Activation of a Delayed-Early Gene Encoding MHR3 by the Ecdysone Receptor Heterodimer EcR-B1–USP-1 but Not by EcR-B1–USP-2

• DOI: 10.1128/mcb.19.7.4897
• Publication Date: 2015-10-26T10:19:28Z
• Authors: Que Lan; Kiyoshi Hiruma; Xiao Hu; Marek Jindra; Lynn M. Riddiford
• Keywords: Ecdysone receptor; 20-Hydroxyecdysone; Orphan receptor; Manduca; Chloramphenicol acetyltransferase; Retinoid X receptor

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