Beyond 5mC: 6mA Joins the Epigenetic Cast

While we have traditionally focused on 5-methylcytosine DNA (5mC) methylation in the laboratory, we started exploring 6-methyladenine (6mA) some years ago with Pedro Romero Charria, the lab's second PhD student. The field was a bit messy since there have been high profile reports saying 6mA is present in all sorts of model systems, including mammals, plants or flies. However, this was also heavily contested. We decided to check for ourselves, since the scattered reports from ciliates, early diverging fungi and the green algae Chlamydomonas suggested that 6mA was probably more widespread. In a new publication in Nature Genetics, we trace the evolution of 6mA to the Last Eukaryotic Common Ancestor, and find that in contrast to 5mC, its patterns are surprisingly conserved throughout evolution. Still, 6mA and its main enzymatic complement, the AMT1 methyltransferase complex, have been lost in many branches of the tree of life, including all complex multicellular lineages (animals, plants, fungi, brown and red algae). Importantly, 6mA is a integral part of the ancestral eukaryotic chromatin, as it is inheritable (deposited in symmetric ApT dinucleotides) and co-localises with H3K4me3. In this regard, multicellular lineages are simpler than their unicellular ancestors, which is a fascinating question we want to explore.

We also got to write a Research Briefing summarising the findings and telling a bit of the behind the paper story.

Diversity of eukaryotes profiled in the study. Expansion microscopy images with DNA (blue) and microtubules (magenta) stained. Pictures and composition courtesy to Dr. Omaya Dudin.

Based on the discoveries of this paper, we got granted a Wellcome Trust Discovery Award to keep exploring 6mA evolution. There are many mysteries that still surround this mark, so if you're curious and want to join our quest or collaborate, feel free to reach out!

Methylation goes viral: from curiosity to pattern?

After discovering that DNA methylation plays a key role in silencing hundreds of viral insertions in the protist Amoebidium, we began to question whether this was a rare exception or a widespread but overlooked phenomenon across eukaryotes. To explore this, we sought to expand our observations to a broader range of lineages. The challenge, however, lies in identifying species that have retained functional 5mC machinery, as the loss of DNA methylation is surprisingly common across the eukaryotic tree.

We focused on three species from distinct eukaryotic supergroups: Acanthamoeba castellanii (Amoebozoa), Naegleria gruberi (Heterolobosea), and Cyanophora paradoxa (Glaucophyta). In all three, we found that 5mC acts as a repressive mark, primarily targeting transposable elements and transcriptionally silent genes. As we extended our analysis to additional lineages, we repeatedly observed that viral insertions tend to localise within methylated, transcriptionally repressed regions of the genome. This pattern supports a broader model in which epigenetic silencing contributes to the genomic containment of potentially harmful, horizontally acquired DNA. This has been published in MBE:

https://academic.oup.com/mbe/advance-article/doi/10.1093/molbev/msaf176/8213644

Figure showing enrichment of genes of viral origin in the methylated fraction of the genome across a diverse set of eukaryotes.

Focusing on Acanthamoeba, we collaborated with John Archibald’s group at Dalhousie University. By comparing two high-quality, chromosome-scale assemblies (Neff and C3 strains), we observed extensive macrosynteny between the genomes, yet found that strain-specific regions were consistently enriched for giant virus insertions. These insertions derived from nuclear-replicating viruses, so we hypothesize they are the kind most likely to integrate accidentally into the host genome. This work can be found in BMC Biology:

https://bmcbiol.biomedcentral.com/articles/10.1186/s12915-025-02280-1

Ctenophore methylation and chromatin looping

Today the paper led by our collaborators Iana Kim and Arnau Sebe-Pedrós lab in Barcelona got published in Nature: https://www.nature.com/articles/s41586-025-08960-w

Unsurprisingly, our contribution to this work, which deals with the origins of chromatin looping in animals, lies in DNA methylation. In a previous study, we sequenced the methylome of the ctenophore Mnemiopsis leidyi, which was quite puzzling. Unlike other invertebrates, gene bodies are not particularly enriched for 5mCG, and promoters are not unmethylated, which is typical of H3K4me3 marked regions. Instead, genes could have methylated "promoters" (proximal upstream regions to the start position of the gene), which were characterised with high repeat/Transposable Element content. Having methylated repeats in the promoter didn't seem to affect gene expression, as there were genes at all levels of expression that had these weird promoters.

Adapted from de Mendoza et al 2020, Nat Ecol Evol

At some point, I thought this was just an artifact of poor gene prediction, as perhaps those "promoters" were not really promoters but truncated gene models, but back then we didn't have the data to proof that....

Some years later, when Arnau's group obtained a new chromosome scale version of the Mnemiopsis leidyi genome, we improved gene annotation with a comprehensive approach and new methods, and checked methylation again. Still, 5mCG was found on those repeat-rich "promoter" regions. So how does that work? Well, it seems Ctenophores have a lot of chromatin loops, which put these genes with repeat-rich methylated promoters in contact with H3K4me3 marked distal "enhancers", which perhaps are playing the role of "promoters" in this species.

So how did ctenophores end up with this weird genomic configuration? It could be that methylation was targeted to repeats in general, also those colonising promoters, silencing them. This accumulation of junk on a sensitive regulatory region was then tolerated by looping over them. It could also be that the looping requires methylation, as the structural CTCF-like zinc fingers that mediate these loops seem to be methyl-sensitive. In sum, ctenophores represent a very interesting example of animals with methylation restricted to transposable elements, which show further links to 3D chromatin structure and genome regulation.