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Analysis of 5-Carboxylcytosine Distribution Using DNA Immunoprecipitation

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DNA Modifications

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2198))

Abstract

DNA methylation (5-methylcytosine, 5mC) is involved in regulation of a wide range of biological processes. TET proteins can oxidize 5mC to 5-hydroxymethylcytosine, 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Although both 5fC and 5caC serve as intermediates in active demethylation pathway, growing body of experimental evidence indicate that these DNA modifications may also interact with specific sets of reader proteins and therefore may represent bona fide epigenetic marks. Despite a number of single-base resolution techniques have recently been proposed for 5fC/5caC mapping, antibody-based approaches still represent a relatively simple and plausible alternative for the analysis of genomic distribution of these DNA modifications. Here, we describe a protocol for 5caC DNA immunoprecipitation (5caC DIP) that can be used for both locus-specific and genome-wide assessment of 5caC distribution. In combination with mass spectrometry–based techniques and single base resolution mapping methods, this approach may contribute to elucidating the role of 5caC in development, differentiation, and tumorigenesis.

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References

  1. Smith ZD, Meissner A (2013) DNA methylation: roles in mammalian development. Nat Rev Genet 14(3):204–220. https://doi.org/10.1038/nrg3354

    Article  CAS  PubMed  Google Scholar 

  2. Li E, Bestor TH, Jaenisch R (1992) Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69:915–926. https://doi.org/10.1016/0092-8674(92)90611-f

    Article  CAS  PubMed  Google Scholar 

  3. Okano M, Bell DW, Haber DA et al (1999) DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 99:247–257. https://doi.org/10.1016/s0092-8674(00)81656-6

    Article  CAS  PubMed  Google Scholar 

  4. Bestor TH (1988) Cloning of a mammalian DNA methyltransferase. Gene 74(1):9–12. https://doi.org/10.1016/0378-1119(88)90238-7

    Article  CAS  PubMed  Google Scholar 

  5. Karpf AR, Matsui S (2005) Genetic disruption of cytosine DNA methyltransferase enzymes induces chromosomal instability in human cancer cells. Cancer Res 65:8635–8639. https://doi.org/10.1158/0008-5472.CAN-05-1961

    Article  CAS  PubMed  Google Scholar 

  6. Dodge JE, Okano M, Dick F et al (2005) Inactivation of Dnmt3b in mouse embryonic fibroblasts results in DNA hypomethylation, chromosomal instability, and spontaneous immortalization. J Biol Chem 280:17986–17991. https://doi.org/10.1074/jbc.M413246200

    Article  CAS  PubMed  Google Scholar 

  7. Walsh CP, Chaillet JR, Bestor TH (1998) Transcription of IAP endogenous retroviruses is constrained by cytosine methylation. Nat Genet 20:116–117. https://doi.org/10.1038/2413

    Article  CAS  PubMed  Google Scholar 

  8. Mayer W, Niveleau A, Walter J et al (2000) Demethylation of the zygotic paternal genome. Nature 403(6769):501–502. https://doi.org/10.1038/35000656

    Article  CAS  PubMed  Google Scholar 

  9. Hajkova P, Erhardt S, Lane N et al (2002) Epigenetic reprogramming in mouse primordial germ cells. Mec Dev 117(1–2):15–23. https://doi.org/10.1016/s0925-4773(02)00181-8

    Article  CAS  Google Scholar 

  10. Wu H, Zhang Y (2014) Reversing DNA methylation: mechanisms, genomics, and biological functions. Cell 156(1–2):45–68. https://doi.org/10.1016/j.cell.2013.12.019

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Wu SC, Zhang Y (2010) Active DNA demethylation: many roads lead to Rome. Nat Rev Mol Cell Biol 11(9):607–620. https://doi.org/10.1038/nrm2950

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Bruniquel D, Schwartz RH (2003) Selective, stable demethylation of the interleukin-2 gene enhances transcription by an active process. Nat Immunol 4(3):235–240. https://doi.org/10.1038/ni887

    Article  CAS  PubMed  Google Scholar 

  13. Martinowich K, Hattori D, Wu H et al (2003) DNA methylation-related chromatin remodeling in activity-dependent BDNF gene regulation. Science 302(5646):890–893. https://doi.org/10.1126/science.1090842

    Article  CAS  PubMed  Google Scholar 

  14. Kriaucionis S, Heintz N (2009) The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and the brain. Science 324:929–930. https://doi.org/10.1126/science.1169786

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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. https://doi.org/10.1126/science.1170116

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Ito S, D’Alessio AC, Taranova OV et al (2010) Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification. Nature 466:1129–1133. https://doi.org/10.1038/nature09303

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Pfaffeneder T, Hackner B, Truss M et al (2011) The discovery of 5-formylcytosine in embryonic stem cell DNA. Angew Chem Int Ed 50:7008–7012. https://doi.org/10.1002/anie.201103899

    Article  CAS  Google Scholar 

  18. 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. https://doi.org/10.1126/science.1210597

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. He YF, Li BZ, Li Z et al (2011) Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA. Science 333:1303–1307. https://doi.org/10.1126/science.1210944

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Maiti A, Drohat AC (2011) Thymine DNA glycosylase can rapidly excise 5-formylcytosine and 5-carboxylcytosine: potential implications for active demethylation of CpG sites. J Biol Chem 286:35334–35338. https://doi.org/10.1074/jbc.C111.284620

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Weber AR, Krawczyk C, Robertson AB et al (2016) Biochemical reconstitution of TET1-TDG-BER-dependent active DNA demethylation reveals a highly coordinated mechanism. Nat Commun 7:10806. https://doi.org/10.1038/ncomms10806

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Moler E, Abakir A, Eleftheriou M et al (2018) Population epigenomics: advancing understanding of phenotypic plasticity, acclimation, adaptation and diseases. In: Rajora O (ed) Population genomics. Springer, Cham. https://doi.org/10.1007/13836_2018_59

    Chapter  Google Scholar 

  23. Spruijt CG, Gnerlich F, Smits AH et al (2013) Resource dynamic readers for 5- (hydroxy) methylcytosine and its oxidized derivatives. Cell 152:1146–1159. https://doi.org/10.1016/j.cell.2013.02.004

    Article  CAS  PubMed  Google Scholar 

  24. Bachman M, Uribe-lewis S, Yang X et al (2015) 5-Formylcytosine can be a stable DNA modification in mammals. Nat Chem Biol 11:3–6. https://doi.org/10.1038/nchembio.1848

    Article  CAS  Google Scholar 

  25. Su M, Kirchner A, Stazzoni S et al (2016) 5-formylcytosine could be a semipermanent base in specific genome sites. Angew Chem Int Ed Engl 55:11797–11800. https://doi.org/10.1002/anie.201605994

    Article  CAS  PubMed  Google Scholar 

  26. Iurlaro M, Mcinroy GR, Burgess HE et al (2016) In vivo genome-wide profiling reveals a tissue-specific role for 5-formylcytosine. Genome Biol 1(9). https://doi.org/10.1186/s13059-016-1001-5

  27. Li F, Zhang Y, Bai J et al (2017) 5-formylcytosine yields DNA—protein cross-links in nucleosome core particles. J Am Chem Soc 139:10617–10620. https://doi.org/10.1021/jacs.7b05495

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Raiber E, Portella G, Cuesta SM et al (2018) 5-Formylcytosine organizes nucleosomes and forms Schiff base interactions with histones in mouse embryonic stem cells. Nat Chem 10:1258–1266. https://doi.org/10.1038/s41557-018-0149-x

    Article  CAS  PubMed  Google Scholar 

  29. Ji S, Fu I, Naldiga S et al (2018) 5-Formylcytosine mediated DNA—protein cross-links block DNA replication and induce mutations in human cells. Nucleic Acids Res 46:6455–6469. https://doi.org/10.1093/nar/gky444

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Ji XS, Park D, Kropachev K et al (2019) 5-Formylcytosine-induced DNA—peptide cross-links reduce transcription efficiency , but do not cause transcription errors in human cells. J Biol Chem 294:18387–18397. https://doi.org/10.1074/jbc.RA119.009834

    Article  CAS  PubMed  Google Scholar 

  31. Kellinger MW, Song C, Chong J et al (2012) 5-formylcytosine and 5-carboxylcytosine reduce rate and substrate specificity of RNA polymerase II transcription. Nat Struct Mol Biol 19:831–834. https://doi.org/10.1038/nsmb.2346

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. 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 U S A 89(5):1827–1831. https://doi.org/10.1073/pnas.89.5.1827

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Nestor C, Ruzov A, Meehan R et al (2010) Enzymatic approaches and bisulfite sequencing cannot distinguish between 5-methylcytosine and 5-hydroxymethylcytosine in DNA. BioTechniques 48(4):317–319. https://doi.org/10.2144/000113403

    Article  CAS  PubMed  Google Scholar 

  34. Yu M, Hon GC, Szulwach KE et al (2012) Base-resolution analysis of 5-hydroxymethylcytosine in the mammalian genome. Cell 149(6):1368–1380. https://doi.org/10.1016/j.cell.2012.04.027

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Booth MJ, Branco MR, Ficz G et al (2012) Quantitative sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution. Science 336(6083):934–937. https://doi.org/10.1126/science.1220671

    Article  CAS  PubMed  Google Scholar 

  36. Song CX, Szulwach KE, Dai Q et al (2013) Genome-wide profiling of 5-formylcytosine reveals its roles in epigenetic priming. Cell 153(3):678–691. https://doi.org/10.1016/j.cell.2013.04.001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Lu X, Song CX, Szulwach K et al (2013) Chemical modification-assisted bisulfite sequencing (CAB-Seq) for 5-carboxylcytosine detection in DNA. J Am Chem Soc 135(25):9315–9317. https://doi.org/10.1021/ja4044856

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Neri F, Incarnato D, Krepelova A (2016) Methylation-assisted bisulfite sequencing to simultaneously map 5fC and 5caC on a genome-wide scale for DNA demethylation analysis. Nat Protoc 11(7):1191–1205. https://doi.org/10.1038/nprot.2016.063

    Article  CAS  PubMed  Google Scholar 

  39. Song CX, Clark TA, Lu XY et al (2011) Sensitive and specific single-molecule sequencing of 5-hydroxymethylcytosine. Nat Methods 9(1):75–77. https://doi.org/10.1038/nmeth.1779

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Liu Q, Fang L, Yu G et al (2019) Detection of DNA base modifications by deep recurrent neural network on Oxford nanopore sequencing data. Nat Commun 10:2449. https://doi.org/10.1038/s41467-019-10168-2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Shen L, Wu H, Diep D et al (2013) Genome-wide analysis reveals TET- and TDG-dependent 5-methylcytosine oxidation dynamics. Cell 153(3):692–706. https://doi.org/10.1016/j.cell.2013.04.002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Iurlaro M, McInroy GR, Burgess HE, Dean W et al (2016) In vivo genome-wide profiling reveals a tissue-specific role for 5-formylcytosine. Genome Biol 17:1474. https://doi.org/10.1186/s13059-016-1001-5

    Article  Google Scholar 

  43. Lewis LC, Lo PC, Foster JM, Dai N et al (2017) Dynamics of 5-carboxylcytosine during hepatic differentiation: potential general role for active demethylation by DNA repair in lineage specification. Epigenetics 12(4):277–286. https://doi.org/10.1080/15592294.2017.1292189

    Article  PubMed  PubMed Central  Google Scholar 

  44. Wheldon LM, Abakir A, Ferjentsik Z et al (2014) Transient accumulation of 5-carboxylcytosine indicates involvement of active demethylation in lineage specification of neural stem cells. Cell Rep 7:1353–1361. https://doi.org/10.1016/j.celrep.2014.05.003

    Article  CAS  PubMed  Google Scholar 

  45. Zhang HY, Xiong J, Qi BL et al (2016) The existence of 5-hydroxymethylcytosine and 5-formylcytosine in both DNA and RNA in mammals. Chem Commun 52:737–740. https://doi.org/10.1039/C5CC07354E

    Article  CAS  Google Scholar 

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Acknowledgments

We thank Lara Lewis for technical assistance. A.R.’s lab is supported by Biotechnology and Biological Sciences Research Council [grant number BB/N005759/1] to A.R. A.A. is supported by Medical Research Council IMPACT DTP PhD Studentship [grant number MR/N013913/1] to A.A.

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Authors and Affiliations

  1. Division of Cancer and Stem Cells, School of Medicine, Biodiscovery Institute, University of Nottingham, Nottingham, UK

    Abdulkadir Abakir, Fahad Alenezi & Alexey Ruzov

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  1. Abdulkadir Abakir
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  2. Fahad Alenezi
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  3. Alexey Ruzov
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Correspondence to Abdulkadir Abakir or Alexey Ruzov .

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Editors and Affiliations

  1. Centre for Biomolecular Sciences, University Park, University of Nottingham, Nottingham, UK

    Alexey Ruzov

  2. School of Life Sciences, Queen’s Medical Centre, University of Nottingham, Nottingham, UK

    Martin Gering

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Abakir, A., Alenezi, F., Ruzov, A. (2021). Analysis of 5-Carboxylcytosine Distribution Using DNA Immunoprecipitation. In: Ruzov, A., Gering, M. (eds) DNA Modifications. Methods in Molecular Biology, vol 2198. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0876-0_24

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  • DOI: https://doi.org/10.1007/978-1-0716-0876-0_24

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0875-3

  • Online ISBN: 978-1-0716-0876-0

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