Tissue-specific versus pleiotropic enhancers within thebric-a-bractandem gene duplicates display differential regulatory activity and evolutionary conservation

Abstract

AbstractDuring animal evolution, de novo emergence and modifications of pre-existing transcriptional enhancers have contributed to biological innovations, by implementing gene regulatory networks. TheDrosophila melanogaster bric-a-brac(bab) complex, comprising the tandem paralogous genesbab1-2, 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) governingbab2expression in leg and antennal tissues. We show here that LAE activity also regulatesbab1. CRISPR/Cas9-mediated LAE excision reveals its critical role forbab2-specific expression along the proximo-distal leg axis, likely through paralog-specific interaction with thebab2gene promoter. Furthermore, LAE appears involved but not strictly required forbab1-2co-expression in leg tissues. Phenotypic rescue experiments, chromatin features and a gene reporter assay reveal a large “pleiotropic”bab1enhancer (termed BER) including a series ofcis-regulatory elements active in the leg, antennal, wing, haltere and gonadal tissues. Phylogenomics analyses indicate that (i)bab2originates frombab1duplication within the Muscomorpha sublineage, (ii) LAE andbab1promoter 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 paralogousbabgenes 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. TheDrosophila bric-a-braclocus, comprising the tandemly-duplicated genesbab1-2, provides a good paradigm to address these issues. The twinbabgenes 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 forbab2-specific expression. Our results also reveal the existence of partly-redundant regulatory functions of a large pleiotropic enhancer which contributes to co-regulate thebabgenes in distal leg tissues. Phylogenomics analyses indicate that theDrosophila bablocus originates from duplication of a dipteranbab1-related gene, which occurred within the Brachycera (true flies) lineage.babenhancer 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.