Drosophila distal-less and Rotund bind a single enhancer ensuring reliable and robust bric-a-brac2 expression in distinct limb morphogenetic fields

PLoS Genet. 2013 Jun;9(6):e1003581. doi: 10.1371/journal.pgen.1003581. Epub 2013 Jun 27.

Abstract

Most identified Drosophila appendage-patterning genes encode DNA-binding proteins, whose cross-regulatory interactions remain to be better characterized at the molecular level, notably by studying their direct binding to tissue-specific transcriptional enhancers. A fine-tuned spatio-temporal expression of bric-a-brac2 (bab2) along concentric rings is essential for proper proximo-distal (P-D) differentiation of legs and antennae. However, within the genetic interaction landscape governing limb development, no transcription factor directly controlling bab2 expression has been identified to date. Using site-targeted GFP reporter assay and BAC recombineering, we show here that restricted bab2 expression in leg and antennal imaginal discs relies on a single 567-bp-long cis-regulatory module (CRM), termed LAE (for leg and antennal enhancer). We show that this CRM (i) is necessary and sufficient to ensure normal bab2 activity in developing leg and antenna, and (ii) is structurally and functionally conserved among Drosophilidae. Through deletion and site-directed mutagenesis approaches, we identified within the LAE essential sequence motifs required in both leg and antennal tissues. Using genetic and biochemical tests, we establish that in the LAE (i) a key TAAT-rich activator motif interacts with the homeodomain P-D protein Distal-less (Dll) and (ii) a single T-rich activator motif binds the C2H2 zinc-finger P-D protein Rotund (Rn), leading to bab2 up-regulation respectively in all or specifically in the proximal-most ring(s), both in leg and antenna. Joint ectopic expression of Dll and Rn is sufficient to cell-autonomously activate endogenous bab2 and LAE-driven reporter expression in wing and haltere cells. Our findings indicate that accuracy, reliability and robustness of developmental gene expression do not necessarily require cis-regulatory information redundancy.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Body Patterning
  • DNA-Binding Proteins / genetics
  • Drosophila Proteins / genetics*
  • Drosophila Proteins / metabolism
  • Drosophila melanogaster / genetics*
  • Drosophila melanogaster / growth & development
  • Enhancer Elements, Genetic
  • Extremities / growth & development
  • Gene Expression Regulation, Developmental
  • Homeodomain Proteins / genetics*
  • Insect Proteins / genetics
  • Morphogenesis / genetics*
  • Mutagenesis, Site-Directed
  • Transcription Factors / genetics*
  • Transcription Factors / metabolism
  • Wings, Animal / growth & development

Substances

  • BAB2 protein, Drosophila
  • Bric-a-brac protein 1, Drosophila
  • DNA-Binding Proteins
  • Drosophila Proteins
  • Homeodomain Proteins
  • Insect Proteins
  • Transcription Factors
  • distal-less protein, insect
  • rn protein, Drosophila

Grant support

This work was supported by grants from the French governmental agency for Research (CNRS), the Paul Sabatier University (UPS) and the “Association pour la Recherche sur le Cancer” (ARC). AB was supported by the CNRS and the Midi Pyrenees Region. LHM-V was supported by the Mexican CONACYT. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.