PIWI proximity proteome reveals Set1-mediated piRNA biogenesis for transposon silencing at telomeres.

Silencing complexes formed by PIWI-clade Argonaute (Ago) proteins and PIWI-interacting RNAs (piRNAs) are essential guardians of genome integrity, restricting the activities of transposable elements (TEs) in the animal germline. However, our understanding of PIWI-piRNA-directed TE silencing remains incomplete. Here, we systemically characterize the proximity proteome of the PIWI members Piwi, Aubergine (Aub), and Ago3 in the germline of Drosophila ovaries. Functional screening identifies previously uncharacterized factors involved in TE silencing, including the H3K4me3 writer and transcriptional coactivator Set1. Transcriptome analysis reveals that Set1 acts as an indispensable repressor of TEs, particularly of those at telomeres. Set1 is required for the production of antisense, TE-targeting piRNAs. Genome-wide chromatin profiling by CUT&Tag demonstrates that Set1 preferentially associates with TE sequences, including their 3'UTRs, and is localized at subtelomeric piRNA-producing loci. It also controls the accumulation of Rhino, a key activator of piRNA precursor transcription, at these sites. Notably, the catalytic activity of Set1 is dispensable. Our findings uncover a noncanonical function of Set1 in Piwi-mediated TE silencing in germline nuclei.