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

Abstract

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.

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Figure 1: FOXO1 is essential for the expression of hESC pluripotency markers.
Figure 2: Reversible effect of loss of FOXO1 on pluripotency and differentiation of hESCs.
Figure 3: Foxo1 and Foxo3 regulate pluripotency of mESCs.
Figure 4: FOXO1 knockdown does not impact hESC proliferation or redox status.
Figure 5: FOXO1 activates the expression of OCT4 and SOX2 pluripotency genes in hESCs by binding directly to their regulatory regions.

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Acknowledgements

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).

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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.

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Correspondence to Saghi Ghaffari.

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Zhang, X., Yalcin, S., Lee, DF. et al. FOXO1 is an essential regulator of pluripotency in human embryonic stem cells. Nat Cell Biol 13, 1092–1099 (2011). https://doi.org/10.1038/ncb2293

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