Locus-Specific DNA Methylation Assays to Study Glutamate Receptor Regulation

  • Jordan A. Brown
  • J. David Sweatt
  • Garrett A. KaasEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1941)


Recent findings indicate that glutamate receptors are regulated at the epigenetic level through the posttranslational modification of histones and through DNA methylation. Furthermore, dysregulation of these marks in the context of neurological disease has been shown to influence glutamate receptor function. Over the past two decades, an appreciation for the essential role epigenetic mechanisms play in nervous system function has led to the development of many methods and tools to map, quantitate, and manipulate these chromatin marks. Here we describe two popular methods used to quantitate DNA methylation levels at the gene or nucleotide level. The first, cloning-based bisulfite sequencing involves modification of DNA samples using the chemical sodium bisulfite (BS), which deaminates all unmethylated cytosines to form uracil. Subsequent PCR amplification converts the uracils to thymine, leaving any cytosines in the PCR product representative of methylation. Fragments are then cloned and sequenced to quantitate the percentage of methylation at each cytosine. The second technique, methyl-binding domain capture (MBDCap), involves shearing the genomic DNA into fragments via sonication. Samples are then incubated with magnetic beads conjugated to methyl-binding domain (MBD) peptides to bind and enrich fragments containing methylated CpGs. Quantitation of DNA methylation levels are then measured indirectly using qRT-PCR with primers specific to the region of interest. Because these methods do not require advanced technical knowledge and can be performed with common laboratory equipment, they are great options for interrogating DNA methylation patterns at the level of the gene, the regulatory region, or in the case of bisulfite sequencing, the nucleotide.

Key words

DNA methylation Bisulfite Glutamate receptors Epigenetics MBDCap CpG AMPA NMDA 



The authors would like to thank Celeste B. Greer for her help with MBD-seq analysis used to locate appropriate targets for MBDCap and cloning-based BS-sequencing of the Grin2A locus. Work presented in this chapter was supported by NIMH grant MH57014 to J.D.S.


  1. 1.
    Riedel G, Platt B, Micheau J (2003) Glutamate receptor function in learning and memory. Behav Brain Res 140:1–47CrossRefGoogle Scholar
  2. 2.
    Bowie D (2008) Ionotropic glutamate receptors & CNS disorders. CNS Neurol Disord Drug Targets 7:129–143. CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Bai G, Hoffman PW. Transcriptional Regulation of NMDA Receptor Expression. In: Van Dongen AM, editor. Biology of the NMDA Receptor. Boca Raton (FL): CRC Press/Taylor & Francis; 2009. Chapter 5.CrossRefGoogle Scholar
  4. 4.
    Harraz MM, Eacker SM, Wang X et al (2012) MicroRNA-223 is neuroprotective by targeting glutamate receptors. Proc Natl Acad Sci 109:18962–18967. CrossRefPubMedGoogle Scholar
  5. 5.
    Lussier MP, Sanz-Clemente A, Roche KW (2015) Dynamic regulation of NMDA and AMPA receptors by posttranslational modifications. J Biol Chem jbc.R115.652750. CrossRefGoogle Scholar
  6. 6.
    Straub C, Tomita S (2012) The regulation of glutamate receptor trafficking and function by TARPs and other transmembrane auxiliary subunits. Curr Opin Neurobiol 22:488–495CrossRefGoogle Scholar
  7. 7.
    Goo MS, Scudder SL, Patrick GN (2015) Ubiquitin-dependent trafficking and turnover of ionotropic glutamate receptors. Front Mol Neurosci 8.
  8. 8.
    Sweatt JD (2016) Dynamic DNA methylation controls glutamate receptor trafficking and synaptic scaling. J Neurochem 137:312–330CrossRefGoogle Scholar
  9. 9.
    Meadows JP, Guzman-Karlsson MC, Phillips S et al (2015) DNA methylation regulates neuronal glutamatergic synaptic scaling. Sci Signal 8. CrossRefGoogle Scholar
  10. 10.
    Qiang M, Denny A, Chen J et al (2010) The site specific demethylation in the 5′-regulatory area of NMDA receptor 2B subunit gene associated with CIE-induced up-regulation of transcription. PLoS One 5. CrossRefGoogle Scholar
  11. 11.
    Gulchina Y, Xu SJ, Snyder MA et al (2017) Epigenetic mechanisms underlying NMDA receptor hypofunction in the prefrontal cortex of juvenile animals in the MAM model for schizophrenia. J Neurochem 143:320–333. CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Frommer M, McDonald LE, Millar DS et al (1992) A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci 89:1827–1831. CrossRefPubMedGoogle Scholar
  13. 13.
    Kriaucionis S, Heintz N (2009) The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and the brain. Science 324:929–930. CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Guo JU, Su Y, Zhong C et al (2011) Hydroxylation of 5-methylcytosine by TET1 promotes active DNA demethylation in the adult brain. Cell 145:423–434. CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Tahiliani M, Koh KP, Shen Y et al (2009) Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 324:930–935. CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Booth MJ, Ost TWB, Beraldi D et al (2013) Oxidative bisulfite sequencing of 5-methylcytosine and 5-hydroxymethylcytosine. Nat Protoc 8:1841–1851. CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Huang Y, Pastor WA, Shen Y et al (2010) The behaviour of 5-hydroxymethylcytosine in bisulfite sequencing. PLoS One 5. CrossRefGoogle Scholar
  18. 18.
    Nestor CE, Meehan RR (2014) Hydroxymethylated DNA immunoprecipitation (hmeDIP). Methods Mol Biol 1094:259–267. CrossRefPubMedGoogle Scholar
  19. 19.
    Bock C, Reither S, Mikeska T et al (2005) BiQ analyzer: visualization and quality control for DNA methylation data from bisulfite sequencing. Bioinformatics 21:4067–4068. CrossRefPubMedGoogle Scholar
  20. 20.
    Hsu HK, Weng YI, Hsu PY et al (2014) Detection of DNA methylation by MeDIP and MBDCap assays: an overview of techniques. Methods Mol Biol 1105:61–70. CrossRefPubMedGoogle Scholar
  21. 21.
    Aberg KA, Xie L, Chan RF et al (2015) Evaluation of methyl-binding domain based enrichment approaches revisited. PLoS One 10. CrossRefGoogle Scholar
  22. 22.
    Bustin SA, Benes V, Garson JA et al (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622. CrossRefPubMedGoogle Scholar
  23. 23.
    Rao X, Huang X, Zhou Z, Lin X (2013) An improvement of the 2̂(−delta delta CT) method for quantitative real-time polymerase chain reaction data analysis. Biostat Bioinforma Biomath 3:71–85. CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative CT method. Nat Protoc 3:1101–1108. CrossRefPubMedGoogle Scholar
  25. 25.
    Kennedy AJ, Rahn EJ, Paulukaitis BS et al (2016) Tcf4 regulates synaptic plasticity, DNA methylation, and memory function. Cell Rep 16:2666–2685. CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Jones PA (2012) Functions of DNA methylation: islands, start sites, gene bodies and beyond. Nat Rev Genet 13:484–492CrossRefGoogle Scholar
  27. 27.
    Yu M., Han D., Hon G.C., He C. (2018) Tet-Assisted Bisulfite Sequencing (TAB-seq). In: Tost J. (eds) DNA Methylation Protocols. Methods in Molecular Biology, vol 1708. Humana Press, New York, NYGoogle Scholar
  28. 28.
    Yu M, Hon GC, Szulwach KE et al (2012) Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine. Nat Protoc 7:2159–2170. CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Ito S, Shen L, Dai Q et al (2011) Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science 333:1300–1303. CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Wu H, Wu X, Zhang Y (2016) Base-resolution profiling of active DNA demethylation using MAB-seq and caMAB-seq. Nat Protoc 11:1081–1100. CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Neri F, Incarnato D, Krepelova A et al (2015) Single-base resolution analysis of 5-formyl and 5-carboxyl cytosine reveals promoter DNA methylation dynamics. Cell Rep 10:674–683. CrossRefGoogle Scholar
  32. 32.
    Wu H, Wu X, Shen L, Zhang Y (2014) Single-base resolution analysis of active DNA demethylation using methylase-assisted bisulfite sequencing. Nat Biotechnol 32:1231–1240. CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Jang HS, Shin WJ, Lee JE, Do JT (2017) CpG and non-CpG methylation in epigenetic gene regulation and brain function. Genes (Basel) 8:2–20Google Scholar
  34. 34.
    Guo JU, Su Y, Shin JH et al (2014) Distribution, recognition and regulation of non-CpG methylation in the adult mammalian brain. Nat Neurosci 17:215–222. CrossRefPubMedGoogle Scholar
  35. 35.
    Chen Z, Riggs AD (2011) DNA methylation and demethylation in mammals. J Biol Chem 286:18347–18353. CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Tost J, Gut IG (2007) DNA methylation analysis by pyrosequencing. Nat Protoc 2:2265–2275. CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Jordan A. Brown
    • 1
  • J. David Sweatt
    • 1
  • Garrett A. Kaas
    • 1
    Email author
  1. 1.Department of PharmacologyVanderbilt UniversityNashvilleUSA

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