Direct and Base Excision Repair-Mediated Regulation of a GC-Rich cis-Element in Response to 5-Formylcytosine and 5-Carboxycytosine.

Stepwise oxidation of the epigenetic mark 5-methylcytosine and base excision repair (BER) of the resulting 5-formylcytosine (5-fC) and 5-carboxycytosine (5-caC) may provide a mechanism for reactivation of epigenetically silenced genes; however, the functions of 5-fC and 5-caC at defined gene elements are scarcely explored. We analyzed the expression of reporter constructs containing either 2'-deoxy-(5-fC/5-caC) or their BER-resistant 2'-fluorinated analogs, asymmetrically incorporated into CG-dinucleotide of the GC box cis-element (5'-TGGGCGGAGC) upstream from the RNA polymerase II core promoter. In the absence of BER, 5-caC caused a strong inhibition of the promoter activity, whereas 5-fC had almost no effect, similar to 5-methylcytosine or 5-hydroxymethylcytosine. BER of 5-caC caused a transient but significant promoter reactivation, succeeded by silencing during the following hours. Both responses strictly required thymine DNA glycosylase (TDG); however, the silencing phase additionally demanded a 5'-endonuclease (likely APE1) activity and was also induced by 5-fC or an apurinic/apyrimidinic site. We propose that 5-caC may act as a repressory mark to prevent premature activation of promoters undergoing the final stages of DNA demethylation, when the symmetric CpG methylation has already been lost. Remarkably, the downstream promoter activation or repression responses are regulated by two separate BER steps, where TDG and APE1 act as potential switches.

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.

Unified Description of Ultrafast Excited State Decay Processes in Epigenetic Deoxycytidine Derivatives.

Epigenetic DNA modifications play a fundamental role in modulating gene expression and regulating cellular and developmental biological processes, thereby forming a second layer of information in DNA. The epigenetic 2'-deoxycytidine modification 5-methyl-2'-deoxycytidine, together with its enzymatic oxidation products (5-hydroxymethyl-2'-deoxycytidine, 5-formyl-2'-deoxycytidine, and 5-carboxyl-2'-deoxycytidine), are closely related to deactivation and reactivation of DNA transcription. Here, we combine sub-30-fs transient absorption spectroscopy with high-level correlated multiconfigurational CASPT2/MM computational methods, explicitly including the solvent, to obtain a unified picture of the photophysics of deoxycytidine-derived epigenetic DNA nucleosides. We assign all the observed time constants and identify the excited state relaxation pathways, including the competition of intersystem crossing and internal conversion for 5-formyl-2'-deoxycytidine and ballistic decay to the ground state for 5-carboxy-2'-deoxycytidine. Our work contributes to shed light on the role of epigenetic derivatives in DNA photodamage as well as on their possible therapeutic use.