Letter | Published:

A protein interaction network for pluripotency of embryonic stem cells

Naturevolume 444pages364368 (2006) | Download Citation



Embryonic stem (ES) cells are pluripotent1,2 and of therapeutic potential in regenerative medicine3,4. Understanding pluripotency at the molecular level should illuminate fundamental properties of stem cells and the process of cellular reprogramming. Through cell fusion the embryonic cell phenotype can be imposed on somatic cells, a process promoted by the homeodomain protein Nanog5, which is central to the maintenance of ES cell pluripotency6,7. Nanog is thought to function in concert with other factors such as Oct4 (ref. 8) and Sox2 (ref. 9) to establish ES cell identity. Here we explore the protein network in which Nanog operates in mouse ES cells. Using affinity purification of Nanog under native conditions followed by mass spectrometry, we have identified physically associated proteins. In an iterative fashion we also identified partners of several Nanog-associated proteins (including Oct4), validated the functional relevance of selected newly identified components and constructed a protein interaction network. The network is highly enriched for nuclear factors that are individually critical for maintenance of the ES cell state and co-regulated on differentiation. The network is linked to multiple co-repressor pathways and is composed of numerous proteins whose encoding genes are putative direct transcriptional targets of its members. This tight protein network seems to function as a cellular module dedicated to pluripotency.

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  1. 1

    Martin, G. R. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc. Natl Acad. Sci. USA 78, 7634–7638 (1981)

  2. 2

    Evans, M. J. & Kaufman, M. H. Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154–156 (1981)

  3. 3

    Donovan, P. J. & Gearhart, J. The end of the beginning for pluripotent stem cells. Nature 414, 92–97 (2001)

  4. 4

    Prelle, K., Zink, N. & Wolf, E. Pluripotent stem cells—model of embryonic development, tool for gene targeting, and basis of cell therapy. Anat. Histol. Embryol. 31, 169–186 (2002)

  5. 5

    Silva, J., Chambers, I., Pollard, S. & Smith, A. Nanog promotes transfer of pluripotency after cell fusion. Nature 441, 997–1001 (2006)

  6. 6

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

  7. 7

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

  8. 8

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

  9. 9

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

  10. 10

    de Boer, E. et al. Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice. Proc. Natl Acad. Sci. USA 100, 7480–7485 (2003)

  11. 11

    Krogan, N. J. et al. Global landscape of protein complexes in the yeast Saccharomyces cerevisiae.. Nature 440, 637–643 (2006)

  12. 12

    Niakan, K. K. et al. Novel role for the orphan nuclear receptor Dax1 in embryogenesis, different from steroidogenesis. Mol. Genet. Metab. 88, 261–271 (2006)

  13. 13

    Sakaki-Yumoto, M. et al. The murine homolog of SALL4, a causative gene in Okihiro syndrome, is essential for embryonic stem cell proliferation, and cooperates with Sall1 in anorectal, heart, brain and kidney development. Development 133, 3005–3013 (2006)

  14. 14

    Mackler, S. A., Homan, Y. X., Korutla, L., Conti, A. C. & Blendy, J. A. The mouse nac1 gene, encoding a cocaine-regulated Bric-a-brac Tramtrac Broad complex/Pox virus and Zinc finger protein, is regulated by AP1. Neuroscience 121, 355–361 (2003)

  15. 15

    Mackler, S. A. et al. NAC-1 is a brain POZ/BTB protein that can prevent cocaine-induced sensitization in the rat. J. Neurosci. 20, 6210–6217 (2000)

  16. 16

    Law, D. J., Du, M., Law, G. L. & Merchant, J. L. ZBP-99 defines a conserved family of transcription factors and regulates ornithine decarboxylase gene expression. Biochem. Biophys. Res. Commun. 262, 113–120 (1999)

  17. 17

    Thompson, J. R. & Gudas, L. J. Retinoic acid induces parietal endoderm but not primitive endoderm and visceral endoderm differentiation in F9 teratocarcinoma stem cells with a targeted deletion of the Rex-1 (Zfp-42) gene. Mol. Cell. Endocrinol. 195, 119–133 (2002)

  18. 18

    Batagelj, V. & Mrvar, A. Pajek—program for large network analysis. Connections 21, 47–57 (1998)

  19. 19

    Loh, Y. H. et al. The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nature Genet. 38, 431–440 (2006)

  20. 20

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

  21. 21

    Albert, R., Jeong, H. & Barabasi, A. L. Error and attack tolerance of complex networks. Nature 406, 378–382 (2000)

  22. 22

    Lauberth, S. M. & Rauchman, M. A conserved twelve amino acid motif in sall1 recruits nuRD. J. Biol. Chem. 281, 23922–23931 (2006)

  23. 23

    Korutla, L., Wang, P. J. & Mackler, S. A. The POZ/BTB protein NAC1 interacts with two different histone deacetylases in neuronal-like cultures. J. Neurochem. 94, 786–793 (2005)

  24. 24

    Niwa, H., Miyazaki, J. & Smith, A. G. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nature Genet. 24, 372–376 (2000)

  25. 25

    Hatano, S. Y. et al. Pluripotential competence of cells associated with Nanog activity. Mech. Dev. 122, 67–79 (2005)

  26. 26

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

  27. 27

    Lee, J., Rhee, B. K., Bae, G. Y., Han, Y. M. & Kim, J. Stimulation of Oct-4 activity by Ewing’s sarcoma protein. Stem Cells 23, 738–751 (2005)

  28. 28

    Perez-Iratxeta, C. et al. Study of stem cell function using microarray experiments. FEBS Lett. 579, 1795–1801 (2005)

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We thank A. Cantor for biotin tagging constructs and for advice on affinity purification and chromatography; Y. Fujiwara and M. Kaku for technical assistance and for Oct4–GFP ES cells; T. De Lange for Rif1 antibody; S. Mackler for Nac1 antibody; S. Lowe and S. Elledge for shRNA vectors; M. Vidal and A.-L. Barabasi for advice and discussion on networks; and R. Tomaino and S. Gygi for performing LC–MS/MS and for providing advice and assistance in data collection and analysis. S.H.O. is an Investigator of the HHMI. Author Contributions S.R. and J.C. contributed equally to this study. J.W. and S.H.O. conceived and initiated the study. J.W., J.C., X.S., D.N.L. and T.W.T. performed the experiments. S.R. and J.W. analysed data and bioinformatics of the network. J.W. and S.H.O. wrote the manuscript.

Author information


  1. Division of Hematology–Oncology, Children’s Hospital and Dana Farber Cancer Institute, Harvard Medical School, Harvard Stem Cell Institute

    • Jianlong Wang
    • , Sridhar Rao
    • , Jianlin Chu
    • , Xiaohua Shen
    • , Dana N. Levasseur
    • , Thorold W. Theunissen
    •  & Stuart H. Orkin
  2. Howard Hughes Medical Institute, Boston, Massachusetts, 02115, USA

    • Stuart H. Orkin


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Competing interests

Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.

Corresponding author

Correspondence to Stuart H. Orkin.

Supplementary information

  1. Supplementary Notes

    This file contains Supplementary Methods, Supplementary Figure Legends and Supplementary Tables 1–3. (DOC 207 kb)

  2. Supplementary Figures

    This file contains Supplementary Figures 1–8. (PDF 1871 kb)

  3. Supplementary Data 1

    Common background proteins in BirA samples identified by MS. (XLS 1469 kb)

  4. Supplementary Data 2

    List of all specific proteins identified by MS from bioNanog samples (XLS 52 kb)

  5. Supplementary Data 3

    A list of the key genes used in this study. (DOC 19 kb)

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