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

putzig 在维持果蝇种系和体细胞基因组稳定性中的作用

• 批准号: 446721799
• 负责人: Dr. Anja Christina Nagel
• 金额: --
• 依托单位: Fachgebiet Allgemeine Genetik (190g)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/446721799?language=en
• 关键词: role; putzig; sustaining; genome; stability

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.

真后生动物基因组的稳定性和完整性是其生存、繁殖和正常发育的基础。转座子对基因组稳定性构成巨大风险。古老的Piwi-piRNA网络通过特异性沉默动物种系内的转座元件来保护基因组的完整性。我们的初步工作归因于基因putzig（pzg）的模式系统果蝇的这一过程。作为各种多蛋白复合物的一部分，Pzg参与基因的表观遗传和转录调节。pzg活性的丧失导致细胞和组织的稳态紊乱，并最终导致来自生殖系和索马的细胞死亡。缺乏pzg的生殖细胞显示增加的DNA双链断裂，这可能是由于观察到的转座子活性升高。此外，Piwi蛋白定位被干扰，因为是异染色质形成，这表明Pzg在Piwi依赖性机制中的作用。Pzg蛋白与卵巢中含有Piwi的蛋白质复合物的物理关联进一步证实了这一工作假设。该提案的目的是从分子上破译Pzg在Piwi介导的转座子沉默和表观遗传调控中的作用。在第一步中，我们询问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，系统地解决这些细胞内进一步转座子家族的活性，以及去阻遏转座子位点的染色质状态。结果使我们能够比较pzg耗竭的结果与已经发表的piwi突变体。此外，我们计划通过分析潜在的Pzg-Piwi-PRC 2蛋白复合物以及转录读出的预期变化来探索Pzg在Piwi介导的PRC 2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，我们询问Pzg是否是引导异染色质皮尔纳簇转录的启动子非依赖性转录机制的组成部分和/或Pzg是否是其正确启动子募集的前提。pzg缺陷型生殖细胞中的qRT-PCR分析将揭示皮尔纳前体转录是否受到影响。第三部分阐述了pzg突变体种系中小piRNA的生物发生，从那些已知在卵巢中富集并在piwi突变体中发生变化的小piRNA开始。一个这样的实例是3R-TAS 1皮尔纳，其也在体细胞中表达。最后，我们的目标是对pzg突变幼虫进行RNA-seq分析，以获得包括小RNA在内的转录组信息，并与来自种系的信息进行比较。pzg的人类直系同源物（即Znf 711）与罕见的人类疾病的联系预示着我们的研究也为人类的新诊断方法奠定了基础。