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Two adjacent cis-regulatory elements are required for ecdysone response of ecdysone receptor (EcR) B1 transcription.

ABSTRACT: Three distinct classes of nuclear receptors, EcR, E75, and HR3, are key regulators in the ecdysone-inducible gene activation cascade in insects. The transcription of these genes is induced by ecdysone (20E) differently, although the detailed mechanisms underlying their responses to 20E are largely unknown. We identified ecdysone response elements (EcREs) present in the promoters of genes coding BmEcR-B1, BmE75-A, and BHR3-B isoforms from Bombyx mori employing luciferase reporter assays in an ecdysteroid-responsive cultured cell line, NIAS-Bm-aff3 (aff3). The EcRE of BmEcR-B1 at -2800 comprises of two adjacent elements separated by 5 bp, E1 (15 bp) and E2 (21 bp), both of which are required for the 20E response. Further analysis using electrophoretic mobility shift assays showed that E1 binds to the EcR/USP heterodimer and that E2 may bind to the E-box (CACGTG) binding factor such as bHLH protein. The unique E1+E2-type EcRE is also detected in the promoter upstream regions of EcR-B1 from seven lepidopteran species studied. In contrast, both a 20 bp EcRE identified in the promoter of BmE75-A and a 18 bp EcRE identified in the BHR3-B promoter, contained only E1-type EcR/USP binding element but the E2 type element was not in the promoter regions of these genes. The combination of presence of the E2 element or other cis-regulatory elements in promoter regions explains the different 20E response of each class of nuclear receptor genes. Furthermore, the E1+E2 structure for EcR-B1 can be involved in a possible cross-talk between ecdysteroid and other regulatory pathways.

SUBMITTER: Shirai H

PROVIDER: S-EPMC3498158 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Two adjacent cis-regulatory elements are required for ecdysone response of ecdysone receptor (EcR) B1 transcription.

Shirai Hiroyuki H Kamimura Manabu M Yamaguchi Junichi J Imanishi Shigeo S Kojima Tetsuya T Fujiwara Haruhiko H

PloS one 20121114 11

Three distinct classes of nuclear receptors, EcR, E75, and HR3, are key regulators in the ecdysone-inducible gene activation cascade in insects. The transcription of these genes is induced by ecdysone (20E) differently, although the detailed mechanisms underlying their responses to 20E are largely unknown. We identified ecdysone response elements (EcREs) present in the promoters of genes coding BmEcR-B1, BmE75-A, and BHR3-B isoforms from Bombyx mori employing luciferase reporter assays in an ecd ...[more]

PMID: 23166644

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Project description: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.

Dataset Information

Two adjacent cis-regulatory elements are required for ecdysone response of ecdysone receptor (EcR) B1 transcription.

Publications

Two adjacent cis-regulatory elements are required for ecdysone response of ecdysone receptor (EcR) B1 transcription.

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