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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References
Kriaucionis, S. & Heintz, N. The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and the brain. Science 324, 929–930 (2009).
Penn, N.W., Suwalski, R., O′Riley, C., Bojanowski, K. & Yura, R. The presence of 5-hydroxymethylcytosine in animal deoxyribonucleic acid. Biochem. J. 126, 781–790 (1972).
Tahiliani, M. et al. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 324, 930–935 (2009).
Huang, Y. et al. The behaviour of 5-hydroxymethylcytosine in bisulfite sequencing. PLoS ONE 5, e8888 (2010).
Globisch, D. et al. Tissue distribution of 5-hydroxymethylcytosine and search for active demethylation intermediates. PLoS ONE 5, e15367 (2010).
Deaton, A.M. & Bird, A. CpG islands and the regulation of transcription. Genes Dev. 25, 1010–1022 (2011).
Jin, S.-G., Wu, X., Li, A.X. & Pfeifer, G.P. Genomic mapping of 5-hydroxymethylcytosine in the human brain. Nucleic Acids Res. 39, 5015–5024 (2011).
Song, C.-X. Selective chemical labeling reveals the genome-wide distribution of 5-hydroxymethylcytosine. Nat. Biotechnol. 29, 68–72 (2011).
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).
He, Y.F. et al. Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA. Science 333, 1303–1307 (2011).
Ito, S. et al. Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science 333, 1300–1303 (2011).
Zhang, L. et al. Thymine DNA glycosylase specifically recognizes 5-carboxylcytosine-modified DNA. Nat. Chem. Biol. 8, 328–330 (2012).
Huang, L.H., Farnet, C.M., Ehrlich, K.C. & Ehrlich, M. Digestion of highly modified bacteriophage DNA by restriction endonucleases. Nucleic Acids Res. 10, 1579–1591 (1982).
Josse, J. & Kornberg, A. Glucosylation of deoxyribonucleic acid. III. α- and β-glucosyl transferases from T4-infected Escherichia coli. J. Biol. Chem. 237, 1968–1976 (1962).
Liu, X.S. Getting started in tiling microarray analysis. PLOS Comput. Biol. 3, 1842–1844 (2007).
Potter, D.P., Yan, P., Huang, T.H.M. & Lin, S. Probe signal correction for differential methylation hybridization experiments. BMC Bioinformatics 9, 453 (2008).
Lattin, J.E. et al. Expression analysis of G protein-coupled receptors in mouse macrophages. Immunome Res. 4, 5 (2008).
Huang, D.W., Sherman, B.T. & Lempicki, R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44–57 (2009).
Cahoy, J.D. et al. A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function. J. Neurosci. 28, 264–278 (2008).
Balsa-Canto, E. & Banga, J.R. AMIGO, a toolbox for advanced model identification in systems biology using global optimization. Bioinformatics 27, 2311–2313 (2011).
Chodavarapu, R.K. et al. Relationship between nucleosome positioning and DNA methylation. Nature 466, 388–392 (2010).
Laurent, L. et al. Dynamic changes in the human methylome during differentiation. Genome Res. 20, 320–331 (2010).
Torrey, E.F., Webster, M., Knable, M., Johnston, N. & Yolken, R.H. The Stanley Foundation brain collection and neuropathology consortium. Schizophr. Res. 44, 151–155 (2000).
Hart, C. et al. Single-molecule sequencing: sequence methods to enable accurate quantitation. Methods Enzymol. 472, 407–430 (2010).
Ou, J.N. et al. Histone deacetylase inhibitor trichostatin A induces global and gene-specific DNA demethylation in human cancer cell lines. Biochem. Pharmacol. 73, 1297–1307 (2007).
Hnilicová, J. et al. Histone deacetylase activity modulates alternative splicing. PLoS ONE 6, e16727 (2011).
Brawand, D. et al. The evolution of gene expression levels in mammalian organs. Nature 478, 343–348 (2011).
Davis, T. & Vaisvila, R. High sensitivity 5-hydroxymethylcytosine detection in Balb/C brain tissue. J. Vis. Exp. 48, e2661 (2011).
Jin, S.-G., Kadam, S. & Pfeifer, G.P. Examination of the specificity of DNA methylation profiling techniques towards 5-methylcytosine and 5-hydroxymethylcytosine. Nucleic Acids Res. 38, e125 (2010).
Kondo, E., Gu, Z., Horii, A. & Fukushige, S. The thymine DNA glycosylase MBD4 represses transcription and is associated with methylated p16(INK4a) and hMLH1 genes. Mol. Cell Biol. 25, 4388–4396 (2005).
Valinluck, V. et al. Oxidative damage to methyl-CpG sequences inhibits the binding of the methyl-CpG binding domain (MBD) of methyl-CpG binding protein 2 (MeCP2). Nucleic Acids Res. 32, 4100–4108 (2004).
Feng, J. et al. Dnmt1 and Dnmt3a maintain DNA methylation and regulate synaptic function in adult forebrain neurons. Nat. Neurosci. 13, 423–430 (2010).
Miller, C.A. & Sweatt, J.D. Covalent modification of DNA regulates memory formation. Neuron 53, 857–869 (2007).
Szulwach, K.E. et al. 5-hmC-mediated epigenetic dynamics during postnatal neurodevelopment and aging. Nat. Neurosci. 14, 1607–1616 (2011).
Chodavarapu, R.K. et al. Relationship between nucleosome positioning and DNA methylation. Nature 466, 388–392 (2010).
Hodges, E. et al. High definition profiling of mammalian DNA methylation by array capture and single molecule bisulfite sequencing. Genome Res. 19, 1593–1605 (2009).
Kelly, W.K. et al. Phase I study of an oral histone deacetylase inhibitor, suberoylanilide hydroxamic acid, in patients with advanced cancer. J. Clin. Oncol. 23, 3923–3931 (2005).
Kolasinska-Zwierz, P. et al. Differential chromatin marking of introns and expressed exons by H3K36me3. Nat. Genet. 41, 376–381 (2009).
Schwartz, S., Meshorer, E. & Ast, G. Chromatin organization marks exon-intron structure. Nat. Struct. Mol. Biol. 16, 990–995 (2009).
Tilgner, H. et al. Nucleosome positioning as a determinant of exon recognition. Nat. Struct. Mol. Biol. 16, 996–1001 (2009).
Luco, R.F. et al. Regulation of alternative splicing by histone modifications. Science 327, 996–1000 (2010).
Andersson, R., Enroth, S., Rada-Iglesias, A., Wadelius, C. & Komorowski, J. Nucleosomes are well positioned in exons and carry characteristic histone modifications. Genome Res. 19, 1732–1741 (2009).
Kolasinska-Zwierz, P. et al. Differential chromatin marking of introns and expressed exons by H3K36me3. Nat. Genet. 41, 376–381 (2009).
Shukla, S. et al. CTCF-promoted RNA polymerase II pausing links DNA methylation to splicing. Nature 479, 74–79 (2011).
Irimia, M. & Blencowe, B.J. Alternative splicing: decoding an expansive regulatory layer. Curr. Opin. Cell Biol. 3, 323–332 (2012).
Luco, R.F., Allo, M., Schor, I.E., Kornblihtt, A.R. & Misteli, T. Epigenetics in alternative pre-mRNA splicing. Cell 144, 16–26 (2011).
Jin, S.-G., Kadam, S. & Pfeifer, G.P. Examination of the specificity of DNA methylation profiling techniques towards 5-methylcytosine and 5-hydroxymethylcytosine. Nucleic Acids Res. 38, e125 (2010).
Tardy-Planechaud, S., Fujimoto, J., Lin, S.S. & Sowers, L.C. Solid phase synthesis and restriction endonuclease cleavage of oligodeoxynucleotides containing 5-(hydroxymethyl)-cytosine. Nucleic Acids Res. 25, 553–559 (1997).
Larivière, L., Kurzeck, J., Aschke-Sonnenborn, U., Ruger, W. & Morera, S. Crystallization and preliminary crystallographic study of a ternary complex between the T4 phage beta-glucosyltransferase, uridine diphosphoglucose and a DNA fragment containing an abasic site. Acta Crystallogr. D Biol. Crystallogr. 58, 1484–1486 (2002).
Ichiyanagi, K. Inhibition of MspI cleavage activity by hydroxymethylation of the CpG site: a concern for DNA modification studies using restriction endonucleases. Epigenetics 7, 131–136 (2012).
Gentleman, R.C. et al. Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 5, R80 (2004).
Vittinghoff, E., Glidden, D.V., Shiboski, S.C. & McCulloch, C.E. Repeated Measures Analysis, in Regression Methods in Biostatistics Ch. 8, 253–290 (Springer, 2005).
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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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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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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DOI: https://doi.org/10.1038/nsmb.2372
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