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Subtelomeric hotspots of aberrant 5-hydroxymethylcytosine-mediated epigenetic modifications during reprogramming to pluripotency

Nature Cell Biology volume 15, pages 700–711 (2013)Cite this article

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Abstract

Mammalian somatic cells can be directly reprogrammed into induced pluripotent stem cells (iPSCs) by introducing defined sets of transcription factors. Somatic cell reprogramming involves epigenomic reconfiguration, conferring iPSCs with characteristics similar to embryonic stem cells (ESCs). Human ESCs (hESCs) contain 5-hydroxymethylcytosine (5hmC), which is generated through the oxidation of 5-methylcytosine by the TET enzyme family. Here we show that 5hmC levels increase significantly during reprogramming to human iPSCs mainly owing to TET1 activation, and this hydroxymethylation change is critical for optimal epigenetic reprogramming, but does not compromise primed pluripotency. Compared with hESCs, we find that iPSCs tend to form large-scale (100 kb–1.3 Mb) aberrant reprogramming hotspots in subtelomeric regions, most of which exhibit incomplete hydroxymethylation on CG sites. Strikingly, these 5hmC aberrant hotspots largely coincide (∼ 80%) with aberrant iPSC–ESC non-CG methylation regions. Our results suggest that TET1-mediated 5hmC modification could contribute to the epigenetic variation of iPSCs and iPSC–hESC differences.

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Figure 1: TET1 is associated with increased hydroxymethylation during human iPSC reprogramming.
Figure 2: Reprogramming confers a 5hmC epigenome in a pattern with a bias towards telomere-proximal regions in autosomes.
Figure 3: 5hmC is associated with gene activity and pluripotency regulatory networks in stem cells.
Figure 4: Aberrant 5hmC reprogramming hotspots cluster at subtelomeric regions.
Figure 5: 5hmC DhMRs largely overlap with non-CG-DMRs in a large-scale pattern.
Figure 6: Large-scale incomplete hydroxymethylation hotspots are characteristics of human iPSCs.
Figure 7: Large-scale hotspots are caused predominantly by aberrant CpG hydroxymethylation.

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Acknowledgements

We thank J. Suhl, M. Santoro, S. Bray and C. Strauss for critical reading of the manuscript. We thank X. Huang from the Viral Vector Core of the Emory Neuroscience NINDS Core Facilities for preparing the retrovirus/lentivirus used in this study. We are grateful to J. Mowrey, V. Patel, C. Street and S. Namburi for support on Illumina Hiseq2000/Miseq sequencing. This study was supported in part by the National Institutes of Health (NS079625 and HD073162 to P.J.; MH089606 and HD24064 to S.T.W.), the Emory Genetics Discovery Fund, and the Autism Speaks grant (#7660 to X.L.).

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Authors and Affiliations

  1. Department of Human Genetics, Emory University School of Medicine, Atlanta, Georgia 30322, USA

    Tao Wang, Yujing Li, Keith E. Szulwach, Li Lin, Xuekun Li, Ian S. Goldlust, Ann Dodd, He Gong, M. Katharine Rudd, Stephen T. Warren & Peng Jin

  2. Genetics and Molecular Biology Graduate Program, Emory University, Atlanta, Georgia 30322, USA

    Tao Wang

  3. Department of Biostatistics and Bioinformatics, Emory University Rollins School of Public Health, Atlanta, Georgia 30322, USA

    Hao Wu

  4. Department of Oral Health and Diagnostic Sciences, School of Dental Medicine, University of Connecticut Health Center, Farmington, Connecticut 06030, USA

    I-Ping Chen

  5. Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, Connecticut 06030, USA

    Stormy J. Chamberlain

  6. Department of Genetics and Neurology, Waisman Center, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA

    Gene Ananiev & Qiang Chang

  7. Department of Medicine, Division of Cardiology, Emory University School of Medicine, Atlanta, Georgia 30322, USA

    Ji Woong Han & Young-sup Yoon

  8. Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, Chicago, Illinois 60637, USA

    Miao Yu, Chun-Xiao Song & Chuan He

  9. Department of Biochemistry and Pediatrics, Emory University School of Medicine, Atlanta, Georgia 30322, USA

    Stephen T. Warren

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Contributions

T.W., S.T.W. and P.J. designed the study and interpreted the results. T.W. and H.W. analysed the data. T.W. performed most experiments; Y.L., L.L., X.L. performed 5hmC capture and parts of library preparation. M.Y. C-X.S, H.G. and C.H. assisted with the TAB-seq experiment and 5hmC capture experiment. A.D. and K.E.S. contributed to the Illumina sequencing; I.S.G. and M.K.R. contributed array CGH experiments. I-P.C., S.J.C., J.W.H., G.A., Y-s.Y. and Q.C. provided some of the hESC and hiPSC lines. T.W., S.T.W. and P.J. wrote the paper with assistance from H.W.

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Correspondence to Stephen T. Warren or Peng Jin.

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Wang, T., Wu, H., Li, Y. et al. Subtelomeric hotspots of aberrant 5-hydroxymethylcytosine-mediated epigenetic modifications during reprogramming to pluripotency. Nat Cell Biol 15, 700–711 (2013). https://doi.org/10.1038/ncb2748

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