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.

The multi-subunit Mediator complex plays a critical role in gene expression by bridging enhancer-bound transcription factors and the RNA polymerase II machinery. Although experimental case studies suggest differential roles of Mediator subunits, a comprehensive view of the specific set of genes regulated by individual subunits in a developing tissue is still missing. Here we address this fundamental question by focusing on the Med19 subunit and using the Drosophila wing imaginal disc as a developmental model. By coupling auxin-inducible degradation of endogenous Med19 in vivo with RNA-seq, we got access to the early consequences of Med19 elimination on gene expression. Differential gene expression analysis reveals that Med19 is not globally required for mRNA transcription but specifically regulates positively or negatively less than a quarter of the expressed genes. By crossing our transcriptomic data with those of Drosophila gene expression profile database, we found that Med19-dependent genes are highly enriched with spatially-regulated genes while the expression of most constitutively expressed genes is not affected upon Med19 loss. Whereas globally downregulation does not exceed upregulation, we identified a functional class of genes encoding spatially-regulated transcription factors, and more generally developmental regulators, responding unidirectionally to Med19 loss with an expression collapse. Moreover, we show in vivo that the Notch-responsive wingless and the E(spl)-C genes require Med19 for their expression. Combined with experimental evidences suggesting that Med19 could function as a direct transcriptional effector of Notch signaling, our data support a model in which Med19 plays a critical role in the transcriptional activation of developmental genes in response to cell signaling pathways.

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 summary Gene 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.

The evolutionarily conserved multiprotein Mediator complex (MED) serves as an interface between DNA-bound transcription factors (TFs) and the RNA Pol II machinery. It has been proposed that each TF interacts with a dedicated MED subunit to induce specific transcriptional responses. But are these binary partnerships sufficient to mediate TF functions? We have previously established that the Med1 Mediator subunit serves as a cofactor of GATA TFs in Drosophila, as shown in mammals. Here, we observe mutant phenotype similarities between another subunit, Med19, and the Drosophila GATA TF Pannier (Pnr), suggesting functional interaction. We further show that Med19 physically interacts with the Drosophila GATA TFs, Pnr and Serpent (Srp), in vivo and in vitro through their conserved C-zinc finger domains. Moreover, Med19 loss of function experiments in vivo or in cellulo indicate that it is required for Pnr- and Srp-dependent gene expression, suggesting general GATA cofactor functions. Interestingly, Med19 but not Med1 is critical for the regulation of all tested GATA target genes, implying shared or differential use of MED subunits by GATAs depending on the target gene. Lastly, we show a direct interaction between Med19 and Med1 by GST pulldown experiments indicating privileged contacts between these two subunits of the MED middle module. Together, these findings identify Med19/Med1 as a composite GATA TF interface and suggest that binary MED subunit-TF partnerships are probably oversimplified models. We propose several mechanisms to account for the transcriptional regulation of GATA-targeted genes.

The evolutionarily-conserved multiprotein Mediator complex (MED) serves as an interface between DNA-bound transcription factors (TFs) and the RNA Polymerase II machinery. It has been proposed that each TF interacts with a dedicated MED subunit to induce specific transcriptional responses. However, binary MED subunit - TF partnerships are probably oversimplified models. Using Drosophila TFs of the GATA family - Pannier (Pnr) and Serpent (Srp) - as a model, we have previously established GATA cofactor evolutionarily-conserved function for the Med1 Mediator subunit. Here, we show that another subunit, Med19, is required for GATA-dependent gene expression and interacts physically with Pnr and Srp in cellulo, in vivo and in vitro through their conserved C-zinc finger (ZF), indicating general GATA co-activator functions. Interestingly, Med19 is critical for the regulation of all tested GATA target genes which is not the case for Med1, suggesting differential use of MED subunits by GATAs depending on the target gene. Lastly, despite their presumed distant position within the MED middle module, both subunits interact physically. In conclusion, our data shed new light first on the MED complex, engaging several subunits to mediate TF-driven transcriptional responses and second, on GATA TFs, showing that ZF DNA-binding domain also serves for transactivation.

DNA-bound transcription factors (TFs) governing developmental gene regulation have been proposed to recruit polymerase II machinery at gene promoters through specific interactions with dedicated subunits of the evolutionarily conserved Mediator (MED) complex. However, whether such MED subunit-specific functions and partnerships have been conserved during evolution has been poorly investigated. To address this issue, we generated the first Drosophila melanogaster loss-of-function mutants for Med1, known as a specific cofactor for GATA TFs and hormone nuclear receptors in mammals. We show that Med1 is required for cell proliferation and hematopoietic differentiation depending on the GATA TF Serpent (Srp). Med1 physically binds Srp in cultured cells and in vitro through its conserved GATA zinc finger DNA-binding domain and the divergent Med1 C terminus. Interestingly, GATA-Srp interaction occurs through the longest Med1 isoform, suggesting a functional diversity of MED complex populations. Furthermore, we show that Med1 acts as a coactivator for the GATA factor Pannier during thoracic development. In conclusion, the Med1 requirement for GATA-dependent regulatory processes is a common feature in insects and mammals, although binding interfaces have diverged. Further work in Drosophila should bring valuable insights to fully understand GATA-MED functional partnerships, which probably involve other MED subunits depending on the cellular context.

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.

Hox genes in species across the metazoa encode transcription factors (TFs) containing highly-conserved homeodomains that bind target DNA sequences to regulate batteries of developmental target genes. DNA-bound Hox proteins, together with other TF partners, induce an appropriate transcriptional response by RNA Polymerase II (PolII) and its associated general transcription factors. How the evolutionarily conserved Hox TFs interface with this general machinery to generate finely regulated transcriptional responses remains obscure. One major component of the PolII machinery, the Mediator (MED) transcription complex, is composed of roughly 30 protein subunits organized in modules that bridge the PolII enzyme to DNA-bound TFs. Here, we investigate the physical and functional interplay between Drosophila melanogaster Hox developmental TFs and MED complex proteins. We find that the Med19 subunit directly binds Hox homeodomains, in vitro and in vivo. Loss-of-function Med19 mutations act as dose-sensitive genetic modifiers that synergistically modulate Hox-directed developmental outcomes. Using clonal analysis, we identify a role for Med19 in Hox-dependent target gene activation. We identify a conserved, animal-specific motif that is required for Med19 homeodomain binding, and for activation of a specific Ultrabithorax target. These results provide the first direct molecular link between Hox homeodomain proteins and the general PolII machinery. They support a role for Med19 as a PolII holoenzyme-embedded "co-factor" that acts together with Hox proteins through their homeodomains in regulated developmental transcription.