Genome evolution and the formation of piRNA clusters in Drosophila

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Almost all life forms find themselves in a constant evolutionary arms race with transposable elements (TEs) in order to protect their genomes against uncontrolled proliferation of these mobile genetic units, as they pose a threat to genome integrity and stability. Since this is particularly critical in the germline where genomic changes are hereditary, organisms have evolved specialised defence systems, such as the metazoan PIWI/piRNA pathway. PIWI proteins are guided by small PIWI-interacting (pi-) RNAs through sequence complementarity to their TE targets and direct post-transcriptional and transcriptional silencing. In virtually all animals, large TE rich genomic loci, called piRNA clusters, produce the vast majority of piRNAs. It is commonly assumed that piRNA clusters play a central role in TE silencing as the main source for TE-targeting piRNAs.In our preliminary work, we studied the evolution of piRNA clusters in the Drosophila genus and found that fly piRNA clusters are exceptionally short-lived and often emerge at genomic loci that are marked by recurrent inversion breakpoints, which are regions that have been proposed to accumulate TE insertions in general. Since this lack of conservation is at odds with a critical function, we have put the model to the test by deleting three of the largest piRNA clusters of Drosophila melanogaster, which alone produce the majority of piRNAs. Surprisingly, this did not affect germ cell viability or lead to reactivation of any TEs, challenging the assumption of their functional relevance. This, however, poses the question why piRNA clusters, which appear to be highly ordered and distinct structures, emerge and how they develop, as they are a ubiquitous and recurrent characteristic of metazoan genomes.In this project, I will examine the influence of inversion-causing DNA fragility on the emergence of piRNA clusters, and the underlying impact of chromosome conformation and packaging, using Hi-C, ATAC-seq and ChIP-seq, across Drosophila species. Here, piRNA clusters will serve as a model study that will be expanded towards the broad-scale impact of genome conformation on genomic structural evolution. Further, I will bioinformatically analyse TE structures of piRNA clusters across species to understand their initial formation and development over time. Finally, since the PIWI system apparently functions independently of piRNA clusters, I will explore how new TEs are recognized without prior existence of complementary piRNAs, by introducing foreign TE copies into fly genomes followed by DNA and small RNA sequencing. Altogether, this project will provide valuable insights into the mutual impact of genomes and transposable elements and a deep view of genomic architecture evolution on an unprecedented scale.