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.

1. 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

2. 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."

3. 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."