NANOG-dependent function of TET1 and TET2 in establishment of pluripotency

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Abstract

Molecular control of the pluripotent state is thought to reside in a core circuitry of master transcription factors including the homeodomain-containing protein NANOG1,2, which has an essential role in establishing ground state pluripotency during somatic cell reprogramming3,4. Whereas the genomic occupancy of NANOG has been extensively investigated, comparatively little is known about NANOG-associated proteins5 and their contribution to the NANOG-mediated reprogramming process. Using enhanced purification techniques and a stringent computational algorithm, we identify 27 high-confidence protein interaction partners of NANOG in mouse embryonic stem cells. These consist of 19 previously unknown partners of NANOG that have not been reported before, including the ten-eleven translocation (TET) family methylcytosine hydroxylase TET1. We confirm physical association of NANOG with TET1, and demonstrate that TET1, in synergy with NANOG, enhances the efficiency of reprogramming. We also find physical association and reprogramming synergy of TET2 with NANOG, and demonstrate that knockdown of TET2 abolishes the reprogramming synergy of NANOG with a catalytically deficient mutant of TET1. These results indicate that the physical interaction between NANOG and TET1/TET2 proteins facilitates reprogramming in a manner that is dependent on the catalytic activity of TET1/TET2. TET1 and NANOG co-occupy genomic loci of genes associated with both maintenance of pluripotency and lineage commitment in embryonic stem cells, and TET1 binding is reduced upon NANOG depletion. Co-expression of NANOG and TET1 increases 5-hydroxymethylcytosine levels at the top-ranked common target loci Esrrb and Oct4 (also called Pou5f1), resulting in priming of their expression before reprogramming to naive pluripotency. We propose that TET1 is recruited by NANOG to enhance the expression of a subset of key reprogramming target genes. These results provide an insight into the reprogramming mechanism of NANOG and uncover a new role for 5-methylcytosine hydroxylases in the establishment of naive pluripotency.

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Figure 1: Identification of TET1 as a novel partner of NANOG.
Figure 2: Synergy between NANOG and TET1 during reprogramming.
Figure 3: Synergy between NANOG and TET1/TET2 during reprogramming is dependent upon catalytic activity of TET1/TET2.
Figure 4: Mechanism and genome-wide significance of the NANOG–TET1 interaction.

References

  1. 1

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

  2. 2

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

  3. 3

    Silva, J. et al. Nanog is the gateway to the pluripotent ground state. Cell 138, 722–737 (2009)

  4. 4

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

  5. 5

    Wang, J. et al. A protein interaction network for pluripotency of embryonic stem cells. Nature 444, 364–368 (2006)

  6. 6

    Kim, J., Cantor, A. B., Orkin, S. H. & Wang, J. Use of in vivo biotinylation to study protein–protein and protein–DNA interactions in mouse embryonic stem cells. Nature Protocols 4, 506–517 (2009)

  7. 7

    Ding, J., Xu, H., Faiola, F., Ma’ayan, A. & Wang, J. Oct4 links multiple epigenetic pathways to the pluripotency network. Cell Res. 22, 155–167 (2011)

  8. 8

    Rees, J. S. et al. In vivo analysis of proteomes and interactomes using parallel affinity capture (iPAC) coupled to mass spectrometry. Mol. Cell Proteomics http://dx.doi.org/10.1074/mcp.M110.002386 (2011)

  9. 9

    Ito, S. et al. Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification. Nature 466, 1129–1133 (2010)

  10. 10

    Tahiliani, M. et al. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 324, 930–935 (2009)

  11. 11

    Theunissen, T. W. et al. Nanog overcomes reprogramming barriers and induces pluripotency in minimal conditions. Curr. Biol. 21, 65–71 (2011)

  12. 12

    Dawlaty, M. M. et al. Tet1 is dispensable for maintaining pluripotency and its loss is compatible with embryonic and postnatal development. Cell Stem Cell 9, 166–175 (2011)

  13. 13

    Pereira, C. F. & Fisher, A. G. Heterokaryon-based reprogramming for pluripotency. Curr. Protoc. Stem Cell Biol. 9, 4B.1.1–4B.1.14 (2009)

  14. 14

    Hanna, J. et al. Direct cell reprogramming is a stochastic process amenable to acceleration. Nature 462, 595–601 (2009)

  15. 15

    Ficz, G. et al. Dynamic regulation of 5-hydroxymethylcytosine in mouse ES cells and during differentiation. Nature 473, 398–402 (2011)

  16. 16

    Doege, C. A. et al. Early-stage epigenetic modification during somatic cell reprogramming by Parp1 and Tet2. Nature 488, 652–655 (2012)

  17. 17

    Chen, X. et al. Integration of external signaling pathways with the core transcriptional network in embryonic stem cells. Cell 133, 1106–1117 (2008)

  18. 18

    Marson, A. et al. Connecting microRNA genes to the core transcriptional regulatory circuitry of embryonic stem cells. Cell 134, 521–533 (2008)

  19. 19

    Williams, K. et al. TET1 and hydroxymethylcytosine in transcription and DNA methylation fidelity. Nature 473, 343–348 (2011)

  20. 20

    Wu, H. et al. Dual functions of Tet1 in transcriptional regulation in mouse embryonic stem cells. Nature 473, 389–393 (2011)

  21. 21

    Das, S., Jena, S. & Levasseur, D. N. Alternative splicing produces Nanog protein variants with different capacities for self-renewal and pluripotency in embryonic stem cells. J. Biol. Chem. 286, 42690–42703 (2011)

  22. 22

    Yu, M. et al. Base-resolution analysis of 5-hydroxymethylcytosine in the mammalian genome. Cell 149, 1368–1380 (2012)

  23. 23

    Kallin, E. M. et al. Tet2 facilitates the derepression of myeloid target genes during CEBPα-induced transdifferentiation of pre-B cells. Mol. Cell 48, 266–276 (2012)

  24. 24

    Silva, J. et al. Promotion of reprogramming to ground state pluripotency by signal inhibition. PLoS Biol. 6, e253 (2008)

  25. 25

    Wang, J., Levasseur, D. N. & Orkin, S. H. Requirement of Nanog dimerization for stem cell self-renewal and pluripotency. Proc. Natl Acad. Sci. USA 105, 6326–6331 (2008)

  26. 26

    Liang, J. et al. Nanog and Oct4 associate with unique transcriptional repression complexes in embryonic stem cells. Nature Cell Biol. 10, 731–739 (2008)

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Acknowledgements

We thank W. Mansfield for blastocyst injections, A. Radzisheuskaya for cell culture assistance, and R. Jaenisch for Tet1−/− embryonic stem cells. This study was supported by a grant from the NIH (1R01-GM095942-01A1), a grant from New York state Department of Health (NYSTEM#C026420), and a seed fund from the Black Family Stem Cell Institute to J.W., by the Wellcome Trust Fellowship (WT086692MA) and the Isaac Newton Trust Grant (11.19(ad)) to J.C.R.S., who is a Wellcome Trust Career Development Fellow, by the BBSRC, the MRC, the Wellcome Trust, and EU Epigenesys and BLUEPRINT to W.R., and by the Wellcome Trust Fellowship WT079249 to T.W.T.

Author information

J.C.R.S. and J.W. conceived the project, designed the experiments, prepared and approved the manuscript. T.W.T. designed and performed experiments and wrote the manuscript draft. Y.C., J.D., F.F., M.F. and A.S. designed and performed experiments and prepared the manuscript. T.A.H. designed and performed experiments. P.V.S. performed interactomics data analysis. M.L. provided technical assistance. S.Di. provided bioinformatic analysis. S.Da., D.N.L., Z.L. and M.X. contributed to the reagents. W.R. designed experiments and contributed to the reagents. J.C.R.S. and J.W. are equal senior and corresponding authors.

Correspondence to José C. R. Silva or Jianlong Wang.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Methods, Supplementary Figures 1-16, Supplementary Tables 1-3 and 5-6 (see separate excel file for Supplementary Table 4), and Supplementary References. (PDF 3821 kb)

Supplementary Table 4

This file contains the genomic coordinates of NANOG and TET1 co-bound sites. (XLS 743 kb)

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Costa, Y., Ding, J., Theunissen, T. et al. NANOG-dependent function of TET1 and TET2 in establishment of pluripotency. Nature 495, 370–374 (2013) doi:10.1038/nature11925

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