DNA-binding properties of the ecdysteroid receptor-complex (EcR/USP) of the epithelial cell line from Chironomus tentans.
DNA-binding features of EcR and USP were investigated using a 0.4 M NaCl extract of the epithelial cell line of Chironomus tentans by means of electrophoretic mobility shift assays (EMSAs). It is shown that the DNA-binding is enhanced by hormone administration and that in the hormone dependent shift, both EcR and USP, are present. Furthermore, we demonstrate that under these conditions, EcR/USP form a unique complex on inverted repeat elements (PAL1 and hsp27-EcRE), while on direct repeat elements (DR1-5), a second complex with higher mobility is formed. In this second complex, neither EcR nor USP are present. Thus, an additional difference between PAL1 and DR-elements is the competition of other factors for DR-elements, modulating its function as an EcRE. A competition EMSA, using PAL1 as radiolabeled probe, reveals the following order of binding strength: PAL1>DR4/5>DR1>DR2/3/hsp27. Surprisingly, using DR1 as radiolabeled probe, shows a different order of binding strength: DR1>DR2>DR3/4/5/PAL1>hsp27. This indicates that the complexes formed on PAL1 are not identical to the ones formed on DR1 and that both are not easily convertible. Furthermore, the affinity of the EcR/USP complex may be altered under various conditions or by interaction with cofactors. Upon hormone administration, DNA binding of the receptor complex is enhanced, but the difference to hormone-free binding reactions decreases in course of time, indicating an additional hormone independent activation. Arch.
Elke
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Abteilung Allgemeine Zoologie und Endokrinologie, Universität Ulm, Ulm, Germany. carsten.elke@biologie.uni-ulm.de
Rauch
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Spindler-Barth
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Spindler
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D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002683:Chironomidae / Q000166:cytology; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004847:Epithelial Cells; D008969:Molecular Sequence Data; D011485:Protein Binding; D011987:Receptors, Steroid / Q000378:metabolism*
D004268:DNA-Binding Proteins; D011987:Receptors, Steroid; C034808:ecdysteroid receptor; D004247:DNA