Understanding how the piRNA pathway mediates epigenetic transposon silencing during foetal germ cell reprogramming

了解 piRNA 通路如何在胎儿生殖细胞重编程过程中介导表观遗传转座子沉默

• 批准号: 388942497
• 负责人: Dr. Ansgar Zoch
• 金额: --
• 依托单位: Centre for Regenerative Medicine
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2017
• 资助国家: 德国
• 起止时间: 2016-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/388942497?language=en
• 关键词: Understanding; how; piRNA; pathway; mediates

Foetal germ cells undergo extensive epigenetic reprogramming during embryonic development to revert somatic lineage specifications and reset paternal imprinting. However, this genome-wide methylation erasure also releases transposable elements from silencing DNA methylation marks. Active transposons pose a serious risk to genome integrity by threatening random mutagenic insertions. Thus, transposon silencing during reprogramming is vital to protect germ cell development and genetic integrity of the gametes. A major defensive system of the germ cell against transposon activity is the piRNA pathway, which recognizes and cleaves transposon transcripts. It also directs formation of an epigenetic memory of active transposon loci through a unique RNA-guided DNA methylation mechanism involving the piRNA binding protein MIWI2 and the DNA methyltransferase interacting protein DNMT3L. Yet, how MIWI2 couples piRNA recognition of transposons to recruitment of the DNMT3L dependent de novo DNA methylation machinery is not known.Here we propose to take full advantage of modern state-of-the-art ‘omics’ approaches to unravel the mechanism by which the piRNA pathway targets transposon loci for silencing. To this end, we will employ mice carrying endogenous epitope-tagged or null-alleles of Miwi2 and Dnmt3l that allow us to couple confocal microscopy, mass-spectrometry and high-throughput sequencing analyses. Specifically, we will study the temporal and local pattern of action of MIWI2 and DNMT3L during reprogramming and resolve how these are interdependent. In addition, we aim to discover the composition of the recognition and silencing complexes through immune-precipitation coupled mass-spectrometry analysis. Finally, we aim to understand the individual contribution of MIWI2 and DNMT3L to alterations of the chromatin landscape during reprogramming by elucidating the hierarchy of repressive silencing marks, i.e. histone modifications and DNA methylation, and their impact on chromatin accessibility at transposon loci. The proposed study thus has the potential to greatly contribute to our understanding of the cellular transposon defence and silencing system that safeguards genome integrity in the critical developmental window of germ cell reprogramming.

胎儿生殖细胞在胚胎发育过程中经历广泛的表观遗传重编程，以恢复体细胞谱系规格并重置父系印记。然而，这种全基因组甲基化擦除也从沉默DNA甲基化标记中释放了转座因子。活性转座子通过威胁随机诱变插入对基因组完整性构成严重风险。因此，在重编程过程中，转座子沉默对于保护生殖细胞发育和配子的遗传完整性至关重要。生殖细胞针对转座子活性的主要防御系统是皮尔纳途径，其识别并切割转座子转录物。它还通过涉及皮尔纳结合蛋白MIWI 2和DNA甲基转移酶相互作用蛋白DNMT 3L的独特RNA引导的DNA甲基化机制指导活性转座子基因座的表观遗传记忆的形成。然而，MIWI 2如何将转座子的皮尔纳识别与DNMT3L依赖的从头DNA甲基化机制的募集结合起来尚不清楚。在这里，我们建议充分利用现代最先进的“组学”方法来阐明皮尔纳途径靶向转座子位点沉默的机制。为此，我们将采用携带Miwi2和Dnmt3l的内源性表位标记或空等位基因的小鼠，这使我们能够耦合共聚焦显微镜，质谱和高通量测序分析。具体来说，我们将研究MIWI 2和DNMT 3L在重编程过程中的时间和局部作用模式，并解决这些是如何相互依赖的。 此外，我们的目标是通过免疫沉淀耦合质谱分析发现识别和沉默复合物的组成。最后，我们的目标是通过阐明抑制性沉默标记的层次结构，即组蛋白修饰和DNA甲基化，以及它们对转座子位点染色质可及性的影响，来了解MIWI 2和DNMT 3L在重编程过程中对染色质景观改变的单独贡献。因此，拟议的研究有可能大大有助于我们理解细胞转座子防御和沉默系统，该系统在生殖细胞重编程的关键发育窗口中保护基因组完整性。

项目成果

TEX15 is an essential executor of MIWI2-directed transposon DNA methylation and silencing

• DOI: 10.1038/s41467-020-17372-5
• 发表时间: 2020-07
• 期刊: Nature Communications
• 影响因子: 16.6
• 作者: Theresa Schöpp;Ansgar Zoch;Rebecca V Berrens;Tania Auchynnikava;Y. Kabayama;L. Vasiliauskaitė;J. Rappsilber;R. Allshire;D. O’Carroll