Abstract
The transcription factor FoxO1 (forkhead box O1) regulates genes that are involved in development, metabolism, cellular innovation, longevity, and stress responses. Assessment of FoxO1 activity is therefore critical to understand the regulatory network of this transcription factor. FoxO1 transactivation activity relies on its ability to bind to the promoters of target genes, which is controlled by posttranslational modifications (e.g., dephosphorylation or phosphorylation) that may promote nuclear translocation or exclusion of FoxO1. In this chapter we describe the protocols for FoxO1 activity assessment using Western blotting analysis of the posttranslational modification of FoxO1 in whole cell lysates and ELISA of DNA binding activity of FoxO1 in nuclear extracts.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Cheng Z (2019) The FoxO-autophagy axis in health and disease. Trends Endocrinol Metab 30(9):658–671. https://doi.org/10.1016/j.tem.2019.07.009
Cheng Z (2015) FoxO1: mute for a tuned metabolism? Trends Endocrinol Metab 26(7):402–403. https://doi.org/10.1016/j.tem.2015.06.006
Calissi G, Lam EW, Link W (2021) Therapeutic strategies targeting FOXO transcription factors. Nat Rev Drug Discov 20(1):21–38. https://doi.org/10.1038/s41573-020-0088-2
Cheng Z, White MF (2011) Targeting Forkhead box O1 from the concept to metabolic diseases: lessons from mouse models. Antioxid Redox Signal 14(4):649–661. https://doi.org/10.1089/ars.2010.3370
Van Der Heide LP, Hoekman MF, Smidt MP (2004) The ins and outs of FoxO shuttling: mechanisms of FoxO translocation and transcriptional regulation. Biochem J 380(Pt 2):297–309. https://doi.org/10.1042/BJ20040167
Aoki M, Jiang H, Vogt PK (2004) Proteasomal degradation of the FoxO1 transcriptional regulator in cells transformed by the P3k and Akt oncoproteins. Proc Natl Acad Sci U S A 101(37):13613–13617. https://doi.org/10.1073/pnas.0405454101
Yamagata K, Daitoku H, Takahashi Y et al (2008) Arginine methylation of FOXO transcription factors inhibits their phosphorylation by Akt. Mol Cell 32(2):221–231. https://doi.org/10.1016/j.molcel.2008.09.013
Zou P, Liu L, Zheng L et al (2014) Targeting FoxO1 with AS1842856 suppresses adipogenesis. Cell Cycle 13(23):3759–3767. https://doi.org/10.4161/15384101.2014.965977
Wang S, Xia P, Huang G et al (2016) FoxO1-mediated autophagy is required for NK cell development and innate immunity. Nat Commun 7:11023. https://doi.org/10.1038/ncomms11023
Park CH, Skarra DV, Rivera AJ, Arriola DJ, Thackray VG (2014) Constitutively active FOXO1 diminishes activin induction of Fshb transcription in immortalized gonadotropes. PLoS One 9(11):e113839. https://doi.org/10.1371/journal.pone.0113839
Brent MM, Anand R, Marmorstein R (2008) Structural basis for DNA recognition by FoxO1 and its regulation by posttranslational modification. Structure 16(9):1407–1416. https://doi.org/10.1016/j.str.2008.06.013
Langlet F, Haeusler RA, Linden D et al (2017) Selective inhibition of FOXO1 activator/repressor balance modulates hepatic glucose handling. Cell 171(4):824–835. e818. https://doi.org/10.1016/j.cell.2017.09.045
Zanella F, Rosado A, Garcia B, Carnero A, Link W (2009) Using multiplexed regulation of luciferase activity and GFP translocation to screen for FOXO modulators. BMC Cell Biol 10:14. https://doi.org/10.1186/1471-2121-10-14
Liu L, Tao Z, Zheng LD et al (2016) FoxO1 interacts with transcription factor EB and differentially regulates mitochondrial uncoupling proteins via autophagy in adipocytes. Cell Death Discovery 2:16066. https://doi.org/10.1038/cddiscovery.2016.66
Chatterjee S, Daenthanasanmak A, Chakraborty P et al (2018) CD38-NAD(+)Axis regulates immunotherapeutic anti-tumor T cell response. Cell Metab 27(1):85–100. e108. https://doi.org/10.1016/j.cmet.2017.10.006
Chakraborty P, Vaena SG, Thyagarajan K et al (2019) Pro-survival lipid Sphingosine-1-phosphate metabolically programs T cells to limit anti-tumor activity. Cell Rep 28(7):1879–1893. e1877. https://doi.org/10.1016/j.celrep.2019.07.044
Tao Z, Shi L, Parke J et al (2021) Sirt1 coordinates with ERalpha to regulate autophagy and adiposity. Cell Death Discovery 7(1):53. https://doi.org/10.1038/s41420-021-00438-8
Tao Z, Liu L, Zheng LD, Cheng Z (2019) Autophagy in adipocyte differentiation. Methods Mol Biol 1854:45–53. https://doi.org/10.1007/7651_2017_65
Tao Z, Zheng LD, Smith C et al (2018) Estradiol signaling mediates gender difference in visceral adiposity via autophagy. Cell Death Dis 9(3):309. https://doi.org/10.1038/s41419-018-0372-9
Acknowledgments
This work was supported in part by the American Heart Association Grant (18TPA34230082 to Z.C.) and the USDA National Institute of Food and Agriculture Grant (1020373 to Z.C).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Shi, L., Tao, Z., Cheng, Z. (2023). Assessing the Activity of Transcription Factor FoxO1. In: Song, Q., Tao, Z. (eds) Transcription Factor Regulatory Networks. Methods in Molecular Biology, vol 2594. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2815-7_8
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2815-7_8
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2814-0
Online ISBN: 978-1-0716-2815-7
eBook Packages: Springer Protocols