Ecdysone response elements in the distal promoter of the Bombyx Broad‐Complex gene, BmBR ‐ C

The B ombyx mori silkworm's homologue of the Broad‐Complex gene ( BmBR ‐ C ) is transcribed from two promoters: a distal promoter ( P dist) and a proximal promoter ( P prox). As determined by a luciferase assay, the transcriptional activity of P dist, but not P prox, was activated by ecdysone. Further analyses using reporters driven by sequential deletion P dist mutants indicated that two regions, ecdysone responsive element ( EcRE) ‐ D and EcRE ‐ P , −4950 bp and −3480 bp upstream from the distal transcription start site, respectively, were important in the responsiveness of P dist to 20‐hydroxyecdysone (20 E ); however, no significant sequence similarities were found between the canonical EcRE and the EcRE ‐ D or EcRE ‐ P regions. Electrophoretic mobility shift assays showed that both the EcRE ‐ D and ‐ P sequences specifically bound to Bombyx protein(s). Sequence analyses and competition assays suggested that the protein(s) bound to EcRE ‐ P might include components other than the ecdysone receptor ( EcR ), suggesting that BmBR ‐ C transcription was indirectly activated by ecdysone through the EcRE ‐ P . Remarkably, protein binding to the mid‐region of the EcRE ‐ D , EcRE ‐ D b, was competitively inhibited by an oligonucleotide containing the D rosophila hsp 27 EcRE sequence. Furthermore, an anti‐ EcR antibody interfered with the formation of the protein‐ EcRE ‐ D b complex. These results indicated that a functional Bombyx ecdysone receptor binds to EcRE ‐ D and activates the expression of BmBR ‐ C .