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
Open Access articles citing this article.
Nature Communications Open Access 10 January 2022
Experimental & Molecular Medicine Open Access 11 June 2021
Nature Communications Open Access 12 March 2021
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Thomson, J. A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998)
Evans, M. J. & Kaufman, M. H. Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154–156 (1981)
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)
Yu, J. & Thomson, J. A. Pluripotent stem cell lines. Genes Dev. 22, 1987–1997 (2008)
Van Hoof, D. et al. A quest for human and mouse embryonic stem cell-specific proteins. Mol. Cell. Proteomics 5, 1261–1273 (2006)
Pera, M. F. & Trounson, A. O. Human embryonic stem cells: prospects for development. Development 131, 5515–5525 (2004)
Brons, I. G. et al. Derivation of pluripotent epiblast stem cells from mammalian embryos. Nature 448, 191–195 (2007)
Tesar, P. J. et al. New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature 448, 196–199 (2007)
Joshi-Tope, G. et al. Reactome: a knowledgebase of biological pathways. Nucleic Acids Res. 33, D428–D432 (2005)
Conaway, R. C. & Conaway, J. W. The INO80 chromatin remodeling complex in transcription, replication and repair. Trends Biochem. Sci. 34, 71–77 (2009)
Casamassimi, A. & Napoli, C. Mediator complexes and eukaryotic transcription regulation: an overview. Biochimie 89, 1439–1446 (2007)
Chamovitz, D. A. Revisiting the COP9 signalosome as a transcriptional regulator. EMBO Rep. 10, 352–358 (2009)
Albright, S. R. & Tjian, R. TAFs revisited: more data reveal new twists and confirm old ideas. Gene 242, 1–13 (2000)
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)
Rino, J. & Carmo-Fonseca, M. The spliceosome: a self-organized macromolecular machine in the nucleus? Trends Cell Biol. 19, 375–384 (2009)
Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007)
Park, I. H. et al. Reprogramming of human somatic cells to pluripotency with defined factors. Nature 451, 141–146 (2008)
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)
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)
Boyer, L. A. et al. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 122, 947–956 (2005)
Tsuneyoshi, N. et al. PRDM14 suppresses expression of differentiation marker genes in human embryonic stem cells. Biochem. Biophys. Res. Commun. 367, 899–905 (2008)
Yamaji, M. et al. Critical function of Prdm14 for the establishment of the germ cell lineage in mice. Nature Genet. 40, 1016–1022 (2008)
Sharov, A. A. & Ko, M. S. Exhaustive search for over-represented DNA sequence motifs with CisFinder. DNA Res. 16, 261–273 (2009)
Hanna, J. et al. Metastable pluripotent states in NOD-mouse-derived ESCs. Cell Stem Cell 4, 513–524 (2009)
Nordhoff, V. et al. Comparative analysis of human, bovine, and murine Oct-4 upstream promoter sequences. Mamm. Genome 12, 309–317 (2001)
Yeom, Y. I. et al. Germline regulatory element of Oct-4 specific for the totipotent cycle of embryonal cells. Development 122, 881–894 (1996)
Ivanova, N. et al. Dissecting self-renewal in stem cells with RNA interference. Nature 442, 533–538 (2006)
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)
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)
Hu, G. et al. A genome-wide RNAi screen identifies a new transcriptional module required for self-renewal. Genes Dev. 23, 837–848 (2009)
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.
The authors declare no competing financial interests.
This file contains Supplementary Figures 1-22 with legends, Supplementary Methods, Supplementary Discussions 1-2 and additional references. (PDF 9436 kb)
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)
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)
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)
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)
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)
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)
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)
Counter-screens: Gene list of positive hits scored by EF1a-GFP, b-ACTIN or GAPDH (Supplementary fig. 6) (XLS 85 kb)
Binding sites of PRDM14 (Coordinates 7,002 ChIP-seq binding peaks defined by MACS). (XLS 459 kb)
This table contains genes associated with PRDM14 bound sites (2,755 RefSeq genes and coordinates of nearest PRDM14 ChIP-seq peak). (XLS 493 kb)
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)
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)
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
This article is cited by
Nature Communications (2022)
Nature Communications (2021)
Nature Materials (2021)
Experimental & Molecular Medicine (2021)
Nature Chemical Biology (2021)