The role of putzig in sustaining genome stability in the germline and in somatic cells of Drosophila

维持基因组的稳定性和完整性对真生动物的生存、成功繁殖和正常发育至关重要。转座子对基因组的稳定性构成了巨大的风险。古老的Piwi-piRNA网络通过沉默动物种系中的转座因子来保护基因组的完整性。我们的初步工作将基因putzig （pzg）归因于模型系统果蝇的这一过程。Pzg是多种多蛋白复合物的一部分，参与基因的表观遗传和转录调控。pzg活性的丧失导致细胞和组织稳态紊乱，并最终导致生殖系和体细胞细胞的死亡。缺乏pzg的生殖细胞显示DNA双链断裂增加，这可能是由转座子活性升高引起的。此外，Piwi蛋白的定位和异染色质的形成一样受到干扰，这表明Pzg在Piwi依赖机制中发挥了作用。Pzg蛋白与卵巢中含有piwi的蛋白复合物的物理关联进一步证实了这一工作假设。本提案的目的是分子解译Pzg在piwi介导的转座子沉默和表观遗传调控中的作用。在第一步，我们问，Pzg是否参与Piwi-piRNA介导的表观遗传抑制。为此，我们希望更详细地研究pzg突变生殖细胞的异染色质状态，系统地研究这些细胞中转座子家族的活性，以及转座子去抑制位点上的染色质状态。结果使我们能够将pzg耗尽的结果与已经发表的piwi突变体的结果进行比较。此外，我们计划通过分析潜在的Pzg- piwi -PRC2蛋白复合物以及转录读出的预期变化，探索Pzg在表观遗传沉默过程中piwi介导的PRC2活性抑制中的作用。其次，我们关注Pzg在pirna转录调控中的潜在作用。在这里，我们想知道Pzg是否是启动子独立的转录机制的一个组成部分，该机制指导异色piRNA簇的转录，以及/或者Pzg是否是启动子正确募集的前提。pzg缺陷生殖细胞的qRT-PCR分析将揭示piRNA前体转录是否受到影响。第三部分讨论了pzg突变种系中小pirna的生物发生，从那些已知在卵巢中富集而在piwi突变体中发生变化的pirna开始。其中一个例子是3R-TAS1 piRNA，它也在体细胞中表达。最后，我们的目标是对pzg突变体幼虫进行RNA-seq分析，以获得包括小rna在内的转录组信息，并与种系信息进行比较。pzg的人类同源物（即Znf711）与罕见的人类疾病的联系预示着我们的研究将为人类新的诊断方法奠定基础。

The maintenance of genome stability and integrity is fundamental to survival, successful reproduction as well as proper development of eumetazoa. Transposons pose a massive risk to genome stability. The ancient Piwi-piRNA network protects the integrity of the genome by silencing transposable elements specifically within the animal germline. Our preliminary work ascribes the gene putzig (pzg) to this process in the model system Drosophila. Being part of various multi-protein complexes, Pzg is involved in the epigenetic and transcriptional regulation of genes. Loss of pzg activity results in a disturbed cell and tissue homeostasis and eventually in the death of cells from the germline and the soma as well. Germ cells devoid of pzg display increased DNA double strand breaks that may result from an observed raise in transposon activity. Moreover, Piwi protein localization is disturbed as is heterochromatin formation, suggesting a role for Pzg in Piwi-dependent mechanisms. This working hypothesis is further corroborated by a physical association of Pzg protein with Piwi-containing protein complexes in ovaries. The aim of this proposal is to molecularly decipher the role of Pzg in Piwi-mediated transposon silencing and epigenetic regulation. In a first step we ask, whether Pzg takes part in the Piwi-piRNA mediated epigenetic repression. To this end, we want to investigate the heterochromatin status of pzg mutant germ cells in greater detail, systematically address the activity of further transposon families within these cells, as well as the chromatin status specifically at de-repressed transposon loci. Results allow us to compare the outcome of pzg depletion with that already published for piwi mutants. Moreover, we plan to explore the role of Pzg in Piwi-mediated repression of PRC2 activity during epigenetic silencing by analyzing potential Pzg-Piwi-PRC2 protein complexes as well as the expected changes in the transcriptional read out. Secondly, we focus on the potential role of Pzg in the transcriptional regulation of piRNAs. Here we ask whether Pzg is an integral part of the promoter-independent transcription machinery directing transcription from the heterochromatic piRNA clusters and/or whether Pzg is a premise for its correct promoter recruitment. qRT-PCR analyses in pzg deficient germ cells will reveal, whether piRNA precursor transcription is affected. The third part addresses the biogenesis of small piRNAs in the pzg mutant germline, starting with those known to be enriched in ovaries and otherwise changed in piwi mutants. One such example is 3R-TAS1 piRNA, which is also expressed in somatic cells. Finally, we aim to perform RNA-seq analyses on pzg mutant larvae to gain information on the transcriptome including small RNAs to be compared with the information from the germline. The link of human orthologues of pzg (i.e. Znf711) to rare human diseases predicts our studies to set the stage for novel diagnostic approaches also in human.