The BmE75 Nuclear Receptors Function as Dominant Repressors of the Nuclear Receptor BmHR3A

Abstract

The orphan nuclear receptors BmE75 and BmHR3 are induced by 20-hydroxyecdysone in the ovary of the silk moth Bombyx mori at the beginning of pupation and show stage-specific expression in ovarian follicles during pharate adult development. To analyze the function of these receptors, we have developed a transactivation assay based on the transcriptional stimulation of a retinoic acid receptor-related receptor response element (RORE)-linked promoter-reporter construct. Co-transfection of a Bombyx cell line with a BmHR3A expression construct results in constitutive activation of the reporter, whereas expression of BmE75 has no measurable effects on reporter expression. However, when the BmE75 receptors are co-introduced with BmHR3A into the cells, the BmHR3A-mediated transactivation is repressed. Repression of BmHR3A by BmE75 occurs by two distinct mechanisms. Increasing doses of BmE75 efficiently displace BmHR3A bound to the RORE target site in gel retardation assays, indicating that both receptors compete for common DNA target sites. However, analysis of the function of deletion mutants of BmE75 in the transactivation assay indicates that repression can also occur in the absence of the DNA-binding domain and that the C-terminal F domain is sufficient for repression. In gel retardation assays, the two receptor types form a ternary complex on a single RORE, suggesting that repression is also mediated by protein interactions on the DNA target site. Yeast two-hybrid assays show that BmHR3A interacts with BmE75 and that this interaction is dependent on the C terminus of BmHR3A and the F domain of BmE75. Because the C terminus of BmHR3A contains a strong activation domain, we predict that BmE75 blocks activation by BmHR3A through competition for co-activator binding sites located at the C terminus of BmHR3A. Our data also indicate that the transcriptional activities of BmHR3A and BmE75 are integrated in such a way that activation of RORE-linked target genes depends on the relative expression levels of the two receptor types.The orphan nuclear receptors E75 and HR3 were originally isolated because of their ubiquitous activation as early-response genes by 20-hydroxyecdysone (20E) 1 in target tissues of insects (1). The mRNA of E75 is typically induced within 30 min of hormone treatment, and the induction also occurs in the presence of protein synthesis inhibitors, suggesting a direct control of E75 gene transcription by the hormone-bound ecdysone receptor complex (2-4). The accumulation of HR3 mRNA, on the other hand, occurs more slowly (starting 2-3 h after hormone exposure) and requires synthesis of additional factors for maximal expression (5-7). Ecdysone-induced E75 and HR3 proteins are subsequently thought to function as transcription factors and play essential roles in the implementation of the 20E-induced gene expression cascade (8).The ovary of the silk moth Bombyx mori is a target of 20E during early pupation (9, 10). Administration of 20E to developmentally arrested abdomens causes abrupt induction of BmE75 a...

Vitellogenesis and Post-Vitellogenic Maturation of the Insect Ovarian Follicle

“…Because BmGATAb and BmHR3A show reciprocal expression patterns during follicle development, it has been suggested that BmHR3A could act as a repressor of BmGATAb expression (Eystathioy et al, 2001). Although BmHR3A is known to act as a constitutive activator, as was mentioned earlier, its activation function can be repressed by the co-expression of BmE75A (Swevers et al, 2002b). Thus, a possible mechanism for the silencing of the BmGATAb gene during vitellogenesis may involve the recruitment of BmHR3A/BmE75A complexes to the RORE sites in the BmGATAb promoter.…”

“…The subsequent (ecdysteroid-independent) increase in the expression of BmE75C mRNA (and protein) at stages À12 to þ12 is consistent with a function as a regulator of the transition from vitellogenesis to choriogenesis. Because BmE75C acts as a transcriptional repressor in tissue culture cells (Figure 15; Swevers et al, 2002b), its function in follicular cells may be related to the shut-down of the vitellogenic program rather than the onset of choriogenesis. BmSH3, a member of a family of conserved adaptor proteins characterized by three SH3 domains at the C-terminus and a lipid raft-targeting ''SoHo'' domain located at the N-terminus, was isolated in a yeast two-hybrid screen using as bait the prolinerich N-terminus of BmE75C (K. Ito, L. Swevers, and K. Iatrou, unpublished data).…”

“…Moreover, in transient expression experiments employing Bombyx-derived tissue culture cells, BmHR3A and BmE75A function as activators and repressors, respectively, of reporter genes that harbor retinoic acid receptor-related orphan receptor elements (ROREs; binding elements for the HR3 and E75 orphan nuclear receptors) in their upstream regions (Figure 15; Swevers et al, 2002b). Because of the antagonistic activities displayed by BmHR3A and BmE75A, it was proposed that activation of RORE-dependent target genes depends on the relative expression levels of BmHR3A and BmE75A.…”

The molecular mechanisms of cuticular melanization: The ecdysone cascade leading to dopa decarboxylase expression in Manduca sexta

“…3). In B. mori, E75 is known to heterodimerize with BHR3 through an interaction between the F domain of E75 and the Activation Factor 2 (AF2) domain of BHR3 (Swevers et al, 2002). Such an interaction might prevent HR3 binding to the MRE and/or its activity.…”

Cell-Based Screening Systems for Insecticides

“…Basal reporter cassettes are engineered with identi fi ed binding sites for the nuclear receptor (for native receptors) or for the Gal4 activator (for fusions with Gal4 DNA-binding domain). An example is the reporter system for the Bombyx HR3 and E75 receptors (Swevers et al 2002 ) .…”