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H3K9 methylation is a barrier during somatic cell reprogramming into iPSCs

Abstract

The induction of pluripotent stem cells (iPSCs) by defined factors is poorly understood stepwise. Here, we show that histone H3 lysine 9 (H3K9) methylation is the primary epigenetic determinant for the intermediate pre-iPSC state, and its removal leads to fully reprogrammed iPSCs. We generated a panel of stable pre-iPSCs that exhibit pluripotent properties but do not activate the core pluripotency network, although they remain sensitive to vitamin C for conversion into iPSCs. Bone morphogenetic proteins (BMPs) were subsequently identified in serum as critical signaling molecules in arresting reprogramming at the pre-iPSC state. Mechanistically, we identified H3K9 methyltransferases as downstream targets of BMPs and showed that they function with their corresponding demethylases as the on/off switch for the pre-iPSC fate by regulating H3K9 methylation status at the core pluripotency loci. Our results not only establish pre-iPSCs as an epigenetically stable signpost along the reprogramming road map, but they also provide mechanistic insights into the epigenetic reprogramming of cell fate.

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Figure 1: Generation and characterization of stable cell lines arrested at the pre-iPSC stage.
Figure 2: FBS and BMPs arrest reprogramming at the pre-iPSC stage.
Figure 3: The BMP signaling pathway is the major inhibitory signal in FBS for reprogramming.
Figure 4: Global profiling of BMP and FBS gene targets during reprogramming.
Figure 5: BMPs inhibit reprogramming through H3K9 methylation on core pluripotency loci.
Figure 6: Setdb1 is a direct downstream target of BMPs that blocks reprogramming at the pre-iPSC stage.
Figure 7: Kdm family H3K9 demethylases promote the further reprogramming of pre-iPSCs.

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Acknowledgements

We are grateful to X. Shu for careful reading and critical comments of the manuscript. We appreciate the contributions of J. Nie to microarray data analysis. We thank H. Zheng, X. Liu, J. Zhao, H. Wu, F. Li, D. Qin, R. Li, M. Esteban, G. Xu and B. Qin for constructive criticism. We also thank L. Zeng, G. Zhu, S. Sun, Y. Wu, K. Lai, H. Pang, H. Zhang, W. He, A. Jiang, T. Zhou, J. Li, S. Huang, J. Xu and D. Yang for technical assistance. We deeply appreciate the assistance of H. Song, K. Mo and S. Chu in blastocyst injection. This work is supported in part by the Strategic Priority Research Program of the Chinese Academy of Sciences (grants XDA01020401, XDA01020202 and XDA01020302), the National Basic Research Program of China (grants 2009CB941102, 2011CBA01106, 2011CB965204 and 2012CB966802), the National Natural Science Foundation of China (grants 31271357 and 91213304), the Ministry of Science and Technology International Technology Cooperation Program (grants 2010DFB30430 and 2012DFH30050), the National Science and Technology Major Special Project on Major New Drug Innovation (grant 2011ZX09102-010), the Bureau of Science and Technology of Guangzhou Municipality, China (grant 2010U1-E00521), the Guangdong Science and Technology Project (grants 2010A090603001 and 2011A08030002), Science and Technology Planning Project of Guangdong Province, China (grant 2011A060901019), the One Hundred Person Project of The Chinese Academy of Sciences to D.P., the Youth Innovation Promotion Association of the Chinese Academy of Sciences and the Pearl River Nova program (grant 2012J2200070). We wish to thank all members of our laboratories for their support.

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Authors

Contributions

Jiekai Chen initiated the study, designed and performed the experiments, analyzed the data and wrote the manuscript. H. Liu performed the experiments and analyzed the data. J.L., Jing Chen and L.G. performed reprogramming experiments. J.Q., J.Y. and Y.W. constructed plasmids and performed experiments on histone modification. B.W., T.P., Y.Z. and D.L. performed coimmunoprecipitation experiments. J.Y. performed qPCR analyses and chromatin immunoprecipitation experiments. H. Liang constructed plasmids and analyzed Smad phosphorylation. Y.C. derived iX pre-iPSCs and screened small molecule compounds on pre-iPSCs. J.Y., Jing Chen, J.Z. and X.L. identified the pre-iPSCs and iPSCs. X.Z. performed blastocyst injection. S.C. isolated hepatocytes. T.W. cloned histone demethylases. D.P. conceived and supervised the whole study and wrote the manuscript.

Corresponding author

Correspondence to Duanqing Pei.

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Supplementary Figures 1–8 and Supplementary Tables 1 and 3–7 (PDF 4839 kb)

Supplementary Table 2

481 overlap genes (XLS 96 kb)

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Chen, J., Liu, H., Liu, J. et al. H3K9 methylation is a barrier during somatic cell reprogramming into iPSCs. Nat Genet 45, 34–42 (2013). https://doi.org/10.1038/ng.2491

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