DNA‐binding properties of Drosophila ecdysone receptor isoforms and their modification by the heterodimerization partner ultraspiracle

Abstract

Transcriptional activity of ecdysone receptor (EcR) isoforms varies considerably and is modified further by the heterodimerization partner and hormone treatment. To investigate whether differences in DNA binding of receptor complexes are responsible for these variations in transcriptional activity, interaction of Drosophila EcR isoforms, and variants of Ultraspiracle (Usp), the orthologue of RXR, with the ecdysone response elements (EcRE) hsp 27, PAL-1, and DR-1, were determined by electrophoretic mobility shift assays. Receptor proteins were expressed in vertebrate cells (CHO-K1) in order to rule out an influence of endogenous receptor proteins. In the absence of a heterodimerization partner, weak DNA binding of EcR was detected even without hormone with EcR-A and -B1, but not EcR-B2. In the presence of hormone, all three isoforms show increased binding to the hsp 27 EcRE. The heterodimerization partner Usp increased DNA binding considerably. The hormone effect of heterodimers is more pronounced with both EcR-B isoforms compared to EcR-A. Two specific bands were obtained for EcR-A and B1 but only one band is visible with EcR-B2. Deletion of the C-domain of Usp still allows basal DNA binding of the heterodimer, but in contrast to full-length Usp, addition of hormone decreases the intensity of the retarded receptor band of all EcR isoforms and the EcREs hsp27 and DR-1 considerably, whereas interaction with the EcRE PAL-1 is only slightly affected. Synergistic effects on transcriptional activity are associated with the formation of different receptor DNA-complexes observed with 1xhsp27 and 3xhsp27. Comparison of DNA-binding properties of EcR isoforms and EcR/Usp heterodimers revealed that binding of receptor complexes to hsp 27 EcRE is dependent on the AB domain of EcR and the AB-, C-, and D-domains of the heterodimerization partner. Interaction with the hsp 27 EcRE correlates neither with ligand binding nor with transcriptional activity of the various receptor complexes. We, therefore, conclude that the different receptor functions are regulated separately, for example, by interaction with co-modulators or post-transcriptional modifications.

Ecdysteroid hormone action

“…A close contact of LBD and DBD is documented for the vertebrate nuclear receptor PPAR [38]. As mentioned above, ecdysteroids increase DNA-binding [22,25], but in the presence of hormone response elements ligand binding is also enhanced. This is certainly partially due to improved dimerisation, but ligand affinity is additionally altered as mentioned above [36].…”

“…EcR isoforms already interact with DNA to various extents in the absence of ultraspiracle [22]. This interaction is enhanced in the presence of the Usp-DBD [22,25]. The intensity of DNA binding does not parallel with transcriptional activity, an indication that transactivation potency is regulated by interaction with comodulators.…”

“…This is confirmed by EMSA experiments: they revealed weak interaction with DNA, which is reinforced by addition of hormone. EcR isoforms already interact with DNA to various extents in the absence of ultraspiracle [22]. This interaction is enhanced in the presence of the Usp-DBD [22,25].…”

“…enhanced interaction with DNA [22] and chromatin [23] or shuttling of the ecdysteroid receptor into the nucleus [24]. Muristerone A stimulates transcriptional activity in vertebrate cells devoid of endogenous EcR and Usp, which are transfected with dmEcR to different degrees depending on the isoform used [25,26].…”

“…The differences in DNA-binding of EcR isoforms and the corresponding EcR/Usp receptor complexes [22,25] clearly indicate an interaction between the DNAbinding domain and the AB-domains of EcR. From vertebrate nuclear receptors, it is known that the functional 3-D structure of the AB-domain is formed only in contact with the DNA binding domain [61].…”