Abstract
Methylation of DNA at the 5′ carbon of cytosine (5mC) is a key epigenetic regulator of gene expression required for the proper development and function of the central nervous system. Observations of experience-dependent fluctuations in the patterns of 5mC at neuronal gene enhancers, promoters and intragenic regions have prompted great interest in the role of DNA methylation mechanisms in the brain. In addition, the recent discovery of 5-hydroxymethylcytosine (5hmC), an oxidized derivative of 5mC that acts as a DNA demethylation intermediate has offered mechanistic insight into how these changes in 5mC levels are achieved. As a result, numerous techniques have now been adapted or newly developed to quantify and profile 5mC and 5hmC in the genome. Here we describe a method using high performance liquid chromatography electrospray ionization tandem mass spectrometry with multiple reaction monitoring (HPLC-ESI-MS/MS-MRM) for the simultaneous detection and quantification of global 5mC and 5hmC levels present in genomic DNA from nervous-system derived samples. HPLC-ESI-MS/MS-MRM analysis offers the highest levels of sensitivity, selectivity, and precision available for the determination of global changes in these epigenetic modifications.
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References
MacDonald JL, Roskams AJ (2009) Epigenetic regulation of nervous system development by DNA methylation and histone deacetylation. Prog Neurobiol 88:170
Rudenko A, Tsai LH (2014) Epigenetic modifications in the nervous system and their impact upon cognitive impairments. Neuropharmacology 80:70
Jiang Y et al (2008) Epigenetics in the nervous system. J Neurosci 28:11753
Moore LD, Le T, Fan G (2013) DNA methylation and its basic function. Neuropsychopharmacology 38:23
Jaenisch R, Bird A (2003) Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat Genet 33(Suppl):245
Bonasio R, Tu S, Reinberg D (2010) Molecular signals of epigenetic states. Science 330:612
Feng S, Jacobsen SE, Reik W (2010) Epigenetic reprogramming in plant and animal development. Science 330:622
Day JJ et al (2013) DNA methylation regulates associative reward learning. Nat Neurosci 16:1445
Miller CA, Sweatt JD (2007) Covalent modification of DNA regulates memory formation. Neuron 53:857
Lubin FD, Roth TL, Sweatt JD (2008) Epigenetic regulation of BDNF gene transcription in the consolidation of fear memory. J Neurosci 28:10576
Ma DK et al (2009) Neuronal activity-induced Gadd45b promotes epigenetic DNA demethylation and adult neurogenesis. Science 323:1074
Guo JU et al (2011) Neuronal activity modifies the DNA methylation landscape in the adult brain. Nat Neurosci 14:1345
Guo JU, Su Y, Zhong C, Ming GL, Song H (2011) Hydroxylation of 5-methylcytosine by TET1 promotes active DNA demethylation in the adult brain. Cell 145:423
Kriaucionis S, Heintz N (2009) The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and the brain. Science 324:929
Tahiliani M et al (2009) Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 324:930
Ito S et al (2011) Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science 333:1300
He YF et al (2011) Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA. Science 333:1303
Bhutani N, Burns DM, Blau HM (2011) DNA demethylation dynamics. Cell 146:866
Branco MR, Ficz G, Reik W (2012) Uncovering the role of 5-hydroxymethylcytosine in the epigenome. Nat Rev Genet 13:7
Globisch D et al (2010) Tissue distribution of 5-hydroxymethylcytosine and search for active demethylation intermediates. PLoS One 5:e15367
Koh KP et al (2011) Tet1 and Tet2 regulate 5-hydroxymethylcytosine production and cell lineage specification in mouse embryonic stem cells. Cell Stem Cell 8:200
Li W, Liu M (2011) Distribution of 5-hydroxymethylcytosine in different human tissues. J Nucleic Acids 2011:870726
Kaas GA et al (2013) TET1 controls CNS 5-methylcytosine hydroxylation, active DNA demethylation, gene transcription, and memory formation. Neuron 79:1086
Le T, Kim KP, Fan G, Faull KF (2011) A sensitive mass spectrometry method for simultaneous quantification of DNA methylation and hydroxymethylation levels in biological samples. Anal Biochem 412:203
Munzel M et al (2010) Quantification of the sixth DNA base hydroxymethylcytosine in the brain. Angew Chem Int Ed Engl 49:5375
Hahn MA et al (2013) Dynamics of 5-hydroxymethylcytosine and chromatin marks in Mammalian neurogenesis. Cell Rep 3:291
Kraus TF et al (2012) Low values of 5-hydroxymethylcytosine (5hmC), the “sixth base,” are associated with anaplasia in human brain tumors. Int J Cancer 131:1577
Feng J et al (2010) Dnmt1 and Dnmt3a maintain DNA methylation and regulate synaptic function in adult forebrain neurons. Nat Neurosci 13:423
Szulwach KE et al (2011) 5-hmC-mediated epigenetic dynamics during postnatal neurodevelopment and aging. Nat Neurosci 14:1607
Song L, James SR, Kazim L, Karpf AR (2005) Specific method for the determination of genomic DNA methylation by liquid chromatography-electrospray ionization tandem mass spectrometry. Anal Chem 77:504
Shen L, Zhang Y (2012) Enzymatic analysis of Tet proteins: key enzymes in the metabolism of DNA methylation. Methods Enzymol 512:93
Liu S et al (2013) Quantitative assessment of Tet-induced oxidation products of 5-methylcytosine in cellular and tissue DNA. Nucleic Acids Res 41:6421
Acknowledgements
This work was supported by NIMH grant MH57014. Primary neuronal culture and brain images were provided by Mikael C. Guzman-Karlsson. We would like to thank Jeremy Day and J. David Sweatt for comments and suggestions in the editing of this manuscript.
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Ross, D.L., Kaas, G.A. (2016). Simultaneous Quantification of Global 5mC and 5hmC Levels in the Nervous System Using an HPLC/MS Method. In: Karpova, N. (eds) Epigenetic Methods in Neuroscience Research. Neuromethods, vol 105. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2754-8_5
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DOI: https://doi.org/10.1007/978-1-4939-2754-8_5
Publisher Name: Humana Press, New York, NY
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