title: Neural precursor-specific expression of multiple Drosophila genes is driven by dual enhancer modules with overlapping function.
abstract: Transcriptional cis-regulatory modules (CRMs), or enhancers, are responsible for directing gene expression in specific territories and cell types during development. In some instances, the same gene may be served by two or more enhancers with similar specificities. Here we show that the utilization of dual, or "shadow", enhancers is a common feature of genes that are active specifically in neural precursor (NP) cells in Drosophila. By genome-wide computational discovery of statistically significant clusters of binding motifs for both proneural activator (P) proteins and basic helix-loop-helix (bHLH) repressor (R) factors (a "P+R" regulatory code), we have identified NP-specific enhancer modules associated with multiple genes expressed in this cell type. These CRMs are distinct from those previously identified for the corresponding gene, establishing the existence of a dual-enhancer arrangement in which both modules reside close to the gene they serve. Using wild-type and mutant reporter gene constructs in vivo, we show that P sites in these modules mediate activation by proneural factors in "proneural cluster" territories, whereas R sites mediate repression by bHLH repressors, which serves to restrict expression specifically to NP cells. To our knowledge, our results identify the first direct targets of these bHLH repressors. Finally, using genomic rescue constructs for neuralized (neur), we demonstrate that each of the gene's two NP-specific enhancers is sufficient to rescue neur function in the lateral inhibition process by which adult sensory organ precursor (SOP) cells are specified, but that deletion of both enhancers results in failure of this event.
authors.affiliation: Division of Biological Sciences/CDB, University of California at San Diego, La Jolla, CA 92093.
authors.forename: Steven W
authors.initials: SW
authors.lastname: Miller
authors.affiliation: Division of Biological Sciences/CDB, University of California at San Diego, La Jolla, CA 92093.
authors.forename: Mark
authors.initials: M
authors.lastname: Rebeiz
authors.affiliation: Division of Biological Sciences/CDB, University of California at San Diego, La Jolla, CA 92093.
authors.forename: Jenny E
authors.initials: JE
authors.lastname: Atanasov
authors.affiliation: Division of Biological Sciences/CDB, University of California at San Diego, La Jolla, CA 92093 jposakony@ucsd.edu.
authors.forename: James W
authors.identifier: http://orcid.org/0000-0001-6377-1732
authors.initials: JW
authors.lastname: Posakony
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D029721:Drosophila Proteins / Q000235:genetics* / Q000378:metabolism; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D018507:Gene Expression Regulation, Developmental; D007150:Immunohistochemistry; D018613:Microscopy, Confocal; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D058953:Neural Stem Cells / Q000378:metabolism*; D055495:Neurogenesis / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D012679:Sense Organs / Q000254:growth & development / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D029721:Drosophila Proteins; D009687:Nuclear Proteins; D014157:Transcription Factors; D044767:Ubiquitin-Protein Ligases; C092070:phyl protein, Drosophila; C413162:sens protein, Drosophila; C071006:neur protein, Drosophila
keywords: bHLH repressors; cis-regulatory code; dual enhancer modules; neural precursors; proneural proteins