Activation of a delayed-early gene encoding MHR3 by the ecdysone receptor heterodimer EcR-B1-USP-1 but not by EcR-B1-USP-2

Abstract

MHR3, a homolog of the retinoid orphan receptor (ROR), is a transcription factor in the nuclear hormone receptor family that is induced by 20-hydroxyecdysone (20E) in the epidermis of the tobacco hornworm, Manduca sexta. Its 2.7-kb 5' flanking region was found to contain four putative ecdysone receptor response elements (EcREs) and a monomeric (GGGTCA) nuclear receptor binding site. Activation of this promoter fused to a chloramphenicol acetyltransferase (CAT) reporter by 2 micrograms of 20E per ml in Manduca GV1 cells was similar to that of endogenous MHR3, with detectable CAT by 3 h. When the ecdysone receptor B1 (EcR-B1) and Ultraspiracle 1 (USP-1) were expressed at high levels under the control of a constitutive promoter, CAT levels after a 3-h exposure to 20E increased two- to sixfold. In contrast, high expression of EcR-B1 and USP-2 caused little increase in CAT levels in response to 20E. Moreover, expression of USP-2 prevented activation by EcR-B1-USP-1. Deletion experiments showed that the upstream region, including the three most proximal putative EcREs, was responsible for most of the 20E activation, with the EcRE3 at -671 and the adjacent GGGTCA being most critical. The EcRE1 at -342 was necessary but not sufficient for the activational response but was the only one of the three putative EcREs to bind the EcR-B1-USP-1 complex in gel mobility shift assays and was responsible for the silencing action of EcR-B1-USP-1 in the absence of hormone. EcRE2 and EcRE3 each specifically bound other protein(s) in the cell extract, but not EcR and USP, and so are not EcREs in this cellular context. When cell extracts were used, the EcR-B1-USP-2 heterodimer showed no binding to EcRE1, and the presence of excess USP-2 prevented the binding of EcR-B1-USP-1 to this element. In contrast, in vitro-transcribed-translated USP-1 and USP-2 both formed heterodimeric complexes with EcR-B1 that bound ponasterone A with the same Kd (7 x 10(-10) M) and bound to both EcRE1 and heat shock protein 27 EcRE. Thus, factors present in the cell extract appear to modulate the differential actions of the two USP isoforms.

FIG. 1

Binding of in vitro-transcribed-translated EcR-B1 and USP-1 or USP-2 to ponasterone A. The nonspecific binding was determined by addition of 10 μM muristerone A and subtracted from the total binding, and then the percentage of maximal binding was obtained as described in Materials and Methods. K_d was calculated from all of the data shown.

FIG. 2

Expression of EcR-B1 and USPs in GV1 cells. Immunoblotting of cell extracts with EcR-B1-specific and USP-common antibodies. The leftmost lane in each blot contains extract from nontransfected cells. The right lanes in each blot contain extracts transfected with the pIE1^hr expression vectors for EcR-B1 or USP-1 or USP-2 as designated. Fifteen micrograms of soluble protein was applied.

FIG. 3

The induction of MHR3 mRNA in GV1 cells in 6 h by 2 μg of 20E per ml in the presence of excess EcR-B1, USP-1, USP-2, or various combinations thereof under the control of the pIE1^hr constitutive promoter. The empty pIE1^hr was transfected as a control. Cells were cultured in the presence and absence of 20E hormone. Relative abundance refers to MHR3 RNA induced by 20E in 6 h in cells which contained the empty pIE1^hr expression vector, designated as 10. Means ± standard deviations (error bars) are shown (n = 3 to 5).

FIG. 4

(Top) Endonuclease restriction map of a cloned genomic DNA fragment of the MHR3 gene. B, BamHI; E, EcoRI; S, SalI; and X, XbaI. The transcription start site (+1) and the DNA (capital letters) and amino acid (titlecase) sequences bordering the first intron are indicated. Sequence data for the MHR3 upstream region are deposited in GenBank (AF086951). (Bottom) S1 nuclease protection assay for MHR3 mRNA. The antisense labeled probe is as indicated in the map. Lanes: 1, yeast RNA; 2, total RNA from GV1 cells treated with 2 μg of 20E per ml for 6 h; 3, total RNA from day 2 4th instar larval epidermis treated with 2 μg of 20E per ml for 21 h; 4, total RNA from GV1 cells. A DNA sequence reaction (lanes G, T, A, and C) was used to indicate the precise sizes of the S1 nuclease-protected fragments.

FIG. 5

Transient transfection assays of MHR3 promoter activity as determined by the increase in CAT protein after exposure to 2 μg of 20E per ml for 1 (), 3 (▧), or 6 (■) h over the uninduced level at 0 h (set at 1). The deletion constructs tested are shown in the map above. Stars, putative EcRE; open circle, GGGTCA sequence. Means ± standard deviations (error bars) are shown (n = 3).

FIG. 6

(A) Effects of increased levels of EcR-B1 and either USP-1 or USP-2 on 20E induction of MHR3 promoter activity. The pIE1^hr expression vectors for EcR-B1 and USP-1 or USP-2 were cotransfected with the MHR3 promoter. Solid bars, EcR-B1–USP-1; hatched bars, EcR-B1–USP-2. (B) Reduction of 20E-induced promoter activity by the deletion of EcRE1. EcRE1 was deleted from the −1,216-bp promoter, and then either this or the parent construct was cotransfected with the pIE1^hr expression vectors for EcR-B1 and USP-1. Transfection assays for MHR3 promoter activity were done 48 h after the cotransfection and 3 h after exposure to 2 μg of 20E per ml as described in Materials and Methods. Constructs are shown in the maps above the figures. Stars, putative EcRE; open circle, GGGTCA sequence (MRE). Means ± standard deviations (error bars) are shown (n = 3). Fold increased CAT was determined as in Fig. 5.

FIG. 7

Concentration-response curves for the −2,571-bp MHR3 promoter construct cotransfected with pIE1^hr–EcR-B1 and pIE1^hr–USP-1. Forty-five hours later, cells were treated with the given concentration of 20E for 6 h, and then the CAT level was assessed as in Fig. 5. Means ± standard deviations (error bars) are shown (n = 3).

FIG. 8

GMSAs using the EcRE1 (A), EcRE2 (B), and EcRE3 (C) oligonucleotides and lysate (D) (see Materials and Methods). Cotransfections were as follows: Extract P, empty pIE1^hr/PA; Extract B1+U1, pIE1^hr–EcR-B1 and pIE1^hr–USP-1; Extract B1+U2, pIE1^hr-EcR-B1 and pIE1^hr-USP-2. Five micrograms of the extracts was used. The antibodies used are indicated above the gels.

FIG. 9

Competition GMSAs using 100× excess probe DNA used in Fig. 8 (EcRE1 [A], EcRE2 [B], and EcRE3 [C]) and the combination of the extracts, which show that all of the major shifted bands are probe specific. As a nonspecific competitor, 100× MRE DNA (see Materials and Methods) was used. Five micrograms of the extracts was used. See the legend to Fig. 8 for definitions of the abbreviations.