Genome-wide mapping of epigenetic modification of 5-formylcytosine at single-base resolution by chemical labeling enrichment and deamination sequencing

DNA cytosine methylation (5-methylcytosine, 5mC) is a predominant epigenetic modification that plays critical roles in a variety of biological and pathological processes in mammals. In active DNA demethylation, the ten-eleven translocation (TET) dioxygenases can sequentially oxidize 5mC to generate three modified forms of cytosine, 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Beyond being a demethylation intermediate, recent studies have shown that 5fC has regulatory functions in gene expression and chromatin organization. While some methods have been developed to detect 5fC, genome-wide mapping of 5fC at base resolution are still highly desirable. Herein, we propose a Chemical Labeling Enrichment and Deamination sequencing (CLED-seq) method for detecting 5fC in genomic DNA at single-base resolution. The CLED-seq method utilizes selective labeling and enrichment of 5fC-containing DNA fragments, followed by deamination mediated by A3A and sequencing. In the CLED-seq process, while all C, 5mC, and 5hmC are interpreted as T during sequencing, 5fC is still read as C, enabling the precise detection of 5fC in DNA. Using the proposed CLED-seq method, we accomplished genome-wide mapping of 5fC in mouse embryonic stem cells. The mapping study revealed that promoter regions enriched with 5fC overlapped with H3K4me1, H3K4me3, and H3K27ac marks. These findings suggest a correlation between 5fC marks and active gene expression in mouse embryonic stem cells. In conclusion, CLED-seq is a straightforward, bisulfite-free method that offers a valuable tool for detecting 5fC in genomes at a single-base resolution. In our current study, we have discovered that biotin-labeled 5fC exhibits notable resistance to deamination by A3A. Building on this finding, we propose a novel method, termed Chemical Labeling Enrichment and Deamination sequencing (CLED-seq), for the precise genome-wide mapping of 5fC at the single-base resolution. In the CLED-seq protocol, we employ biotin-ONH2 to selectively label the formyl group of 5fC in fragmented genomic DNA. Subsequently, streptavidin-coated magnetic beads are utilized to enrich the 5fC-containing DNA fragments. Treatment with A3A leads to the deamination of C, 5mC, and 5hmC, resulting in the formation of U, T, and 5hmU, respectively.