Tissue-specific versus pleiotropic enhancers within the bric-a-brac tandem gene duplicates display differential regulatory activity and evolutionary conservation

Abstract

During animal evolution, de novo emergence and modifications of pre-existing transcriptional enhancers have contributed to biological innovations, by implementing gene regulatory networks. The Drosophila melanogaster bric-a-brac (bab) complex, comprising the tandem paralogous genes bab1-2, provides a paradigm to address how enhancers contribute and co-evolve to regulate jointly or differentially duplicated genes. We previously characterized an intergenic enhancer (named LAE) governing bab2 expression in leg and antennal tissues. We show here that LAE activity also regulates bab1. CRISPR/Cas9-mediated LAE excision reveals its critical role for bab2-specific expression along the proximo-distal leg axis, likely through paralog-specific interaction with the bab2 gene promoter. Furthermore, LAE appears involved but not strictly required for bab1-2 co-expression in leg tissues. Phenotypic rescue experiments, chromatin features and a gene reporter assay reveal a large "pleiotropic" bab1 enhancer (termed BER) including a series of cis-regulatory elements active in the leg, antennal, wing, haltere and gonadal tissues. Phylogenomics analyses indicate that (i) bab2 originates from bab1 duplication within the Muscomorpha sublineage, (ii) LAE and bab1 promoter sequences have been evolutionarily-fixed early on within the Brachycera lineage, while (iii) BER elements have been conserved more recently among muscomorphans. Lastly, we identified conserved binding sites for transcription factors known or prone to regulate directly the paralogous bab genes in diverse developmental contexts. This work provides new insights on enhancers, particularly about their emergence, maintenance and functional diversification during evolution. Author summaryGene duplications and transcriptional enhancer emergence/modifications are thought having greatly contributed to phenotypic innovations during animal evolution. However, how enhancers regulate distinctly gene duplicates and are evolutionary-fixed remain largely unknown. The Drosophila bric-a-brac locus, comprising the tandemly-duplicated genes bab1-2, provides a good paradigm to address these issues. The twin bab genes are co-expressed in many tissues. In this study, genetic analyses show a partial co-regulation of both genes in the developing legs depending on tissue-specific transcription factors known to bind a single enhancer. Genome editing and gene reporter assays further show that this shared enhancer is also required for bab2-specific expression. Our results also reveal the existence of partly-redundant regulatory functions of a large pleiotropic enhancer which contributes to co-regulate the bab genes in distal leg tissues. Phylogenomics analyses indicate that the Drosophila bab locus originates from duplication of a dipteran bab1-related gene, which occurred within the Brachycera (true flies) lineage. bab enhancer and promoter sequences have been differentially-conserved among Diptera suborders. This work illuminates how transcriptional enhancers from tandem gene duplicates (i) differentially interact with distinct cognate promoters and (ii) undergo distinct evolutionary changes to diversifying their respective tissue-specific gene expression pattern.