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Putzig safeguards genome integrity by contributing to the Piwi-mediated repression of transposon activity in the female germline of Drosophila in a two-tiered fashion

ABSTRACT: Genome integrity in the germline is jeopardized by the activity of transposable elements (TEs). Transposon activity is kept in check by the Piwi-piRNA pathway, which silences TEs post-transcriptionally in the cytoplasm as well as co-transcriptionally in the nucleus. piRNAs derive from long precursor transcripts originating at piRNA-loci by non-conventional transcription. They are processed in the cytoplasm and loaded into Piwi-piRNA complexes that then enter the nucleus to bind to the target RNA and direct local heterochromatin formation at TE loci, involving several downstream effectors. In this work, we have analyzed the role of Putzig (Pzg) in Piwi-mediated TE silencing in the female germline. Pzg has multi-facetted roles during Drosophila development and is important for DNA integrity, germ cell differentiation and survival. Here, we provide evidence for a two-tier activity of Pzg by serving as a hub for various Piwi-pathway members. Firstly, Pzg assists transcription initiation at piRNA clusters by coupling the Trf2-Moonshiner transcription initiation complex to the Rhino-Deadlock-Cutoff complex, thereby promoting formation of piRNA-precursor transcripts. Secondly, in the context of co-transcriptional gene silencing, Pzg licences heterochromatin formation by linking the Piwi-piRNA silencing machinery and the histone demethylase Lsd1, involved in promoter inactivation.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE324254 | GEO | 2026/03/11

REPOSITORIES: GEO

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Project description:PIWI-clade Argonaute proteins silence transposon expression in animal gonads. Their target specificity is defined by bound ~23-30nt piRNAs that are processed from single-stranded precursor transcripts via two distinct pathways. Primary piRNAs are defined by the endo-nuclease Zucchini, while biogenesis of secondary piRNAs depends on piRNA-guided transcript cleavage and results in piRNA amplification. Here, we analyze the inter-dependencies between these piRNA biogenesis pathways in the developing Drosophila ovary. We show that secondary piRNA-guided target slicing is the predominant mechanism that specifies transcripts—including those from piRNA clusters—as primary piRNA precursors and that defines the spectrum of Piwi-bound piRNAs in germline cells. Post-transcriptional silencing in the cytoplasm therefore enforces nuclear, transcriptional target silencing, which ensures the tight suppression of transposons during oogenesis. As target slicing also defines the nuclear piRNA pool during mouse spermatogenesis, our findings uncover an unexpected conceptual similarity between the mouse and fly piRNA pathways. To understand the hierarchical order of primary versus secondary piRNA biogenesis in Drosophila ovaries, we sequenced piRNAs bound to total-Piwi, germline-Piwi, Aubergine and Argonaute3 from ovaries of germline specific knockdowns of control, piwi, aub, ago3 single knockdowns and aub/ago3 double knockdowns. To determine changes in Transposable Element (TE) transcription or TE RNA steady state in perturbed piRNA pathway conditions, we performed Pol2-ChIP-sequencing and polyA bound RNA-sequencing from ovaries of multiple germline knockdown genotypes. We also sequenced genomic DNA from ovaries of control knockdowns to experimentally estimate the TE copy number in our genetic background. Finally, we used CAP-seq from germline specific Piwi depletions to identify the Transcriptional Start Sites (TSS) in TEs in a deregulated background. Replicates are labeled with R1, R2, R3, R4 where indicated.

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Putzig safeguards genome integrity by contributing to the Piwi-mediated repression of transposon activity in the female germline of Drosophila in a two-tiered fashion

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