Letter | Published:

FOXO1 is an essential regulator of pluripotency in human embryonic stem cells

Nature Cell Biology volume 13, pages 10921099 (2011) | Download Citation


Pluripotency of embryonic stem cells (ESCs) is defined by their ability to differentiate into three germ layers and derivative cell types1,2,3 and is established by an interactive network of proteins including OCT4 (also known as POU5F1; ref. 4), NANOG (refs 5, 6), SOX2 (ref. 7) and their binding partners. The forkhead box O (FoxO) transcription factors are evolutionarily conserved regulators of longevity and stress response whose function is inhibited by AKT protein kinase. FoxO proteins are required for the maintenance of somatic and cancer stem cells8,9,10,11,12,13; however, their function in ESCs is unknown. We show that FOXO1 is essential for the maintenance of human ESC pluripotency, and that an orthologue of FOXO1 (Foxo1) exerts a similar function in mouse ESCs. This function is probably mediated through direct control by FOXO1 of OCT4 and SOX2 gene expression through occupation and activation of their respective promoters. Finally, AKT is not the predominant regulator of FOXO1 in human ESCs. Together these results indicate that FOXO1 is a component of the circuitry of human ESC pluripotency. These findings have critical implications for stem cell biology, development, longevity and reprogramming, with potentially important ramifications for therapy.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1.

    , & Molecular control of pluripotency. Curr. Opin. Genet. Dev. 16, 455–462 (2006).

  2. 2.

    How is pluripotency determined and maintained? Development 134, 635–646 (2007).

  3. 3.

    & Stem cells, the molecular circuitry of pluripotency and nuclear reprogramming. Cell 132, 567–582 (2008).

  4. 4.

    et al. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 95, 379–391 (1998).

  5. 5.

    et al. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 113, 643–655 (2003).

  6. 6.

    et al. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 113, 631–642 (2003).

  7. 7.

    et al. Multipotent cell lineages in early mouse development depend on SOX2 function. Genes Dev. 17, 126–140 (2003).

  8. 8.

    et al. FoxOs are lineage-restricted redundant tumor suppressors and regulate endothelial cell homeostasis. Cell 128, 309–323 (2007).

  9. 9.

    et al. Foxo3a is essential for maintenance of the hematopoietic stem cell pool. Cell Stem Cell 1, 101–112 (2007).

  10. 10.

    et al. Foxo3 is essential for the regulation of Ataxia telangiectasia mutated and oxidative stress-mediated homeostasis of hematopoietic stem cells. J. Biol. Chem. 283, 25692–25705 (2008).

  11. 11.

    et al. FoxO3 regulates neural stem cell homeostasis. Cell Stem Cell 5, 527–539 (2009).

  12. 12.

    et al. TGF-β-FOXO signalling maintains leukaemia-initiating cells in chronic myeloid leukaemia. Nature 463, 676–680.

  13. 13.

    et al. ROS-mediated amplification of AKT/mTOR signalling pathwayleads to myeloproliferative syndrome in Foxo3(-/-) mice. EMBO J. 29, 4118–4131 (2010).

  14. 14.

    et al. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 122, 947–956 (2005).

  15. 15.

    & Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006).

  16. 16.

    et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007).

  17. 17.

    et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917–1920 (2007).

  18. 18.

    et al. The Fork head transcription factor DAF-16 transduces insulin-like metabolic and longevity signals in C. elegans. Nature 389, 994–999 (1997).

  19. 19.

    et al. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 96, 857–868 (1999).

  20. 20.

    et al. Direct control of the Forkhead transcription factor AFX by protein kinase B. Nature 398, 630–634 (1999).

  21. 21.

    et al. FOXO transcription factor activation by oxidative stress mediated by the small GTPase Ral and JNK. EMBO J. 23, 4802–4812 (2004).

  22. 22.

    et al. IκB kinase promotes tumorigenesis through inhibition of forkhead FOXO3a. Cell 117, 225–237 (2004).

  23. 23.

    , & ERK and MDM2 prey on FOXO3a. Nat. Cell Biol. 10, 125–126 (2008).

  24. 24.

    et al. Phosphorylation of forkhead transcription factors by erythropoietin and stem cell factor prevents acetylation and their interaction with coactivator p300 in erythroid progenitor cells. Oncogene 21, 1556–1562 (2002).

  25. 25.

    et al. Stress-dependent regulation of FOXO transcription factors by the SIRT1 deacetylase. Science 303, 2011–2015 (2004).

  26. 26.

    et al. Silent information regulator 2 potentiates Foxo1-mediated transcription through its deacetylase activity. Proc. Natl Acad. Sci. USA 101, 10042–10047 (2004).

  27. 27.

    et al. Skp2 inhibits FOXO1 in tumor suppression through ubiquitin-mediated degradation. Proc. Natl Acad. Sci. USA 102, 1649–1654 (2005).

  28. 28.

    et al. Arginine methylation of FOXO transcription factors inhibits their phosphorylation by Akt. Mol. Cell 32, 221–231 (2008).

  29. 29.

    et al. Redox-sensitive cysteines bridge p300/CBP-mediated acetylation and FoxO4 activity. Nat. Chem. Biol. 5, 664–672 (2009).

  30. 30.

    & Integrating opposing signals toward Forkhead box O. Antioxid Redox Signal 14, 607–621 (2011).

  31. 31.

    , , & Regulation and function of FoxO transcription factors in normal and cancer stem cells: what have we learned? Curr. Drug Targets 12 (2011).

  32. 32.

    , , , & Suppression of ovarian follicle activation in mice by the transcription factor Foxo3a. Science 301, 215–218 (2003).

  33. 33.

    et al. Disruption of forkhead transcription factor (FOXO) family members in mice reveals their functional diversification. Proc. Natl Acad. Sci. USA 101, 2975–2980 (2004).

  34. 34.

    et al. Foxo3 is required for the regulation of oxidative stress in erythropoiesis. J. Clin. Invest. 117, 2133–2144 (2007).

  35. 35.

    Embryonic stem cell differentiation: emergence of a new era in biology and medicine. Genes Dev. 19, 1129–1155 (2005).

  36. 36.

    et al. Transcriptome characterization elucidates signaling networks that control human ES cell growth and differentiation. Nat. Biotechnol. 22, 707–716 (2004).

  37. 37.

    et al. A core Klf circuitry regulates self-renewal of embryonic stem cells. Nat. Cell Biol. 10, 353–360 (2008).

  38. 38.

    , , , & REST maintains self-renewal and pluripotency of embryonic stem cells. Nature 453, 223–227 (2008).

  39. 39.

    et al. Transcriptome profiling of human and murine ESCs identifies divergent paths required to maintain the stem cell state. Stem Cells 23, 166–185 (2005).

  40. 40.

    & Regulatory networks in embryo-derived pluripotent stem cells. Nat. Rev. Mol. Cell Biol. 6, 872–884 (2005).

  41. 41.

    , & Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat. Genet. 24, 372–376 (2000).

  42. 42.

    & Naive and primed pluripotent states. Cell Stem Cell 4, 487–492 (2009).

  43. 43.

    et al. Characterization of human embryonic stem cell lines by the International Stem Cell Initiative. Nat. Biotechnol. 25, 803–816 (2007).

  44. 44.

    , , & Identification of the differential distribution patterns of mRNAs and consensus binding sequences for mouse DAF-16 homologues. Biochem. J. 349, 629–634 (2000).

  45. 45.

    et al. Activation of Akt signaling is sufficient to maintain pluripotency in mouse and primate embryonic stem cells. Oncogene 25, 2697–2707 (2006).

  46. 46.

    et al. Dissecting self-renewal in stem cells with RNA interference. Nature 442, 533–538 (2006).

  47. 47.

    et al. mTOR activation induces tumor suppressors that inhibit leukemogenesis and deplete hematopoietic stem cells after Pten deletion. Cell Stem Cell 7, 593–605 (2010).

  48. 48.

    , & Mammary gland, limb and yolk sac defects in mice lacking Tbx3, the gene mutated in human ulnar mammary syndrome. Development 130, 2263–2273 (2003).

  49. 49.

    et al. TCL1 participates in early embryonic development and is overexpressed in human seminomas. Proc. Natl Acad. Sci. USA 99, 11712–11717 (2002).

  50. 50.

    , , , & Development of the hemangioblast defines the onset of hematopoiesis in human ES cell differentiation cultures. Blood 109, 2679–2687 (2007).

  51. 51.

    , , , & Specificity in transforming growth factor β-induced transcription of the plasminogen activator inhibitor-1 gene: interactions of promoter DNA, transcription factor muE3, and Smad proteins. Proc. Natl Acad. Sci. USA 96, 13130–13135 (1999).

  52. 52.

    et al. Measurement of protein-DNA interactions in vivo by chromatin immunoprecipitation. Methods Mol. Biol. 284, 129–146 (2004).

  53. 53.

    & Activation of Eklf expression during hematopoiesis by Gata2 and Smad5 prior to erythroid commitment. Development 135, 2071–2082 (2008).

Download references


We thank F. Lohmann for advice on the ChIP assay, J. Bieker (Mount Sinai School of Medicine) and G. Blobel (University of Pennsylvania) for critical reading of the manuscript, I. George and M. Grisotto for cell sorting, and the Flow Cytometry Shared Research Facility of Mount Sinai School of Medicine. This work was supported in part by a National Institutes of Health grant RO1 DK077174, an American Cancer Society Research Scholarship (RSG LIB-110480), a Career Enhancement Award (K18 HL76510-01), a Black Family Stem Cell Institute Exploratory Research Award, a New York State Stem Cell Science (NYSTEM) award (CO24408), an Irma Hirschl/Weill-Caulier Trust Research Award and a Roche Foundation for Anemia Research (RoFAR) Award to S.G., and an NIH P20 GM75019 (S.G. CoPI).

Author information

Author notes

    • Markus Landthaler

    Present address: Berlin Institute for Medical Systems Biology at the Max-Delbrück-Center for Molecular Medicine, 13092 Berlin, Germany


  1. Department of Developmental and Regenerative Biology, Mount Sinai School of Medicine, New York, New York 10029, USA

    • Xin Zhang
    • , Safak Yalcin
    • , Dung-Fang Lee
    • , Seung-Min Lee
    • , Jie Su
    • , Sathish Kumar Mungamuri
    • , Pauline Rimmelé
    • , Ihor Lemischka
    •  & Saghi Ghaffari
  2. Research Service, VA San Diego Healthcare System, School of Medicine, University of California, San Diego, La Jolla, California 92093, USA

    • Tsung-Yin J. Yeh
    •  & Nai-Wen Chi
  3. McEwen Center for Regenerative Medicine, University Health Network, Toronto, Ontario M5G 1L7, Canada

    • Marion Kennedy
    •  & Gordon Keller
  4. Department of Pathology, Albert Einstein College of Medicine, New York, New York 10461, USA

    • Rani Sellers
  5. Howard Hughes Medical Institute, Laboratory for RNA Molecular Biology, The Rockefeller University, New York, New York 10065, USA

    • Markus Landthaler
    •  & Thomas Tuschl
  6. Black Family Stem Cell Institute, Mount Sinai School of Medicine, New York, New York 10029, USA

    • Ihor Lemischka
    •  & Saghi Ghaffari
  7. Department of Medicine Division of Hematology, Oncology, Mount Sinai School of Medicine, New York, New York 10029, USA

    • Saghi Ghaffari


  1. Search for Xin Zhang in:

  2. Search for Safak Yalcin in:

  3. Search for Dung-Fang Lee in:

  4. Search for Tsung-Yin J. Yeh in:

  5. Search for Seung-Min Lee in:

  6. Search for Jie Su in:

  7. Search for Sathish Kumar Mungamuri in:

  8. Search for Pauline Rimmelé in:

  9. Search for Marion Kennedy in:

  10. Search for Rani Sellers in:

  11. Search for Markus Landthaler in:

  12. Search for Thomas Tuschl in:

  13. Search for Nai-Wen Chi in:

  14. Search for Ihor Lemischka in:

  15. Search for Gordon Keller in:

  16. Search for Saghi Ghaffari in:


X.Z. and S.G. designed experiments and analysed data; X.Z. carried out most of the experiments, with significant help from S.Y. and some assistance from S-M.L., S.K.M. and P.R.; M.K. helped with the set-up of some critical techniques; R.S. analysed data; D-F.L. and J.S. designed and carried out experiments involving mESCs; N-W.C. designed and carried out antibody calibration experiments with the help of T-Y.J.Y.; M.L. and T.T. contributed key reagents; I.L. and G.K. provided valuable reagents and advice; S.G. conceived the project and wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Saghi Ghaffari.

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    Supplementary Information

Excel files

  1. 1.

    Supplementary Table 1

    Supplementary Information

About this article

Publication history






Further reading