Abstract
Fibroblasts can be reprogrammed into induced pluripotent stem cells (iPSCs) by the application of Yamanaka factors (OSKM), but the mechanisms underlying this reprogramming remain poorly understood. Here, we report that Sox2 directly regulates endogenous microRNA-29b (miR-29b) expression during iPSC generation and that miR-29b expression is required for OSKM- and OSK-mediated reprogramming. Mechanistic studies show that Dnmt3a and Dnmt3b are in vivo targets of miR-29b and that Dnmt3a and Dnmt3b expression is inversely correlated with miR-29b expression during reprogramming. Moreover, the effect of miR-29b on reprogramming can be blocked by Dnmt3a or Dnmt3b overexpression. Further experiments indicate that miR-29b-DNMT signaling is significantly involved in the regulation of DNA methylation-related reprogramming events, such as mesenchymal-to-epithelial transition (MET) and Dlk1-Dio3 region transcription. Thus, our studies not only reveal that miR-29b is a novel mediator of reprogramming factor Sox2 but also provide evidence for a multistep mechanism in which Sox2 drives a miR-29b-DNMT signaling axis that regulates DNA methylation-related events during reprogramming.
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Acknowledgements
This work was supported by grants from the Ministry of Science and Technology (2011CB965100, 2011DFA30480, 2010CB944900, 2010CB945000, and 2011CBA01100), the National Natural Science Foundation of China (91219305, 31071306, 31101061, 31210103905, 90919028, 31000378, 31171432, and 30971451), the Science and Technology Commission of Shanghai Municipality (11ZR1438500 and 11XD1405300), and the Ministry of Education (IRT1168 and 20110072110039). The work was also supported by the “Chen Guang” project, the Shanghai Municipal Education Commission and Shanghai Education Development Foundation (12CG19) and the Fundamental Research Funds for the Central Universities.
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( Supplementary information is linked to the online version of the paper on the Cell Research website.)
Supplementary information
Supplementary information, Figure S1
(A) Relative expression of miR-29 family members in OG-MEFs transduced by OSKM during the reprogramming process and that in iPSCs and ESCs, according to microarray data (Agilent mouse miRNA array). (PDF 57 kb)
Supplementary information, Figure S2
(A) QRT-PCR analyses of pluripotency gene expression (Nanog, Esrrb, Utf1, and Dppa5) in OSKM-iPSC lines and OSKM+miR-29b-derived iPSC lines. (PDF 303 kb)
Supplementary information, Figure S3
(A) OSKM+miR-29b-derived iPSCs form EBs in suspension culture. (PDF 63 kb)
Supplementary information, Figure S4
miR-29b induction efficiency was abrogated by the ectopic expression of Dnmt3a/3b. (PDF 60 kb)
Supplementary information, Figure S5
(A) Bisulfite sequencing of the Cdh2 promoter in OG-MEFs induced by OSKM factors or OSKM in combination with miR-29b on the indicated days (0, 2, 4, 6, and 8) of iPSC induction. (PDF 115 kb)
Supplementary information, Figure S6
(A) Expression analyses of Gtl2 and Rian in an additional three OSKM iPSC lines and seven OSKM+miR-29b-derived iPSC lines. (PDF 150 kb)
Supplementary information, Table S1
Primers used for vector construction. (PDF 54 kb)
Supplementary information, Table S2
Primer sets used in PCR assays. (PDF 14 kb)
Supplementary information, Table S3
Primers used for PCR genotyping (PDF 5 kb)
Supplementary information, Table S4
Primers used for exogenous transgene silencing (PDF 5 kb)
Supplementary information, Table S5
Primers used for promoter bisulfite sequencing (PDF 9 kb)
Supplementary information, Table S6
Primers used to amplify germ layer markers in differentiated EBs (PDF 45 kb)
Supplementary information, Table S7
Primers used for ChIP assays (PDF 4 kb)
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Guo, X., Liu, Q., Wang, G. et al. microRNA-29b is a novel mediator of Sox2 function in the regulation of somatic cell reprogramming. Cell Res 23, 142–156 (2013). https://doi.org/10.1038/cr.2012.180
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DOI: https://doi.org/10.1038/cr.2012.180
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