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

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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.