Within Aim 1 (piRNA-guided heterochromatin formation), we first established powerful methodologies and assay systems to dissect, at the genetic and molecular level, the heterochromatin formation process downstream of nuclear Piwi. This was key to identify the protein complex SFiNX that acts downstream of Piwi. We could show that SFiNX consists of the orphan protein Panoramix, the nuclear mRNA export variant Nxf2-Nxt1, and the homo-dimerization factor Ctp. We systematically characterized SFiNX at the structural, molecular, and functional level, which allowed us to attribute the core silencing function within SFiNX to Panoramix. Multivalent interactions, enabled by SFiNX dimerization are essential for SFiNX function at the level of the nascent target RNA, but is not required for SFiNX-mediated repression when recruited directly to the DNA of the target locus. This allowed us to identify the unstructured N-terminal half of Panoramix (IDR) as the central silencing domain. A small LxxLL containing peptide within the Panoramix IDR was shown to mediate the interaction between SFiNX and the large Zinc finger protein Small Ovaries (Sov), a central component of the cellular heterochromatin machinery. Through genetic, biochemical, and structural approaches we showed that the Panoramix–Sov interaction is coordinated in addition through Panoramix SUMOylation and Sumo interacting motifs in Sov. All in all, our work pioneered the mechanistic understanding of small RNA-guided heterochromatin formation in animals and uncovered several conceptual parallels to the recently identified HUSH complex in mammals.
Within Aim 2, we discovered (A) the molecular machinery that underlies heterochromatin transcription at piRNA source loci, (B) the machinery that exports piRNA precursors to the cytoplasmic piRNA biogenesis sites, and (C) the principle of piRNA cluster definition on chromatin. In all three projects, we combined fly genetics with in vivo studies, cell-biology, molecular biology, and biochemistry to reveal the conceptual logic and the mechanistic principles underlying heterochromatic piRNA clusters. Our work provides major examples of how gene/protein functions can be altered through duplication and subsequent adaptation processes. Within part C, we discovered the first known example of HP1 protein guidance to chromatin via a protein that binds the chromodomain of an HP1 family protein. Finally, we succeeded in establishing the first stable cell line derived from ovarian germline stem cells. This major achievement (publication in preparation) will enable us and the field to study the complete piRNA pathway with new and powerful experimental approaches.
The results from our work have been disseminated in the form of several major primary publications, a methods publication, as well as conference presentations and invited seminars by me or students/postdocs in my group.
