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Chromatin signatures of pluripotent cell lines


Epigenetic genome modifications are thought to be important for specifying the lineage and developmental stage of cells within a multicellular organism. Here, we show that the epigenetic profile of pluripotent embryonic stem cells (ES) is distinct from that of embryonic carcinoma cells, haematopoietic stem cells (HSC) and their differentiated progeny. Silent, lineage-specific genes replicated earlier in pluripotent cells than in tissue-specific stem cells or differentiated cells and had unexpectedly high levels of acetylated H3K9 and methylated H3K4. Unusually, in ES cells these markers of open chromatin were also combined with H3K27 trimethylation at some non-expressed genes. Thus, pluripotency of ES cells is characterized by a specific epigenetic profile where lineage-specific genes may be accessible but, if so, carry repressive H3K27 trimethylation modifications. H3K27 methylation is functionally important for preventing expression of these genes in ES cells as premature expression occurs in embryonic ectoderm development (Eed)-deficient ES cells. Our data suggest that lineage-specific genes are primed for expression in ES cells but are held in check by opposing chromatin modifications.

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Figure 1: Pluripotent ES cells, multipotent HSC and unipotent T lymphocytes have distinct replication timing profiles.
Figure 2: Replication timing profiles of ES and embryonic carcinoma (EC) cells reflect their distinct lineage potential.
Figure 3: Markers of active and repressed chromatin are simultaneously present at silent tissue-specific genes in undifferentiated ES cells.
Figure 4: Eed is required for repressing neural-specific gene expression in undifferentiated ES cells.
Figure 5: Replication timing is not significantly altered in Eed-deficient ES cells despite gene derepression.


  1. Terskikh, A. V. et al. From hematopoiesis to neuropoiesis: evidence of overlapping genetic programs. Proc. Natl Acad. Sci. USA 98, 7934–7939 (2001).

    CAS  Article  Google Scholar 

  2. Ivanova, N. B. et al. A stem cell molecular signature. Science 298, 601–604 (2002).

    CAS  Article  Google Scholar 

  3. Ramalho-Santos, M., Yoon, S., Matsuzaki, Y., Mulligan, R. C. & Melton, D. A. “Stemness”: transcriptional profiling of embryonic and adult stem cells. Science 298, 597–600 (2002).

    CAS  Article  Google Scholar 

  4. Fortunel, N. O. et al. Comment on “'Stemness': transcriptional profiling of embryonic and adult stem cells” and “a stem cell molecular signature”. Science 302, 393 (2003).

    CAS  Article  Google Scholar 

  5. Evsikov, A. V. & Solter, D. Comment on “'Stemness': transcriptional profiling of embryonic and adult stem cells” and “a stem cell molecular signature”. Science 302, 393 (2003).

    CAS  Article  Google Scholar 

  6. Vogel, G. Stem cells. 'Stemness' genes still elusive. Science 302, 371 (2003).

    CAS  Article  Google Scholar 

  7. Bernstein, B. E. et al. Genomic maps and comparative analysis of histone modifications in human and mouse. Cell 120, 169–181 (2005).

    CAS  Article  Google Scholar 

  8. Schubeler, D. et al. The histone modification pattern of active genes revealed through genome-wide chromatin analysis of a higher eukaryote. Genes Dev. 18, 1263–1271 (2004).

    Article  Google Scholar 

  9. Woodfine, K. et al. Replication timing of the human genome. Hum. Mol. Genet. 13, 191–202 (2004).

    CAS  Article  Google Scholar 

  10. Perry, P. et al. A dynamic switch in the replication timing of key regulator genes in embryonic stem cells upon neural induction. Cell Cycle 3, 1645–1650 (2004).

    CAS  Article  Google Scholar 

  11. Hiratani, I., Leskovar, A. & Gilbert, D. M. Differentiation-induced replication-timing changes are restricted to AT-rich/long interspersed nuclear element (LINE)-rich isochores. Proc. Natl Acad. Sci. USA 101, 16861–16866 (2004).

    CAS  Article  Google Scholar 

  12. Gilbert, N. et al. Chromatin architecture of the human genome: gene-rich domains are enriched in open chromatin fibers. Cell 118, 555–566 (2004).

    CAS  Article  Google Scholar 

  13. Rugg-Gunn, P. J., Ferguson-Smith, A. C. & Pedersen, R. A. Epigenetic status of human embryonic stem cells. Nature Genet. 37, 585–587 (2005).

    CAS  Article  Google Scholar 

  14. Evans, M. & Hunter, S. Source and nature of embryonic stem cells. C. R. Biol. 325, 1003–1007 (2002).

    CAS  Article  Google Scholar 

  15. Schubeler, D. et al. Genome-wide DNA replication profile for Drosophila melanogaster: a link between transcription and replication timing. Nature Genet. 32, 438–442 (2002).

    Article  Google Scholar 

  16. Azuara, V. et al. Heritable gene silencing in lymphocytes delays chromatid resolution without affecting the timing of DNA replication. Nature Cell Biol. 5, 668–674 (2003).

    CAS  Article  Google Scholar 

  17. Gilbert, D. M. In search of the holy replicator. Nature Rev. Mol. Cell Biol. 5, 848–855 (2004).

    CAS  Article  Google Scholar 

  18. Spooncer, E., Heyworth, C. M., Dunn, A. & Dexter, T. M. Self-renewal and differentiation of interleukin-3-dependent multipotent stem cells are modulated by stromal cells and serum factors. Differentiation 31, 111–118 (1986).

    CAS  Article  Google Scholar 

  19. Nutt, S. L., Heavey, B., Rolink, A. G. & Busslinger, M. Commitment to the B-lymphoid lineage depends on the transcription factor Pax5. Nature 401, 556–562 (1999).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  21. Hogan, B. L., Taylor, A. & Adamson, E. Cell interactions modulate embryonal carcinoma cell differentiation into parietal or visceral endoderm. Nature 291, 235–237 (1981).

    CAS  Article  Google Scholar 

  22. Jones-Villeneuve, E. M., Rudnicki, M. A., Harris, J. F. & McBurney, M. W. Retinoic acid-induced neural differentiation of embryonal carcinoma cells. Mol. Cell Biol. 3, 2271–2279 (1983).

    CAS  Article  Google Scholar 

  23. Williams, R. R. et al. Neural induction promotes large-scale chromatin reorganisation of the Mash1 locus. J. Cell Sci. 119, 132–140 (2006).

    CAS  Article  Google Scholar 

  24. Montgomery, N. D. et al. The murine polycomb group protein Eed is required for global histone H3 lysine-27 methylation. Curr. Biol. 15, 942–947 (2005).

    CAS  Article  Google Scholar 

  25. Peters, A. H. & Schubeler, D. Methylation of histones: playing memory with DNA. Curr. Opin. Cell Biol. 17, 230–238 (2005).

    CAS  Article  Google Scholar 

  26. Morin-Kensicki, E. M., Faust, C., LaMantia, C. & Magnuson, T. Cell and tissue requirements for the gene eed during mouse gastrulation and organogenesis. Genesis 31, 142–146 (2001).

    CAS  Article  Google Scholar 

  27. Faust, C., Lawson, K. A., Schork, N. J., Thiel, B. & Magnuson, T. The Polycomb-group gene eed is required for normal morphogenetic movements during gastrulation in the mouse embryo. Development 125, 4495–4506 (1998).

    CAS  PubMed  Google Scholar 

  28. Silva, J. et al. Establishment of histone H3 methylation on the inactive X chromosome requires transient recruitment of Eed—Enx1 polycomb group complexes. Dev. Cell 4, 481–495 (2003).

    CAS  Article  Google Scholar 

  29. Plath, K. et al. Role of histone H3 lysine 27 methylation in X inactivation. Science 300, 131–135 (2003).

    CAS  Article  Google Scholar 

  30. Vogelauer, M., Rubbi, L., Lucas, I., Brewer, B. J. & Grunstein, M. Histone acetylation regulates the time of replication origin firing. Mol. Cell 10, 1223–1233 (2002).

    CAS  Article  Google Scholar 

  31. Lin, C. M., Fu, H., Martinovsky, M., Bouhassira, E. & Aladjem, M. I. Dynamic alterations of replication timing in mammalian cells. Curr. Biol. 13, 1019–1028 (2003).

    CAS  Article  Google Scholar 

  32. Zhang, J., Xu, F., Hashimshony, T., Keshet, I. & Cedar, H. Establishment of transcriptional competence in early and late S phase. Nature 420, 198–202 (2002).

    CAS  Article  Google Scholar 

  33. Ballas, N. & Mandel, G. The many faces of REST oversee epigenetic programming of neuronal genes. Curr. Opin. Neurobiol. 15, 500–506 (2005).

    CAS  Article  Google Scholar 

  34. Hansen, R. S., Canfield, T. K., Lamb, M. M., Gartler, S. M. & Laird, C. D. Association of fragile X syndrome with delayed replication of the FMR1 gene. Cell 73, 1403–1409 (1993).

    CAS  Article  Google Scholar 

  35. Gomez, M. & Brockdorff, N. Heterochromatin on the inactive X chromosome delays replication timing without affecting origin usage. Proc. Natl Acad. Sci. USA 101, 6923–6928 (2004).

    CAS  Article  Google Scholar 

  36. Wang, J. et al. Imprinted X inactivation maintained by a mouse Polycomb group gene. Nature Genet. 28, 371–375 (2001).

    CAS  Article  Google Scholar 

  37. Vandesompele, J. et al. Accurate normalization of real-time quantitative RT—PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3, DOI: 10.1186/gb-2002-3-7-research0034 (2002).

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We thank A. Smith for ES cell lines, M. Dexter for the FDCP-mix A4 clone, M. Busslinger for Pax5-deficient pro-B cells, R. Lovell-Badge for the F9-EC line and V. Episkopou for the embryonic carcinoma cell lines F9 and P19. E. O'Connor, R. Brough, G. Reed and R. Newton are thanked for help and advice and A. Allen, M. Harrison, T. Reed and Z. Szarka (at Oxford Gene Technology, Oxford, UK). J. Santos and S. Giadrossi for communicating unpublished information. S. Giadrossi, N. Brockdorff and M. Raff are acknowledged for advice and critical reading of the manuscript. This work was supported by the Medical Research Council (MRC) and by a MRC Collaborative Career Development Fellowship in Stem Cell Research funded by the Parkinson's disease Society (V.A.).

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Correspondence to Véronique Azuara or Amanda G. Fisher.

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Supplementary Figures S1, S2, S3, S4 and Supplementary Methods (PDF 375 kb)

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Azuara, V., Perry, P., Sauer, S. et al. Chromatin signatures of pluripotent cell lines. Nat Cell Biol 8, 532–538 (2006).

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