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The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells

Abstract

Oct4 and Nanog are transcription factors required to maintain the pluripotency and self-renewal of embryonic stem (ES) cells. Using the chromatin immunoprecipitation paired-end ditags method, we mapped the binding sites of these factors in the mouse ES cell genome. We identified 1,083 and 3,006 high-confidence binding sites for Oct4 and Nanog, respectively. Comparative location analyses indicated that Oct4 and Nanog overlap substantially in their targets, and they are bound to genes in different configurations. Using de novo motif discovery algorithms, we defined the cis-acting elements mediating their respective binding to genomic sites. By integrating RNA interference–mediated depletion of Oct4 and Nanog with microarray expression profiling, we demonstrated that these factors can activate or suppress transcription. We further showed that common core downstream targets are important to keep ES cells from differentiating. The emerging picture is one in which Oct4 and Nanog control a cascade of pathways that are intricately connected to govern pluripotency, self-renewal, genome surveillance and cell fate determination.

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Figure 1: Schematic diagram of genome-wide mapping of Oct4 and Nanog binding sites using ChIP-PET.
Figure 2: Distribution of Oct4 and Nanog binding sites.
Figure 3: Oct4 and Nanog binding site configurations at genomic locations.
Figure 4: De novo prediction of motifs that mediate specific transcription factor–DNA interaction.
Figure 5: Genome-wide association of Oct4 and Nanog binding sites with differentiation profiles of mouse ES cells.
Figure 6: Genome-wide association of Oct4 and Nanog binding sites with expression profiles of mouse ES cells depleted of Oct4 or Nanog.
Figure 7: Regulation of pluripotency by downstream targets of Oct4 and Nanog.
Figure 8: Conserved and diverged Oct4 and Nanog circuitries of mouse and human ES cells.

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Acknowledgements

We are grateful to the Biomedical Research Council (BMRC) and Agency for Science, Technology and Research (A*STAR) for funding. Y.-H.L is supported by the A*STAR graduate scholarship. J.-L.C is supported by the Singapore Millennium Foundation graduate scholarship. W.Z. and X.C. are supported by the National University of Singapore graduate scholarship. B.L. is partially supported by grants from the US National Institutes of Health (DK47636 and AI54973). We thank E. Cheung, T. Lufkin, N. Clarke, C.-A. Lim, P. Melamed and J. Buhlman for critical comments on the manuscript. We are grateful to E. Ng, A. Ang and Y.-C. Chong for assistance in annotating the binding sites.

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Correspondence to Yijun Ruan or Huck-Hui Ng.

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Supplementary information

Supplementary Figure 1

Validation of Oct4 ChIP-PET data by real-time PCR. (PDF 26 kb)

Supplementary Figure 2

Profiles of Oct4 binding as shown by by ChIP-PET. (PDF 38 kb)

Supplementary Figure 3

Validation of Nanog ChIP-PET data by real-time PCR. (PDF 27 kb)

Supplementary Figure 4

Validation of ChIP-PET data with epitope-tagged Nanog. (PDF 27 kb)

Supplementary Figure 5

Validation of Nanog binding profiles at Pou5f1, Sox2 and Nanog upstream regulatory regions. (PDF 57 kb)

Supplementary Figure 6

Co-occupancies of Oct4 and Sox2 on target sites. (PDF 38 kb)

Supplementary Figure 7

Binding of Nanog to DNA containing CATT motifs. (PDF 37 kb)

Supplementary Figure 8

Rescue experiments demonstrate the specificity of the Pou5f1 RNAi results. (PDF 69 kb)

Supplementary Figure 9

Specificity of Nanog siRNA. (PDF 107 kb)

Supplementary Figure 10

Locations of ChIP-PET clusters relative to genes that are differentially expressed after Pou5f1 or Nanog RNAi knockdown. (PDF 23 kb)

Supplementary Figure 11

Characterization of Nanog-overexpressing ES cell line. (PDF 100 kb)

Supplementary Figure 12

ES cells expressing scrambled Esrrb or Rif1 siRNA sequences retained non-differentiated cell morphology. (PDF 63 kb)

Supplementary Table 1

Coordinates of loci for validation of Oct4 binding. (XLS 41 kb)

Supplementary Table 2

Coordinates of Oct4 and Nanog binding loci and their associated genes. (XLS 2890 kb)

Supplementary Table 3

Common genes that are bound by both Oct4 and Nanog. (XLS 180 kb)

Supplementary Table 4

Differentiation profiles of ES cells (data set for Fig. 5). (XLS 9944 kb)

Supplementary Table 5

Differentially expressed genes after Pou5f1 or Nanog RNAi (data sets for Figs. 6a,b). (XLS 2096 kb)

Supplementary Table 6

List of differentially expressed genes bound by Oct4 or Nanog (data set for Fig. 6c). (XLS 266 kb)

Supplementary Table 7

Mouse and human targets: location comparison. (XLS 255 kb)

Supplementary Note (PDF 306 kb)

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Loh, YH., Wu, Q., Chew, JL. et al. The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nat Genet 38, 431–440 (2006). https://doi.org/10.1038/ng1760

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