A single H2A variant prevents genome instability through piRNAs biogenesis and replication stress control.
Transposable elements (TEs) pose a major threat to genome integrity in the germline, where the piRNA pathway ensures heritable TEs silencing. How the chromatin environment that enables piRNA biogenesis is first established during oogenesis remains unclear. Here, we identify the histone variant His2Av, the single H2A variant in Drosophila combining H2A.Z (transcriptional) and H2A.X (DNA repair) features, as a critical regulator of the earliest steps of piRNA pathway activation. Germline-specific depletion of His2Av disrupts transcription of piRNA pathway genes, abolishes dual-strand piRNA cluster transcription, and triggers strong TEs derepression. These defects are associated with loss of Rhino recruitment despite intact H3K9me3, suggesting that His2Av contributes to the establishment of a specialized heterochromatin permissive for noncanonical piRNA cluster transcription. His2Av depletion also causes DNA damage, replication stress, and activation of Chk2- and Claspin-dependent checkpoints, leading to oogenesis arrest. Remarkably, overexpression of RNase H, but not a catalytic-dead variant, robustly rescues oocyte development, suggesting that replication stress is a major source of DNA damage in His2Av mutants. Finally, using a separation-of-function approach with a C-terminal truncation inhibiting H2A.X-like activity, we show that the essential germline role of His2Av is transcriptional (H2A.Z-like). Together, our findings reveal that His2Av primes germline chromatin for piRNA pathway initiation while limiting transcription-replication conflicts during early oogenesis.