Project summary The piRNA pathway is an evolutionarily conserved mechanism that acts in the germline of metazoans to repress the activity of transposable elements and ensure genome integrity and fertility. At the core of the pathway lies an Argonaute protein of the Piwi clade and the associated small non-coding RNA, piwi-interacting (pi)RNA, which guides the Piwi protein to its targets. In the cytoplasm Piwi proteins act by destroying the mRNA targets using their intrinsic nuclease activity. piRNA also guide nuclear the Piwi to establish repressive chromatin and induce transcriptional repression of genomic targets. We have found that SUMO and an E3 SUMO ligase play an essential role in piRNA-guided transcriptional repression by linking the nuclear Piwi complex to the chromatin modifier. However, individual steps of piRNA-guided chromatin repression remain poorly understood. In this proposal, we will investigate the molecular mechanism of piRNA-guided repression. We will determine how the SUMO ligase is recruited to genomic piRNA targets and explore the role of SUMO in assembly of the repressive piRNA complexes. We will determine functions of Piwi and piRNA in establishment of repressive chromatin and initiation of piRNA biogenesis. In addition to its function in the germline of adult flies, the maternal Piwi-piRNA complex is deposited into the egg and was proposed to activate piRNA biogenesis in the progeny. However, Piwi is indispensable for oogenesis preventing direct interrogation of its embryonic function. To circumvent this problem and study the function of maternal Piwi in embryogenesis we have developed a strategy for depleting maternal Piwi in the embryo. We will apply this strategy to probe the role of Piwi in specification of piRNA clusters in primordial germ cells. How distinct chromatin domains are demarcated is the central question of chromatin biology. In animals, heterochromatin is disassembled in gametes and re-established during embryogenesis. How heterochromatin is assembled de novo during development remains poorly understood. Transposable elements comprise the bulk of heterochromatin, making Piwi-piRNA complexes an ideal tool for finding and marking heterochromatin sequences. During early zygotic development maternal Piwi-piRNA complexes are localized to somatic nuclei of the embryo. We propose that Piwi-bound piRNAs direct de novo establishment of repressive chromatin domains in somatic cells of the early embryo followed by its piRNA-independent propagation and maintenance during later development. We will explore this model by depleting maternal Piwi and analyzing its effect on chromatin. The proposed work will advance our knowledge of RNA-mediated regulation and of transcription chromatin structure in animals. We explore the function of piRNA in transmission of epigenetic information from generation to generation. Our studies promise to shed new light on how distinct chromatin domains are demarked. A detailed mechanistic...