The first defense system discovered to guide jumping genes "Kipferl"

A large part of our DNA is made up of selfish, repeating DNA elements, some of which can jump from one location in the genome to another, potentially damaging the genome
.
Researchers at the Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA) describe how different types of repeating DNA elements in the ovaries of fruit flies are controlled
by the same silencing mechanism.
At the heart of their discovery is an undescribed protein, which the researchers named "Kipferl," which ensures effective control
of jumping genes.
These findings suggest that different selfish elements are competing for the host genome's defense system, and Kipferl may be the first
in a series of molecules with similar roles that have yet to be discovered.
The findings were published in
the journal eLife.

About half of the human genome and one-fifth of the fruit fly genome are made up of gene-like genetic "parasites" that can self-replicate and insert themselves into random regions of our genome, potentially disrupting normal gene expression
.
To control these so-called transposons, a variety of defense mechanisms
have evolved.
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" generated from transposon-rich sequences in DNA, which are small RNAs that bind to so-called Argonaute proteins in the silencing mechanism to target
transposons that complement their 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 piRNA pathway was first discovered in Drosophila melanogaster using a protein called "Rhino" to look for piRNA clusters
in the genome.
However, how Rhino recognizes piRNA clusters in DNA is still unknown
.
Julius Brennecke, corresponding author of the study and head of the IMBA group, said: "Previous in vitro data have shown that Rhino has an affinity for a specific epigenetic marker
, H3K9me3.
" This modification is a marker for "heterochromatin," a tightly packed DNA in which genes are silent
.
However, H3K9me3 is not specific to piRNA clusters but is often found
in other dense regions of the genome.
There, H3K9me3 binds to the master heterochromatin protein 1 (HP1), a close relative
of Rhino.
Why HP1 and Rhino bind to different heterochromatin subsets, even though they have a comparable affinity for the same chromatin modification, has long been unclear
.
There is no doubt that H3K9me3 is required, but not enough to explain the binding
of Rhino to chromatin.
Therefore, there must be additional molecular cues to help locate Rhino to the piRNA cluster
.

In their search for this molecular clue, the team classified Rhino's direct interacters, looking for a partner protein
that might guide Rhino into the piRNA cluster.
By combining genetic, genomic and imaging methods, the researchers found a protein in the ovaries of fruit flies that contained several zinc finger folds
they called "Kipferl.
" Kipferl can not only bind to Rhino, but also use its zinc finger to specifically bind
guanosine-rich DNA motifs.
The team found that most piRNA clusters were identified by binding of Kipferl's specific DNA binding site to local heterochromatin
.
At these sites, Rhino's interaction with the H3K9me3 epigenetic marker was stabilized by Kipferl, which explains why Rhino binds
to only a small fraction of all heterochromatin found in the genome.

To add complexity, the team knew that Rhino was not only localized to the piRNA cluster
.
Recently, Rhino was shown to be able to bind to
so-called "satellite arrays.
" These are repetitive sequences
of non-coding and non-translocated DNA located near the centromeres of chromosomes.
Lisa Baumgartner, a PhD student and first author in IMBA's Brennnecker lab, said: "When we generated flies that mutated or lacked Kipferl and looked at them under a microscope, we saw a significant effect
on Rhino.
" When Kipferl is mutated, Rhino no longer localizes the entire genome of piRNA clusters
.
Instead, it accumulates strongly in genomic satellite arrays
.
"We saw that Rhino formed a distinctive crescent instead of the tiny dots
scattered around the nucleus.
Based on this first observation, we named the new protein "Kipferl", a
popular Austrian pastry in the shape of a croissant.
"It was later discovered that these structures correspond
to the megabase extension of the satellite array.
Thus, the scientists showed that Kipferl helps to properly assign Rhino to piRNA clusters and avoid it being isolated into satellite arrays
.

The Rhino protein is one of
the fastest evolving proteins in the Drosophila genome.
Brennnecker and his team hypothesized that this rapid evolution was likely due to positive evolutionary pressures
from satellite arrays.
"The satellite array will not shift, but it can be reorganized
.
However, if they do so in an uncontrolled manner, the entire chromosome arm may be lost
.
Therefore, satellite arrays may require a control mechanism that includes Rhino and other piRNA pathway components to help pack them into tight heterochromatin
.
That's probably why satellite arrays seem to want to isolate all the Rhinos they can find," Baumgartner explains
.

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

Based on these observations and analyses, the scientists believe that the satellite array may have used another partner protein like Kipferl to help map Rhino into their DNA
.
"To counter the isolation of Rhino by satellite arrays, we speculate that Kipferl may have evolved to help relocate Rhino to a piRNA cluster
.
" Therefore, our findings suggest that Rhino may be in the crossfire of genetic clashes," Brennecke said
.
In addition, Rhino is expressed in both the testes and ovaries of Drosophila, while Kipferl is only expressed
in the ovaries.
"Kipferl is probably the first of Rhino's guiding factors that has not yet been discovered," Brennecke concludes
.

Lisa Baumgartner, Dominik Handler, Sebastian Wolfgang Platzer, Changwei Yu, Peter Duchek, Julius Brennecke.
The Drosophila ZAD zinc finger protein Kipferl guides Rhino to piRNA clusters.
eLife, 2022; 11