Periodic Reporting for period 4 - PIWI-Chrom (Understanding small RNA-mediated transposon control at the level of chromatin in the animal germline)
Período documentado: 2021-01-01 hasta 2022-06-30
Eukaryotic genomes are littered with various classes of transposons, selfish genetic elements that multiply in the host genome. Due to the mutagenic nature of transposons, host organisms have evolved sophisticated transposon defense systems. In the animal germline, a small RNA-based silencing system, the PIWI-interacting RNA (piRNA) pathway silences all classes of transposable elements. With the ERC project PIWI-Chrom, we studied the intersection between the piRNA pathway and chromatin. We focused our research on the fruitfly Drosophila melanogaster, which is at the forefront of mechanistic piRNA pathway studies. PIWI-Chrom allowed us to answer two fundamental questions:
1. How do nuclear Argonaute proteins, once targeted via small RNAs complementarity to a transposon transcript, orchestrate local heterochromatin formation?
2. How do cells re-interpret heterochromatin at selected loci, where transposon sequence information is stored, in order to transcribe the precursor transcripts for piRNA biogenesis.
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: transposons contribute to genetic and genomic innovation at multiple levels thanks to their rapid diversification and mobility. 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. A deep conceptual and mechanistic understanding of transposon control is therefore of central importance. Despite the fact that genome defense systems in 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.
1. How do nuclear Argonaute proteins, once targeted via small RNAs complementarity to a transposon transcript, orchestrate local heterochromatin formation?
2. How do cells re-interpret heterochromatin at selected loci, where transposon sequence information is stored, in order to transcribe the precursor transcripts for piRNA biogenesis.
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: transposons contribute to genetic and genomic innovation at multiple levels thanks to their rapid diversification and mobility. 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. A deep conceptual and mechanistic understanding of transposon control is therefore of central importance. Despite the fact that genome defense systems in 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.
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.
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.
All proposed Aims and Milestones for PIWI-Chrom were fully reached, and even surpassed. In both project areas, we established not only the biological insight and the molecular concepts, but also critical experimental tools and resources as strong foundation for future work.