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Systems-level dynamic analyses of fate change in murine embryonic stem cells

Nature volume 462, pages 358–362 (2009)Cite this article

Abstract

Molecular regulation of embryonic stem cell (ESC) fate involves a coordinated interaction between epigenetic1,2,3,4, transcriptional5,6,7,8,9,10 and translational11,12 mechanisms. It is unclear how these different molecular regulatory mechanisms interact to regulate changes in stem cell fate. Here we present a dynamic systems-level study of cell fate change in murine ESCs following a well-defined perturbation. Global changes in histone acetylation, chromatin-bound RNA polymerase II, messenger RNA (mRNA), and nuclear protein levels were measured over 5 days after downregulation of Nanog, a key pluripotency regulator13,14,15. Our data demonstrate how a single genetic perturbation leads to progressive widespread changes in several molecular regulatory layers, and provide a dynamic view of information flow in the epigenome, transcriptome and proteome. We observe that a large proportion of changes in nuclear protein levels are not accompanied by concordant changes in the expression of corresponding mRNAs, indicating important roles for translational and post-translational regulation of ESC fate. Gene-ontology analysis across different molecular layers indicates that although chromatin reconfiguration is important for altering cell fate, it is preceded by transcription-factor-mediated regulatory events. The temporal order of gene expression alterations shows the order of the regulatory network reconfiguration and offers further insight into the gene regulatory network. Our studies extend the conventional systems biology approach to include many molecular species, regulatory layers and temporal series, and underscore the complexity of the multilayer regulatory mechanisms responsible for changes in protein expression that determine stem cell fate.

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Figure 1: Measuring changes in the epigenome, the transcriptome and the nuclear proteome after Nanog downregulation.
Figure 2: Comparisons across different molecular regulatory layers.
Figure 3: Dynamic changes in ESC networks.
Figure 4: Interactive visualization of the multilayer dynamic data.

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Acknowledgements

We would like to thank D. Storton for technical support, and E. Wieschaus, Y. Shi, S. Tavazoie and N. Slavov for constructive discussions. We also acknowledge the laboratories of the following people for providing antibodies for western blot: A. Okuda, J. Flint and Y. Kang. This work was supported by the NIH, and in part supported by the BBSRC and Leukaemia Research UK. O.G.T., F.M. and E.M.A. were partially supported by the NIH and US National Science Foundation.

Author Contributions R.L. and I.R.L. designed the experiments. R.L. prepared the cell samples for all the experiments, performed the RNA polymerase II ChIP-chip, the mRNA microarray, and verification experiments such as western blot, ChIP and quantitative PCR. R.D.U. and A.D.W. performed the proteomic experiments and primary analysis on proteomic data. L.A.B. performed the histone acetylation ChIP-chip experiments. R.L., F.M., E.M.A., R.R. and O.G.T. performed general data processing and statistical analyses. R.L. and F.M. plotted Figs 1– 3. A.L., B.D.M. and A.M. developed and plotted interactive Fig. 4a. A.L. and A.M. developed and plotted interactive Fig. 4b. R.L., J.L., F.M. and I.R.L performed network analysis shown in Fig. 3. R.L. and I.R.L. wrote the paper.

Author information

Author notes

  1. Rong Lu, Florian Markowetz, Jeffrey T. Leek, Edoardo M. Airoldi & Roye Rozov

    Present address: Present addresses: Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Beckman Center B261, 279 Campus Drive, Stanford, California 94305, USA (R.L.); Cancer Research UK, Cambridge Research Institute, Cambridge CB2 0RE, UK (F.M.); Johns Hopkins Bloomberg School of Public Health, Department of Biostatistics 615 North Wolfe Street, Baltimore, Maryland 21205, USA (J.T.L.); Department of Statistics, Harvard University, 1 Oxford Street, Cambridge, Massachusetts 02128, USA (E.M.A.); Blavatnik School of Computer Science, Tel Aviv University, 69978 Tel Aviv, Israel (R.R.).,

  2. Florian Markowetz and Richard D. Unwin: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular Biology,,

    Rong Lu & Ihor R. Lemischka

  2. Lewis-Sigler Institute for Integrative Genomics and Department of Computer Science, Princeton University, Princeton, New Jersey 08544, USA,

    Florian Markowetz, Edoardo M. Airoldi & Olga G. Troyanskaya

  3. Stem Cell and Leukaemia Proteomics Laboratory, School of Cancer and Imaging Sciences, Manchester Academic Health Science Centre, University of Manchester, Wolfson Molecular Imaging Centre, Manchester M20 4QL, UK

    Richard D. Unwin & Anthony D. Whetton

  4. Department of Gene and Cell Medicine and The Black Family Stem Cell Institute,,

    Jeffrey T. Leek, Ben D. MacArthur, Roye Rozov & Ihor R. Lemischka

  5. Department of Pharmacology and System Therapeutics and Systems Biology Center New York (SBCNY), Mount Sinai School of Medicine, New York, New York 10029, USA,

    Ben D. MacArthur, Alexander Lachmann & Avi Ma’ayan

  6. Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA,

    Laurie A. Boyer

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Corresponding authors

Correspondence to Rong Lu or Ihor R. Lemischka.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-12 with Legends, Supplementary Data, Legends for Supplementary Movies 1-7 and Supplementary References. Please note that Supplementary Figure 8 is an interactive version of Figure 4 in the main paper and requires Adobe Reader 9 or higher. Alternatively, see Supplementary Movies, files 1-7. This file was replaced on December 24 2009 to add the missing link for the Raw Data files as described on page 18. (PDF 7270 kb)

Supplementary Movie 1

This movie file is an interactive movie of figure 4 a in the main paper. (SWF 1404 kb)

Supplementary Movie 2

This movie file is an interactive movie of figure 4 b in the main paper. (SWF 707 kb)

Supplementary Movie 3

This movie file is an interactive movie of figure 4 b in the main paper. (SWF 706 kb)

Supplementary Movie 4

This movie file is an interactive movie of figure 4 b in the main paper. (SWF 707 kb)

Supplementary Movie 5

This movie file is an interactive movie of figure 4 b in the main paper. (SWF 718 kb)

Supplementary Movie 6

This movie file is an interactive movie of figure 4 b in the main paper. (SWF 719 kb)

Supplementary Movie 7

This movie file is an interactive movie of figure 4 b in the main paper. (SWF 718 kb)

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Lu, R., Markowetz, F., Unwin, R. et al. Systems-level dynamic analyses of fate change in murine embryonic stem cells. Nature 462, 358–362 (2009). https://doi.org/10.1038/nature08575

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