Periodic Reporting for period 3 - PIWI-Chrom (Understanding small RNA-mediated transposon control at the level of chromatin in the animal germline)
Reporting period: 2019-07-01 to 2020-12-31
At the core of this proposal are two fundamental questions:
1. how can a nuclear Argonaute protein, once targeted via small RNA complementarity to a target transcript, orchestrate heterochromatin formation?
2. how can cells re-interpret heterochromatin in order to express selected heterochromatin loci, where transposon sequence information is stored, in order to produce the precursors transcripts for small RNA biogenesis.
Both of these questions are among the most important open questions in the field of eukaryotic genome defense and small RNA pathways.
The co-evolution between eukaryotic genomes and selfish genetic elements such as transposons is one of the oldest genetic conflicts. It is central not only for species survival, but also for evolution as transposons due to their fast evolution and their mobility contribute to genome evolution at multiple levels. A deep mechanistic and genetic understanding of this 'arms-race' is therefore of central importance. It also becomes increasingly clear that transposons have major impact on human biology and disease. Uncontrolled transposon activity in the germline leads to sterility, and transposition events can lead to several human disease phenotypes.
Despite the fact that the genome defense systems employed by eukaryotes vary widely, recent years have uncovered striking similarities in the molecular mechanisms that are being exploited over and over again. This is why studying genome defense pathways in highly established and powerful model systems such as the fruit fly has been and will continue to be key in uncovering these parallels.
Within Aim 1, we established powerful methodologies and assay systems to dissect, at the genetic and molecular detail, the heterochromatin formation process downstream of nuclear Piwi. We identified not only the immediate downstream adaptor proteins and their protein-complexes, but also described for the first time the heterochromatin effector machinery that is employed by this pathway. A key step forward at the technical level was the establishment of genome engineering systems that allow us now to modify a gonad-derived cell line at will in a fast and accurate manner. While similar approaches have been established in mammalian cell-culture systems, the application of such tools in the Drosophila OSC cell line was a major technical challenge. We are now able to employ nearly the entire battery of genome engineering tools in flies, as well as in the fly cell-culture system.
Our work has so far uncovered a novel protein complex downstream of nuclear Piwi, and we are using this insight into the systematic dissection of the silencing process.
Within Aim 2, we discovered the molecular machinery that underlies heterochromatin transcription at piRNA source loci. This major progress paved the way for a mechanistic dissection of the transcriptional and co-transcriptional processes at piRNA clusters. We also discovered the complete nuclear RNA export pathway that delivers piRN A precursor transcripts to the cytoplasmic processing sites.
These two major findings combined point to a unified model of heterochromatin expression, where a dedicated heterochromatin binding protein (Rhino) recruits strategic effector proteins of the gene expression cascade that confer competence for transcription initiation, elongation, and RNA export.
In two areas, we have made more substantial progress than anticipated.
The first area is our attempt to expand the transcriptional silencing project to a mammalian study object. We invested considerable efforts into establishing such systems and are now gearing up to do our first genetic screens in order to find new silencing biology, or to extend our findings from Drosophila to a mammalian cell line. I expect considerable progress beyond the state of the art from this research direction until the end of the funding period.
The second area where progress has been beyond expectations is the establishment of a germline stem cell culture. Such cell lines do not exist for Drosophila (despite considerable efforts in the community) and they are extremely rare outside the insect community. We have succeeded in establishing a new stable cell culture system for Drosophila female germline stem cells. This cell line promises to deliver several major breakthroughs in the area of piRNA research, but also in the broader area of germline stem cell research.
All in all, the project is very much progressing according to expectations. We are confident that we continue to make key discoveries in this area and that we establish study systems and uncover new key questions for future challenges.