Single-Base Resolution Analysis of 5-Formyl and 5-Carboxyl Cytosine Reveals Promoter DNA Methylation Dynamics

Ten eleven translocation (Tet) proteins oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5fC and 5caC can be further excised by thymine-DNA glycosylase (Tdg). Here, we present a genome-wide approach, named methylation-assisted bisulfite sequencing (MAB-seq), that enables single-base resolution mapping of 5fC and 5caC and measures their abundance. Application of this method to mouse embryonic stem cells (ESCs) shows the occurrence of 5fC and 5caC residues on the hypomethylated promoters of highly expressed genes, which is increased upon Tdg silencing, revealing active DNA demethylation on these promoters. Genome-wide mapping of Tdg reveals extensive colocalization with Tet1 on active promoters. These regions were found to be methylated by Dnmt1 and Dnmt3a and demethylated by a Tet-dependent mechanism. Our work demonstrates the DNA methylation dynamics that occurs on the promoters of the expressed genes and provides a genomic reference map of 5fC and 5caC in ESCs. [Display omitted] •We present MAB-seq, a method for mapping genome-wide 5fC and 5caC•5fC and 5caC occur on active promoters and enhancers•5fC and 5caC correlate with Tet1 and TDG, and TDG knockdown increases their levels•Highly expressed genes are continuously involved into methylation and demethylation Ten eleven translocation enzymes convert 5-methylcytosine to 5hmC, 5fC, and 5caC, promoting DNA demethylation through base excision by the thymine-DNA glycosylase. Neri et al. map 5fC and 5caC genome wide and find them on promoters of highly expressed genes, indicating dynamic methylation and demethylation.