Differential control of gene activity by isoforms A, B1 and B2 of the Drosophila ecdysone receptor

Abstract

The steroid hormone ecdysone initiates molting and metamorphosis in Drosophila via a heterodimeric receptor consisting of EcR that binds hormone, and USP, a homolog of the vertebrate RXR receptor. EcR exists in three isoforms EcRA, EcRB1 and EcRB2 that are thought to direct specific physiological responses to ecdysone. These three isoforms differ only in their N-terminal A/B domain that implies that sequences responsible for the differential physiological effects lie within the A/B domains of the EcR isoforms. In the present study, we set out to determine the capability of the three isoforms and their A/B domains to control gene transcription. When full-length EcR plasmids were cotransfected into mammalian cells with a USP expressing and a cognate reporter plasmid, the three EcR isoforms showed striking differences in their ability to control gene transcription, both in the presence and in the absence of hormone. Furthermore, the A/B domains of EcRB1 and of EcRB2 when fused to the GAL4 DNA binding domain are sufficient to activate transcription of a reporter gene, in yeast as well as in mammalian cells. In contrast, a fusion construct containing the A/B domain of EcRA represses basal transcription of the reporter gene. All these findings emphasize the importance of the A/B domains of the three EcR isoforms for differentially controlling gene transcription. Furthermore, they provide evidence for the existence of an autonomous ligand-independent activation function (AF1) in the A/B domains of EcRB1 and EcRB2 and of an inhibitory function (IF) in the A/B domain of EcRA.

Ecdysone-mediated programmed cell death in Drosophila

“…has been difficult to study due to the lack of a specific antibody and mutant. In terms of regulating transcription, EcR-A has an inhibitory function whereas EcR-B1 and EcR-B2 have activation functions (Mouillet et al, 2001).…”

Fine-tuning of secondary arbor development: the effects of the ecdysone receptor on the adult neuronal lineages of theDrosophilathoracic CNS

“…Ecdysone responses are also modulated by the specific blending of the three different EcR isoforms expressed (Talbot et al, 1993;Truman et al, 1994). The N-terminal regions of EcR-B1 and EcR-B2 have a strong ligandindependent activational function Mouillet et al, 2001), and these isoforms promote pruning in remodeling neurons (Lee et al, 2000;Schubiger et al, 1998). EcR-A has an inhibitory domain at its N terminus (Mouillet et al, 2001), and is the most prevalent isoform in maturing neurons.…”

“…The N-terminal regions of EcR-B1 and EcR-B2 have a strong ligandindependent activational function Mouillet et al, 2001), and these isoforms promote pruning in remodeling neurons (Lee et al, 2000;Schubiger et al, 1998). EcR-A has an inhibitory domain at its N terminus (Mouillet et al, 2001), and is the most prevalent isoform in maturing neurons. As a neuron progresses through specific developmental phases, the levels of the different EcR isoforms within the neuron fluctuate (Truman et al, 1994), thereby influencing the response of the neuron to ecdysone during metamorphosis (H. L. D. Brown, PhD thesis, University of Washington, 2007).…”

EcR isoforms inDrosophila: testing tissue-specific requirements by targeted blockade and rescue

“…In Drosophila, there are three EcR isoforms that differ only in their N-terminal regions, the three isoforms being derived from a single structural gene by both alternative promoter usage and alternative splicing . The potencies of the three isoforms have been tested in an EcR-deficient Drosophila cell line (X. H., L. C. and P. C., unpublished), in yeast (Dela Cruz et al, 2000) and in mammalian cells (Mouillet et al, 2001). These studies confirm that the individual isoforms (and their isolated N-terminal regions tested as fusions) differ markedly in their abilities to activate particular test promoters, and that each A/B region contains an AF1 that can activate transcription in an appropriate experimental setting.…”