electron transfer that yields the pyrenyl radical cation and the corresponding pyrimidine radical anion (dU.- and dC.-. By comparing the excited state dynamics of PydC and PydU, we derived information about the energy difference between the two pyrimidine radical anion states. To determine the influence of protonation on the rates of photoinduced intramolecular ET, the spectroscopic investigations were performed in acetonitrile, MeCN, and in water at different pH values. The results show a significant difference in the basicity of the generated pyrimidine radical anions and imply an involvement of proton transfer during electron hopping in DNA. Our studies revealed that the radical anion dC.- is being protonated even in basic aqueous solution on a picosecond time scale (or faster). These results suggest that protonation of dC.- may also occur in DNA. In contrast, efficient ET in PydU could only be observed at low pH values (< 5). In conclusion, we propose--based on the free energy differences and the different basicities--that only dT.- but not dC.- can participate as an intermediate charge carrier for excess electron migration in DNA.     

Quantification of 5-methylcytosine, 5-hydroxymethylcytosine and 5-carboxylcytosine from the blood of cancer patients by an enzyme-based immunoassay

Background

Genome-wide aberrations of the classic epigenetic modification 5-methylcytosine (5mC), considered the hallmark of gene silencing, has been implicated to play a pivotal role in mediating carcinogenic transformation of healthy cells. Recently, three epigenetic marks derived from enzymatic oxidization of 5mC namely 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), have been discovered in the mammalian genome. Growing evidence suggests that these novel bases possess unique regulatory functions and may play critical roles in carcinogenesis.

Methods

To provide a quantitative basis for these rare epigenetic marks, we have designed a biotin–avidin mediated enzyme-based immunoassay (EIA) and evaluated its performance in genomic DNA isolated from blood of patients diagnosed with metastatic forms of lung, pancreatic and bladder cancer, as well as healthy controls. The proposed EIA incorporates spatially optimized biotinylated antibody and a high degree of horseradish-peroxidase (HRP) labeled streptavidin, facilitating signal amplification and sensitive detection.

Results

We report that the percentages of 5mC, 5hmC and 5caC present in the genomic DNA of blood in healthy controls as 1.025 ± 0.081, 0.023 ± 0.006 and 0.001 ± 0.0002, respectively. We observed a significant (p < 0.05) decrease in the mean global percentage of 5hmC in blood of patients with malignant lung cancer (0.013 ± 0.003%) in comparison to healthy controls.

Conclusion

The precise biological roles of these epigenetic modifications in cancers are still unknown but in the past two years it has become evident that the global 5hmC content is drastically reduced in a variety of cancers. To the best of our knowledge, this is the first report of decreased 5hmC content in the blood of metastatic lung cancer patients and the clinical utility of this observation needs to be further validated in larger sample datasets.     

A photo-elutable and template-free isothermal amplification strategy for sensitive fluorescence detection of 5-formylcytosine in genomic DNA

5-Formylcytosine (5fC), as an important epigenetic modification, plays a vital role in diverse biological processes and multiple diseases by regulating gene expression. Owing to the extremely low abundance of 5fC in all mammalian tissues and high structural similarity with other cytosine derivatives, the precise and sensitive detection of 5fC is challenging. Herein, a photo-elutable and template-free isothermal amplification strategy has been proposed for the sensitive detection of 5fC in genomic DNA based on 5fC-specific biotinylation, enrichment, photocleavage, and terminal deoxynucleotidyl transferase (TdT)-assisted fluorescence signal amplification, which is termed 5fC-PTIAS. By introducing the highly specific chemolabeling and the one-step photoelution processes, this strategy possesses a minimal nonspecific background as well as a much higher amplification efficiency. With the high signal-to-noise ratio, this strategy can achieve the accurate quantification of 5fC in various biological samples including mouse brain, kidney, and liver, with a limit of detection (LOD) of 0.025‰ in DNA (S/N = 3). These results not only confirm the widespread distribution of 5fC but also indicate its significant variation in different tissues and ages. The bisulfite- and mass spectrometry-free strategy is highly sensitive, selective, and easily mastered, holding great promise in detecting other epigenetic modifications with much lower levels.     

5-Methylcytosine is Oxidized to the Natural Metabolites of TET Enzymes by a Biomimetic Iron(IV)-Oxo Complex

Ten-eleven-translocation (TET) methyl cytosine dioxygenases play a key role in epigenetics by oxidizing the epigenetic marker 5-methyl cytosine (5mC) to 5-hydroxymethyl cytosine (5hmC), 5-formyl cytosine (5fC), and 5-carboxy cytosine (5cC). Although much of the metabolism of 5mC has been studied closely, certain aspects—such as discrepancies among the observed catalytic activity of TET enzymes and calculated bond dissociation energies of the different cytosine substrates—remain elusive. Here, it is reported that the DNA base 5mC is oxidized to 5hmC, 5fC, and 5cC by a biomimetic iron(IV)-oxo complex, reminiscent of the activity of TET enzymes. Studies show that 5hmC is preferentially turned over compared with 5mC and 5fC and that this is in line with the calculated bond dissociation energies. The optimized syntheses of d₃-5mC and d₂-5hmC are also reported and in the reaction with the biomimetic iron(IV)-oxo complex these deuterated substrates showed large kinetic isotope effects, confirming the hydrogen abstraction as the rate-limiting step. Taken together, these results shed light on the intrinsic reactivity of the C−H bonds of epigenetic markers and the contribution of the second coordination sphere in TET enzymes.     

Structure and function of noncanonical nucleobases

DNA and RNA contain, next to the four canonical nucleobases, a number of modified nucleosides that extend their chemical information content. RNA is particularly rich in modifications, which is obviously an adaptation to their highly complex and variable functions. In fact, the modified nucleosides and their chemical structures establish a second layer of information which is of central importance to the function of the RNA molecules. Also the chemical diversity of DNA is greater than originally thought. Next to the four canonical bases, the DNA of higher organisms contains a total of four epigenetic bases: m(5) dC, hm(5) dC, f(5) dC und ca(5) dC. While all cells of an organism contain the same genetic material, their vastly different function and properties inside complex higher organisms require the controlled silencing and activation of cell-type specific genes. The regulation of the underlying silencing and activation process requires an additional layer of epigenetic information, which is clearly linked to increased chemical diversity. This diversity is provided by the modified non-canonical nucleosides in both DNA and RNA.     

Label‐Free Quantification of 5‐Azacytidines Directly in the Genome

Azacytidines (AzaC and AzadC) are clinically relevant pharmaceuticals that operate at the epigenetic level. They are integrated into the genome as antimetabolites to block DNA methylation events. This leads to a reduction of the 5‐methyl‐2′‐deoxycytidine (m⁵dC) level in the genome, which can activate epigenetically silenced genes. Because of the inherent chemical instability of Aza(d)Cs, their incorporation levels in DNA and RNA are difficult to determine, which hinders correlation of therapeutic effects with incorporation and removal processes. Existing methods involve radioactive labeling and are therefore unsuitable to monitor levels from patients. We report here a new direct chemical method that allows absolute quantification of the levels of incorporated AzaC and AzadC in both RNA and DNA. Furthermore, it clarifies that Aza(d)C accumulates to high levels (up to 12.9 million bases per genome). Although RNA‐based antimetabolites are often 2′‐deoxygenated in vivo and incorporated into DNA, for AzaC we see only limited incorporation into DNA. It accumulates predominantly in RNA where it, however, only leads to insignificant demethylation.     

Efficient synthesis of 5-hydroxymethylcytosine containing DNA

5-Hydroxymethylcytosine ((5-HOMe)dC) was recently discovered as the sixth base in the mammalian genome. The development of a new phosphoramidite building block is reported, which allows efficient synthesis of (5-HOMe)dC containing DNA. Key steps of the synthesis are a palladium-catalyzed formylation and the simultaneous protection of a hydroxyl and amino group as a cyclic carbamate. DNA synthesis is possible under standard conditions, and deprotection can be carried out with dilute NaOH.     

Unnatural Cytosine Bases Recognized as Thymines by DNA Polymerases by the Formation of the Watson–Crick Geometry

The emergence of unnatural DNA bases provides opportunities to demystify the mechanisms by which DNA polymerases faithfully decode chemical information on the template. It was previously shown that two unnatural cytosine bases (termed “M‐fC” and “I‐fC”), which are chemical labeling adducts of the epigenetic base 5‐formylcytosine, can induce C‐to‐T transition during DNA amplification. However, how DNA polymerases recognize such unnatural cytosine bases remains enigmatic. Herein, crystal structures of unnatural cytosine bases pairing to dA/dG in the KlenTaq polymerase‐host–guest complex system and pairing to dATP in the KlenTaq polymerase active site were determined. Both M‐fC and I‐fC base pair with dA/dATP, but not with dG, in a Watson–Crick geometry. This study reveals that the formation of the Watson–Crick geometry, which may be enabled by the A‐rule, is important for the recognition of unnatural cytosines.     

pH‐Independent Recognition of the dG ⋅ dC Base Pair in Triplex DNA: 9‐Deazaguanine N7‐(2′‐Deoxyribonucleoside) and Halogenated Derivatives Replacing Protonated dC

Triplex-forming oligonucleotides (TFOs) containing 9-deazaguanine N⁷-(2′-deoxyribonucleoside) 1a and halogenated derivatives 1b,c were synthesized employing solid-phase oligonucleotide synthesis. For that purpose, the phosphoramidite building blocks 5a – c and 8a – c were synthesized. Multiple incorporations of 1a – c in place of dC were performed within TFOs, which involved the sequence of five consecutive 1a – c ⋅ dG ⋅ dC triplets as well as of three alternating 1a – c ⋅ dG ⋅ dC and dT ⋅ dA ⋅ dT triplets. These TFOs were designed to bind in a parallel orientation to the target duplex. Triplex forming properties of these oligonucleotides containing 1a – c in the presence of Na⁺ and Mg²⁺ were studied by UV/melting-curve analysis and confirmed by circular-dichroism (CD) spectroscopy. The oligonucleotides containing 1a in the place of dC formed stable triplexes at physiological pH in the case of sequence of five consecutive 1a ⋅ dG ⋅ dC triplets as well as three alternating 1a – c ⋅ dG ⋅ dC and dT ⋅ dA ⋅ dT triplets. The replacement of 1a by 9-halogenated derivatives 1b,c further enhanced the stability of DNA triplexes. Nucleosides 1a – c also stabilized duplex DNA.     

Comparative Nucleosomal Reactivity of 5-Formyl-Uridine and 5-Formyl-Cytidine

5-Formyl-deoxyuridine (fdU) and 5-formyl-deoxycytidine (fdC) are formyl-containing nucleosides that are created by oxidative stress in differentiated cells. While fdU is almost exclusively an oxidative stress lesion formed from deoxythymidine (T), the situation for fdC is more complex. Next to formation as an oxidative lesion, it is particularly abundant in stem cells, where it is more frequently formed in an epigenetically important oxidation reaction performed by α-ketoglutarate dependent TET enzymes from 5-methyl-deoxycytidine (mdC). Recently, it was shown that genomic fdC and fdU can react with the ϵ-aminogroups of nucleosomal lysines to give Schiff base adducts that covalently link nucleosomes to genomic DNA. Here, we show that fdU features a significantly higher reactivity towards lysine side chains compared with fdC. This result shows that depending on the amounts of fdC and fdU, oxidative stress may have a bigger impact on nucleosome binding than epigenetics.