Reporter gene assays for screening and identification of novel molting hormone- and juvenile hormone-like chemicals

Abstract

A reporter gene assay (RGA) is used to investigate the activity of synthetic chemicals mimicking the molting hormones (MHs) and juvenile hormones (JHs) of insects, so-called insect growth regulators (IGRs). The MH receptor, a heterodimer of the ecdysone receptor (EcR) and ultraspiracle (USP), and the JH receptor Methoprene-tolerant (Met) are ligand-dependent transcription factors. Ligand-bound EcR-USP and Met bind to specific cis-acting DNA elements, referred to as the ecdysone-responsive element (EcRE) and the JH-responsive element (JHRE), respectively, in order to transactivate target genes. Insect hormone-induced transactivation systems have been reconstituted by the introduction of reporter genes under the control of EcRE and JHRE, or two-hybrid reporter genes, into insect, mammalian, and yeast cells expressing receptor proteins. RGA is easy to use and convenient for examining the MH- and JH-like activities of synthetic chemicals and is suitable for the high-throughput screening of novel structural classes of chemicals targeting EcR-USP and Met.

Keywords: agonist; ecdysone receptor; insect growth regulator; juvenile hormone receptor; reporter gene assay.

Fig. 1. MH-receptor EcR. (a) Domain structure of EcR. NR superfamily proteins including EcR are comprised of six domains (A–F) with conserved functions and sequences. AF: activation function; DBD: DNA-binding domain; H: hinge domain; LBD: ligand-binding domain.¹³⁶⁾ (b) Nucleotide sequence of D. melanogaster hsp27 EcRE.⁴³⁾ The arrows indicate half-site sequences forming a putative palindromic structure. (c) Patterns of typical REs for vertebrate NRs. Half sites with a canonical sequence (AGGTCA) are aligned in three different orientations: inverted repeat (IR), direct repeat (DR), and everted repeat (ER). Two half sites are zero to several nucleotides apart.^137,138) (d) Schematic representation of the molecular mechanism underlying the MH-dependent transactivation of target genes by EcR-USP in D. melanogaster. See the text for details.

Fig. 2. RGA for EcR-USP. (a) Schematic summary of RGA for EcR-USP. RGA using a reporter gene under the control of EcRE, in which multiple copies of EcRE are integrated upstream of the promoter (EcRE×n). Ligand-bound EcR and USP form a heterodimer on EcRE. The interaction of transcriptional coactivator SRC enhances reporter gene expression by recruiting RNA pol II general transcription factors of host cells. (b) Two-hybrid-based RGA for EcR-USP. EcR and USP (LBD or full length) fused to Gal4-DBD and transcriptional activation domain (AD) of Gal4 or VP16, respectively (and vice versa), interact in an MH-dependent manner and induce reporter gene expression in host cells.

Fig. 3. Principle of RGA in yeast strains expressing insect ecdysone receptors EcR and USP. In response to ligands, EcR and USP expressed in yeast cells form a heterodimer and bind to response elements upstream of the minimal promoter of the iso-cytochrome c (CYC) gene. D. melanogaster Tai, a steroid receptor coactivator (SRC), cooperates with EcR-USP to enhance the expression of the lacZ reporter gene by recruiting the RNA pol II general transcription factors of yeast. The expression of β-galactosidase may be visualized and quantified by the development of a yellow color due to the accumulation of ONP in the assay buffer. ONPG: o-nitrophenyl-β-D-galactopyranoside; ONP: o-nitrophenol; RNA pol II: RNA polymerase II; P_CYC: minimal promoter of the CYC gene.⁸⁷⁾

Fig. 4. JH-receptor Met. (a) Domain structures of Met and Gce, members of the bHLH-PAS domain protein family. Met and Gce are comprised of three functional domains—bHLH: a basic helix-loop-helix domain involved in DNA binding; PAS-A: a domain for transcriptional activation; PAS-B: a domain for ligand binding.^26,116) (b) Schematic representation of the JH-dependent transactivation of a target gene by Met/Gce in D. melanogaster. See the text for details. (c) Conservation of the JHRE motif including the E-box in the regulatory regions of the Kr-h1 gene of various insects.^–107)

Fig. 5. RGA for Met. (a) Schematic summary of RGA for Met. RGA using a reporter gene under the control of JHRE, in which multiple copies of JHRE are integrated upstream of the promoter (JHRE×n). Ligand-bound Met and SRC form a heterodimer on JHRE, recruiting RNA pol II general transcription factors of host cells for the induction of the reporter gene. (b) Two-hybrid-based RGA for Met. Met and SRC (partial or full length) fused to Gal4-DBD and AD, respectively (and vice versa), interact in a JH-dependent manner and induce reporter gene expression in host cells.