Enrichment analyses

We performed functional enrichment analyses to determine whether certain Gene Ontology terms were overrepresented among genes showing significant sex-biased expression or inferred to be regulated by Dsx. We characterized protein functions based on homology with described genes in the Gene Ontology database⁶⁰ using the programme Blast2GO (ref. ⁶¹), resulting in functional categories representing gene product properties (for example, contributions to cell structure, their involvement in molecular pathways or their role in a biological process). We used ErmineJ^62,63 to test whether each functional category was enriched for differentially expressed genes relative to all other genes using gene score resampling (scores were based on adjusted P values) to generate a null distribution. We then compared our lists of functional categories that were enriched for either sex-biased or Dsx-mediated genes to the list of functional categories found to be enriched for Dsx targets as identified in D. melanogaster⁹.