Dll and Sp1 ChIP-seq data from Drosophila third instar leg imaginal discs. Dll and Sp1 ChIP-seq data from Drosophila third instar leg imaginal discs

Animal limb development relies on the establishment of organizing centers, which govern limb outgrowth and patterning by regulating the spatial and temporal expression of secreted signaling molecules. On a molecular level the establishment of organizing centers occurs via cis regulatory modules (CRMs), also known as transcriptional enhancers, that integrate upstream temporal and spatial inputs. We elucidated the mechanism that governs the establishment of an Epidermal Growth Factor Receptor (EGFR) organizing center (EOC) during leg development in Drosophila melanogaster. We find that EGFR activation occurs by sequential activation of the EGFR ligand Vein (Vn) and the EGFR ligand-processing protease Rhomboid (Rho), each through single CRMs. These CRMs integrate in a distinct manner inputs from the Wingless (Wg) and Decapentaplegic (Dpp) signaling pathways, and from the leg selector transcription factors Distal-less (Dll) and Sp1. Elimination of the vn (vnE) and rho (rhoE) EOC enhancers eliminates the expression of these genes in the center of the leg imaginal discs, respectively. A vnE rhoE double deficiency, but not single deletions, demonstrates an absolute requirement of these CRMs for specifying the most distal, but not more proximal, leg fates. In addition, the cis-regulatory logic of vnE and rhoE transcends the leg EOC developmental program, because genomic regions with similar inputs, based on predicted and genome-wide binding by the transcription factors that establish the EOC, faithfully predicts novel CRMs active in the distal leg. The combinatorial input of Wg, Dpp, Dll and Sp1 at these CRMs reveals a molecular signature for coordinating gene expression in the Drosophila leg that might be analogous to many other multi-cellular systems. Overall design: Triplicate pools of 100 yw and 100 recombineered Sp1-GFP (BAC) L3 wandering larvae were used to perform independent chromatin IPs. The Sp1-GFP (BAC) is a GFP-tagged Sp1 in BAC clone CH321-64M02 inserted in landing site VK00033 (gift from Dr. Rebecca Spokony). All 6 leg discs from each larva were used as material for each IP. Chromatin from the yw larvae pools was immuno-precipitated with goat anti-Dll antibody (sc-15858, Santa Cruz Biotechnology, 1.5 µg/ml for IP) while chromatin from the Sp1-GFP larvae pools was immuno-precipitated with rabbit anti-GFP antibody (ab290, Abcam, 1∶300 dilution for IP). DNA from non-immunoprecipitated 10% chromatin input was isolated from each pool as reference control.