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.
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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.
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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 EF1–GFP 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.
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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)
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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
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DOI: https://doi.org/10.1038/nature09531
