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.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Gurdon, J. B. Adult frogs derived from the nuclei of single somatic cells. Dev. Biol. 4, 256–273 (1962)
Briggs, R. & King, T. J. Transplantation of living nuclei from blastula cells into enucleated frogs’ eggs. Proc. Natl Acad. Sci. USA 38, 455–463 (1952)
Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007)
Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006)
Wernig, M. et al. In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 448, 318–324 (2007)
Okita, K., Ichisaka, T. & Yamanaka, S. Generation of germline-competent induced pluripotent stem cells. Nature 448, 313–317 (2007)
Blau, H. M., Chiu, C. P. & Webster, C. Cytoplasmic activation of human nuclear genes in stable heterocaryons. Cell 32, 1171–1180 (1983)
Chiu, C. P. & Blau, H. M. Reprogramming cell differentiation in the absence of DNA synthesis. Cell 37, 879–887 (1984)
Chiu, C. P. & Blau, H. M. 5-Azacytidine permits gene activation in a previously noninducible cell type. Cell 40, 417–424 (1985)
Blau, H. M. et al. Plasticity of the differentiated state. Science 230, 758–766 (1985)
Blau, H. M. & Baltimore, D. Differentiation requires continuous regulation. J. Cell Biol. 112, 781–783 (1991)
Baron, M. H. & Maniatis, T. Rapid reprogramming of globin gene expression in transient heterokaryons. Cell 46, 591–602 (1986)
Wright, W. E. Expression of differentiated functions in heterokaryons between skeletal myocytes, adrenal cells, fibroblasts and glial cells. Exp. Cell Res. 151, 55–69 (1984)
Spear, B. T. & Tilghman, S. M. Role of α-fetoprotein regulatory elements in transcriptional activation in transient heterokaryons. Mol. Cell. Biol. 10, 5047–5054 (1990)
Blau, H. M. Differentiation requires continuous active control. Annu. Rev. Biochem. 61, 1213–1230 (1992)
Pavlath, G. K. & Blau, H. M. Expression of muscle genes in heterokaryons depends on gene dosage. J. Cell Biol. 102, 124–130 (1986)
Zhang, F., Pomerantz, J. H., Sen, G., Palermo, A. T. & Blau, H. M. Active tissue-specific DNA demethylation conferred by somatic cell nuclei in stable heterokaryons. Proc. Natl Acad. Sci. USA 104, 4395–4400 (2007)
Park, I. H. et al. Reprogramming of human somatic cells to pluripotency with defined factors. Nature 451, 141–146 (2008)
Yu, J. et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917–1920 (2007)
Aoi, T. et al. Generation of pluripotent stem cells from adult mouse liver and stomach cells. Science 321, 699–702 (2008)
Eminli, S. et al. Differentiation stage determines potential of hematopoietic cells for reprogramming into induced pluripotent stem cells. Nature Genet. 41, 968–976 (2009)
Aasen, T. et al. Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes. Nature Biotechnol. 26, 1276–1284 (2008)
Mikkelsen, T. S. et al. Dissecting direct reprogramming through integrative genomic analysis. Nature 454, 49–55 (2008)
Cowan, C. A., Atienza, J., Melton, D. A. & Eggan, K. Nuclear reprogramming of somatic cells after fusion with human embryonic stem cells. Science 309, 1369–1373 (2005)
Silva, J., Chambers, I., Pollard, S. & Smith, A. Nanog promotes transfer of pluripotency after cell fusion. Nature 441, 997–1001 (2006)
Simonsson, S. & Gurdon, J. DNA demethylation is necessary for the epigenetic reprogramming of somatic cell nuclei. Nature Cell Biol. 6, 984–990 (2004)
Ooi, S. K. & Bestor, T. H. The colorful history of active DNA demethylation. Cell 133, 1145–1148 (2008)
Rai, K. et al. DNA demethylation in zebrafish involves the coupling of a deaminase, a glycosylase, and Gadd45. Cell 135, 1201–1212 (2008)
Muramatsu, M. et al. Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell 102, 553–563 (2000)
Morgan, H. D., Dean, W., Coker, H. A., Reik, W. & Petersen-Mahrt, S. K. Activation-induced cytidine deaminase deaminates 5-methylcytosine in DNA and is expressed in pluripotent tissues: implications for epigenetic reprogramming. J. Biol. Chem. 279, 52353–52360 (2004)
Palermo, A. et al. Nuclear reprogramming in heterokaryons is rapid, extensive, and bidirectional. FASEB J. 23, 1431–1440 (2009)
Ivanova, N. et al. Dissecting self-renewal in stem cells with RNA interference. Nature 442, 533–538 (2006)
Bhattacharya, B. et al. Gene expression in human embryonic stem cell lines: unique molecular signature. Blood 103, 2956–2964 (2004)
Loh, Y. H. et al. The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nature Genet. 38, 431–440 (2006)
Feng, B. et al. Reprogramming of fibroblasts into induced pluripotent stem cells with orphan nuclear receptor Esrrb. Nature Cell Biol. 11, 197–203 (2009)
Wernig, M. et al. A drug-inducible transgenic system for direct reprogramming of multiple somatic cell types. Nature Biotechnol. 26, 916–924 (2008)
Vuong, B. Q. et al. Specific recruitment of protein kinase A to the immunoglobulin locus regulates class-switch recombination. Nature Immunol. 10, 420–426 (2009)
Okazaki, I. M., Kinoshita, K., Muramatsu, M., Yoshikawa, K. & Honjo, T. The AID enzyme induces class switch recombination in fibroblasts. Nature 416, 340–345 (2002)
Deb, K., Sivaguru, M., Yong, H. Y. & Roberts, R. M. Cdx2 gene expression and trophectoderm lineage specification in mouse embryos. Science 311, 992–996 (2006)
Gong, Z. et al. ROS1, a repressor of transcriptional gene silencing in Arabidopsis, encodes a DNA glycosylase/lyase. Cell 111, 803–814 (2002)
Choi, Y. et al. DEMETER, a DNA glycosylase domain protein, is required for endosperm gene imprinting and seed viability in Arabidopsis . Cell 110, 33–42 (2002)
Cortázar, D., Kunz, C., Saito, Y., Steinacher, R. & Schar, P. The enigmatic thymine DNA glycosylase. DNA Repair (Amst.) 6, 489–504 (2007)
Millar, C. B. et al. Enhanced CpG mutability and tumorigenesis in MBD4-deficient mice. Science 297, 403–405 (2002)
Gehring, M., Reik, W. & Henikoff, S. DNA demethylation by DNA repair. Trends Genet. 25, 82–90 (2009)
Conticello, S. G., Langlois, M. A., Yang, Z. & Neuberger, M. S. DNA deamination in immunity: AID in the context of its APOBEC relatives. Adv. Immunol. 94, 37–73 (2007)
Barreto, G. et al. Gadd45a promotes epigenetic gene activation by repair-mediated DNA demethylation. Nature 445, 671–675 (2007)
Jin, S. G., Guo, C. & Pfeifer, G. P. GADD45A does not promote DNA demethylation. PLoS Genet. 4, e1000013 (2008)
Pereira, C. F. et al. Heterokaryon-based reprogramming of human B lymphocytes for pluripotency requires Oct4 but not Sox2. PLoS Genet. 4, e1000170 (2008)
Dahl, J. A. & Collas, P. A rapid micro chromatin immunoprecipitation assay (microChIP). Nature Protocols 3, 1032–1045 (2008)
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.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Supplementary information
Supplementary Information
This file contains Supplementary Figures S1-S15 with Legends and Supplementary Tables 1-4 (PDF 795 kb)
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nature08752
This article is cited by
-
Epigenetics and stroke: role of DNA methylation and effect of aging on blood–brain barrier recovery
Fluids and Barriers of the CNS (2023)
-
Epigenetic regulation in hematopoiesis and its implications in the targeted therapy of hematologic malignancies
Signal Transduction and Targeted Therapy (2023)
-
Gene body methylation in cancer: molecular mechanisms and clinical applications
Clinical Epigenetics (2022)
-
Advances in the DNA methylation hydroxylase TET1
Biomarker Research (2021)
-
Nanosecond pulsed electric fields enhance mesenchymal stem cells differentiation via DNMT1-regulated OCT4/NANOG gene expression
Stem Cell Research & Therapy (2020)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.