The piRNA pathway is central to the transcriptional and post-transcriptional control of transposon activity within animal germ cells. Despite notable advancements, a comprehensive understanding of the molecular mechanisms underlying piRNA biogenesis and function remains elusive. Our prior work demonstrated that depletion of the small ubiquitin-like modifier (SUMO) from the Drosophila female germline leads to substantial gene deregulation, aberrant transposon activation, and infertility. Nevertheless, the specific targets and mechanistic roles of SUMOylation within diverse biological contexts, including in the piRNA pathway, have been hindered by technical challenges.

To investigate the role of protein modification by SUMO in the Drosophila ovary further, we devised a novel transgenic model and compatible diGly proteomics-based strategy for unbiased discovery of SUMO targets with aminoacid-level precision. This approach revealed a comprehensive set of SUMO targets, prominently enriched in proteins connected to the piRNA pathway and heterochromatin (Ninova et al., 2023, Cell Genomics 3, 100329). Intriguingly, while SUMO is known to primarily affect nuclear proteins, we found multiple SUMO modifications on proteins localized within the germ granule-like compartment 'nuage'' - the site of piRNA biogenesis and post-transcriptional transposon cleavage. Furthermore, we found that multiple piRNA pathway proteins undergo regulated multi-SUMOylation, dependent on the central epigenetic silencing effector Piwi. These results collectively indicate a wide-ranging and multifaceted role of protein SUMOylation in chromatin regulation and transposon control extending beyond previously recognized functions, thereby opening new avenues in the field.