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.