The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells

Abstract

Oct4 and Nanog are transcription factors required to maintain the pluripotency and self-renewal of embryonic stem (ES) cells. Using the chromatin immunoprecipitation paired-end ditags method, we mapped the binding sites of these factors in the mouse ES cell genome. We identified 1,083 and 3,006 high-confidence binding sites for Oct4 and Nanog, respectively. Comparative location analyses indicated that Oct4 and Nanog overlap substantially in their targets, and they are bound to genes in different configurations. Using de novo motif discovery algorithms, we defined the cis-acting elements mediating their respective binding to genomic sites. By integrating RNA interference–mediated depletion of Oct4 and Nanog with microarray expression profiling, we demonstrated that these factors can activate or suppress transcription. We further showed that common core downstream targets are important to keep ES cells from differentiating. The emerging picture is one in which Oct4 and Nanog control a cascade of pathways that are intricately connected to govern pluripotency, self-renewal, genome surveillance and cell fate determination.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Schematic diagram of genome-wide mapping of Oct4 and Nanog binding sites using ChIP-PET.
Figure 2: Distribution of Oct4 and Nanog binding sites.
Figure 3: Oct4 and Nanog binding site configurations at genomic locations.
Figure 4: De novo prediction of motifs that mediate specific transcription factor–DNA interaction.
Figure 5: Genome-wide association of Oct4 and Nanog binding sites with differentiation profiles of mouse ES cells.
Figure 6: Genome-wide association of Oct4 and Nanog binding sites with expression profiles of mouse ES cells depleted of Oct4 or Nanog.
Figure 7: Regulation of pluripotency by downstream targets of Oct4 and Nanog.
Figure 8: Conserved and diverged Oct4 and Nanog circuitries of mouse and human ES cells.

Accession codes

Accessions

Gene Expression Omnibus

References

  1. 1

    Smith, A.G. Embryo-derived stem cells: of mice and men. Annu. Rev. Cell Dev. Biol. 17, 435–462 (2001).

  2. 2

    Pera, M.F., Reubinoff, B. & Trounson, A. Human embryonic stem cells. J. Cell Sci. 113, 5–10 (2000).

  3. 3

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

  4. 4

    Loebel, D.A., Watson, C.M., De Young, R.A. & Tam, P.P. Lineage choice and differentiation in mouse embryos and embryonic stem cells. Dev. Biol. 264, 1–14 (2003).

  5. 5

    Scholer, H.R., Ruppert, S., Suzuki, N., Chowdhury, K. & Gruss, P. New type of POU domain in germ line-specific protein Oct-4. Nature 344, 435–439 (1990).

  6. 6

    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).

  7. 7

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

  8. 8

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

  9. 9

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

  10. 10

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

  11. 11

    Pesce, M. & Scholer, H.R. Oct-4: gatekeeper in the beginnings of mammalian development. Stem Cells 19, 271–278 (2001).

  12. 12

    Chambers, I. & Smith, A. Self-renewal of teratocarcinoma and embryonic stem cells. Oncogene 23, 7150–7160 (2004).

  13. 13

    Ng, P. et al. Gene identification signature (GIS) analysis for transcriptome characterization and genome annotation. Nat. Methods 2, 105–111 (2005).

  14. 14

    Wei, C.L. et al. A global map of p53 transcription-factor binding sites in the human genome. Cell 124, 207–219 (2006).

  15. 15

    Chew, J.L. et al. Reciprocal transcriptional regulation of Pou5f1 and Sox2 via the Oct4/Sox2 complex in embryonic stem cells. Mol. Cell. Biol. 25, 6031–6046 (2005).

  16. 16

    Mi, H. et al. The PANTHER database of protein families, subfamilies, functions and pathways. Nucleic Acids Res. 33, D284–D288 (2005).

  17. 17

    Yeom, Y.I. et al. Germline regulatory element of Oct-4 specific for the totipotent cycle of embryonal cells. Development 122, 881–894 (1996).

  18. 18

    Pavesi, G., Mauri, G. & Pesole, G. An algorithm for finding signals of unknown length in unaligned DNA sequences. Bioinformatics 17 (Suppl.), 207–214 (2001).

  19. 19

    Down, T.A. & Hubbard, T.J. NestedMICA: sensitive inference of over-represented motifs in nucleic acid sequence. Nucleic Acids Res. 33, 1445–1453 (2005).

  20. 20

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

  21. 21

    Cartwright, P. et al. LIF/STAT3 controls ES cell self-renewal and pluripotency by a Myc-dependent mechanism. Development 132, 885–896 (2005).

  22. 22

    Cawley, S. et al. Unbiased mapping of transcription factor binding sites along human chromosomes 21 and 22 points to widespread regulation of noncoding RNAs. Cell 116, 499–509 (2004).

  23. 23

    Kim, T.H. et al. A high-resolution map of active promoters in the human genome. Nature 436, 876–880 (2005).

  24. 24

    Pollack, J.R. & Iyer, V.R. Characterizing the physical genome. Nat. Genet. 32, 515–521 (2002).

  25. 25

    Buck, M.J. & Lieb, J.D. ChIP-chip: considerations for the design, analysis, and application of genome-wide chromatin immunoprecipitation experiments. Genomics 83, 349–360 (2004).

  26. 26

    Impey, S. et al. Defining the CREB regulon: a genome-wide analysis of transcription factor regulatory regions. Cell 119, 1041–1054 (2004).

  27. 27

    Kim, J., Bhinge, A.A., Morgan, X.C. & Iyer, V.R. Mapping DNA-protein interactions in large genomes by sequence tag analysis of genomic enrichment. Nat. Methods 2, 47–53 (2005).

  28. 28

    Rodda, D.J. et al. Transcriptional regulation of nanog by OCT4 and SOX2. J. Biol. Chem. 280, 24731–24737 (2005).

  29. 29

    Kuroda, T. et al. Octamer and Sox elements are required for transcriptional cis regulation of Nanog gene expression. Mol. Cell. Biol. 25, 2475–2485 (2005).

  30. 30

    Okumura-Nakanishi, S., Saito, M., Niwa, H. & Ishikawa, F. Oct-3/4 and Sox2 regulate Oct-3/4 gene in embryonic stem cells. J. Biol. Chem. 280, 5307–5317 (2005).

  31. 31

    Ambrosetti, D.C., Scholer, H.R., Dailey, L. & Basilico, C. Modulation of the activity of multiple transcriptional activation domains by the DNA binding domains mediates the synergistic action of Sox2 and Oct-3 on the fibroblast growth factor-4 enhancer. J. Biol. Chem. 275, 23387–23397 (2000).

  32. 32

    Tokuzawa, Y. et al. Fbx15 is a novel target of Oct3/4 but is dispensable for embryonic stem cell self-renewal and mouse development. Mol. Cell. Biol. 23, 2699–2708 (2003).

  33. 33

    Nishimoto, M., Fukushima, A., Okuda, A. & Muramatsu, M. The gene for the embryonic stem cell coactivator UTF1 carries a regulatory element which selectively interacts with a complex composed of Oct-3/4 and Sox-2. Mol. Cell. Biol. 19, 5453–5465 (1999).

  34. 34

    Kornberg, T.B. Understanding the homeodomain. J. Biol. Chem. 268, 26813–26816 (1993).

  35. 35

    Affolter, M., Schier, A. & Gehring, W.J. Homeodomain proteins and the regulation of gene expression. Curr. Opin. Cell Biol. 2, 485–495 (1990).

  36. 36

    Brandenberger, R. et al. MPSS profiling of human embryonic stem cells. BMC Dev. Biol. 4, 10 (2004).

  37. 37

    Wei, C.L. 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).

  38. 38

    Martone, R. et al. Distribution of NF-kappaB-binding sites across human chromosome 22. Proc. Natl. Acad. Sci. USA 100, 12247–12252 (2003).

  39. 39

    Hanna, L.A., Foreman, R.K., Tarasenko, I.A., Kessler, D.S. & Labosky, P.A. Requirement for Foxd3 in maintaining pluripotent cells of the early mouse embryo. Genes Dev. 16, 2650–2661 (2002).

  40. 40

    Guo, Y. et al. The embryonic stem cell transcription factors Oct-4 and FoxD3 interact to regulate endodermal-specific promoter expression. Proc. Natl. Acad. Sci. USA 99, 3663–3667 (2002).

  41. 41

    Dodge, J.E., Kang, Y.K., Beppu, H., Lei, H. & Li, E. Histone H3–K9 methyltransferase ESET is essential for early development. Mol. Cell. Biol. 24, 2478–2486 (2004).

  42. 42

    Luo, J. et al. Placental abnormalities in mouse embryos lacking the orphan nuclear receptor ERR-beta. Nature 388, 778–782 (1997).

  43. 43

    Mitsunaga, K. et al. Loss of PGC-specific expression of the orphan nuclear receptor ERR-beta results in reduction of germ cell number in mouse embryos. Mech. Dev. 121, 237–246 (2004).

  44. 44

    Adams, I.R. & McLaren, A. Identification and characterisation of mRif1: a mouse telomere-associated protein highly expressed in germ cells and embryo-derived pluripotent stem cells. Dev. Dyn. 229, 733–744 (2004).

  45. 45

    Xu, L. & Blackburn, E.H. Human Rif1 protein binds aberrant telomeres and aligns along anaphase midzone microtubules. J. Cell Biol. 167, 819–830 (2004).

  46. 46

    Silverman, J., Takai, H., Buonomo, S.B., Eisenhaber, F. & de Lange, T. Human Rif1, ortholog of a yeast telomeric protein, is regulated by ATM and 53BP1 and functions in the S-phase checkpoint. Genes Dev. 18, 2108–2119 (2004).

Download references

Acknowledgements

We are grateful to the Biomedical Research Council (BMRC) and Agency for Science, Technology and Research (A*STAR) for funding. Y.-H.L is supported by the A*STAR graduate scholarship. J.-L.C is supported by the Singapore Millennium Foundation graduate scholarship. W.Z. and X.C. are supported by the National University of Singapore graduate scholarship. B.L. is partially supported by grants from the US National Institutes of Health (DK47636 and AI54973). We thank E. Cheung, T. Lufkin, N. Clarke, C.-A. Lim, P. Melamed and J. Buhlman for critical comments on the manuscript. We are grateful to E. Ng, A. Ang and Y.-C. Chong for assistance in annotating the binding sites.

Author information

Correspondence to Yijun Ruan or Huck-Hui Ng.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Figure 1

Validation of Oct4 ChIP-PET data by real-time PCR. (PDF 26 kb)

Supplementary Figure 2

Profiles of Oct4 binding as shown by by ChIP-PET. (PDF 38 kb)

Supplementary Figure 3

Validation of Nanog ChIP-PET data by real-time PCR. (PDF 27 kb)

Supplementary Figure 4

Validation of ChIP-PET data with epitope-tagged Nanog. (PDF 27 kb)

Supplementary Figure 5

Validation of Nanog binding profiles at Pou5f1, Sox2 and Nanog upstream regulatory regions. (PDF 57 kb)

Supplementary Figure 6

Co-occupancies of Oct4 and Sox2 on target sites. (PDF 38 kb)

Supplementary Figure 7

Binding of Nanog to DNA containing CATT motifs. (PDF 37 kb)

Supplementary Figure 8

Rescue experiments demonstrate the specificity of the Pou5f1 RNAi results. (PDF 69 kb)

Supplementary Figure 9

Specificity of Nanog siRNA. (PDF 107 kb)

Supplementary Figure 10

Locations of ChIP-PET clusters relative to genes that are differentially expressed after Pou5f1 or Nanog RNAi knockdown. (PDF 23 kb)

Supplementary Figure 11

Characterization of Nanog-overexpressing ES cell line. (PDF 100 kb)

Supplementary Figure 12

ES cells expressing scrambled Esrrb or Rif1 siRNA sequences retained non-differentiated cell morphology. (PDF 63 kb)

Supplementary Table 1

Coordinates of loci for validation of Oct4 binding. (XLS 41 kb)

Supplementary Table 2

Coordinates of Oct4 and Nanog binding loci and their associated genes. (XLS 2890 kb)

Supplementary Table 3

Common genes that are bound by both Oct4 and Nanog. (XLS 180 kb)

Supplementary Table 4

Differentiation profiles of ES cells (data set for Fig. 5). (XLS 9944 kb)

Supplementary Table 5

Differentially expressed genes after Pou5f1 or Nanog RNAi (data sets for Figs. 6a,b). (XLS 2096 kb)

Supplementary Table 6

List of differentially expressed genes bound by Oct4 or Nanog (data set for Fig. 6c). (XLS 266 kb)

Supplementary Table 7

Mouse and human targets: location comparison. (XLS 255 kb)

Supplementary Note (PDF 306 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Loh, Y., Wu, Q., Chew, J. et al. The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nat Genet 38, 431–440 (2006). https://doi.org/10.1038/ng1760

Download citation

Further reading