Protocol for Methylated DNA Immunoprecipitation (MeDIP) Analysis

  • Nina N. KarpovaEmail author
  • Juzoh Umemori
Part of the Neuromethods book series (NM, volume 105)


DNA methylation is a fundamental epigenetic mechanism for silencing gene expression by either modifying chromatin structure to a repressive state or interfering with the transcription factors’ binding. DNA methylation primarily occurs at the position C5 of a cytosine ring mainly in the context of CpG dinucleotides. The modification can be recognized both in vivo and in vitro by the methyl-CpG binding proteins (MBPs) as well as in vitro by an antibody raised against 5-methylcytosine (5mC). This chapter describes different MBPs and introduces a standard methylated DNA immunoprecipitation (MeDIP) method, which is based on using the anti-5mC antibody to isolate methylated DNA fragments for subsequent locus-specific DNA methylation analysis. The MeDIP-generated DNA can be used as well for methylation profiling on a genome scale using array-based (MeDIP-chip) and high-throughput (MeDIP-seq) technologies.

Key words

Brain Methylation Methyl-CpG-binding protein MBD DNMT TET 5-methylcytosine Anti-5mC antibody PCR Bdnf promoter 


  1. 1.
    Bird A (2002) DNA methylation patterns and epigenetic memory. Genes Dev 16:6–21CrossRefPubMedGoogle Scholar
  2. 2.
    Reamon-Buettner SM, Borlak J (2007) A new paradigm in toxicology and teratology: altering gene activity in the absence of DNA sequence variation. Reprod Toxicol 24:20–30CrossRefPubMedGoogle Scholar
  3. 3.
    Klose RJ, Bird AP (2006) Genomic DNA methylation: the mark and its mediators. Trends Biochem Sci 31:89–97CrossRefPubMedGoogle Scholar
  4. 4.
    Lister R, Ecker JR (2009) Finding the fifth base: genome-wide sequencing of cytosine methylation. Genome Res 19:959–966PubMedCentralCrossRefPubMedGoogle Scholar
  5. 5.
    Haines TR, Rodenhiser DI, Ainsworth PJ (2001) Allele-specific non-CpG methylation of the Nf1 gene during early mouse development. Dev Biol 240:585–598CrossRefPubMedGoogle Scholar
  6. 6.
    Lomvardas S, Barnea G, Pisapia DJ et al (2006) Interchromosomal interactions and olfactory receptor choice. Cell 126:403–413CrossRefPubMedGoogle Scholar
  7. 7.
    Bird A, Taggart M, Frommer M et al (1985) A fraction of the mouse genome that is derived from islands of nonmethylated. CpG-rich DNA. Cell 40:91–99CrossRefPubMedGoogle Scholar
  8. 8.
    Weber M, Hellmann I, Stadler MB et al (2007) Distribution, silencing potential and evolutionary impact of promoter DNA methylation in the human genome. Nat Genet 39:457–466CrossRefPubMedGoogle Scholar
  9. 9.
    Tahiliani M, Koh KP, Shen Y et al (2009) Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 324:930–935PubMedCentralCrossRefPubMedGoogle Scholar
  10. 10.
    Guo JU, Su Y, Zhong C et al (2011) Hydroxylation of 5-methylcytosine by TET1 promotes active DNA demethylation in the adult brain. Cell 145:423–434PubMedCentralCrossRefPubMedGoogle Scholar
  11. 11.
    Santoro R, Grummt I (2005) Epigenetic mechanism of rRNA gene silencing: temporal order of NoRC-mediated histone modification, chromatin remodeling, and DNA methylation. Mol Cell Biol 25:2539–2546PubMedCentralCrossRefPubMedGoogle Scholar
  12. 12.
    Yoon HG, Chan DW, Reynolds AB et al (2003) N-CoR mediates DNA methylation-dependent repression through a methyl CpG binding protein Kaiso. Mol Cell 12:723–734CrossRefPubMedGoogle Scholar
  13. 13.
    Zhang Y, Ng HH, Erdjument-Bromage H et al (1999) Analysis of the NuRD subunits reveals a histone deacetylase core complex and a connection with DNA methylation. Genes Dev 13:1924–1935PubMedCentralCrossRefPubMedGoogle Scholar
  14. 14.
    Nan X, Campoy FJ, Bird A (1997) MeCP2 is a transcriptional repressor with abundant binding sites in genomic chromatin. Cell 88:471–481CrossRefPubMedGoogle Scholar
  15. 15.
    Amir RE, Van den Veyver IB, Wan M et al (1999) Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat Genet 23:185–188CrossRefPubMedGoogle Scholar
  16. 16.
    Buck-Koehntop BA, Defossez PA (2013) On how mammalian transcription factors recognize methylated DNA. Epigenetics 8:131–137PubMedCentralCrossRefPubMedGoogle Scholar
  17. 17.
    Klose RJ, Sarraf SA, Schmiedeberg L et al (2005) DNA binding selectivity of MeCP2 due to a requirement for A/T sequences adjacent to methyl-CpG. Mol Cell 19:667–678CrossRefPubMedGoogle Scholar
  18. 18.
    Clouaire T, de Las Heras JI, Merusi C, Stancheva I (2010) Recruitment of MBD1 to target genes requires sequence-specific interaction of the MBD domain with methylated DNA. Nucleic Acids Res 38:4620–4634PubMedCentralCrossRefPubMedGoogle Scholar
  19. 19.
    Scarsdale JN, Webb HD, Ginder GD, Williams DC Jr (2011) Solution structure and dynamic analysis of chicken MBD2 methyl binding domain bound to a target-methylated DNA sequence. Nucleic Acids Res 39:6741–6752PubMedCentralCrossRefPubMedGoogle Scholar
  20. 20.
    Baubec T, Ivánek R, Lienert F, Schübeler D (2013) Methylation-dependent and -independent genomic targeting principles of the MBD protein family. Cell 153:480–492CrossRefPubMedGoogle Scholar
  21. 21.
    Günther K, Rust M, Leers J et al (2013) Differential roles for MBD2 and MBD3 at methylated CpG islands, active promoters and binding to exon sequences. Nucleic Acids Res 41:3010–3021PubMedCentralCrossRefPubMedGoogle Scholar
  22. 22.
    Cramer JM, Scarsdale JN, Walavalkar NM et al (2014) Probing the dynamic distribution of bound states for methylcytosine-binding domains on DNA. J Biol Chem 289:1294–1302PubMedCentralCrossRefPubMedGoogle Scholar
  23. 23.
    Quenneville S, Verde G, Corsinotti A et al (2011) In embryonic stem cells, ZFP57/KAP1 recognize a methylated hexanucleotide to affect chromatin and DNA methylation of imprinting control regions. Mol Cell 44:361–372PubMedCentralCrossRefPubMedGoogle Scholar
  24. 24.
    Sharif J, Muto M, Takebayashi S et al (2007) The SRA protein Np95 mediates epigenetic inheritance by recruiting Dnmt1 to methylated DNA. Nature 450:908–912CrossRefPubMedGoogle Scholar
  25. 25.
    Arita K, Ariyoshi M, Tochio H et al (2008) Recognition of hemi-methylated DNA by the SRA protein UHRF1 by a base-flipping mechanism. Nature 455:818–821CrossRefPubMedGoogle Scholar
  26. 26.
    Avvakumov GV, Walker JR, Xue S et al (2008) Structural basis for recognition of hemi-methylated DNA by the SRA domain of human UHRF1. Nature 455:822–825CrossRefPubMedGoogle Scholar
  27. 27.
    Prokhortchouk A, Hendrich B, Jorgensen H et al (2001) The p120 catenin partner Kaiso is a DNA methylation-dependent transcriptional repressor. Genes Dev 15:1613–1618PubMedCentralCrossRefPubMedGoogle Scholar
  28. 28.
    Frauer C, Hoffmann T, Bultmann S et al (2011) Recognition of 5-hydroxymethylcytosine by the Uhrf1 SRA domain. PLoS One 6:e21306PubMedCentralCrossRefPubMedGoogle Scholar
  29. 29.
    Weber M, Davies JJ, Wittig D et al (2005) Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells. Nat Genet 37:853–862CrossRefPubMedGoogle Scholar
  30. 30.
    Thu KL, Pikor LA, Kennett JY et al (2010) Methylation analysis by DNA immunoprecipitation. J Cell Physiol 222:522–531PubMedGoogle Scholar
  31. 31.
    Ventskovska O, Porkka-Heiskanen T, Karpova NN (2015) Spontaneous sleep-wake cycle and sleep deprivation differently induce Bdnf1, Bdnf4 and Bdnf9a DNA methylation and transcripts levels in the basal forebrain and frontal cortex in rats. J Sleep Res 24(2):124–130. doi: 10.1111/jsr.12242 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Neuroscience CenterUniversity of HelsinkiHelsinkiFinland

Personalised recommendations