CpG Islands pp 157-174 | Cite as

Genome-Wide Profiling of DNA Methyltransferases in Mammalian Cells

  • Massimiliano Manzo
  • Christina Ambrosi
  • Tuncay BaubecEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1766)


Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) is currently the method of choice to determine binding sites of chromatin-associated factors in a genome-wide manner. Here, we describe a method to investigate the binding preferences of mammalian DNA methyltransferases (DNMT) based on ChIP-seq using biotin-tagging. Stringent ChIP of DNMT proteins based on the strong interaction between biotin and avidin circumvents limitations arising from low antibody specificity and ensures reproducible enrichment. DNMT-bound DNA fragments are ligated to sequencing adaptors, amplified and sequenced on a high-throughput sequencing instrument. Bioinformatic analysis gives valuable information about the binding preferences of DNMTs genome-wide and around promoter regions. This method is unconventional due to the use of genetically engineered cells; however, it allows specific and reliable determination of DNMT binding.

Key words

ChIP-seq Immunoprecipitation In vivo biotinylation Next-generation sequencing DNA methyltransferases CpG islands 



We thank Isabel Schwarz and Joël Wirz for carefully reading the manuscript prior to submission. Research in the Baubeclab is supported by an SNSF Professorship (SNF157488) and Special Opportunities Grant (2015_322) to T.B., and by the University of Zurich.


  1. 1.
    Okano M, Bell DW, Haber DA, Li E (1999) DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 99(3):247–257CrossRefGoogle Scholar
  2. 2.
    Smith ZD, Meissner A (2013) DNA methylation: roles in mammalian development. Nat Rev Genet 14(3):204–220. CrossRefGoogle Scholar
  3. 3.
    Suzuki MM, Bird A (2008) DNA methylation landscapes: provocative insights from epigenomics. Nat Rev Genet 9(6):465–476. CrossRefGoogle Scholar
  4. 4.
    Landt SG, Marinov GK, Kundaje A, Kheradpour P, Pauli F, Batzoglou S, Bernstein BE, Bickel P, Brown JB, Cayting P, Chen Y, DeSalvo G, Epstein C, Fisher-Aylor KI, Euskirchen G, Gerstein M, Gertz J, Hartemink AJ, Hoffman MM, Iyer VR, Jung YL, Karmakar S, Kellis M, Kharchenko PV, Li Q, Liu T, Liu XS, Ma L, Milosavljevic A, Myers RM, Park PJ, Pazin MJ, Perry MD, Raha D, Reddy TE, Rozowsky J, Shoresh N, Sidow A, Slattery M, Stamatoyannopoulos JA, Tolstorukov MY, White KP, Xi S, Farnham PJ, Lieb JD, Wold BJ, Snyder M (2012) ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia. Genome Res 22(9):1813–1831. CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Einhauer A, Jungbauer A (2001) The FLAG peptide, a versatile fusion tag for the purification of recombinant proteins. J Biochem Biophys Methods 49(1–3):455–465CrossRefGoogle Scholar
  6. 6.
    Kolodziej KE, Pourfarzad F, de Boer E, Krpic S, Grosveld F, Strouboulis J (2009) Optimal use of tandem biotin and V5 tags in ChIP assays. BMC Mol Biol 10:6. CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Wilbanks EG, Larsen DJ, Neches RY, Yao AI, Wu CY, Kjolby RA, Facciotti MT (2012) A workflow for genome-wide mapping of archaeal transcription factors with ChIP-seq. Nucleic Acids Res 40(10):e74. CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Kidder BL, Hu G, Zhao K (2011) ChIP-Seq: technical considerations for obtaining high-quality data. Nat Immunol 12(10):918–922. CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    de Boer E, Rodriguez P, Bonte E, Krijgsveld J, Katsantoni E, Heck A, Grosveld F, Strouboulis J (2003) Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice. Proc Natl Acad Sci U S A 100(13):7480–7485. CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Green NM (1990) Avidin and streptavidin. Methods Enzymol 184:51–67CrossRefGoogle Scholar
  11. 11.
    Baubec T, Colombo DF, Wirbelauer C, Schmidt J, Burger L, Krebs AR, Akalin A, Schubeler D (2015) Genomic profiling of DNA methyltransferases reveals a role for DNMT3B in genic methylation. Nature 520(7546):243–247. CrossRefPubMedGoogle Scholar
  12. 12.
    Lindqvist Y, Schneider G (1996) Protein-biotin interactions. Curr Opin Struct Biol 6(6):798–803CrossRefGoogle Scholar
  13. 13.
    Schatz PJ (1993) Use of peptide libraries to map the substrate specificity of a peptide-modifying enzyme: a 13 residue consensus peptide specifies biotinylation in Escherichia coli. Biotechnology (N Y) 11(10):1138–1143Google Scholar
  14. 14.
    Kim J, Cantor AB, Orkin SH, Wang J (2009) Use of in vivo biotinylation to study protein–protein and protein–DNA interactions in mouse embryonic stem cells. Nat Protoc 4:506–517CrossRefGoogle Scholar
  15. 15.
    Flemr M, Buhler M (2015) Single-step generation of conditional knockout mouse embryonic stem cells. Cell Rep 12(4):709–716. CrossRefPubMedGoogle Scholar
  16. 16.
    Weber M, Hellmann I, Stadler MB, Ramos L, Paabo S, Rebhan M, Schubeler D (2007) Distribution, silencing potential and evolutionary impact of promoter DNA methylation in the human genome. Nat Genet 39(4):457–466. CrossRefPubMedGoogle Scholar
  17. 17.
    Meyer CA, Liu XS (2014) Identifying and mitigating bias in next-generation sequencing methods for chromatin biology. Nat Rev Genet 15(11):709–721. CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
  19. 19.
    Langmead B, Trapnell C, Pop M, Salzberg SL (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10(3):R25. CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Gaidatzis D, Lerch A, Hahne F, Stadler MB (2015) QuasR: quantification and annotation of short reads in R. Bioinformatics 31(7):1130–1132. CrossRefPubMedGoogle Scholar
  21. 21.
    Lawrence M, Huber W, Pages H, Aboyoun P, Carlson M, Gentleman R, Morgan MT, Carey VJ (2013) Software for computing and annotating genomic ranges. PLoS Comput Biol 9(8):e1003118. CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Robinson MD, McCarthy DJ, Smyth GK (2010) edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26(1):139–140. CrossRefPubMedGoogle Scholar
  23. 23.
    Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15(12):550. CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Gentleman R, Carey V, Huber W, Hahne F (2016) genefilter: methods for filtering genes from high-throughput experiments.
  25. 25.
    Karolchik D, Hinrichs AS, Furey TS, Roskin KM, Sugnet CW, Haussler D, Kent WJ (2004) The UCSC Table Browser data retrieval tool. Nucleic Acids Res 32(Database issue):D493–D496. CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Gardiner-Garden M, Frommer M (1987) CpG islands in vertebrate genomes. J Mol Biol 196(2):261–282CrossRefGoogle Scholar
  27. 27.
    Takai D, Jones PA (2002) Comprehensive analysis of CpG islands in human chromosomes 21 and 22. Proc Natl Acad Sci U S A 99(6):3740–3745. CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Hackenberg M, Carpena P, Bernaola-Galvan P, Barturen G, Alganza AM, Oliver JL (2011) WordCluster: detecting clusters of DNA words and genomic elements. Algorithms Mol Biol 6:2. CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Stadler MB, Murr R, Burger L, Ivanek R, Lienert F, Scholer A, van Nimwegen E, Wirbelauer C, Oakeley EJ, Gaidatzis D, Tiwari VK, Schubeler D (2011) DNA-binding factors shape the mouse methylome at distal regulatory regions. Nature 480(7378):490–495. CrossRefPubMedGoogle Scholar
  30. 30.
    Baubec T, Ivanek R, Lienert F, Schubeler D (2013) Methylation-dependent and -independent genomic targeting principles of the MBD protein family. Cell 153(2):480–492. CrossRefPubMedGoogle Scholar
  31. 31.
    Pagès H, Aboyoun P, Gentleman R, DebRoy S (2016) Biostrings: string objects representing biological sequences, and matching algorithms.
  32. 32.
    Illingworth RS, Gruenewald-Schneider U, Webb S, Kerr AR, James KD, Turner DJ, Smith C, Harrison DJ, Andrews R, Bird AP (2010) Orphan CpG islands identify numerous conserved promoters in the mammalian genome. PLoS Genet 6(9):e1001134. CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Akalin A, Franke V, Vlahovicek K, Mason CE, Schubeler D (2015) Genomation: a toolkit to summarize, annotate and visualize genomic intervals. Bioinformatics 31(7):1127–1129. CrossRefPubMedGoogle Scholar
  34. 34.
    mod/mouse/humanENCODE: blacklisted genomic regions for functional genomics analysis (2014)
  35. 35.
    Manzo M, et al (2017) Isoform-specific localization of DNMT3A regulates DNA methylation fidelity at bivalent CpG islands. EMBO J 36:3421–3434CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Massimiliano Manzo
    • 1
    • 2
  • Christina Ambrosi
    • 1
    • 2
  • Tuncay Baubec
    • 1
    Email author
  1. 1.Department of Molecular Mechanisms of DiseaseUniversity of ZurichZurichSwitzerland
  2. 2.Molecular Life Science PhD Program of the Life Science Zurich Graduate SchoolUniversity of ZurichZurichSwitzerland

Personalised recommendations