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

Abstract

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.

Retracted Article: Divergent synthesis of 5-substituted pyrimidine 2′-deoxynucleosides and their incorporation into oligodeoxynucleotides for the survey of uracil DNA glycosylases

“…Direct deformylation of 5foC can also lead to the restoration of cytosine (C). 6 An alternative proposal involves 5mC deamination by activationinduced deaminase (AID), 7,8 followed by thymine DNA glycosylase (TDG)-initiated base excision repair (BER). 9 Recent ndings also implicate the removal of modied C or uracil (U) derivatives by DNA glycosylase/BER/nucleotide excision repair (NER) as another potential demethylation mechanism.…”

Deformylation of 5‐Formylcytidine in Different Cell Types

“…There is certainly a need for active nucleophiles that are able to attack the C6-position of 2'-F-fdC (1) to promote the reaction. [18] The herein reported data show that the synthesis pathway towards the probe molecule of 2'-F-fdC (1) using only 2'-F-dU intermediates ensures the best possible purity of the compound for biological feeding experiments. Feeding of the newly synthesized compound shows that deformylation of 2'-F-fdC (1) to 2'-F-dC (2) occurs inside the genome and that it is a common process in a variety of cell lines that is inversely correlated with the speed of replication.…”

5‐Hydroxymethyl‐, 5‐Formyl‐ and 5‐Carboxydeoxycytidines as Oxidative Lesions and Epigenetic Marks

“…This whole process of dC methylation by dedicated DNA methyltransferases (Dnmt enzymes), Tet‐induced oxidation to xdC and Tdg‐initiated conversion of fdC and cadC to dC sites, creates a dynamic active methylation and demethylation circle depicted in Figure 1 [11–14] …”