Improved Dual-Luciferase Reporter Assays for Nuclear Receptors

Abstract

Nuclear receptors play important roles in many cellular functions through control of gene transcription. It is also a large target class for drug discovery. Luciferase reporter assays are frequently used to study nuclear receptor function because of their wide dynamic range, low endogenous activity, and ease of use. Recent improvements of luciferase genes and vectors have further enhanced their utilities. Here we applied these improvements to two reporter formats for studying nuclear receptors. The first assay contains a Murine Mammary Tumor Virus promoter upstream of a destabilized luciferase. The presence of response elements for nuclear hormone receptor in this promoter allows the studies of endogenous and/or exogenous full length receptors. The second assay contains a ligand binding domain (LBD) of a nuclear receptor fused to the GAL4 DNA binding domain (DBD) on one vector and multiple Gal4 Upstream Activator Sequences (UAS) upstream of luciferase reporter on another vector. We showed that codon optimization of luciferase reporter genes increased expression levels in conjunction with the incorporation of protein destabilizing sequences into luciferase led to a larger assay dynamic range in both formats. The optimum number of UAS to generate the best response was determined. The expression vector for nuclear receptor LBD/GAL4 DBD fusion also constitutively expresses a Renilla luciferase-neoR fusion protein, which provides selection capability (G418 resistance, neoR) as well as an internal control (Renilla luciferase). This dual-luciferase format allowed detecting compound cytotoxicity or off-target change in expression during drug screening, therefore improved data quality. These luciferase reporter assays provided better research and drug discovery tools for studying the functions of full length nuclear receptors and ligand binding domains.

Two Panels of Steroid Receptor Luciferase Reporter Cell Lines for Compound Profiling

“…The resulting reporter assay (U2OS-ER G420C -LBD/9xGal4UAS) has 10 times higher fold induction (data not shown) and higher Z´-factor (0.76). In addition the Gal4/LBD reporter assays can benefit from the constitutive expression of a Renilla luciferase-neo R fusion gene which can be used as an internal control in the HTS and can be helpful in compensating for variability between samples caused by difference in cell number for example by random pipetting errors or by nonspecific interference with the luciferase reporter readout and cytotoxic effects of compounds [13].…”

“…The second reporter cell line was described recently [13] and carries one amino acid substitution causing higher fold induction of the luciferase activity compared to wild type ER -LBD (data not shown) but also decreases the affinity of ligands to receptors by 0.5 -1.5 logs depending on the particular ligand (Table 1a and 1b). There is very little difference between the response from U2OS-ER /3xERE and U2OS-ER wt -LBD in both agonist and antagonist mode as illustrated by few examples in the Fig.…”

“…Expression vectors encoding chimeric receptors consisting of Gal4-DBD and of the ligand binding domain (LBD) of the human steroid receptor: Creation of pBIND-ER G420C and pBIND-GR in pFN26A (BIND) vector was described earlier [13].…”

“…Several luciferase reporter systems have been developed recently using different cell lines [11][12][13][14]. Here we report on a development of two panels of U2OS-based luciferase steroid receptor reporter cell lines using two different reporter formats.…”

“…This construct was cotransfected to the cells together with reporter vector containing 9 copies of Gal4 Upstream Activator Sequences (UAS) coupled to minimal promoter upstream of the luc2P gene coding for the luciferase with PEST destabilizing sequence (pGL4.35 [luc2P/9XGAL4UAS/Hygro]). This system was described recently, and its utility was illustrated by preparation of ER and GR reporter assays in HEK293 cells [13]. In this work we extended this system to all members of the steroid receptor family and built a panel of selective reporter cell-lines in U2OS cells.…”