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

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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.