Genome evolution and the formation of piRNA clusters in Drosophila

几乎所有的生命形式都发现自己处于与转座因子（te）的持续进化军备竞赛中，以保护自己的基因组免受这些移动遗传单位的不受控制的增殖，因为它们对基因组的完整性和稳定性构成了威胁。由于这在基因组变化具有遗传性的种系中尤为重要，生物已经进化出专门的防御系统，例如后生动物PIWI/piRNA途径。PIWI蛋白是由小的PIWI相互作用（pi-） rna通过与其TE靶点的序列互补以及直接的转录后和转录沉默来引导的。在几乎所有的动物中，大量富含TE的基因组位点，称为piRNA簇，产生了绝大多数的piRNA。通常认为，作为TE靶向piRNA的主要来源，piRNA簇在TE沉默中起着核心作用。在我们的初步工作中，我们研究了果蝇属piRNA簇的进化，发现果蝇piRNA簇非常短暂，并且经常出现在以反复反转断点为标志的基因组位点上，这些区域通常被认为是TE插入的累积区域。由于这种保守性的缺乏与一个关键功能不一致，我们通过删除黑腹果蝇的三个最大的piRNA簇来测试该模型，它们单独产生大多数piRNA。令人惊讶的是，这并没有影响生殖细胞的活力或导致任何te的再激活，挑战了它们功能相关性的假设。然而，这就提出了一个问题，为什么piRNA集群似乎是高度有序和独特的结构，以及它们是如何发展的，因为它们是后生动物基因组中普遍存在和反复出现的特征。在这个项目中，我将使用Hi-C、ATAC-seq和ChIP-seq，研究反转引起的DNA脆性对piRNA簇出现的影响，以及染色体构象和包装的潜在影响。在这里，piRNA集群将作为一个模型研究，将扩展到基因组构象对基因组结构进化的大范围影响。此外，我将从生物信息学上分析不同物种piRNA簇的TE结构，以了解它们的初始形成和发展。最后，由于PIWI系统显然独立于piRNA簇发挥作用，我将通过将外源TE拷贝引入果蝇基因组，然后进行DNA和小RNA测序，探索在没有互补piRNA存在的情况下如何识别新的TE。总之，该项目将为基因组和转座因子的相互影响提供有价值的见解，并在前所未有的规模上深入了解基因组结构的进化。

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.