Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

A genome-wide RNAi screen reveals determinants of human embryonic stem cell identity

Abstract

The derivation of human ES cells (hESCs) from human blastocysts represents one of the milestones in stem cell biology1. The full potential of hESCs in research and clinical applications requires a detailed understanding of the genetic network that governs the unique properties of hESCs. Here, we report a genome-wide RNA interference screen to identify genes which regulate self-renewal and pluripotency properties in hESCs. Interestingly, functionally distinct complexes involved in transcriptional regulation and chromatin remodelling are among the factors identified in the screen. To understand the roles of these potential regulators of hESCs, we studied transcription factor PRDM14 to gain new insights into its functional roles in the regulation of pluripotency. We showed that PRDM14 regulates directly the expression of key pluripotency gene POU5F1 through its proximal enhancer. Genome-wide location profiling experiments revealed that PRDM14 colocalized extensively with other key transcription factors such as OCT4, NANOG and SOX2, indicating that PRDM14 is integrated into the core transcriptional regulatory network. More importantly, in a gain-of-function assay, we showed that PRDM14 is able to enhance the efficiency of reprogramming of human fibroblasts in conjunction with OCT4, SOX2 and KLF4. Altogether, our study uncovers a wealth of novel hESC regulators wherein PRDM14 exemplifies a key transcription factor required for the maintenance of hESC identity and the reacquisition of pluripotency in human somatic cells.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Genome-wide screen for regulators that maintain hESC identity.
Figure 2: Pathway analyses.
Figure 3: PRDM14 and NFRKB can enhance reprogramming of human fibroblasts to iPSCs.
Figure 4: PRDM14 is integrated into the core hESC transcriptional regulatory network.

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

Microarray and ChIP-seq data are deposited at the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) under accession numbers GSE22792, GSE22795 and GSE22767.

References

  1. Thomson, J. A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998)

    ADS  CAS  Article  PubMed  Google Scholar 

  2. Evans, M. J. & Kaufman, M. H. Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154–156 (1981)

    ADS  CAS  Article  PubMed  Google Scholar 

  3. Martin, G. R. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc. Natl Acad. Sci. USA 78, 7634–7638 (1981)

    ADS  CAS  Article  PubMed  PubMed Central  Google Scholar 

  4. Yu, J. & Thomson, J. A. Pluripotent stem cell lines. Genes Dev. 22, 1987–1997 (2008)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  5. Van Hoof, D. et al. A quest for human and mouse embryonic stem cell-specific proteins. Mol. Cell. Proteomics 5, 1261–1273 (2006)

    CAS  Article  PubMed  Google Scholar 

  6. Pera, M. F. & Trounson, A. O. Human embryonic stem cells: prospects for development. Development 131, 5515–5525 (2004)

    CAS  Article  PubMed  Google Scholar 

  7. Brons, I. G. et al. Derivation of pluripotent epiblast stem cells from mammalian embryos. Nature 448, 191–195 (2007)

    ADS  CAS  Article  PubMed  Google Scholar 

  8. Tesar, P. J. et al. New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature 448, 196–199 (2007)

    ADS  CAS  Article  PubMed  Google Scholar 

  9. Joshi-Tope, G. et al. Reactome: a knowledgebase of biological pathways. Nucleic Acids Res. 33, D428–D432 (2005)

    CAS  Article  PubMed  Google Scholar 

  10. Conaway, R. C. & Conaway, J. W. The INO80 chromatin remodeling complex in transcription, replication and repair. Trends Biochem. Sci. 34, 71–77 (2009)

    CAS  Article  PubMed  Google Scholar 

  11. Casamassimi, A. & Napoli, C. Mediator complexes and eukaryotic transcription regulation: an overview. Biochimie 89, 1439–1446 (2007)

    CAS  Article  PubMed  Google Scholar 

  12. Chamovitz, D. A. Revisiting the COP9 signalosome as a transcriptional regulator. EMBO Rep. 10, 352–358 (2009)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. Albright, S. R. & Tjian, R. TAFs revisited: more data reveal new twists and confirm old ideas. Gene 242, 1–13 (2000)

    CAS  Article  PubMed  Google Scholar 

  14. Jackson, R. J., Hellen, C. U. & Pestova, T. V. The mechanism of eukaryotic translation initiation and principles of its regulation. Nature Rev. Mol. Cell Biol. 11, 113–127 (2010)

    CAS  Article  Google Scholar 

  15. Rino, J. & Carmo-Fonseca, M. The spliceosome: a self-organized macromolecular machine in the nucleus? Trends Cell Biol. 19, 375–384 (2009)

    CAS  Article  PubMed  Google Scholar 

  16. Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007)

    CAS  Article  PubMed  Google Scholar 

  17. Park, I. H. et al. Reprogramming of human somatic cells to pluripotency with defined factors. Nature 451, 141–146 (2008)

    ADS  CAS  Article  PubMed  Google Scholar 

  18. Samavarchi-Tehrani, P. et al. Functional genomics reveals a BMP-driven mesenchymal-to-epithelial transition in the initiation of somatic cell reprogramming. Cell Stem Cell 7, 64–77 (2010)

    CAS  Article  PubMed  Google Scholar 

  19. Assou, S. et al. A meta-analysis of human embryonic stem cells transcriptome integrated into a web-based expression atlas. Stem Cells 25, 961–973 (2007)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  20. Boyer, L. A. et al. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 122, 947–956 (2005)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. Tsuneyoshi, N. et al. PRDM14 suppresses expression of differentiation marker genes in human embryonic stem cells. Biochem. Biophys. Res. Commun. 367, 899–905 (2008)

    CAS  Article  PubMed  Google Scholar 

  22. Yamaji, M. et al. Critical function of Prdm14 for the establishment of the germ cell lineage in mice. Nature Genet. 40, 1016–1022 (2008)

    CAS  Article  PubMed  Google Scholar 

  23. Sharov, A. A. & Ko, M. S. Exhaustive search for over-represented DNA sequence motifs with CisFinder. DNA Res. 16, 261–273 (2009)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. Hanna, J. et al. Metastable pluripotent states in NOD-mouse-derived ESCs. Cell Stem Cell 4, 513–524 (2009)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. Nordhoff, V. et al. Comparative analysis of human, bovine, and murine Oct-4 upstream promoter sequences. Mamm. Genome 12, 309–317 (2001)

    CAS  Article  PubMed  Google Scholar 

  26. Yeom, Y. I. et al. Germline regulatory element of Oct-4 specific for the totipotent cycle of embryonal cells. Development 122, 881–894 (1996)

    CAS  PubMed  Google Scholar 

  27. Ivanova, N. et al. Dissecting self-renewal in stem cells with RNA interference. Nature 442, 533–538 (2006)

    ADS  CAS  Article  PubMed  Google Scholar 

  28. Fazzio, T. G., Huff, J. T. & Panning, B. An RNAi screen of chromatin proteins identifies Tip60-p400 as a regulator of embryonic stem cell identity. Cell 134, 162–174 (2008)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. Ding, L. et al. A genome-scale RNAi screen for Oct4 modulators defines a role of the Paf1 complex for embryonic stem cell identity. Cell Stem Cell 4, 403–415 (2009)

    CAS  Article  PubMed  Google Scholar 

  30. Hu, G. et al. A genome-wide RNAi screen identifies a new transcriptional module required for self-renewal. Genes Dev. 23, 837–848 (2009)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We are grateful to the Biomedical Research Council (BMRC), Agency for Science, Technology and Research (A*STAR) and Singapore Stem Cell Consortium for funding. We are grateful to K. Kuay, L.-P. Yaw, C.-K. Tong and C.-W. Chang for technical assistance. We acknowledge V. Cacheux-Rataboul for karyotype analysis and the GTB group for sequencing. We are grateful to A. Surani, P. Tesar and R. Mckay for gift of EpiSCs and Q. Yu for plasmids. We thank A. Colman, A. Hutchins and T. Huber for comments on the manuscript.

Author information

Authors and Affiliations

Authors

Contributions

N.-Y.C. conducted the genetic screen, generated the POU5F1–GFP line and performed the secondary screens. ChIP experiments and EMSA were conducted by Y.-S.C. and M.-S.L. Reprogramming experiments were done by B.F. and L.Y. Luciferase experiments and target validations were carried out by X.L. Bioinformatics analyses were performed by Y.L.O., P.K., M.H. and N.D.C.; D.M. printed the siRNA plates. P.K. and T.L. supported the in vivo mouse work. B.-S.S. and P.L. generated the EF1GFP reporter cells. H.-H.N., F.B. and N.-Y.C. wrote the manuscript with contributions from Y.-S.C., B.F., B.L. and J.J.; N.-Y.C., H.-H.N. and F.B. designed the experiments.

Corresponding authors

Correspondence to Frederic Bard or Huck-Hui Ng.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-22 with legends, Supplementary Methods, Supplementary Discussions 1-2 and additional references. (PDF 9436 kb)

Supplementary Table 1

Gene list sorted by Fav score. F1: z-score of GFP fluorescence change for replicate 1, F2: z-score of GFP fluorescence change for replicate 2, Fav: average z-score of the GFP fluorescence change of the duplicates. (XLS 4897 kb)

Supplementary Table 2

Gene list sorted by Nav score. N1: z-score of nucleic number change for replicate 1, N2: z-score of nuclei number change for replicate 2, Fav: average z-score of the nuclei number change of the duplicates. (XLS 4897 kb)

Supplementary Table 3

Table of genes for the enriched categories obtained from Reactome analysis. The genes identified in the functional categories as shown in Figure 2a can also be found in this excel file. (XLS 39 kb)

Supplementary Table 4

Secondary screen data: Deconvoluted siRNA screen data for the 200 genes in all three hESC lines. The sequences for the 800 siRNAs can also be found in this excel file. (XLS 277 kb)

Supplementary Table 5

This table contains a gene list of positive hits scored by all the different stemness markers of assessment for each of the three hESCs lines (Supplementary fig. 5b). (XLS 34 kb)

Supplementary Table 6

This table contains a gene list of consolidated positive hits identified by OCT4 reduction in all three hESC lines (Supplementary fig. 5c). (XLS 21 kb)

Supplementary Table 7

This table contains a Gene list of consolidated positive hits identified by NANOG reduction in all three hESC lines (Supplementary fig. 5c) (XLS 18 kb)

Supplementary Table 8

Counter-screens: Gene list of positive hits scored by EF1a-GFP, b-ACTIN or GAPDH (Supplementary fig. 6) (XLS 85 kb)

Supplementary Table 9

Binding sites of PRDM14 (Coordinates 7,002 ChIP-seq binding peaks defined by MACS). (XLS 459 kb)

Supplementary Table 10

This table contains genes associated with PRDM14 bound sites (2,755 RefSeq genes and coordinates of nearest PRDM14 ChIP-seq peak). (XLS 493 kb)

Supplementary Table 11

This table contains genes activated by PRDM14 (Genes that are associated with PRDM14 binding (see Supplementary table 10) and defined as down-regulated at 3 days after PRDM14 knockdown). (XLS 44 kb)

Supplementary Table 12

This table contains genes repressed by PRDM14 (Genes that are associated with PRDM14 binding (see Supplementary table 10) and defined as up-regulated at 3 days after PRDM14 knockdown). (XLS 67 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Chia, NY., Chan, YS., Feng, B. et al. A genome-wide RNAi screen reveals determinants of human embryonic stem cell identity. Nature 468, 316–320 (2010). https://doi.org/10.1038/nature09531

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature09531

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing