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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.

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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

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  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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