5-Formylcytosine can be a stable DNA modification in mammals

Abstract

5-Formylcytosine (5fC) is a rare base found in mammalian DNA and thought to be involved in active DNA demethylation. Here, we show that developmental dynamics of 5fC levels in mouse DNA differ from those of 5-hydroxymethylcytosine (5hmC), and using stable isotope labeling in vivo, we show that 5fC can be a stable DNA modification. These results suggest that 5fC has functional roles in DNA that go beyond being a demethylation intermediate.

Figure 1. Dynamics of global levels of 5fC during mouse development are distinct from those of 5hmC.

(a) Metabolism of cytosine modifications in DNA. Although the majority of 5mC and 5hmC was known to persist in the genomic DNa, the stability of 5fC and 5caC in vivo was unknown. Feeding [methyl-¹³CD₃]l-methionine can be used as a means to measure the lifetime of cytosine modifications in cells and in vivo. Labeling pattern is indicated in red. See also Figure 2. (b) Global levels of 5fC and 5caC in genomic DNA from mouse embryos (embryonic day 11.5, E11.5). Shown are mean ± s.e.m. of three animals. Each sample was analyzed in technical duplicate and the mean value was used. WT, wild type. (c–e) Changes of global 5fC (c), 5mC (d) and 5hmC (e) levels during development in selected C57Bl/6 mouse tissues (further data in Supplementary Fig. 6). shown are mean ± s.e.m. of three embryos (E11.5) (data from Fig. 1b), three newborns (1 d old), and two adolescent (21 d old) and two adult (15 week old) mice. see also Supplementary Figures 7 and 8.

Figure 2. 5fC can be a stable DNA modification in vivo.

(a) Labeling ratios of 5mC, 5hmC and 5fC in the genomic DNA of mEs cells cultured in the presence of [methyl-¹³CD₃]l-methionine. Shown are single measurements; total labeling time is given in brackets. (b–d) Labeling ratios of 5mC, 5hmC and 5fC in the genomic DNA of C57Bl/6 mice fed with the [methyl-¹³CD₃]l-methionine diet. (b) 6-d-old pups labeled from 1 week before birth (total labeling time of 13 d) or 1-d-old newborns (total labeling time of 22 d, parents on labeled diet for 52 d before conception). Shown are mean ± s.e.m. of two animals (6 d old pups) or two technical replicates (1 d old pup). (c,d) Mice labeled in adulthood. Shown are mean ± s.e.m. of at least two technical replicates from individual mice, and total labeling time is shown in brackets. The absence of 5fC[+2] in the brain (d), where 5fC is most abundant (see Fig. 1 and Supplementary Fig. 6), indicates minimal or no further generation of 5fC once placed in postmitotic tissues. Moreover, if 5fC was involved in cycles of methylation and demethylation, its labeling ratio would be similar to that of 5mC in RNA (d). See also Supplementary Figure 9 and Supplementary Table 1.