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Reprogramming towards pluripotency requires AID-dependent DNA demethylation

Abstract

Reprogramming of somatic cell nuclei to yield induced pluripotent stem (iPS) cells makes possible derivation of patient-specific stem cells for regenerative medicine. However, iPS cell generation is asynchronous and slow (2–3 weeks), the frequency is low (<0.1%), and DNA demethylation constitutes a bottleneck. To determine regulatory mechanisms involved in reprogramming, we generated interspecies heterokaryons (fused mouse embryonic stem (ES) cells and human fibroblasts) that induce reprogramming synchronously, frequently and fast. Here we show that reprogramming towards pluripotency in single heterokaryons is initiated without cell division or DNA replication, rapidly (1 day) and efficiently (70%). Short interfering RNA (siRNA)-mediated knockdown showed that activation-induced cytidine deaminase (AID, also known as AICDA) is required for promoter demethylation and induction of OCT4 (also known as POU5F1) and NANOG gene expression. AID protein bound silent methylated OCT4 and NANOG promoters in fibroblasts, but not active demethylated promoters in ES cells. These data provide new evidence that mammalian AID is required for active DNA demethylation and initiation of nuclear reprogramming towards pluripotency in human somatic cells.

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Figure 1: Absence of cell division and DNA replication in heterokaryons.
Figure 2: Time course of human fibroblast pluripotency gene expression in heterokaryons at the single-cell level.
Figure 3: Time course of DNA demethylation at pluripotency gene promoters in heterokaryons.
Figure 4: Requirement of AID-dependent DNA demethylation for initiation of reprogramming towards pluripotency in heterokaryons.
Figure 5: AID binding to pluripotency gene promoters by chromatin immunoprecipitation.
Figure 6: Model for AID-dependent active DNA demethylation in reprogramming.

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Acknowledgements

We thank O. Alkan for generating retroviral constructs for GFP and DsRed expression; R. Doyonnas for helping to standardize FACS isolation of heterokaryons; Y. Liao for performing the initial experiments showing demethylation in heterokaryons; M. Pajçini for technical assistance with the BrdU experiments; D. Schatz and S. Unniraman for kindly providing the human AID construct; F. Alt for his generous provision of antibody to AID; J. A. Dahl for helpful discussions about chromatin immunoprecipitation assays; H. Chang for use of the Bioruptor sonicator; and M. Wernig, G. Sen, C.-Z. Chen and J. Pomerantz for insightful comments on the manuscript. This work was supported by a National Science Foundation Graduate Research Fellowship awarded to J.J.B., National Institutes of Health (NIH) training grant AI007328 to M.D., and NIH grants AG009521, AG024987 and support from the Baxter Foundation to H.M.B.

Author Contributions N.B. and H.M.B. designed the research, N.B., J.J.B. and M.D. performed the experiments and analysed the data. M.D. assisted with western blots, and conducted the ChIP analyses. A.S. and S.Y.C. performed FACS isolation of heterokaryons and provided expert help in figure preparation. N.B., J.J.B. and H.M.B. discussed the results and wrote the paper.

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Correspondence to Helen M. Blau.

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Bhutani, N., Brady, J., Damian, M. et al. Reprogramming towards pluripotency requires AID-dependent DNA demethylation. Nature 463, 1042–1047 (2010). https://doi.org/10.1038/nature08752

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