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Transcriptional Activity of FOXO Transcription Factors Measured by Luciferase Assays

  • Antonia Ávila-FloresEmail author
  • Javier Arranz-Nicolás
  • Isabel Mérida
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1890)

Abstract

The Forkhead box O (FOXO) family of transcription factors translates environmental cues into gene expression. FOXO factors are crucial for the maintenance of cell homeostasis, with important roles in cell fate decisions and differentiation. Identification of FOXO target genes requires strict validation by several methods. Luciferase-based reporters are a valuable starting point for determining the transcription-promoting capacity of potential FOXO-binding sites in candidate genes. Luciferase, an enzyme found in bioluminescent organisms catalyzes oxidation of luciferin to produce oxyluciferin together with light, which can be easily detected and measured with a luminometer. Due to their many advantages, transcriptional assays based on luciferase activity are widely used; they are easy, highly reproducible, and very sensitive. Continued improvements in luciferase-based vectors and measurement reagents confer considerable versatility. Luciferase-based reporters are also a reliable approach in the search for unknown components in the signaling pathways that control FOXO factor activity.

We previously reported that FOXO transcription factors control expression of the enzyme diacylglycerol kinase α (DGKα) in T cells. DGKα consumes diacylglycerol, a lipid that activates several mitogenic pathways. Here, we describe the use of a luciferase-based promoter bearing the FOXO-binding sites of the DGKα gene to explore the relationship between the expression of this enzyme and stress conditions in NIH3T3 mouse fibroblasts. Our data support a role for FOXO factors in promoting high DGKα levels in conditions of growth factor deprivation. DGKα regulation by FOXO factors correlates with the reported alterations in DGKα expression during cell transformation and cancer progression.

Key words

FOXO factor Transcriptional regulation Luciferase Reporter gene PI3K AKT Diacylglycerol kinase α 

Notes

Acknowledgments

We thank Job García-Liévana and Mónica Martínez-Moreno, with whom we started the DGKα Expression Regulation Project. We thank Raquel Arcos Pérez and Alejandra Cordero Cantos for technical assistance, and Catherine Mark for editorial assistance. JAN holds a pre-doctoral FPI fellowship from the Spanish Ministry of Economy and Competitiveness (MINECO). This work was supported in part by grants from the MINECO (BFU2016-77207-R), Spanish Ministry of Health (Instituto de Salud Carlos III; RD12/0036/0059) and the Madrid regional government (IMMUNOTHERCAM Consortium B2017/BMD3733).

References

  1. 1.
    Alam J, Cook JL (1990) Reporter genes: application to the study of mammalian gene transcription. Anal Biochem 188(2):245–254CrossRefGoogle Scholar
  2. 2.
    Pance A (2013) Tailoring the modes of transcription. Int J Mol Sci 14:7583–7597CrossRefGoogle Scholar
  3. 3.
    de Wet JR, Wood KW, DeLuca M, Helinski DR, Subramani S (1987) Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol 7(2):725–737CrossRefGoogle Scholar
  4. 4.
    Allard STM, Kopish K (2008) Luciferase reporter assays: powerful, adaptable tools for cell biology research. Cell Notes Promega 21:23–26Google Scholar
  5. 5.
    Kaskova ZM, Tsarkova AS, Yampolsky IV (2016) 1001 lights: luciferins, luciferases, their mechanisms of action and applications in chemical analysis, biology and medicine. Chem Soc Rev 45(21):6048–6077CrossRefGoogle Scholar
  6. 6.
    Dual-Luciferase Reporter Assay System Technical Manual #TM040. Promega CorporationGoogle Scholar
  7. 7.
    pGL3 Luciferase Reporter Vectors Technical Manual #TM033. Promega CorporationGoogle Scholar
  8. 8.
    pGL4 Luciferase Reporter Vectors Technical Manual #TM259. Promega CorporationGoogle Scholar
  9. 9.
    Matthews JC, Hori K, Cormier MJ (1977) Purification and properties of Renilla reniformis luciferase. Biochemistry 16(1):85–91CrossRefGoogle Scholar
  10. 10.
    Sherf BA (1996) Dual luciferase reporter assay. Promega Notes 57:2–9Google Scholar
  11. 11.
    Farr A, Roman A (1992) A pitfall of using a second plasmid to determine transfection efficiency. Nucleic Acids Res 20(4):920CrossRefGoogle Scholar
  12. 12.
    Shifera AS, Hardin JA (2010) Factors modulating expression of Renilla luciferase from control plasmids used in luciferase reporter gene assays. Anal Biochem 396(2):167–172CrossRefGoogle Scholar
  13. 13.
    pRL Renilla Luciferase Reporter Vectors Technical Bulleting #TB550. Promega CorporationGoogle Scholar
  14. 14.
    Throne N, Inglese J, Auld DS (2010) Illuminating insights into firefly luciferase and other bioluminescent reporters used in chemical biology. Cell 17:646–655Google Scholar
  15. 15.
    Yun C, DasGupta R (2014) Luciferase reporter assay in Drosophila and mammalian tissue culture cells. Curr Protoc Chem Biol 6(1):7–23CrossRefGoogle Scholar
  16. 16.
    Cevenini L, Calabretta MM, Calabria D, Roda A, Michelini E (2016) Luciferase genes as reporter reactions: how to use them in molecular biology? Adv Biochem Eng Biotechnol 154:3–17PubMedGoogle Scholar
  17. 17.
    Martinez-Moreno M, Garcia-Lievana J, Soutar D, Torres-Ayuso P, Andrada E, Zhong XP, Koretzky GA, Merida I, Avila-Flores A (2012) FoxO-dependent regulation of diacylglycerol kinase alpha gene expression. Mol Cell Biol 32(20):4168–4180CrossRefGoogle Scholar
  18. 18.
    Merida I, Andrada E, Gharbi SI, Avila-Flores A (2015) Redundant and specialized roles for diacylglycerol kinases alpha and zeta in the control of T cell functions. Sci Signal 8(374):re6CrossRefGoogle Scholar
  19. 19.
    Merida I, Avila-Flores A (2014) DGKA (diacylglycerol kinase, alpha 80 kDa). Atlas Genet Cytogenet Oncol Haematol 18(8):545–549Google Scholar
  20. 20.
    Merida I, Avila-Flores A, Garcia J, Merino E, Almena M, Torres-Ayuso P (2009) Diacylglycerol kinase alpha, from negative modulation of T cell activation to control of cancer progression. Adv Enzym Regul 49(1):174–188CrossRefGoogle Scholar
  21. 21.
    Gu J, Iyer VR (2006) PI3K signaling and miRNA expression during the response of quiescent human fibroblasts to distinct proliferative stimuli. Genome Biol 7(5):R42CrossRefGoogle Scholar
  22. 22.
    Merida I, Torres-Ayuso P, Avila-Flores A, Arranz-Nicolás J, Andrada E, Tello-Lafoz M, Liébana R, Arcos R (2017) Diacylglycerol kinases in cancer. Adv Enzym Regul 63:22–31CrossRefGoogle Scholar
  23. 23.
    Brunet A, Bonni A, Zigmond M, Zin M, Jou P, Hu L, Andrson M, Arden K, Blenis J, Greenberg M (1999) Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 96:857–868CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Antonia Ávila-Flores
    • 1
    Email author
  • Javier Arranz-Nicolás
    • 1
  • Isabel Mérida
    • 1
  1. 1.Department of Immunology and OncologyCentro Nacional de Biotecnología (CNB-CSIC)MadridSpain

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