Constraints on the evolution of a doublesex target gene arising from doublesex’s pleiotropic deployment

Significance Most sexually dimorphic features of Drosophila melanogaster are specified by the action of sex-specific transcription factors encoded by the doublesex ( dsx ) gene. Evolutionary changes in such sexually dimorphic features are often a result of changes in the cis -regulatory sequences of the DSX target genes. When a particular target gene is directly regulated by DSX in multiple tissues, evolutionarily conflicting constraints may be generated. The research we present here reveals that such conflict can be solved by deploying different cis -regulatory modules (sharing the DSX-binding site) for different tissues. This mechanism could also apply to other transcription factors. “Regulatory evolution,” that is, changes in a gene’s expression pattern through changes at its regulatory sequence, rather than changes at the coding sequence of the gene or changes of the upstream transcription factors, has been increasingly recognized as a pervasive evolution mechanism. Many somatic sexually dimorphic features of Drosophila melanogaster are the results of gene expression regulated by the doublesex ( dsx ) gene, which encodes sex-specific transcription factors (DSX F in females and DSX ᴹ in males). Rapid changes in such sexually dimorphic features are likely a result of changes at the regulatory sequence of the target genes. We focused on the Flavin-containing monooxygenase-2 ( Fmo-2 ) gene, a likely direct dsx target, to elucidate how sexually dimorphic expression and its evolution are brought about. We found that dsx is deployed to regulate the Fmo-2 transcription both in the midgut and in fat body cells of the spermatheca (a female-specific tissue), through a canonical DSX-binding site in the Fmo-2 regulatory sequence. In the melanogaster group, Fmo-2 transcription in the midgut has evolved rapidly, in contrast to the conserved spermathecal transcription. We identified two cis -regulatory modules (CRM-p and CRM-d) that direct sexually monomorphic or dimorphic Fmo-2 transcription, respectively, in the midguts of these species. Changes of Fmo-2 transcription in the midgut from sexually dimorphic to sexually monomorphic in some species are caused by the loss of CRM-d function, but not the loss of the canonical DSX-binding site. Thus, conferring transcriptional regulation on a CRM level allows the regulation to evolve rapidly in one tissue while evading evolutionary constraints posed by other tissues.