Abstract
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.
Highlights
EWG is a member of a family of related proteins that includes the human nuclear respiratory factor 1 (NRF-1), chicken IBR/F, zebrafish Nrf, and sea urchin P3A2 proteins [2]
EWG Is a Sequence-specific DNA-binding Protein—EWG is highly related to human NRF-1, chicken IBR/F, and sea urchin P3A2 proteins in regions that correspond to the nuclear localization, DNA binding, and dimerization domains identified in these homologs [7, 9, 17, 18]
Using a gel mobility shift assay, we observed full-length EWG bound to a 24-base pair oligonucleotide containing a palindromic NRF-1 consensus site (Fig. 2A) [8, 9]
Summary
EWG is a member of a family of related proteins that includes the human NRF-1 ( called ␣-Pal), chicken IBR/F, zebrafish Nrf, and sea urchin P3A2 proteins [2]. These proteins share regions of significant homology, diverse activities have been reported for EWG homologs in different species. In this study we show that EWG functions as a transcriptional activator and that evolutionarily conserved regions of EWG contribute both positively and negatively to transcriptional regulation. Our studies suggest that EWG participates in neurogenesis and indirect flight muscle development by directly binding to and regulating the expression of target genes important for differentiation or maintenance of these tissues. The finding that evolutionarily conserved regions of EWG contribute to transcriptional regulation suggests that EWG and its homologs in other species have maintained common mechanisms of action and regulation across evolution
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