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5-hmC in the brain is abundant in synaptic genes and shows differences at the exon-intron boundary

Nature Structural & Molecular Biology volume 19, pages 1037–1043 (2012)Cite this article

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Abstract

The 5-methylcytosine (5-mC) derivative 5-hydroxymethylcytosine (5-hmC) is abundant in the brain for unknown reasons. Here we characterize the genomic distribution of 5-hmC and 5-mC in human and mouse tissues. We assayed 5-hmC by using glucosylation coupled with restriction-enzyme digestion and microarray analysis. We detected 5-hmC enrichment in genes with synapse-related functions in both human and mouse brain. We also identified substantial tissue-specific differential distributions of these DNA modifications at the exon-intron boundary in human and mouse. This boundary change was mainly due to 5-hmC in the brain but due to 5-mC in non-neural contexts. This pattern was replicated in multiple independent data sets and with single-molecule sequencing. Moreover, in human frontal cortex, constitutive exons contained higher levels of 5-hmC relative to alternatively spliced exons. Our study suggests a new role for 5-hmC in RNA splicing and synaptic function in the brain.

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Figure 1: Measurement assay for 5-hmC, array validation and relationship to steady-state mRNA levels.
Figure 2: Levels of 5-hmC in mouse and human brains is higher in synapse-related genes than in other genes.
Figure 3: 5-hmC marks exon-intron boundaries in the human brain, and 5-mC marks those in the human liver.
Figure 4: 5-hmC marks exon-intron boundaries in the mouse brain but not in mouse organs of non-neural origin.
Figure 5: Constitutive exons have higher 5-hmC levels than do alternatively-spliced exons in human brain.

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Acknowledgements

We thank N. Miller and C. Austin (National Center for Advancing Translational Sciences, US National Institutes of Health, Bethesda, Maryland, USA) for providing cell lines for the SAHA experiment. We also thank J.-S. Doucet (Centre for Addiction and Mental Health Toronto, Canada) for assistance with mouse brain sample preparation. This research has been supported by the Canadian Institutes of Health Research (grants 199170 and 186007) and the US National Institutes of Health (grants MH074127, MH088413, DP3DK085698, HG005758 and HG004535) to A.P. and Canadian Institutes of Health Research grants to B.J.B., by the Research Council of Lithuania and the French Ministry of Foreign and European affairs under the Lithuania-France bilateral collaboration program Gilibert (grant TAP-13/2011–2012) to S.M. and S.K., and by the European Social Fund under the Global Grant measure (grant VP1-3.1-ŠMM-07-K-01-105) to S.K. A.P. is supported as a Tapscott Chair in Schizophrenia Studies at the University of Toronto and by the Ontario Mental Health Foundation. M.I. is supported by a Human Frontier Science Program Organization Long Term Fellowship.

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Author notes

  1. Tarang Khare and Shraddha Pai: These authors contributed equally to this work.

Authors and Affiliations

  1. The Krembil Family Epigenetics Laboratory, The Campbell Family Mental Health Research Institute, Centre for Addiction and Mental Health, Toronto, Ontario, Canada

    Tarang Khare, Shraddha Pai, Karolis Koncevicius, Mrinal Pal, Peixin Jia, Carolyn Ptak, Sun Chong Wang, Viviane Labrie & Arturas Petronis

  2. Institute of Mathematics and Informatics, Vilnius University, Vilnius, Lithuania

    Karolis Koncevicius

  3. Department of Biological DNA Modification, Institute of Biotechnology, Vilnius University, Vilnius, Lithuania

    Edita Kriukiene, Zita Liutkeviciute & Saulius Klimasauskas

  4. The Donnelly Centre, University of Toronto, Toronto, Ontario, Canada

    Manuel Irimia & Benjamin J Blencowe

  5. National Center for Advancing Translational Sciences, US National Institutes of Health, Bethesda, Maryland, USA

    Menghang Xia

  6. Division of the National Toxicology Program Biomolecular Screening Branch, National Institute of Environmental Health Sciences, Durham, North Carolina, USA

    Raymond Tice

  7. Department of Neuropsychiatry, Graduate School of Medicine, University of Tokyo, Tokyo, Japan

    Mamoru Tochigi

  8. Laboratoire d'Enzymologie et Biochimie Structurales, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France

    Solange Moréra

  9. Department of Physiology, University of Toronto, Ontario, Canada

    Anaies Nazarians & Denise Belsham

  10. Department of Medicine, University of Toronto, Toronto, Ontario, Canada

    Anaies Nazarians & Denise Belsham

  11. Neuroscience Department, Centre for Addiction and Mental Health, Toronto, Ontario, Canada

    Albert H C Wong

  12. Institute of Systems Biology and Bioinformatics, National Central University, Jhongli, Taiwan

    Sun Chong Wang

  13. St. Laurent Institute, Cambridge, Massachusetts, USA

    Philipp Kapranov

  14. Dalla Lana School of Public Health, University of Toronto, Toronto, Ontario, Canada

    Rafal Kustra

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Contributions

S.K., A.P., T.K. and S.P. conceived of the study and designed the experiments. S.M., E.K., Z.L. and S.K. developed and verified the methods for 5-hmC detection. T.K., M.P., M.T., P.J., C.P., V.L., A.N., D.B. and A.H.C.W. conducted the microarray experiments. S.P., K.K., P.K., S.C.W. and R.K. developed bioinformatics methods and analyzed microarray data. P.K., T.K., S.P. and A.P. contributed to the design and data analysis of the Helicos sequencing experiment. M.X. and R.T. generated SAHA-treated cell lines. M.I. and B.J.B. generated the lists of alternative and constitutive exons and performed 5-hmC comparisons. T.K., S.P., V.L., S.K. and A.P. wrote the manuscript. All authors reviewed the manuscript.

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Correspondence to Saulius Klimasauskas or Arturas Petronis.

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Khare, T., Pai, S., Koncevicius, K. et al. 5-hmC in the brain is abundant in synaptic genes and shows differences at the exon-intron boundary. Nat Struct Mol Biol 19, 1037–1043 (2012). https://doi.org/10.1038/nsmb.2372

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