Effects of Tet-mediated Oxidation Products of 5-Methylcytosine on DNA Transcription in vitro and in Mammalian Cells

5-methylcytosine (5-mC) is a well-characterized epigenetic regulator in mammals. Recent studies showed that Ten-eleven translocation (Tet) proteins can catalyze the stepwise oxidation of 5-mC to produce 5-hydroxymethylcytosine (5-HmC), 5-formylcytosine (5-FoC) and 5-carboxylcytosine (5-CaC). The exciting discovery of these novel cytosine modifications has stimulated substantial research interests about their roles in epigenetic regulation. Here we systematically examined the effects of the oxidized 5-mC derivatives on the efficiency and fidelity of DNA transcription using a recently developed competitive transcription and adduct bypass assay. Our results showed that, when located on the transcribed strand, 5-FoC and 5-CaC exhibited marginal mutagenic and modest inhibitory effects on DNA transcription mediated by single-subunit T7 RNA polymerase or multi-subunit human RNA polymerase II in vitro and in human cells. 5-HmC displayed relatively milder blocking effects on transcription, and no mutant transcript could be detectable for 5-HmC in vitro or in cells. The lack of considerable mutagenic effects of the oxidized 5-mC derivatives on transcription was in agreement with their functions in epigenetic regulation. The modest blocking effects on transcription suggested that 5-FoC and 5-CaC may function in transcriptional regulation. These findings provided new evidence for the potential functional interplay between cytosine methylation status and transcription.