Abstract
Renilla luciferase reporter is a widely used internal control in dual luciferase reporter assay system, where its transcription is driven by a constitutively active promoter. However, the authenticity of the Renilla luciferase response in some experimental settings has recently been questioned. Testicular receptor 4 (TR4, also known as NR2C2) belongs to the subfamily 2 of nuclear receptors. TR4 binds to a direct repeat regulatory element in the promoter of a variety of target genes and plays a key role in tumorigenesis, lipoprotein regulation, and central nervous system development. In our experimental system using murine pituitary corticotroph tumor AtT20 cells to investigate TR4 actions on POMC transcription, we found that overexpression of TR4 resulted in reduced Renilla luciferase expression whereas knockdown TR4 increased Renilla luciferase expression. The TR4 inhibitory effect was mediated by the TR4 DNA-binding domain and behaved similarly to the GR and its agonist, Dexamethasone. We further demonstrated that the chimeric intron, commonly present in various Renilla plasmid backbones such as pRL-Null, pRL-SV40, and pRL-TK, was responsible for TR4’s inhibitory effect. The results suggest that an intron-free Renilla luciferase reporter may provide a satisfactory internal control for TR4 at certain dose range. Our findings advocate caution on the use of Renilla luciferase as an internal control in TR4-directed studies to avoid misleading data interpretation.
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Acknowledgements
We thank Dr. Michael R. Downes and Dr. Ronald M. Evans (The Salk Institute for Biological Studies, San Diego, CA) for their gifts of plasmids of V5-mTR4 and V5-mGR; Dr. Jerome F Strauss III (Virginia Commonwealth University School of Medicine) for the gift of pRL-TK Delta238 plasmid.
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Zhang, D., Atlasi, S.S., Patel, K.K. et al. False responses of Renilla luciferase reporter control to nuclear receptor TR4. Mol Cell Biochem 430, 139–147 (2017). https://doi.org/10.1007/s11010-017-2961-9
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DOI: https://doi.org/10.1007/s11010-017-2961-9
Keywords
- Beta-Galactosidase (β-gal)
- Cytomegalovirus (CMV)
- DNA-binding domain (DBD)
- Dexamethasone (Dex)
- Direct repeat (DR)
- Glucocorticoid (GC)
- Glucocorticoid receptor (GR)
- Herpes simplex virus thymidine kinase promoter (HSV-TK)
- Ligand-binding domain (LBD)
- Renilla luciferase plasmid (pRL)
- Simian virus 40 early promoter (SV40)
- Testicular receptor 4 (TR4)