C. Elke et al., DNA-binding properties of the ecdysteroid receptor-complex (EcR/USP) of the epithelial cell line from Chironomus tentans, ARCH INS B, 46(1-2), 2001, pp. 1-10
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>DR3/415/PAL1>hsp27. This indicates that the complexe
s formed on PALI 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.