Functional characterization of ecdysone receptor gene switches in mammalian cells.
Regulated expression of transgene is essential in basic research as well as for many therapeutic applications. The main purpose of the present study is to understand the functioning of the ecdysone receptor (EcR)-based gene switch in mammalian cells and to develop improved versions of EcR gene switches. We utilized EcR mutants to develop new EcR gene switches that showed higher ligand sensitivity and higher magnitude of induction of reporter gene expression in the presence of ligand. We also developed monopartite versions of EcR gene switches with reduced size of the components that are accommodated into viral vectors. Ligand binding assays revealed that EcR alone could not bind to the nonsteroidal ligand, RH-2485. The EcR's heterodimeric partner, ultraspiracle, is required for efficient binding of EcR to the ligand. The essential role of retinoid X receptor (RXR) or its insect homolog, ultraspiracle, in EcR function is shown by RXR knockdown experiments using RNAi. Chromatin immunoprecipitation assays demonstrated that VP16 (activation domain, AD):GAL4(DNA binding domain, DBD):EcR(ligand binding domain, LBD) or GAL4(DBD):EcR(LBD) fusion proteins can bind to GAL4 response elements in the absence of ligand. The VP16(AD) fusion protein of a chimera between human and locust RXR could heterodimerize with GAL4(DBD):EcR(LBD) in the absence of ligand but the VP16(AD) fusion protein of Homo sapiens RXR requires ligand for its heterodimerization with GAL4(DBD):EcR(LBD).
Panguluri
Siva K
SK
Department of Entomology, College of Agriculture, University of Kentucky, Lexington, KY 40546, USA.
Kumar
Prasanna
P
Palli
Subba R
SR
D000818:Animals; D005786:Gene Expression Regulation; D005812:Genes, Switch / Q000235:genetics*; D006801:Humans; D008024:Ligands; D051379:Mice; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D041681:NIH 3T3 Cells; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000502:physiology; D011987:Receptors, Steroid / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D047488:Retinoid X Receptors / Q000235:genetics / Q000502:physiology*; D019076:Transgenes / Q000235:genetics / Q000502:physiology*
D008024:Ligands; D034741:RNA, Small Interfering; D011987:Receptors, Steroid; D011993:Recombinant Fusion Proteins; D047488:Retinoid X Receptors; C055469:ecdysone receptor