Tài liệu Báo cáo khoa học: Improved ecdysone receptor-based inducible gene regulation system doc

Improved ecdysone receptor-based inducible gene regulation system

Subba R. Palli1

, Mariana Z. Kapitskaya2

, Mohan B. Kumar2 and Dean E. Cress2

1

Department of Entomology, College of Agriculture, University of Kentucky, KY, USA; 2

RHeoGene LLC, Spring House, PA, USA

To develop an ecdysone receptor (EcR)-based inducible

gene regulation system, several constructs were prepared by

fusing DEF domains of Choristoneura fumiferana EcR

(CfEcR), C. fumiferana ultraspiracle (CfUSP), Mus muscu￾lus retinoid X receptor (MmRXR) to either GAL4 DNA

binding domain (DBD) or VP16 activation domain. These

constructs were tested in mammalian cells to evaluate their

ability to transactivate luciferase gene placed under the

control of GAL4 response elements and synthetic TATAA

promoter. A two-hybrid format switch, where GAL4 DBD

was fused to CfEcR (DEF) and VP16 AD was fused to

MmRXR (EF) was found to be the best combination. It had

the lowest background levels of reporter gene activity in the

absence of a ligand and the highest level of reporter gene

activity in the presence of a ligand. Both induction and turn￾off responses were fast. A 16-fold induction was observed

within 3 h of ligand addition and increased to 8942-fold by

48 h after the addition of ligand. Withdrawal of the ligand

resulted in 50% and 80% reduction in reporter gene activity

by 12 h and 24 h, respectively.

Keywords: gene switch; ponasterone A; receptors; EcR;

RXR.

Twenty hydroxyecdysone (20E) is a steroid hormone that

regulates molting, metamorphosis, reproduction and vari￾ous other developmental processes in insects. Ecdysone

functions through a heterodimeric receptor complex com￾prised of ecdysone receptor (EcR) and ultraspiracle (USP).

Both EcR and USP cDNAs have been cloned from

Drosophila melanogaster and several other insects [1] and

were shown to be members of the steroid hormone receptor

superfamily. Members of this superfamily are characterized

by the presence of five modular domains, A/B (transacti￾vation), C(DNA binding/heterodimerization), D (hinge,

heterodimerization), E (ligand binding, heterodimerization,

transactivation) and F (transactivation). Crystallographic

studies on the E domain structures of several nuclear

receptors showed a conserved fold composed of 11 helices

(H1 and H3–H12) and two short strands (s1 and s2) [2].

Recently, the crystal structure of USP was solved by two

groups [3,4], both structures showed a long H1-H3 loop and

an insert between H5 and H6. These structures appear to

lock USP in an inactive conformation by displacing helix 12

from agonist conformation. In both crystal structures USP

had a large hydrophobic cavity, which contained phos￾pholipid ligands. The crystal structure of the EcR has yet

to be determined; however, homology models for CtEcR

(Chironomus tentans EcR) [5], and CfEcR (Choristoneura

fumiferana EcR) [6] have been generated [7,8].

Ecdysone receptors are found in insects and other related

invertebrates [1]. Ecdysteroids and related compounds have

been identified in plants, insects and other related inverte￾brates. EcR and its ligands are not detected in vertebrates

such as humans, therefore they are very good candidates for

developing gene regulation systems for use in vertebrates.

Insect EcR can heterodimerize with retinoid X receptor

(RXR) and transactivate genes that are placed under the

control of ecdysone response elements (EcRE) in various

cellular backgrounds including mammalian cells. The EcR￾based gene switch is being developed for use in various

applications including gene therapy, expression of toxic

proteins in cell lines as well as for cell-based drug discovery

assays [9–17].

After initial reports [18,19] on the function of EcR as an

ecdysteroid dependent transcription factor in cultured

mammalian cells, No et al. [20] used D. melanogaster EcR

(DmEcR) and human RXRa to develop an ecdysone

inducible gene expression system that can function in

mammalian cells and mice. Later, Suhr et al. [21] showed

that the nonsteroidal ecdysone agonist, tebufenozide,

induced high level of transactivation of reporter genes in

mammalian cells through Bombyx mori EcR (BmEcR) [22]

and endogenous RXR. Hoppe et al. [23] combined DmEcR

and BmEcR systems and created a chimeric Drosophila/

Bombyx EcR (DBEcR) that had combined positive aspects

of both systems, i.e. the chimeric receptor bound to

modified ecdysone response elements and functioned

without exogenous RXR. Recent improvements to the

EcR-based gene switch include expression of both EcR

and RXR in a bicistronic vector [24] and the discovery that

the RXR ligands enhance the ligand dependent activity of

the EcR-based gene switch [25].

An optimal gene regulation system should have the

following characteristics: (a) low or no basal expression in

the absence of an inducer (b) high induced expression in the

presence of a wide range of inducer concentration (c) rapid

Correspondence to S. R. Palli, Department of Entomology, College

of Agriculture, University of Kentucky, Lexington KY 40546.

Fax:+ 1 859 3231120, Tel.:+ 1 859 2574962,

E-mail: [email protected]

Abbreviations: 20E, twenty hydroxyecdysone; EcR, ecdysone receptor;

LBD, ligand binding domain; RXR, retinoid X receptor;

USP, ultrapiracle.

(Received 11 December 2002, revised 21 January 2003,

accepted 5 February 2003)

Eur. J. Biochem. 270, 1308–1315 (2003)
FEBS 2003 doi:10.1046/j.1432-1033.2003.03501.x