Both proteins and DNA can carry marks, called epigenetic markers, that instruct when and which genes are expressed. Although many such markers have been described in proteins, epigenetic marks in DNA are more elusive. Until recently, only one bona fide marker had been identified in DNA: an addition of a methyl group called 5-methylcytosine (5mC). Now a new study showed that one derivative of 5mC, 5-formylcytosine (5fC) also functions as a DNA epigenetic marker and influences expression of genes during early development.1 The finding, published in Cell, expands scientists’ understanding of how DNA modifications, in addition to protein modifications, can influence cellular processes.
“This study brings back the spotlight to DNA modifications in a major way,” said Alfonso Bellacosa, a geneticist at Temple University who was not involved in the study.
For decades, scientists believed that 5mC was the primary epigenetic mark on DNA, acting to silence genes through methylation. Although scientists initially believed the oxidized derivatives of 5mC, including 5fC, to be merely intermediates in the process of DNA demethylation, they questioned whether these marks could also play a role in gene regulation.2
“If they had a life of their own was still unclear,” said coauthor Christof Niehrs, a molecular biologist at the Institute of Molecular Biology in Mainz.
To characterize the role of these derivatives, Niehrs’ team studies the development of Xenopus frog embryos. Specifically, they examined a process called zygotic genome activation, during which the oocyte transitions from using maternal genes to expressing the embryo’s own genes.
The team used immunofluorescence to observe how the two other derivates (5caC and 5hmC) accumulated throughout the embryo to see if they might have any influence over gene expression. Early during development, the team found that 5fC, but not the other derivatives, accumulated into structures called chromocenters. These chromocenters were located in a region known to be associated with RNA polymerase III (Pol III) transcription. When the team investigated where 5fC accumulated on the genome, they found that it was uniquely enriched on tRNA genes that, when transcribed, recruit Pol III and promote protein biosynthesis.
The team then manipulated the levels of 5fC to see how that would impact Pol III binding. To do this, they used TET enzymes, which are responsible for oxidizing 5mC to form 5fC and the enzyme thymine DNA glycosylase (TDG), which removes 5fC. Injecting embryos with morpholinos that impaired 5fC synthesis and production, the levels of 5fC and Pol III binding to target genes and tRNA expression decreased. Similarly, lowering 5fC levels by TDG overexpression impaired Pol III binding to tRNA genes and inhibited, rather than activated, their expression.
The discovery of 5fC as just the second DNA epigenetic mark expands researchers’ understanding of just how influential these changes can be. This could lead to new avenues of research into how different DNA modifications influence cellular processes. For example, the finding that 5fC is essential for the activation of tRNA genes during zygotic genome activation in early embryos suggests that it may play a critical role in regulating gene expression programs required for proper embryonic development. And since 5fC is enriched at active regulatory regions, it may be important for maintaining stem cell identity and regulating differentiation pathways.
Additionally, 5fC may be involved in regulating oncogenes or tumor suppressor genes, Bellacosa pointed out. “It would be interesting to see if similar mechanisms apply in in that case,” he said. If it turns out that 5fC also regulates other processes, altered 5fC patterns may serve as epigenetic signatures that could be used as diagnostic or prognostic biomarkers for various diseases.
It will be important to see if this mechanism generalizes to animals beyond the frog, Bellacosa added. Finally, exactly how an intermediate product actively influences gene expression still remains to be resolved, Bellacosa added. “The genius is in the details.”
- Parasyraki E, et al. 5-Formylcytosine is an activating epigenetic mark for RNA Pol III during zygotic reprogramming. Cell. 2024;187(21):6088-6103.e18.
- Hill PWS, et al. DNA demethylation, Tet proteins and 5-hydroxymethylcytosine in epigenetic reprogramming: An emerging complex story. Genomics. 2014;104(5):324-333.
