Active turnover of genomic methylcytosine in pluripotent cells.

Epigenetic plasticity underpins cell potency, but the extent to which active turnover of DNA methylation contributes to such plasticity is not known, and the underlying pathways are poorly understood. Here we use metabolic labeling with stable isotopes and mass spectrometry to quantitatively address the global turnover of genomic 5-methyl-2'-deoxycytidine (mdC), 5-hydroxymethyl-2'-deoxycytidine (hmdC) and 5-formyl-2'-deoxycytidine (fdC) across mouse pluripotent cell states. High rates of mdC/hmdC oxidation and fdC turnover characterize a formative-like pluripotent state. In primed pluripotent cells, the global mdC turnover rate is about 3-6% faster than can be explained by passive dilution through DNA synthesis. While this active component is largely dependent on ten-eleven translocation (Tet)-mediated mdC oxidation, we unveil additional oxidation-independent mdC turnover, possibly through DNA repair. This process accelerates upon acquisition of primed pluripotency and returns to low levels in lineage-committed cells. Thus, in pluripotent cells, active mdC turnover involves both mdC oxidation-dependent and oxidation-independent processes.

Deformylation of 5-Formylcytidine in Different Cell Types.

Epigenetic programming of cells requires methylation of deoxycytidines (dC) to 5-methyl-dC (mdC) followed by oxidation to 5-hydroxymethyl-dC (hmdC), 5-formyl-dC (fdC), and 5-carboxy-dC (cadC). Subsequent transformation of fdC and cadC back to dC by various pathways establishes a chemical intra-genetic control circle. One of the discussed pathways involves the Tdg-independent deformylation of fdC directly to dC. Here we report the synthesis of a fluorinated fdC feeding probe (F-fdC) to study direct deformylation to F-dC. The synthesis was performed along a novel pathway that circumvents any F-dC as a reaction intermediate to avoid contamination interference. Feeding of F-fdC and observation of F-dC formation in vivo allowed us to gain insights into the Tdg-independent removal process. While deformylation was shown to occur in stem cells, we here provide data that prove deformylation also in different somatic cell types. We also investigated active demethylation in a non-dividing neurogenin-inducible system of iPS cells that differentiate into bipolar neurons.

Intragenomic Decarboxylation of 5-Carboxy-2'-deoxycytidine.

Cellular DNA is composed of four canonical nucleosides (dA, dC, dG and T), which form two Watson-Crick base pairs. In addition, 5-methylcytosine (mdC) may be present. The methylation of dC to mdC is known to regulate transcriptional activity. Next to these five nucleosides, the genome, particularly of stem cells, contains three additional dC derivatives, which are formed by stepwise oxidation of the methyl group of mdC with the help of Tet enzymes. These are 5-hydroxymethyl-dC (hmdC), 5-formyl-dC (fdC), and 5-carboxy-dC (cadC). It is believed that fdC and cadC are converted back into dC, which establishes an epigenetic control cycle that starts with methylation of dC to mdC, followed by oxidation and removal of fdC and cadC. While fdC was shown to undergo intragenomic deformylation to give dC directly, a similar decarboxylation of cadC was postulated but not yet observed on the genomic level. By using metabolic labelling, we show here that cadC decarboxylates in several cell types, which confirms that both fdC and cadC are nucleosides that are directly converted back to dC within the genome by C-C bond cleavage.

Biomimetic Iron Complex Achieves TET Enzyme Reactivity*.

The epigenetic marker 5-methyl-2'-deoxycytidine (5mdC) is the most prevalent modification to DNA. It is removed inter alia via an active demethylation pathway: oxidation by Ten-Eleven Translocation 5-methyl cytosine dioxygenase (TET) and subsequent removal via base excision repair or direct demodification. Recently, we have shown that the synthetic iron(IV)-oxo complex [FeIV (O)(Py5 Me2 H)]2+ (1) can serve as a biomimetic model for TET by oxidizing the nucleobase 5-methyl cytosine (5mC) to its natural metabolites. In this work, we demonstrate that nucleosides and even short oligonucleotide strands can also serve as substrates, using a range of HPLC and MS techniques. We found that the 5-position of 5mC is oxidized preferably by 1, with side reactions occurring only at the strand ends of the used oligonucleotides. A detailed study of the reactivity of 1 towards nucleosides confirms our results; that oxidation of the anomeric center (1') is the most common side reaction.

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.

1H NMR Chemical Exchange Techniques Reveal Local and Global Effects of Oxidized Cytosine Derivatives.

5-Carboxycytosine (5caC) is a rare epigenetic modification found in nucleic acids of all domains of life. Despite its sparse genomic abundance, 5caC is presumed to play essential regulatory roles in transcription, maintenance and base-excision processes in DNA. In this work, we utilize nuclear magnetic resonance (NMR) spectroscopy to address the effects of 5caC incorporation into canonical DNA strands at multiple pH and temperature conditions. Our results demonstrate that 5caC has a pH-dependent global destabilizing and a base-pair mobility enhancing local impact on dsDNA, albeit without any detectable influence on the ground-state B-DNA structure. Measurement of hybridization thermodynamics and kinetics of 5caC-bearing DNA duplexes highlighted how acidic environment (pH 5.8 and 4.7) destabilizes the double-stranded structure by ∼10-20 kJ mol-1 at 37 °C when compared to the same sample at neutral pH. Protonation of 5caC results in a lower activation energy for the dissociation process and a higher barrier for annealing. Studies on conformational exchange on the microsecond time scale regime revealed a sharply localized base-pair motion involving exclusively the modified site and its immediate surroundings. By direct comparison with canonical and 5-formylcytosine (5fC)-edited strands, we were able to address the impact of the two most oxidized naturally occurring cytosine derivatives in the genome. These insights on 5caC's subtle sensitivity to acidic pH contribute to the long-standing questions of its capacity as a substrate in base excision repair processes and its purpose as an independent, stable epigenetic mark.

Synthesis and structure elucidation of the human tRNA nucleoside mannosyl-queuosine.

Queuosine (Q) is a structurally complex, non-canonical RNA nucleoside. It is present in many eukaryotic and bacterial species, where it is part of the anticodon loop of certain tRNAs. In higher vertebrates, including humans, two further modified queuosine-derivatives exist - galactosyl- (galQ) and mannosyl-queuosine (manQ). The function of these low abundant hypermodified RNA nucleosides remains unknown. While the structure of galQ was elucidated and confirmed by total synthesis, the reported structure of manQ still awaits confirmation. By combining total synthesis and LC-MS-co-injection experiments, together with a metabolic feeding study of labelled hexoses, we show here that the natural compound manQ isolated from mouse liver deviates from the literature-reported structure. Our data show that manQ features an α-allyl connectivity of its sugar moiety. The yet unidentified glycosylases that attach galactose and mannose to the Q-base therefore have a maximally different constitutional connectivity preference. Knowing the correct structure of manQ will now pave the way towards further elucidation of its biological function.

Copper(i)-catalysed transfer hydrogenations with ammonia borane.

Highly Z-selective alkyne transfer semihydrogenations and conjugate transfer hydrogenations of enoates can be effected by employing a readily available and air-stable copper(i)/N-heterocyclic carbene (NHC) complex, [IPrCuOH]. As an easy to handle and potentially recyclable H2 source, ammonia borane (H3NBH3) is used.