Third Natural Eukaryotic Epigenetic Mark Found

DNA carries the blueprint to build the body, but it is a living document: the design can be adjusted by epigenetic markers. In humans and other eukaryotes, two major epigenetic marks are known.

A group at the Marine Biology Laboratory (MBL) now found a third epigenetic mark in this freshwater invertebrate, Adineta vaga, which has previously been found only in bacteria. For the first time, a horizontally transferred gene has been shown to remodel gene regulatory systems in eukaryotes.

"We found that vermicularis rotifers were very good at capturing foreign genes as early as 2008," said study director Dr. Irina Arkhipova. “What we found here is that about 60 million years ago, rotifers accidentally captured a bacterial gene that led them to introduce a new epigenetic mark that did not previously exist."

Dr. Fernando Rodriguez, a research scientist at the Arkhipova laboratory and co-first author of the team's paper published in Nature Communications, said: "The CRISPR-Cas system in bacteria is a good comparison and it began as a basic research finding. CRISPR-Cas9 is now widely used for gene editing tools in other organisms. It’s a new system. Does it have applications and implications for future research? It's hard to say."

They point out in the text, "We combined multiple lines of evidence to determine that 4mC modifications can be used as epigenetic marks in eukaryotic genomes, and our work shows how a horizontally transferred gene becomes part of a complex regulatory system that is maintained by selection over tens of millions of years of evolution."

Epigenetic marks are modifications to the bases of DNA that do not change the underlying genetic code but "write" additional information on it that can be inherited with the genome. In two epigenetic marks known in eukaryotes, methyl groups are added to DNA bases, either cytosine or adenine. Epigenetic marks often regulate the expression of genes—they turn genes on or off—especially during early development or when the body is under stress. They can also repress "jumping genes," which are transposable elements that threaten genome integrity.

“Eukaryotes mostly use base modifications for regulation, and 5mC is the main form of epigenetic modification in eukaryotic genomes.” The team added: "5mC, commonly referred to as the 'fifth base', plays an important role in genome defense against mobile genetic elements and is frequently associated with transcriptional silencing, establishment of closed chromatin configurations and repressive histone modifications."

4mC has not been shown to act as an epigenetic mark in eukaryotes, scientists say, "and most claims about 4mC in eukaryotes lack the confirmation of orthogonal methods and do not identify the components of the enzyme." In fact, 4mC is also cytosine modified, but its methyl group is located similarly to bacteria, which essentially recapitulates evolutionary events more than 2 billion years ago, when traditional epigenetic marks emerged in early eukaryotes.

Vermicularis rotifer is a highly adaptable animal, as discovered over the years by the Arkhipova and David Mark Welch laboratories at MBL. These organisms can dry completely over a period of weeks or months and then resume vitality when there is water. During their drying phase, the DNA of R. vermicularis breaks down into many fragments. "When they rehydrate or otherwise make their DNA ends accessible, this may be an opportunity to transfer foreign DNA fragments from ingested bacteria, fungi, or microalgae into the genome of rotifers," Arkhipova said. They found that approximately 10% of the genome of rotifers comes from non-metazoans.

Nevertheless, the Arkhipova laboratory was surprised to find that the rotifer genome is similar to bacterial methyltransferases (methyltransferases catalyze the transfer of methyl groups to DNA). "We hypothesize that this gene confers a new function to this repressed transposon, and we have spent the past 6 years demonstrating that this is indeed the case," Arkhipova said. As the authors comment, "We found N4CMT, a bacteria-derived horizontal transferase,” the researchers said in the paper, "Our results show that non-native DNA methyl groups can remodel the epigenetic system, silence transposons, and show the potential of horizontal gene transfer to drive regulatory innovation in eukaryotes."

"Quite unusual, not previously reported," added Arkhipova. “Horizontally transferred genes are considered as operational genes rather than regulatory genes. Imagine how a single, horizontally transferred gene forms a new regulatory system because the existing regulatory system is already very complex."

"This is almost incredible," said Dr. Irina Yushenova, a research scientist and co-first author at the Arkhipova laboratory. “Try to imagine that sometime in the past, a piece of bacterial DNA happened to fuse with a piece of eukaryotic DNA. They all join the rotifer's genome and form a functional enzyme. It's not easy to do, even in the lab, it happens naturally. This complex enzyme then created this magical regulatory system, and vermicularis began to use it to control all these jumping transposons. It's like magic."

"You don't want transposons to jump around in your genome," Rodriguez said. “They're gonna screw it up, so you gotta control them. The epigenetic system that achieves the goal is different in different animals. In this case, horizontal gene transfer from bacteria to Bdelloid rotifers creates a new epigenetic system in animals that has not been previously described."

"Bdelloid rotifers, in particular, have to control their transposons because they mainly reproduce asexually," Arkhipova points out. “Asexual ancestry has fewer means of inhibiting deleterious transposon proliferation, so adding an additional layer of protection can prevent the collapse of mutations. In fact, the transposon content in leeches is much lower than that in sexual eukaryotes, which do not have this additional epigenetic layer in their genomic defense system."

These novel findings may open the door to new tools and research directions for studying genome function and adaptability in rotifer systems. As the authors summarize, "Overall, our findings help solve a fascinating evolutionary mystery: how do DNA bacterial enzymes with non-epigenetic modifications penetrate eukaryotic gene silencing systems and are preserved in tens of millions of years of natural selection?"

They added: "The system shows that horizontal gene transfer can reshape the complex regulatory circuits of metazoans, thereby driving major evolutionary innovations including epigenetic control systems. The role of horizontal gene transfer in the evolution of eukaryotic regulation has been a topic of intense debate."