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miR-34 miRNAs provide a barrier for somatic cell reprogramming

Abstract

Somatic reprogramming induced by defined transcription factors is a low-efficiency process that is enhanced by p53 deficiency1,2,3,4,5. So far, p21 is the only p53 target shown to contribute to p53 repression of iPSC (induced pluripotent stem cell) generation1,3, indicating that additional p53 targets may regulate this process. Here, we demonstrate that miR-34 microRNAs (miRNAs), particularly miR-34a, exhibit p53-dependent induction during reprogramming. Mir34a deficiency in mice significantly increased reprogramming efficiency and kinetics, with miR-34a and p21 cooperatively regulating somatic reprogramming downstream of p53. Unlike p53 deficiency, which enhances reprogramming at the expense of iPSC pluripotency, genetic ablation of Mir34a promoted iPSC generation without compromising self-renewal or differentiation. Suppression of reprogramming by miR-34a was due, at least in part, to repression of pluripotency genes, including Nanog, Sox2 and Mycn (also known as N-Myc). This post-transcriptional gene repression by miR-34a also regulated iPSC differentiation kinetics. miR-34b and c similarly repressed reprogramming; and all three miR-34 miRNAs acted cooperatively in this process. Taken together, our findings identified miR-34 miRNAs as p53 targets that play an essential role in restraining somatic reprogramming.

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Figure 1: Generation of Mir34a and Mir34b/c knockout MEFs.
Figure 2: Deficiency of miR-34 miRNAs increases reprogramming efficiency.
Figure 3: miR-34a and p21 cooperate to repress iPSC generation.
Figure 4: M i r 34 a−/− iPSCs functionally resemble wild-type iPSCs.
Figure 5: Mir34a represses Nanog, Sox2 and N-Myc expression post-transcriptionally.

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Acknowledgements

We thank B. Zaghi, A. Basila, A. Perez, G. Lai, M. Foth, A. Kemp, A. Fresnoza, A. Valeros, A. Fritz, D. Schichnes and H. Noller for technical assistance and A. Economides for discussions and input. We also thank J. M. Halbleib, D. Schaffer and R. M. Harland for reading the manuscripts, and I. Lemischka, Q. Li, M. Hemann and B. Vogelstein for sharing reagents. In particular, we are grateful for the pathological expertise R. Van Andel offered us. L.H. is a Searle Scholar, and is supported by a new faculty award by California Institute for Regenerative Medicine (RN2-00923-1) and an R01 (R01 CA139067) and an R00 grant (R00 CA126186) from the National Cancer Institute (NCI). G.J.H. is a Howard Hughes Medical Institute investigator, and is supported by a program project grant from the NCI. C-P.L. is supported by a Siebel postdoctoral fellowship.

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Authors

Contributions

Y.J.C., C-P.L. and L.H. designed all experiments and carried out the majority of the experiments shown in all figures and supplementary figures. J.J.H. and Y.Z. carried out immunofluorescence analyses and teratoma analyses to characterize the pluripotency of the iPSCs. L.H., X.H., P.B. and G.J.H. generated knockout constructs for Mir34a and Mir34b/c, and identified the correctly targeted ESC clones for Mir34a. N.O. and P.B. identified the correctly targeted ESC clone for Mir34b/c, validated Mir34a and Mir34b/c targeting in mice by Southern blotting and generated M i r 34 a−/−, M i r 34 b/c−/− and Mir34 triple knockout MEFs. S.Y.K. and G.J.H. carried out the blastocyst injection for M i r 34 a+/− and M i r 34 b/c+/− ESC clones. A.O., S.Y.K. and G.G.H. characterized the pluripotency of iPSCs using chimaera assays. M.J.B. and C.C. contributed to the identification of miR-34a targets.

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Correspondence to Lin He.

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Choi, Y., Lin, CP., Ho, J. et al. miR-34 miRNAs provide a barrier for somatic cell reprogramming. Nat Cell Biol 13, 1353–1360 (2011). https://doi.org/10.1038/ncb2366

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