A protein interaction network for pluripotency of embryonic stem cells

Abstract

Embryonic stem (ES) cells are pluripotent1,2 and of therapeutic potential in regenerative medicine3,4. Understanding pluripotency at the molecular level should illuminate fundamental properties of stem cells and the process of cellular reprogramming. Through cell fusion the embryonic cell phenotype can be imposed on somatic cells, a process promoted by the homeodomain protein Nanog5, which is central to the maintenance of ES cell pluripotency6,7. Nanog is thought to function in concert with other factors such as Oct4 (ref. 8) and Sox2 (ref. 9) to establish ES cell identity. Here we explore the protein network in which Nanog operates in mouse ES cells. Using affinity purification of Nanog under native conditions followed by mass spectrometry, we have identified physically associated proteins. In an iterative fashion we also identified partners of several Nanog-associated proteins (including Oct4), validated the functional relevance of selected newly identified components and constructed a protein interaction network. The network is highly enriched for nuclear factors that are individually critical for maintenance of the ES cell state and co-regulated on differentiation. The network is linked to multiple co-repressor pathways and is composed of numerous proteins whose encoding genes are putative direct transcriptional targets of its members. This tight protein network seems to function as a cellular module dedicated to pluripotency.

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Figure 1: Protein complexes containing Nanog protein in mouse ES cells.
Figure 2: Confirmation of Nanog association by co-immunoprecipitation in ES cells.
Figure 3: Functional validation by RNA-mediated interference.
Figure 4: A protein interaction network in ES cells.

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Acknowledgements

We thank A. Cantor for biotin tagging constructs and for advice on affinity purification and chromatography; Y. Fujiwara and M. Kaku for technical assistance and for Oct4–GFP ES cells; T. De Lange for Rif1 antibody; S. Mackler for Nac1 antibody; S. Lowe and S. Elledge for shRNA vectors; M. Vidal and A.-L. Barabasi for advice and discussion on networks; and R. Tomaino and S. Gygi for performing LC–MS/MS and for providing advice and assistance in data collection and analysis. S.H.O. is an Investigator of the HHMI. Author Contributions S.R. and J.C. contributed equally to this study. J.W. and S.H.O. conceived and initiated the study. J.W., J.C., X.S., D.N.L. and T.W.T. performed the experiments. S.R. and J.W. analysed data and bioinformatics of the network. J.W. and S.H.O. wrote the manuscript.

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Correspondence to Stuart H. Orkin.

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Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.

Supplementary information

Supplementary Notes

This file contains Supplementary Methods, Supplementary Figure Legends and Supplementary Tables 1–3. (DOC 207 kb)

Supplementary Figures

This file contains Supplementary Figures 1–8. (PDF 1871 kb)

Supplementary Data 1

Common background proteins in BirA samples identified by MS. (XLS 1469 kb)

Supplementary Data 2

List of all specific proteins identified by MS from bioNanog samples (XLS 52 kb)

Supplementary Data 3

A list of the key genes used in this study. (DOC 19 kb)

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Wang, J., Rao, S., Chu, J. et al. A protein interaction network for pluripotency of embryonic stem cells. Nature 444, 364–368 (2006). https://doi.org/10.1038/nature05284

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