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Targeted DNA demethylation and activation of endogenous genes using programmable TALE-TET1 fusion proteins

Nature Biotechnology volume 31, pages 1137–1142 (2013)Cite this article

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Abstract

Genome-wide studies have defined cell type–specific patterns of DNA methylation1 that are important for regulating gene expression in both normal development2 and disease3. However, determining the functional significance of specific methylation events remains challenging, owing to the lack of methods for removing such modifications in a targeted manner. Here we describe an approach for efficient targeted demethylation of specific CpGs in human cells using fusions of engineered transcription activator–like effector (TALE) repeat arrays and the TET1 hydroxylase catalytic domain. Using these TALE-TET1 fusions, we demonstrate that modification of critical methylated promoter CpG positions can lead to substantial increases in the expression of endogenous human genes. Our results delineate a strategy for understanding the functional significance of specific CpG methylation marks in the context of endogenous gene loci and validate programmable DNA demethylation reagents with potential utility for research and therapeutic applications.

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Figure 1: TALE-TET1 fusion proteins induce targeted demethylation in human cells.
Figure 2: Targeted demethylation and activation of human RHOXF2 by TALE-TET1 fusion proteins.
Figure 3: Targeted demethylation and activation of the human HBB gene by TALE-TET1 fusion proteins.

References

  1. Jones, P.A. Functions of DNA methylation: islands, start sites, gene bodies and beyond. Nat. Rev. Genet. 13, 484–492 (2012).

    Article  CAS  Google Scholar 

  2. Smith, Z.D. & Meissner, A. DNA methylation: roles in mammalian development. Nat. Rev. Genet. 14, 204–220 (2013).

    Article  CAS  Google Scholar 

  3. Brena, R.M. & Costello, J.F. Genome-epigenome interactions in cancer. Hum. Mol. Genet. 16 Spec No 1, R96–R105 (2007).

    Article  CAS  Google Scholar 

  4. Bird, A. DNA methylation patterns and epigenetic memory. Genes Dev. 16, 6–21 (2002).

    Article  CAS  Google Scholar 

  5. Suzuki, M.M. & Bird, A. DNA methylation landscapes: provocative insights from epigenomics. Nat. Rev. Genet. 9, 465–476 (2008).

    Article  CAS  Google Scholar 

  6. Reik, W., Dean, W. & Walter, J. Epigenetic reprogramming in mammalian development. Science 293, 1089–1093 (2001).

    Article  CAS  Google Scholar 

  7. Tahiliani, M. et al. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 324, 930–935 (2009).

    Article  CAS  Google Scholar 

  8. Guo, J.U., Su, Y., Zhong, C., Ming, G.-L. & Song, H. Hydroxylation of 5-methylcytosine by TET1 promotes active DNA demethylation in the adult brain. Cell 145, 423–434 (2011).

    Article  CAS  Google Scholar 

  9. Ito, S. et al. Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science 333, 1300–1303 (2011).

    Article  CAS  Google Scholar 

  10. He, Y.F. et al. Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA. Science 333, 1303–1307 (2011).

    Article  CAS  Google Scholar 

  11. Xu, Y. et al. Genome-wide regulation of 5hmC, 5mC, and gene expression by Tet1 hydroxylase in mouse embryonic stem cells. Mol. Cell 42, 451–464 (2011).

    Article  CAS  Google Scholar 

  12. Williams, K. et al. TET1 and hydroxymethylcytosine in transcription and DNA methylation fidelity. Nature 473, 343–348 (2011).

    Article  CAS  Google Scholar 

  13. Ito, S. et al. Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification. Nature 466, 1129–1133 (2010).

    Article  CAS  Google Scholar 

  14. Joung, J.K. & Sander, J.D. TALENs: a widely applicable technology for targeted genome editing. Nat. Rev. Mol. Cell Biol. 14, 49–55 (2013).

    Article  CAS  Google Scholar 

  15. Song, H.W. et al. The RHOX homeobox gene cluster is selectively expressed in human oocytes and male germ cells. Hum. Reprod. 28, 1635–1646 (2013).

    Article  CAS  Google Scholar 

  16. Mabaera, R. et al. Developmental- and differentiation-specific patterns of human gamma- and beta-globin promoter DNA methylation. Blood 110, 1343–1352 (2007).

    Article  CAS  Google Scholar 

  17. Maiti, A. & Drohat, A.C. Thymine DNA glycosylase can rapidly excise 5-formylcytosine and 5-carboxylcytosine: potential implications for active demethylation of CpG sites. J. Biol. Chem. 286, 35334–35338 (2011).

    Article  CAS  Google Scholar 

  18. Miller, J.C. et al. A TALE nuclease architecture for efficient genome editing. Nat. Biotechnol. 29, 143–148 (2011).

    Article  CAS  Google Scholar 

  19. Tan, S. et al. Zinc-finger protein-targeted gene regulation: genomewide single-gene specificity. Proc. Natl. Acad. Sci. USA 100, 11997–12002 (2003).

    Article  CAS  Google Scholar 

  20. Mendenhall, E.M. et al. Locus-specific editing of histone modifications at endogenous enhancers. Nat. Biotechnol. doi:10.1038/nbt.2701 (8 September 2013).

  21. Konermann, S. et al. Optical control of mammalian endogenous transcription and epigenetic states. Nature 500, 472–476 (2013).

    Article  CAS  Google Scholar 

  22. Snowden, A.W., Gregory, P.D., Case, C.C. & Pabo, C.O. Gene-specific targeting of H3K9 methylation is sufficient for initiating repression in vivo. Curr. Biol. 12, 2159–2166 (2002).

    Article  CAS  Google Scholar 

  23. Xu, G.L. & Bestor, T.H. Cytosine methylation targetted to pre-determined sequences. Nat. Genet. 17, 376–378 (1997).

    Article  CAS  Google Scholar 

  24. McNamara, A.R., Hurd, P.J., Smith, A.E.F. & Ford, K.G. Characterisation of site-biased DNA methyltransferases: specificity, affinity and subsite relationships. Nucleic Acids Res. 30, 3818–3830 (2002).

    Article  CAS  Google Scholar 

  25. Carvin, C.D., Parr, R.D. & Kladde, M.P. Site-selective in vivo targeting of cytosine-5 DNA methylation by zinc-finger proteins. Nucleic Acids Res. 31, 6493–6501 (2003).

    Article  CAS  Google Scholar 

  26. Smith, A.E. & Ford, K.G. Specific targeting of cytosine methylation to DNA sequences in vivo. Nucleic Acids Res. 35, 740–754 (2007).

    Article  CAS  Google Scholar 

  27. Nomura, W. & Barbas, C.F. In vivo site-specific DNA methylation with a designed sequence-enabled DNA methylase. J. Am. Chem. Soc. 129, 8676–8677 (2007).

    Article  CAS  Google Scholar 

  28. Li, F. et al. Chimeric DNA methyltransferases target DNA methylation to specific DNA sequences and repress expression of target genes. Nucleic Acids Res. 35, 100–112 (2007).

    Article  Google Scholar 

  29. Rivenbark, A.G. et al. Epigenetic reprogramming of cancer cells via targeted DNA methylation. Epigenetics 7, 350–360 (2012).

    Article  CAS  Google Scholar 

  30. Siddique, A.N. et al. Targeted methylation and gene silencing of VEGF-A in human cells by using a designed Dnmt3a-Dnmt3L single-chain fusion protein with increased DNA methylation activity. J. Mol. Biol. 425, 479–491 (2013).

    Article  CAS  Google Scholar 

  31. Reyon, D. et al. FLASH assembly of TALENs for high-throughput genome editing. Nat. Biotechnol. 30, 460–465 (2012).

    Article  CAS  Google Scholar 

  32. Maeder, M.L. et al. Rapid “open-source” engineering of customized zinc-finger nucleases for highly efficient gene modification. Mol. Cell 31, 294–301 (2008).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by a National Institutes of Health (NIH) Director's Pioneer Award DP1 GM105378 (to J.K.J.), NIH P50 HG005550 (to J.K.J.), NIH R01 HD053808 (to M.F.W.), NIH R01 HD045595 (to M.F.W.), NIH R01 CA169316 (to J.F.C.), NIH F32 GM105189 (to S.Q.T.), Defense Advanced Research Projects Agency (DARPA) W911NF-11-2-0056 (to J.K.J.), the Karen Osney Brownstein Endowed Chair (to J.F.C.), the Jim and Ann Orr Massachusetts General Hospital (MGH) Research Scholar Award (to J.K.J.) and a US National Science Foundation Graduate Research Fellowship (to M.L.M.). We thank the MGH Nucleic Acid Quantitation Core (supported by NIH P30 NS45776) for use of their real-time PCR machine.

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Author notes

  1. Morgan L Maeder and James F Angstman: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Pathology and Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts, USA

    Morgan L Maeder, James F Angstman, Samantha J Linder, Vincent M Cascio, Shengdar Q Tsai, Quan H Ho, Jeffry D Sander, Deepak Reyon, Bradley E Bernstein & J Keith Joung

  2. Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA

    Morgan L Maeder, James F Angstman, Samantha J Linder, Vincent M Cascio, Shengdar Q Tsai, Jeffry D Sander, Deepak Reyon & J Keith Joung

  3. Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, Massachusetts, USA

    Morgan L Maeder & J Keith Joung

  4. Department of Molecular and Cellular Biology, Graduate Program in Molecules, Cells and Organisms, Harvard University, Cambridge, Massachusetts, USA

    James F Angstman

  5. Department of Reproductive Medicine, School of Medicine, University of California, San Diego, La Jolla, California, USA

    Marcy E Richardson & Miles F Wilkinson

  6. Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA

    Shengdar Q Tsai, Jeffry D Sander, Deepak Reyon, Bradley E Bernstein & J Keith Joung

  7. Howard Hughes Medical Institute, Chevy Chase, Maryland, USA

    Bradley E Bernstein

  8. Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA

    Bradley E Bernstein

  9. Department of Neurological Surgery, University of California, San Francisco, San Francisco, California, USA

    Joseph F Costello

  10. Institute for Genomic Medicine, University of California, San Diego, La Jolla, California, USA

    Miles F Wilkinson

Authors
  1. Morgan L Maeder
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Contributions

M.L.M., J.F.A., M.E.R., B.E.B., J.F.C., M.F.W. and J.K.J. designed experiments. M.L.M., J.F.A., M.E.R., S.J.L., V.M.C., S.Q.T., Q.H.H., J.D.S. and D.R. conducted experiments. M.L.M., J.F.A., M.E.R., B.E.B., J.F.C., M.F.W. and J.K.J. wrote the paper.

Corresponding author

Correspondence to J Keith Joung.

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Competing interests

M.L.M., J.F.A. and J.K.J. have filed a provisional patent application covering the TALE-TET fusion proteins. J.K.J. has a financial interest in Transposagen Biopharmaceuticals. J.K.J.'s interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–13, Supplementary Results and Supplementary Table 2 (PDF 1825 kb)

Supplementary Table 1

Primers used in this study. All primers listed 5′ to 3′ (XLSX 12 kb)

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Maeder, M., Angstman, J., Richardson, M. et al. Targeted DNA demethylation and activation of endogenous genes using programmable TALE-TET1 fusion proteins. Nat Biotechnol 31, 1137–1142 (2013). https://doi.org/10.1038/nbt.2726

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