Understanding how the piRNA pathway mediates epigenetic transposon silencing during foetal 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和重置父系印记。然而，这种全基因组甲基化清除也会从沉默的DNA甲基化标记中释放转座因子。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威胁。因此，重编程过程中的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。生殖细胞对转座子活性的主要防御系统是piRNA通路，它识别和切割转座子转录物。它还通过独特的rna引导的DNA甲基化机制，包括piRNA结合蛋白MIWI2和DNA甲基转移酶相互作用蛋白DNMT3L，指导活性转座子位点表观遗传记忆的形成。然而，MIWI2如何将转座子的piRNA识别与DNMT3L依赖性的从头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”方法来揭示piRNA途径靶向转座子位点沉默的机制。为此，我们将使用携带内源性Miwi2和Dnmt3l表位标记或零等位基因的小鼠，使我们能够将共聚焦显微镜，质谱分析和高通量测序分析结合起来。具体来说，我们将研究MIWI2和DNMT3L在重编程过程中的时间和局部作用模式，并解决它们是如何相互依赖的。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沉默标记（组蛋白修饰和DNA甲基化）的层次结构，以及它们对转座子位点染色质可及性的影响，了解MIWI2和DNMT3L在重编程过程中对染色质景观改变的个体贡献。因此，提出的研究有可能极大地促进我们对细胞转座子防御和沉默系统的理解，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。

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.