Insulin signaling regulates R2 retrotransposon expression to orchestrate transgenerational rDNA copy number maintenance

Preserving a large number of essential yet highly unstable ribosomal DNA (rDNA) repeats is critical for the germline to perpetuate the genome through generations. Spontaneous rDNA loss must be countered by rDNA copy number (CN) expansion. Germline rDNA CN expansion is best understood in Drosophila melanogaster, which relies on unequal sister chromatid exchange (USCE) initiated by DNA breaks at rDNA. The rDNA-specific retrotransposon R2 responsible for USCE-inducing DNA breaks is typically expressed only when rDNA CN is low to minimize the danger of DNA breaks; however, the underlying mechanism of R2 regulation remains unclear. Here we identify the insulin receptor (InR) as a major repressor of R2 expression, limiting unnecessary R2 activity. Through single-cell RNA sequencing we find that male germline stem cells (GSCs), the major cell type that undergoes rDNA CN expansion, have reduced InR expression when rDNA CN is low. Reduced InR activity in turn leads to R2 expression and CN expansion. We further find that dietary manipulation alters R2 expression and rDNA CN expansion activity. This work reveals that the insulin pathway integrates rDNA CN surveying with environmental sensing, revealing a potential mechanism by which diet exerts heritable changes to genomic content. Overall design: 50 testes were hand dissected from 1 to 5-day-old flies in 1x PBS and transferred immediately into tubes with cold 1x PBS on ice. Tissue dissociation was performed. Cell viability and density was determined on a hemocytometer using Trypan Blue stain and DIC imaging. Cells were processed for library preparation using the 10 X Genomics Chromium Controller and Chromium Single Cell Library and Gel Bead Kit following standard manufacturer's protocol. Amplified cDNA libraries were quantified by bioanalyzer, size selected by AMPure beads, and sequenced on a NovaSeq SP.