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

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 13. 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.

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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)

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Supplementary Movie 5

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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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