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Dissecting self-renewal in stem cells with RNA interference

Abstract

We present an integrated approach to identify genetic mechanisms that control self-renewal in mouse embryonic stem cells. We use short hairpin RNA (shRNA) loss-of-function techniques to downregulate a set of gene products whose expression patterns suggest self-renewal regulatory functions. We focus on transcriptional regulators and identify seven genes for which shRNA-mediated depletion negatively affects self-renewal, including four genes with previously unrecognized roles in self-renewal. Perturbations of these gene products are combined with dynamic, global analyses of gene expression. Our studies suggest specific biological roles for these molecules and reveal the complexity of cell fate regulation in embryonic stem cells.

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Figure 1: Identification of self-renewal regulators in ES cells.
Figure 2: Genetic complementation to rescue shRNA-induced self-renewal defects.
Figure 3: Global gene expression changes after downregulation of individual self-renewal regulators.
Figure 4: Nanog rescue of phenotypes caused by shRNA directed against other self-renewal regulators.
Figure 5: Provisional model of cell fate regulatory interactions in ES cells in vitro.

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Acknowledgements

We thank A. Smith for E14/T cells and the episomal expression system, G. Daley for Aniv15 cells, and D. Baltimore for the FUGW lentiviral expression system. We are grateful to K. A. Moore and members of the Lemischka laboratory for their assistance at various stages of these studies. We also thank A. Smith for constructive criticisms. This work was supported by funding from the NIDDK. Author Contributions N.I. and I.R.L. designed the experiments. N.I., R.L., I.K., J.L., C.D., X.S. and Y.L. performed the experiments. N.I., R.D. and I.R.L. analysed the data. N.I. and I.R.L. wrote the paper.

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Correspondence to Natalia Ivanova or Ihor R. Lemischka.

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

Supplementary information

Supplementary Notes

This file contains Supplementary Materials, Supplementary Tables 1–4, Supplementary Figures 1–18 and additional references. (PDF 2301 kb)

Supplementary Data 1

MAS5.0 normalized microarray data (ZIP 16494 kb)

Supplementary Data 2

MIAME description of microarray data (DOC 55 kb)

Supplementary Data 3

Description of microarray files (XLS 27 kb)

Supplementary Data 4

Non-redundant probes used in microarray analysis (XLS 3771 kb)

Supplementary Data 5

Probes affected at least in one shRNA time-course (XLS 503 kb)

Supplementary Data 6

901 probes down-regulated in RA differentiation time-course (XLS 732 kb)

Supplementary Data 7

Information for genes used in the over-expression studies (XLS 43 kb)

Supplementary Data 8

List of genes affected by Nanog shRNA (XLS 252 kb)

Supplementary Data 9

List of genes affected by Oct4 shRNA (XLS 189 kb)

Supplementary Data 10

List of genes affected by Sox2 shRNA (XLS 228 kb)

Supplementary Data 11

List of genes affected by Esrrb shRNA (XLS 149 kb)

Supplementary Data 12

List of genes affected by Tbx3 shRNA (XLS 136 kb)

Supplementary Data 13

List of genes affected by Tcl1 shRNA (XLS 100 kb)

Supplementary Data 14

List of genes affected by Mm.343880 shRNA (XLS 111 kb)

Supplementary Data 15

List of genes affected by Dppa4 shRNA (XLS 74 kb)

Supplementary Data 16

List of genes down-regulated during the Dppa4 shRNA time-course only. (XLS 20 kb)

Supplementary Data 17

List of genes down-regulated during the Mm.343880 shRNA time-course only. (XLS 17 kb)

Supplementary Data 18

List of genes up-regulated during the Mm.343880 shRNA time-course only. (XLS 11 kb)

Supplementary Data 19

List of genes down-regulated during the Nanog and Oct4 shRNA time-courses only. (XLS 13 kb)

Supplementary Data 20

List of genes down-regulated during the Nanog shRNA time-course only. (XLS 36 kb)

Supplementary Data 21

List of genes up-regulated during the Nanog shRNA time-course only. (XLS 25 kb)

Supplementary Data 22

List of genes down-regulated during the Oct4 shRNA time-course only. (XLS 21 kb)

Supplementary Data 23

List of genes up-regulated during the Oct4 shRNA time-course only. (XLS 20 kb)

Supplementary Data 24

List of genes up-regulated during the Oct4 and Sox2 shRNA time-courses only. (XLS 14 kb)

Supplementary Data 25

List of genes down-regulated in Pattern1 (XLS 27 kb)

Supplementary Data 26

List of genes up-regulated in Pattern1 (XLS 116 kb)

Supplementary Data 27

List of genes down-regulated in Pattern2 (XLS 48 kb)

Supplementary Data 28

List of genes up-regulated in Pattern2 (XLS 48 kb)

Supplementary Data 29

List of genes down-regulated in Pattern3 (XLS 15 kb)

Supplementary Data 30

List of genes up-regulated in Pattern3 (XLS 49 kb)

Supplementary Data 31

List of genes down-regulated during the Sox2 shRNA time-course only (XLS 16 kb)

Supplementary Data 32

List of genes up-regulated during the Sox2 shRNA time-course only (XLS 19 kb)

Supplementary Data 33

List of genes down-regulated during the Tbx3 shRNA time-course only (XLS 18 kb)

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Ivanova, N., Dobrin, R., Lu, R. et al. Dissecting self-renewal in stem cells with RNA interference. Nature 442, 533–538 (2006). https://doi.org/10.1038/nature04915

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