Iki, T.; Kai, T.; Isshiki, W.; Kozuka-Hata, H.; Oyama, M.

Silencing complexes formed by PIWI-clade Argonaute (Ago) proteins and PIWI-interacting RNAs (piRNAs) are essential guardians of genome integrity, controlling the deleterious activities of transposable elements (TEs) in animal germline. However, our understanding of PIWI-piRNA-directed TE silencing remains incomplete. Here, we systemically characterize the proximity proteome of PIWI members, Piwi, Aubergine (Aub), and Ago3 in the germline of Drosophila ovaries. Functional screening identifies previously uncharacterized factors involved in TE silencing, including H3K4me3 writer and transcriptional coactivator Set1. Transcriptome analysis reveals that Set1 acts as an indispensable repressor for TEs, particularly those forming telomeres. The involvement of Set1 in Piwi pathway is further supported by its critical role in the production of antisense, TE-targeting piRNAs. Notably, catalytic activity of Set1 is dispensable for TE silencing. Genome-wide chromatin binding analysis using CUT&Tag demonstrates that Set1 preferentially associates with TE sequences and localizes to subtelomeric piRNA cluster loci, suggesting a role in promoting piRNA precursor transcription through direct binding. Collectively, these findings uncover a noncanonical function of Set1 in Piwi-mediated TE silencing and telomere control in germline nuclei.-