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 ex tract of the epithelial cell line of Chironomus tentans by means of electro phoretic 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 the se conditions, EcR/USP form a unique complex on inverted repeat elements (P AL1 and hsp27-EcRE), while on direct repeat elements (DR1-5), a second comp lex with higher mobility is formed. In this second complex, neither EcR nor USP are present. Thus, an additional difference between PAL1 and DR-elemen ts is the competition of other factors for DR-elements, modulating its func tion as an EcRE. A competition EMSA, using PAL1 as radiolabeled probe, reve als the following order of binding strength: PAL1>DR4/5>DR1>DR2/3/hsp27. Su rprisingly, using DR1 as radiolabeled probe, shows a different order of bin ding strength: DR1>DR2>DR3/4/5/PAL1>hsp27. This indicates that the complexe s 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 comple x may be altered under various conditions or by interaction with cofactors. Upon hormone administration, DNA binding of the receptor complex is enhance d, but the difference to hormone-free binding reactions decreases in course of time, indicating an additional hormone independent activation. (C) 1999 Wiley-Liss, Inc.