5-Formlycytosine (5fC): A new base?
DNA methylation and hydroxymethylation are well-established key players in gene regulation. Although many would agree that 5hmC also plays a critical role, there is still some debate over whether it serves a functional role, or is merely an artifact of DNA de-methylation. Even still, other intermediates of de-methylation are actively being investigated. In a recent paper by Iuilaro et al. 2016, a group of scientists argue that 5-formlycytosine (5fC), an intermediate of DNA de-methylation, is a stable and detectable base within the genome and may have an epigenetic function in its own right.
Active DNA de-methylation in mammals involves oxidation of 5-methylcytosine (5mC) by one or more of the TET enzymes to generate 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). 5fC and 5caC are then excised by the thymine DNA glycosylase (TDG) enzyme, initiating base excision repair (BER). 5fC has been detected in a variety of tissues and cell systems, at relatively low levels compared to 5mC and 5hmC. Recently, scientists at The Babraham Institute have shown that not only is 5fC is detectable and stable in vivo, but that several proteins have been found to specifically bind 5fC in various cell lines.
The group used both WT and Tdg null mouse embryos to identify sites marks by TET/TDG-driven active de-methylation. They first quantified genomic levels of 5fC in mouse embryos at various stages of development using LC/MS. Tdg null embryos lacking the TDG enzyme had roughly a seven-fold increase of 5fC compared to WT, however general genome-wide patterns did not differ much. The highest levels of 5fC were detected in the embryos’ hindbrind, and the lowest levels found in liver. Using a pull-down technique to profile 5fC marks genome wide, 5fC was found to be enriched in exons, 5’UTRs, and 3’UTRs, with lower presence in introns and gene bodies, and nearly absent in intergenic regions. The authors also observed 5fC enrichment over exon/intron boundaries in both WT and Tdg null embryos, suggesting a role in regulating splicing.
The group next analyzed 5fC distribution in relation to ENCODE histone datasets. They found that in all embryonic tissue, 5fC was enriched over distal regulatory regions marked by H3K4me1, as well as active enhancers, marked by H3K4me1 and H3K27ac. In contrast, 5fC was not enriched in regions marked by activating histone modifications. Tissue comparisons showed that 5fC-enhancer enrichment was largely tissue specific, with 5fC being enriched in brain-specific enhancers in hindbrain, and heart-specific enhancers in cardiac tissue. Taken together, the authors claim that this shows a specific enrichment of 5fC at active enhancer regions during embryonic development.
Iurlaro M, McInroy GR, Burgess HE, Dean W, Raiber EA, Bachman M, Beraldi D, Balasubramanian S, & Reik W (2016). In vivo genome-wide profiling reveals a tissue-specific role for 5-formylcytosine. Genome biology, 17 (1) PMID: 27356509