5fC

5-formylcytosine (5fC) is a chemically modified form of cytosine that arises in DNA as part of the active DNA demethylation pathway. It is generated when 5-methylcytosine (5mC) is oxidized by the TET family of dioxygenases, progressing from 5mC to 5-hydroxymethylcytosine (5hmC) to 5fC, and then to 5-carboxylcytosine (5caC). In this sequence, 5fC represents an intermediate state rather than a stable, long-lived epigenetic mark.

In mammals, 5fC is typically processed through the base excision repair pathway. Thymine DNA glycosylase (TDG) recognizes and removes 5fC (and 5caC), generating an abasic site that is repaired to unmodified cytosine. This sequence of oxidation and repair completes the active demethylation cycle, restoring a cytosine without a methyl group.

5fC is found at relatively low abundance compared with 5mC and 5hmC, but its presence is dynamic. Genomic studies show enrichment of 5fC at regulatory regions such as promoters and enhancers and in developmental contexts, including embryonic stages and neural tissue, where DNA methylation landscapes undergo remodeling.

The functional significance of 5fC extends beyond a mere intermediate. As a reactive aldehyde, it can influence DNA structure and accessibility, and it serves as a substrate for TDG-mediated demethylation. Some evidence suggests 5fC may contribute to transcriptional regulation and may interact with DNA repair and chromatin-modulating processes, though its roles as a stable epigenetic signal remain a subject of ongoing research.

Detection and quantification of 5fC employ chemical labeling approaches that exploit its aldehyde group, as well as sequencing-based methods and mass spectrometry. Together, these techniques enable mapping of 5fC distribution and dynamics across genomes.