A shared ancient enhancer element differentially regulates the bric-a-brac tandem gene duplicates in the developing Drosophila leg.

Gene duplications and transcriptional enhancer emergence/modifications are thought having greatly contributed to phenotypic innovations during animal evolution. Nevertheless, little is known about how enhancers evolve after gene duplication and how regulatory information is rewired between duplicated genes. The Drosophila melanogaster bric-a-brac (bab) complex, comprising the tandem paralogous genes bab1 and bab2, provides a paradigm to address these issues. We previously characterized an intergenic enhancer (named LAE) regulating bab2 expression in the developing legs. We show here that bab2 regulators binding directly the LAE also govern bab1 expression in tarsal cells. LAE excision by CRISPR/Cas9-mediated genome editing reveals that this enhancer appears involved but not strictly required for bab1 and bab2 co-expression in leg tissues. Instead, the LAE enhancer is critical for paralog-specific bab2 expression along the proximo-distal leg axis. Chromatin features and phenotypic rescue experiments indicate that LAE functions partly redundantly with leg-specific regulatory information overlapping the bab1 transcription unit. Phylogenomics analyses indicate that (i) the bab complex originates from duplication of an ancestral singleton gene early on within the Cyclorrhapha dipteran sublineage, and (ii) LAE sequences have been evolutionarily-fixed early on within the Brachycera suborder thus predating the gene duplication event. This work provides new insights on enhancers, particularly about their emergence, maintenance and functional diversification during evolution.

Tissue-specific enhancer repression through molecular integration of cell signaling inputs.

Drosophila leg morphogenesis occurs under the control of a relatively well-known genetic cascade, which mobilizes both cell signaling pathways and tissue-specific transcription factors. However, their cross-regulatory interactions, deployed to refine leg patterning, remain poorly characterized at the gene expression level. Within the genetically interacting landscape that governs limb development, the bric-à-brac2 (bab2) gene is required for distal leg segmentation. We have previously shown that the Distal-less (Dll) homeodomain and Rotund (Rn) zinc-finger activating transcription factors control limb-specific bab2 expression by binding directly a single critical leg/antennal enhancer (LAE) within the bric-à-brac locus. By genetic and molecular analyses, we show here that the EGFR-responsive C15 homeodomain and the Notch-regulated Bowl zinc-finger transcription factors also interact directly with the LAE enhancer as a repressive duo. The appendage patterning gene bab2 is the first identified direct target of the Bowl repressor, an Odd-skipped/Osr family member. Moreover, we show that C15 acts on LAE activity independently of its regular partner, the Aristaless homeoprotein. Instead, we find that C15 interacts physically with the Dll activator through contacts between their homeodomain and binds competitively with Dll to adjacent cognate sites on LAE, adding potential new layers of regulation by C15. Lastly, we show that C15 and Bowl activities regulate also rn expression. Our findings shed light on how the concerted action of two transcriptional repressors, in response to cell signaling inputs, shapes and refines gene expression along the limb proximo-distal axis in a timely manner.