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The oncogene c-Jun impedes somatic cell reprogramming

Nature Cell Biology volume 17, pages 856–867 (2015)Cite this article

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A Corrigendum to this article was published on 28 August 2015

This article has been updated

Abstract

Oncogenic transcription factors are known to mediate the conversion of somatic cells to tumour or induced pluripotent stem cells (iPSCs). Here we report c-Jun as a barrier for iPSC formation. c-Jun is expressed by and required for the proliferation of mouse embryonic fibroblasts (MEFs), but not mouse embryonic stem cells (mESCs). Consistently, c-Jun is induced during mESC differentiation, drives mESCs towards the endoderm lineage and completely blocks the generation of iPSCs from MEFs. Mechanistically, c-Jun activates mesenchymal-related genes, broadly suppresses the pluripotent ones, and derails the obligatory mesenchymal to epithelial transition during reprogramming. Furthermore, inhibition of c-Jun by shRNA, dominant-negative c-Jun or Jdp2 enhances reprogramming and replaces Oct4 among the Yamanaka factors. Finally, Jdp2 anchors 5 non-Yamanaka factors (Id1, Jhdm1b, Lrh1, Sall4 and Glis1) to reprogram MEFs into iPSCs. Our studies reveal c-Jun as a guardian of somatic cell fate and its suppression opens the gate to pluripotency.

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Figure 1: c-Jun is induced during ESC differentiation and required for proliferation of MEFs, but not ESCs.
Figure 2: c-Jun induces ESC differentiation and inhibits iPSC generation.
Figure 3: c-Jun suppresses pluripotent genes but induces EMT genes.
Figure 4: c-Jun regulates MET genes directly.
Figure 5: c-Jun dominant-negative mutants c-JunDN, Jdp2 and c-Jun shRNA can enhance the efficiency of iPSC generation.
Figure 6: c-Jun dominant-negative mutants c-JunDN, Jdp2 and c-Jun shRNA can substitute Oct4 partially.
Figure 7: c-JunDN and Jdp2 target similar genes as Oct4 during reprogramming.
Figure 8: Induction of pluripotent stem cells from MEFs by non-Yamanaka factors.

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Change history

  • 10 August 2015

    In the version of this Article originally published, panels from Fig. 8e were mistakenly reproduced as Fig. 6g. The correct panels for Fig. 6g (KS/Jdp2-iPSC) have been amended in all online versions of the Article.

  • 28 August 2015

    Nat. Cell Biol. 17, 856–867 (2015); published online 22 June 2015; corrected after print 10 August 2015 In the version of this Article originally published, panels from Fig. 8e were mistakenly reproduced as Fig. 6g. The correct panels for Fig. 6g (KS/Jdp2-iPSC) are shown here and have been amended in all online versions of the Article.

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Acknowledgements

We thank H. Zheng, M. Esteban, B. Qin and X. Shu for their critical comments. We appreciate the contributions of K. Mo, S. Chu, J. Xie, X. Zhao, H. Pang, S. Huang, R. Luo, Y. Li and K. Lai for technical assistance. We are grateful to other members in our laboratory for their supporting work. This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (grant XDA01020401), the National Basic Research Program of China (grants 2014CB965200, 2012CB966802, 2015CB964800), the National Natural Science Foundation of China (grants 31421004, 31401264, 91419310, 31271357, 91413110, 31471210), Guangdong Natural Science Funds for Distinguished Young Scholars (grant S2013050014040), and Bureau of Science and Technology of Guangzhou Municipality (grants 2014J2200077, 2012J2200070).

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Authors and Affiliations

  1. Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China

    Jing Liu, Qingkai Han, Tianran Peng, Meixiu Peng, Bei Wei, Dongwei Li, Xiaoshan Wang, Shengyong Yu, Jiaqi Yang, Shangtao Cao, Kaimeng Huang, Andrew Paul Hutchins, He Liu, Junqi Kuang, Zhiwei Zhou, Jing Chen, Haoyu Wu, Lin Guo, Yongqiang Chen, You Chen, Xuejia Li, Hongling Wu, Baojian Liao, Wei He, Hong Song, Hongjie Yao, Guangjin Pan, Jiekai Chen & Duanqing Pei

  2. Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China

    Jing Liu, Qingkai Han, Tianran Peng, Meixiu Peng, Bei Wei, Dongwei Li, Xiaoshan Wang, Jiaqi Yang, Shangtao Cao, Kaimeng Huang, Andrew Paul Hutchins, He Liu, Zhiwei Zhou, Jing Chen, Lin Guo, Yongqiang Chen, You Chen, Xuejia Li, Hongling Wu, Baojian Liao, Wei He, Hong Song, Hongjie Yao, Guangjin Pan, Jiekai Chen & Duanqing Pei

  3. Guangzhou Branch of the Supercomputing Center of Chinese Academy of Sciences, Guangzhou 510530, China

    Xiaoshan Wang, Andrew Paul Hutchins, Jiekai Chen & Duanqing Pei

  4. School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, China

    Shengyong Yu & Junqi Kuang

  5. Department of Biological Engineering, College of Pharmacy, Jilin University, 1266 Fu Jin Road Changchun 130021, China,

    Haoyu Wu

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  1. Jing Liu
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Contributions

J.L. designed and performed the experiments, and analysed the data. Q.H. initiated the study and performed reprogramming experiments with M.P., S.Y. and S.C. T.P. D.L. and K.H. performed ChIP experiments. B.W. and D.L. performed ESC differentiation. J.Y., H.L. and W.H. performed qPCR analyses and RNA-seq. X.W. and A.P.H. analysed the RNA-seq and ChIP-seq data. J.K., Q.H. and D.L. constructed plasmids. Haoyu W., Hongling W. and B.L. performed the gene editing. Z.Z. performed the zebrafish experiment. Yongqiang C. and H.S. performed blastocyst injection. L.G. and You C. cultured the cells and identified the iPSC lines. Jing C. and X.L. screened the compounds. H.Y. supervised the ChIP-seq experiment. G.P. supervised the gene editing and sequencing. J.L., Jiekai C. and D.P. wrote the manuscript. Jiekai C. and D.P. conceived and supervised the whole study. D.P. approved the final version.

Corresponding authors

Correspondence to Jiekai Chen or Duanqing Pei.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Relative expression of AP-1 family genes on ESCs and somatic cells.

(a) Immunostaining of c-JUN and NANOG in MEFs and ESCs. ESCs were cultured in feeder free (up panel) or feeder (middle panel) condition, respectively. Scale bars, 100 μm. (b) Relative expression of AP-1 family members in MEFs, iPSCs and ESCs (Mean ± s.e.m., Two-tailed, unpaired t-test; n = 3 independent experiments, Junb ∗∗P = 0024, Fra1 ∗P = 0.0483, Fra1 ∗P = 0.021). (c) Time course analysis of AP-1 family members during RA induced ESCs differentiation by RT–qPCR. Mouse ESCs were cultured in mES+RA (1 μM). (Mean ± s.d., n = 3 independent experiments). (d) Time course analysis of the pluripotent genes and AP-1 family members during EB formation by RT–qPCR (Mean ± s.d., n = 3 independent experiments). (e,f) Time course analysis of c-Jun expression during embryo development of Zebra fish (e) and EB formation of human ESCs (f) (e,f, n = 2 independent experiments). (g) Relative expression of the pluripotent genes in WT and Jun-deficient ESCs. Cells were culture in 2i+LIF medium (n = 2 independent experiments). (h) Relative expression of germ layer markers during the EB generation in WT and Jun-deficient ESCs (Mean ± s.d., n = 3 independent experiments). For source data, see Supplementary Table 3.

Supplementary Figure 2 The function of AP-1 family members on somatic reprogramming.

(a) Phase and GFP images of iPSC colonies generated with OKS+Ctrl, OKS+c-Jun, OKSM+Ctrl, and OKSM+c-Jun. Cells were culture in iCD1 medium. Scale bars, 250 μm. (b) c-Jun inhibit OKSM induced reprogramming (Mean ± s.d., Two-tailed, unpaired t-test, n = 9 wells from 3 independent experiments, ∗∗∗P < 0.001). (c) The effect of AP-1 family members to OKS induced reprogramming in mES+Vc medium (Mean ± s.d., Two-tailed, unpaired t-test; n = 7 wells (Mock, Vector, Fos, Fra1, Fra2) or 6 wells (JunB, ATF2, ATF3) from 3 independent experiment, Vector-Fos ∗∗∗p < 0.001, Vector-Fra2 ∗∗∗P < 0.001, Vector-Fra1 ∗∗∗P < 0.001). (d) The effect of c-Jun on reprogramming at different time windows. OKS, Retrovirus; c-JunTetOn, lenti-virus carrying an inducible c-Jun code sequence under the control of TetOn promoter. Reprogramming cells were cultured in iCD1 medium, Doxycycline (2 μg ml−1) was administrated at different time windows as indicated (n = 4 wells from 2 independent experiments). The initial cell number for reprogramming is 2 × 104/well (p12) for OKS protocol, or 1.5 × 104/well (p12) for OKSM protocol. For source data, see Supplementary Table 6.

Supplementary Figure 3 c-Jun suppresses pluripotent genes and actives EMT relative genes.

(a) Time course analysis the expression of EMT relative genes Ets1, Smad3 and Tgfb2 by RT–qPCR (mean ± s.d., Two-tailed, unpaired t-test; n = 3 independent experiments, Ets1 ∗∗P = 0.004, Smad3 ∗∗P = 0.006, Tgfb2 ∗∗P = 0.002). (b) Time course analysis the expression of pluripotent related genes Oct4, Rex1 and Sall4 by RT–qPCR. c-JunTetOn ESCs were cultured in 2i+LIF medium with Dox (2 μg ml−1) treatment (mean ± s.d., One-way ANOVA, Dunnett’s multiple comparisons test; n = 4 independent experiments, Oct4 ∗q = 4.922, Rex1 ∗q = 6.844, Sall4 ∗q = 7.263). For source data, see Supplementary Table 6.

Supplementary Figure 4 c-Jun actives EMT relative genes directly.

(a) The bindings of c-Jun on the Oct4, Rex1 and Sall4 loci were determined by ChIP-PCR. Arrows indicate the predicted binding site containing the conserved AP binding motif (mean ± s.d., n = 3 independent experiments). (b) ChIP-seq result of c-Jun at EMT related genes Zeb2, Dusp5, Smad3, Thy1, Ets1 and Tgfb2. For source data, see Supplementary Table 6.

Supplementary Figure 5 c-JunDN and Jdp2 enhance reprogramming efficiency.

(a) The construction of different c-Jun mutants. Basic motif, Leucine-Zipper and Transactivation domain were shown as yellow rectangle, light green rectangle and Red rectangle, respectively. (b) Effect of various c-Jun mutants on reprogramming (mean ± s.d., One-way ANOVA, Dunnett’s multiple comparisons test; n = 6 wells pooled from 3 independent experiments, a.a1-334 ∗∗∗q = 20.35, a.a170-334 ∗∗∗q = 17.03, a.a254-334 ∗∗∗q = 13.19). (c) The construction of different Jdp2 mutants. (d) Effect of various Jdp2 mutants on reprogramming (mean ± s.d., One-way ANOVA, Dunnett’s multiple comparisons test; n = 6 wells pooled from 3 independent experiments, a.a1-163 ∗∗q = 7.349, a.a50-163 ∗∗∗q = 10.29, a.a50-163 ∗∗q = 8.639). For iPSC generation, the initial cell number was 2 × 104/well (p12), Oct4-GFP+ colonies were quantified at post-infection Day12 in mES+Vc medium For source data, see Supplementary Table 6.

Supplementary Figure 6 c-Jun antagonists can replace Oct4 in somatic reprogramming.

(a) Normal karyotypes of representative KSM/c-JunDN, KS/c-JunDN, KSM/Jdp2, KSM/Jdp2 iPSCs colonies. (b) Immunofluorescence analysis the expression of pluripotent markers of indicated iPSCs colonies. Scale bars, 100 μm. (c) Bisulfite genomic sequencing of the Nanog and Oct4 promoter regions in indicated iPSCs colonies. (d) PCR analysis show the KSM-Jdp2 iPSC colonies contributed to all three germ layer in chimeric mice. (e) Normal karyotypes of representative KSM/c-Jun shRNA iPSCs colonies.

Supplementary Figure 7 c-Jun antagonists regulated different genes with Oct4 in MEFs.

(a) Heatmap for RNA-seq data from MEFs infected with control, Oct4, c-JunDN and Jdp2. MEFs, cultured in 10%FBS for 72 h after virus infection, were harvested for analysis. (b) c-JunWT promotes, but c-JunDN, Jdp2 and Oct4 inhibit, the proliferation of MEFs. Cells were cultured in 10%FBS (mean ± s.d., n = 3 wells, this experiment were repeated twice, one of the represented experiment was showed). For source data, see Supplementary Table 5 (a) and Supplementary Table 6 (b).

Supplementary Figure 8 non-Yamanaka factors reprogramming.

(a) Id1 and Id3 enhance OS induced reprogramming in iCD1 or iCD1+BMP medium. The initial cell number was 2 × 104 per well (p12), cells were cultured in iCD1 or iCD1+BMP4 (10ng/ml) medium. Oct4-GFP+ colonies were quantitated at post-infection Day16 (mean ± s.d., n = 4 wells pooled from 2 independent experiments). (b) Phase and GFP images of iPSC colonies generated with Oct4/Sox2/Id1 and Oct4/Jhdm1b/Id1. Scale bars, 250 μm. (c) The replacement of Oct4 by indicated genes. The initial cell number was 2 × 104 per well (p12). Cells were cultured in iCD1 medium. Oct4-GFP+ colonies were quantitated at post-infection Day16 (mean ± s.d., n = 6 wells pooled from 2 independent experiments). (d) Retroviral integrations of 6F-iPSCs analysed by PCR with genomic DNA. (e) Normal karyotypes of representative 6F iPSCs. (f) Retroviral integrations of 6F-iPSCs analysed by PCR with genomic DNA. For source data, see Supplementary Table 3.

Supplementary Figure 9 Uncropped western blot images.

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Liu, J., Han, Q., Peng, T. et al. The oncogene c-Jun impedes somatic cell reprogramming. Nat Cell Biol 17, 856–867 (2015). https://doi.org/10.1038/ncb3193

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