Epigenetic Requirements for Triggering Heterochromatinization and Piwi-Interacting RNA Production from Transgenes in the Drosophila Germline

Transgenes containing a fragment of the I retrotransposon represent a powerful model of piRNA cluster de novo formation in the Drosophila germline. We revealed that the same transgenes located at different genomic loci form piRNA clusters with various capacity of small RNA production. Transgenic piRNA clusters are not established in piRNA pathway mutants. However, in the wild-type context, the endogenous ancestral I-related piRNAs heterochromatinize and convert the I-containing transgenes into piRNA-producing loci. Here, we address how the quantitative level of piRNAs influences the heterochromatinization and piRNA production. We show that a minimal amount of maternal piRNAs from ancestral I-elements is sufficient to form the transgenic piRNA clusters. Supplemental piRNAs stemming from active I-element copies do not stimulate additional chromatin changes or piRNA production from transgenes. Therefore, chromatin changes and piRNA production are initiated by a minimum threshold level of complementary piRNAs, suggesting a selective advantage of prompt cell response to the lowest level of piRNAs. It is noteworthy that the weak piRNA clusters do not transform into strong ones after being targeted by abundant I-specific piRNAs, indicating the importance of the genomic context for piRNA cluster establishment. Analysis of ovarian transcription profiles suggests that regions facilitating convergent transcription favor the formation of transgenic piRNA clusters.