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Distribution, recognition and regulation of non-CpG methylation in the adult mammalian brain

Nature Neuroscience 17, 215–222 (2014) | Download Citation

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Abstract

DNA methylation has critical roles in the nervous system and has been traditionally considered to be restricted to CpG dinucleotides in metazoan genomes. Here we show that the single base–resolution DNA methylome from adult mouse dentate neurons consists of both CpG (∼75%) and CpH (∼25%) methylation (H = A/C/T). Neuronal CpH methylation is conserved in human brains, enriched in regions of low CpG density, depleted at protein-DNA interaction sites and anticorrelated with gene expression. Functionally, both methylated CpGs (mCpGs) and mCpHs can repress transcription in vitro and are recognized by methyl-CpG binding protein 2 (MeCP2) in neurons in vivo. Unlike most CpG methylation, CpH methylation is established de novo during neuronal maturation and requires DNA methyltransferase 3A (DNMT3A) for active maintenance in postmitotic neurons. These characteristics of CpH methylation suggest that a substantially expanded proportion of the neuronal genome is under cytosine methylation regulation and provide a new foundation for understanding the role of this key epigenetic modification in the nervous system.

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Acknowledgements

We thank S. Baylin, D. Ginty and members of the Song and Ming laboratories for comments and suggestions and Y. Cai and L. Liu for technical support. This work was supported by the US National Institutes of Health (NIH) (NS047344, ES021957 and MH087874), the Simons Foundation Autism Research Initiative and NARSAD to H.S., by the NIH (HD069184 and NS048271), the Maryland Stem Cell Research Fund (MSCRF), the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation and NARSAD to G.-l.M., by the Lieber Institute fund to Y.G., by NIH (HD064743, HD066560) to Q.C. and by NIH (NS072924) to G.F.; J.U.G. is a Damon Runyon Fellow supported by the Damon Runyon Cancer Research Foundation. Y.S. and C.Z. were supported by MSCRF postdoctoral fellowships; J.S. was supported by a Samsung Scholarship.

Author information

    • Junjie U Guo

    Present address: Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA.

    • Junjie U Guo
    •  & Yijing Su

    These authors contributed equally to this work.

Affiliations

  1. Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

    • Junjie U Guo
    • , Yijing Su
    • , Jaehoon Shin
    • , Chun Zhong
    • , Yuan Gao
    • , Guo-li Ming
    •  & Hongjun Song

  2. The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

    • Junjie U Guo
    • , Guo-li Ming
    •  & Hongjun Song

  3. Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

    • Junjie U Guo
    • , Yijing Su
    • , Chun Zhong
    • , Guo-li Ming
    •  & Hongjun Song

  4. Lieber Institute for Brain Development, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

    • Joo Heon Shin
    • , Bin Xie
    •  & Yuan Gao

  5. Graduate Program in Cellular and Molecular Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

    • Jaehoon Shin
    • , Guo-li Ming
    •  & Hongjun Song

  6. Waisman Center, University of Wisconsin-Madison, Madison, Wisconsin, USA.

    • Hongda Li
    •  & Qiang Chang

  7. Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

    • Shaohui Hu
    •  & Heng Zhu

  8. Department of Human Genetics, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, USA.

    • Thuc Le
    •  & Guoping Fan

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Contributions

J.U.G. and Y.S. conducted most of the experiments. Y.S. constructed the libraries, and J.U.G. performed the bioinformatic analyses. J.H.S., B.X. and Y.G. assisted with high-throughput sequencing. J.S. contributed to the EMSA and ChIP experiments. H.L. and Q.C. provided the MeCP2-ChIP samples. C.Z. performed the shRNA experiment. S.H. and H.Z. assisted with the EMSA experiments. T.L. and G.F. provided the DNMT-cTKO samples. Y.G., G.-l.M. and H.S. supervised the project. J.U.G., G.-l.M. and H.S. wrote the manuscript. All of the authors discussed results and commented on the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Hongjun Song.

Integrated supplementary information

Supplementary figures

  1. 1.

    Global levels of CpG and CpH methylation across mouse chromosomes.

  2. 2.

    Inter-sample correlation of CpG and CpH methylation.

  3. 3.

    CpH methylation is spatially associated with CpG methylation.

  4. 4.

    Mouse neuronal CpG and CpH methylation on two opposite DNA strands.

  5. 5.

    Distance analysis of CHG and CHH methylation.

  6. 6.

    Neuronal CpG and CpH methylation around ESC-specific transcription factor binding sites.

  7. 7.

    Anti-correlation between CpG-far CpH methylation in regulatory regions and gene expression.

  8. 8.

    In vitro methylated reporter assay in HEK293 cells.

  9. 9.

    Effects of CpH methylation on MBD2b-DNA interaction.

  10. 10.

    Knock-down efficiency of shRNAs.

  11. 11.

    Lack of effects of DNMT3A knock-down on unmethylated and CpG-methylated CpH-unmethylated regions.

  12. 12.

    DNMT3A binds to CpH-methylated regions in adult dentate gyrus in vivo.

  13. 13.

    Dnmt gene expression in adult mouse tissues.

  14. 14.

    Summary of similarities and differences between CpG and CpH methylation in ESCs in vitro and neurons in vivo.

Supplementary information

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  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–14, Supplementary Tables 1 and 2

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DOI

https://doi.org/10.1038/nn.3607

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