Differential responsiveness of BmBR-C promoters to ecdysone signals

Abstract

The Bombyx mori homolog of the Broad-Complex gene (BmBR-C) is transcribed from two different promoters separated by 101kbp. In our previous report where we used semi-quantitative RT-PCR to amplify the total RNA prepared from tissues at different stages, the distal (Pdist) and the proximal (Pprox) promoters appeared to respond differentially to ecdysone signals. To explore the expression control mechanism of BmBR-C in response to ecdysone, transfection assays using Bombyx BM-N cells were performed with luciferase reporter genes under the control of various Pdist or Pprox fragments. In these experiments, the promoter regions located between 3 and 5kbp upstream from the proximal transcription start site (TSS), and between 2 and 5kbp upstream from the distal TSS, appeared to function in the down-regulation of Pprox and the up-regulation of Pdist, respectively, in response to 20-hydroxyecdysone (20E). In electrophoretic mobility shift assays (EMSAs), proteins bound to the probe containing the candidate ecdysone-responsive element (cEcRE), located 3 kbp upstream from the distal TSS, were detected predominantly in the nuclear extract prepared from 20E-treated cells; however oligonucleotides containing the typical EcRE (that of the hsp27 or fbp1 genes of Drosophila) could not compete with probe for binding to these proteins. Furthermore, the level of responsiveness of Pdist to 20E declined, but did not disappear, after deletion of the cEcRE. These results suggest that the functional ecdysone receptor (EcR) may bind to elements other than the cEcRE, or that transcription from Pdist is activated by an ecdysone-responsive factor, such as βFTZ-F1, rather than a functional EcR.