Conserved regions of the Drosophila erect wing protein contribute both positively and negatively to transcriptional activity.
Genetic studies of the Drosophila erect wing (ewg) gene have revealed that ewg has an essential function in the embryonic nervous system and is required for the specification of certain muscle cells. We have found that EWG is a site-specific transcriptional activator, and we report here that evolutionarily conserved regions of EWG contribute both positively and negatively to transcriptional activity. Using gel mobility shift assays, we have shown that an EWG dimer binds specifically to DNA. In transfection assays, EWG activated expression of a reporter gene bearing specific binding sites. Analysis of deletion mutants and fusions of EWG to the Gal4 DNA binding domain has identified a transcriptional activation domain in the C terminus of EWG. Deletion analysis also revealed a novel inhibitory region in the N terminus of EWG. Strikingly, both the activation domain and the inhibitory region are conserved in EWG homologs including human nuclear respiratory factor 1 (NRF-1) and the sea urchin P3A2 protein. The strong conservation of elements that determine transcriptional activity suggests that the EWG, NRF-1, and P3A2 family of proteins shares common mechanisms of action and has maintained common functions across evolution.
Department of Pathology, Harvard Medical School, Boston, Massachussetts 02115, USA.
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D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D001665:Binding Sites; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry; D019281:Dimerization; D004330:Drosophila; D029721:Drosophila Proteins; D019143:Evolution, Molecular; D017353:Gene Deletion; D017930:Genes, Reporter; D006801:Humans; D015151:Immunoblotting; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D051265:NF-E2-Related Factor 1; D009479:Neuropeptides / Q000737:chemistry* / Q000502:physiology*; D009687:Nuclear Proteins / Q000737:chemistry* / Q000502:physiology*; D051266:Nuclear Respiratory Factor 1; D051264:Nuclear Respiratory Factors; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012617:Sea Urchins; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators / Q000737:chemistry; D014157:Transcription Factors; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection
D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; C081334:EWG protein, Drosophila; D051265:NF-E2-Related Factor 1; C495633:NRF1 protein, human; D009479:Neuropeptides; D009687:Nuclear Proteins; D051266:Nuclear Respiratory Factor 1; D051264:Nuclear Respiratory Factors; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C072372:P3A2 regulatory protein, Strongylocentrotus purpuratus; D004247:DNA