The first to discover the guided defense jump gene: "Kipferl"

A large part of our DNA is made up of selfish duplicate DNA elements, some of which can jump from one location in the genome to another, potentially destroying the genome
.
Researchers at the Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA) have described how different types of duplicate DNA elements in Drosophila ovaries are controlled by the
same silencing mechanism.
At the heart of their discovery is an uncharacteristic protein that the researchers named "Kipferl
.
" , to ensure the effective control
of the jumping gene.
The findings suggest that different selfish elements compete for the host genome's e-defense system, and Kipferl may be the first
in a series of molecules with similar effects that have not yet been discovered.
The findings were published in the journal eLife
.

About half of the human genome and one-fifth of the Drosophila genome are made up of genetically similar genetic parasites that can replicate themselves and insert themselves into random regions of our genome, potentially disrupting normal gene expression
.
In order to control these so-called transposons, people have evolved a variety of defense mechanisms
.
One such mechanism is an RNA interference system
called the piRNA pathway.
The piRNA pathway is a small RNA silencing pathway that is conserved throughout the animal kingdom, from sponges to mammals
.
This silencing mechanism uses piRNAs called "piRNA clusters" produced from transposon-rich sequences in DNA
.
"PiRNAs are small RNAs that bind to the so-called Argonaute protein of the silencing mechanism and target
transposons with complementary sequences.
Thus, these piRNAs serve as a blueprint for the recognition and silencing of transposons with complementary sequences in the genome, no matter how far
they can jump.

The study uses a protein called "Rhino" to look for piRNA clusters
in the genome.
However, how Rhino recognizes piRNA clusters in DNA is still unknown
.
"Previous in vitro data showed that Rhino had an affinity for a specific epigenetic marker, chromatin-modified H3K9me3, said
Julius Brennecke, head of the IMBA team and corresponding author of the study.
This modification is a marker of "heterochromatin," a tightly packed line of DNA in which genes are silenced
.
However, H3K9me3 is not specific to piRNA clusters, but is also often found
in other dense regions of the genome.
There, H3K9me3 binds to master heterochromatin protein 1 (HP1), a close relative
of Rhino.
Why HP1 and Rhino bind to different heterochromatin subsets, even though they have considerable affinity for the same chromatin modification, has long been unclear
.
"There is no doubt that H3K9me3 is necessary, but not enough to explain Rhino's binding
to chromatin.
So we knew there must be other molecular cues that helped localize Rhino to piRNA clusters," Brennecke added
.

In the search for this molecular cue, the team classified Rhino's direct interactors, looking for a partner protein
that might guide Rhino into the piRNA cluster.
By binding genes, genomes and imaging methods, the researchers found a "mate" of rhinos in the ovaries of fruit flies: a protein that contains several zinc finger folds
they call "Kipferl.
" Kipferl can not only bind to Rhino, but also use its zinc finger to specifically bind
to guanosine-rich DNA motifs.
The team found that most piRNA clusters are determined by binding to local heterochromatin at Kipferl's
specific DNA-binding site.
At these sites, Rhino's interaction with the H3K9me3 epigenetic marker was stabilized by Kipferl, which explains why Rhino binds
only to a small fraction of all heterochromatin found in the genome.

To add to the complexity, the team knew that Rhino wasn't just localized into piRNA clusters
.
Recently, rhinos have been shown to bind to so-called "satellite arrays
.
" These are repeats of non-coding and non-transposed DNA located near chromosomal
centromeres.
"When we produce fruit flies with mutations or deletions of Kipferl and look at them under the microscope, we see amazing results on Rhino," said
Lisa Baumgartner, a doctoral student and first author in IMBA's Brennecker lab.
When Kipferl mutates, Rhino is no longer positioned in piRNA clusters
throughout the genome.
Instead, it accumulates
strongly in genomic satellite arrays.
"Instead of the small dots that are distributed around the nucleus, what we see is the formation of a distinctive crescent
.
Based on this first observation, we named the new protein "Kipferl", named after a popular croissant-shaped Austrian pastry
.
We didn't find out until much later that these structures corresponded
to the giant base extension of the satellite array.
Therefore, the scientists showed that Kipferl helps to properly assign Rhino to piRNA clusters and avoid being isolated to satellite arrays
.

Rhino protein is one of
the fastest evolving proteins in the fly genome.
Brenneck and his team hypothesized that this rapid evolution is most likely due to positive evolutionary pressures
from satellite arrays.
"Satellite arrays cannot be shifted, but they can be reassembled
.
However, if they do so in an uncontrolled manner, the entire chromosome arm may be lost
.
Therefore, the satellite array may require a control mechanism that includes Rhino and other piRNA pathway components to help package them into tight heterochromatin
.
That's probably why the satellite array seems to want to isolate all the rhinos they can find," Felix explained
.

Baumgartner believes that Rhino and piRNA pathways may play very different roles
in interactions with satellite arrays or piRNA clusters and transposons.
"The jump and proliferation of transposons pose a threat to the function of the genome, so it is necessary to silence it through the piRNA pathway," she said
.
Thus, in the transposon's view, piRNA pathways are the "enemy"
that prevents them from spreading in the genome.
Satellite arrays, on the other hand, require only an extra layer of control to ensure that they can maintain high copy numbers without disrupting the genome
through unnecessary recombination.
So, in the eyes of the satellite array, I think rhinos are a factor in ensuring their survival," she said
.

Based on these observations and analyses, the scientists believe the satellite array may have used another partner protein like Kipferl to help localize Rhino into their DNA
.
"To counter the satellite array's isolation of Rhino, we speculate that Kipferl's evolution may have been to help relocate Rhino to piRNA clusters
.
" Therefore, our findings suggest that rhinos may be in the crossfire of genetic conflict," Brennecke said
.
In addition, Rhino is expressed in both the testicles and ovaries of fruit flies, while Kipferl is expressed
only in the ovaries.
"Kipferl may be the first rhino guiding factor that has yet to be discovered," Brennecke concluded
.

The Drosophila ZAD zinc finger protein Kipferl guides Rhino to piRNA clusters