Advertisement

Rewriting DNA Methylation Signatures at Will: The Curable Genome Within Reach?

  • Sabine Stolzenburg
  • Désirée Goubert
  • Marianne G. RotsEmail author
Chapter
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 945)

Abstract

Epigenetic regulation of gene expression is vital for the maintenance of genome integrity and cell phenotype. In addition, many different diseases have underlying epigenetic mutations, and understanding their role and function may unravel new insights for diagnosis, treatment, and even prevention of diseases. It was an important breakthrough when epigenetic alterations could be gene-specifically manipulated using epigenetic regulatory proteins in an approach termed epigenetic editing. Epigenetic editors can be designed for virtually any gene by targeting effector domains to a preferred sequence, where they write or erase the desired epigenetic modification. This chapter describes the tools for editing DNA methylation signatures and their applications. In addition, we explain how to achieve targeted DNA (de)methylation and discuss the advantages and disadvantages of this approach. Silencing genes directly at the DNA methylation level instead of targeting the protein and/or RNA is a major improvement, as repression is achieved at the source of expression, potentially eliminating the need for continuous administration. Re-expression of silenced genes by targeted demethylation might closely represent the natural situation, in which all transcript variants might be expressed in a sustainable manner. Altogether epigenetic editing, for example, by rewriting DNA methylation, will assist in realizing the curable genome concept.

Keywords

Zinc Finger Zinc Finger Protein SOX2 Promoter Heterochromatic Gene CDKN2A Locus 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Abbreviations

ATF

Artificial transcription factor

ChIP

Chromatin immunoprecipitation

CpG

Cytosine–phosphate–guanine

CRISPRs

Clustered regulatory interspaced palindromic repeats

DNMT

DNA methyltransferase

ncRNA

Nonprotein-coding RNA

sgRNA

Single-guide RNA

TALEs

Transcription activator-like effectors

TDG

Thymidine–DNA glycosylase

TET

Ten–eleven translocation

ZF

Zinc finger

Notes

Acknowledgments

We would like to acknowledge the EU funding for D.G. (H2020-MSCA-ITN-2014-ETN 642691 EpiPredict). M.G.R. serves as vice-chair of H2020-COST CM1406, and her team is partially funded by NWO-Vidi-91786373 and EU-FP7-SNN-4D22C-T2007.

References

  1. Bashtrykov P, Kungulovski G, Jeltsch A. Correction of aberrant imprinting by allele specific epigenome editing. Clin Pharmacol Ther. 2015;99(5):482–4.CrossRefPubMedGoogle Scholar
  2. Beltran A, Parikh S, Liu Y, Cuevas BD, Johnson GL, Futscher BW, et al. Re-activation of a dormant tumor suppressor gene maspin by designed transcription factors. Oncogene. 2007;26(19):​2791–8.CrossRefPubMedGoogle Scholar
  3. Bernstein DL, Le Lay JE, Ruano EG, Kaestner KH. TALE-mediated epigenetic suppression of CDKN2A increases replication in human fibroblasts. J Clin Invest. 2015;125(5):1998–2006.CrossRefPubMedPubMedCentralGoogle Scholar
  4. Bird A, Taggart M, Frommer M, Miller OJ, Macleod D. A fraction of the mouse genome that is derived from islands of nonmethylated, CpG-rich DNA. Cell. 1985;40(1):91–9.CrossRefPubMedGoogle Scholar
  5. Boch J, Bonas U. Xanthomonas AvrBs3 family-type III effectors: discovery and function. Annu Rev Phytopathol. 2010;48:419–36.CrossRefPubMedGoogle Scholar
  6. Bostick M, Kim JK, Esteve PO, Clark A, Pradhan S, Jacobsen SE. UHRF1 plays a role in maintaining DNA methylation in mammalian cells. Science. 2007;317(5845):1760–4.CrossRefPubMedGoogle Scholar
  7. Boyes J, Bird A. Repression of genes by DNA methylation depends on CpG density and promoter strength: evidence for involvement of a methyl-CpG binding protein. EMBO J. 1992;11(1):327–33.PubMedPubMedCentralGoogle Scholar
  8. Briggs AW, Rios X, Chari R, Yang L, Zhang F, Mali P, et al. Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers. Nucleic Acids Res. 2012;40(15):e117.CrossRefPubMedPubMedCentralGoogle Scholar
  9. Cano-Rodriguez D, Gjaltema RA, Jilderda LJ, Jellema P, Dokter-Fokkens J, Ruiters MH, Rots MG. Writing of H3K4Me3 overcomes epigenetic silencing in a sustained but context-dependent manner. Nat Commun. 2016;7:12284. (doi: 10.1038/ncomms12284. PubMed PMID: 27506838).
  10. Carvin CD, Parr RD, Kladde MP. Site-selective in vivo targeting of cytosine-5 DNA methylation by zinc-finger proteins. Nucleic Acids Res. 2003;31(22):6493–501.CrossRefPubMedPubMedCentralGoogle Scholar
  11. Cermak T, Doyle EL, Christian M, Wang L, Zhang Y, Schmidt C, et al. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res. 2011;39(12):e82.CrossRefPubMedPubMedCentralGoogle Scholar
  12. Chen H, Kazemier HG, de Groote ML, Ruiters MH, Xu GL, Rots MG. Induced DNA demethylation by targeting Ten-Eleven Translocation 2 to the human ICAM-1 promoter. Nucleic Acids Res. 2014;42(3):1563–74.CrossRefPubMedGoogle Scholar
  13. Chen T, Hevi S, Gay F, Tsujimoto N, He T, Zhang B, et al. Complete inactivation of DNMT1 leads to mitotic catastrophe in human cancer cells. Nat Genet. 2007;39(3):391–6.CrossRefPubMedGoogle Scholar
  14. Choo Y, Sanchez-Garcia I, Klug A. In vivo repression by a site-specific DNA-binding protein designed against an oncogenic sequence. Nature. 1994;372(6507):642–5.CrossRefPubMedGoogle Scholar
  15. Choudhury SR, Cui Y, Lubecka K, Stefanska B, Irudayaraj J. CRISPR-dCas9 mediated TET1 targeting for selective DNA demethylation at BRCA1 promoter. Oncotarget. 2016. (Epub ahead of print) (doi: 10.18632/oncotarget.10234. PubMed PMID: 27356740).
  16. Cui C, Gan Y, Gu L, Wilson J, Liu Z, Zhang B, et al. P16-specific DNA methylation by engineered zinc finger methyltransferase inactivates gene transcription and promotes cancer metastasis. Genome Biol. 2015;16(1):252.CrossRefPubMedPubMedCentralGoogle Scholar
  17. de Groote ML, Verschure PJ, Rots MG. Epigenetic Editing: targeted rewriting of epigenetic marks to modulate expression of selected target genes. Nucleic Acids Res. 2012;40(21):10596–613.CrossRefPubMedPubMedCentralGoogle Scholar
  18. Dekker AD, De Deyn PP, Rots MG. Epigenetics: the neglected key to minimize learning and memory deficits in Down syndrome. Neurosci Biobehav Rev. 2014;45:72–84.CrossRefPubMedGoogle Scholar
  19. Ehrlich M, Gama-Sosa MA, Huang LH, Midgett RM, Kuo KC, McCune RA, et al. Amount and distribution of 5-methylcytosine in human DNA from different types of tissues of cells. Nucleic Acids Res. 1982;10(8):2709–21.CrossRefPubMedPubMedCentralGoogle Scholar
  20. Falahi F, Huisman C, Kazemier HG, van der Vlies P, Kok K, Hospers GA, et al. Towards sustained silencing of HER2/neu in cancer by epigenetic editing. Mol Cancer Res. 2013;11(9):1029–39.CrossRefPubMedGoogle Scholar
  21. Falahi F, Sgro A, Blancafort P. Epigenome engineering in cancer: fairytale or a realistic path to the clinic? Front Oncol. 2015;5:22.CrossRefPubMedPubMedCentralGoogle Scholar
  22. Gaj T, Gersbach CA, Barbas 3rd CF. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol. 2013;31(7):397–405.CrossRefPubMedPubMedCentralGoogle Scholar
  23. Gowher H, Liebert K, Hermann A, Xu G, Jeltsch A. Mechanism of stimulation of catalytic activity of Dnmt3A and Dnmt3B DNA-(cytosine-C5)-methyltransferases by Dnmt3L. J Biol Chem. 2005;280(14):13341–8.CrossRefPubMedGoogle Scholar
  24. Gregory DJ, Zhang Y, Kobzik L, Fedulov AV. Specific transcriptional enhancement of inducible nitric oxide synthase by targeted promoter demethylation. Epigenetics. 2013;8(11):1205–12.CrossRefPubMedGoogle Scholar
  25. Hilton IB, D'Ippolito AM, Vockley CM, Thakore PI, Crawford GE, Reddy TE, et al. Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat Biotechnol. 2015;33(5):510–7.CrossRefPubMedPubMedCentralGoogle Scholar
  26. Holliday R, Pugh JE. DNA modification mechanisms and gene activity during development. Science. 1975;187(4173):226–32.CrossRefPubMedGoogle Scholar
  27. Huisman C, van der Wijst MG, Falahi F, Overkamp J, Karsten G, Terpstra MM, et al. Prolonged re-expression of the hypermethylated gene EPB41L3 using artificial transcription factors and epigenetic drugs. Epigenetics. 2015a;10(5):384–96.CrossRefPubMedPubMedCentralGoogle Scholar
  28. Huisman C, van der Wijst MG, Schokker M, Blancafort P, Terpstra MM, Kok K, et al. Re-expression of selected epigenetically silenced candidate tumor suppressor genes in cervical cancer by TET2-directed demethylation. Mol Ther. 2015b;24(3):536–47.CrossRefPubMedGoogle Scholar
  29. Huisman C, Wisman GB, Kazemier HG, van Vugt MA, van der Zee AG, Schuuring E, et al. Functional validation of putative tumor suppressor gene C13ORF18 in cervical cancer by Artificial Transcription Factors. Mol Oncol. 2013;7(3):669–79.CrossRefPubMedGoogle Scholar
  30. Jones PA, Takai D. The role of DNA methylation in mammalian epigenetics. Science. 2001;293(5532):1068–70.CrossRefPubMedGoogle Scholar
  31. Jurkowski TP, Ravichandran M, Stepper P. Synthetic epigenetics-towards intelligent control of epigenetic states and cell identity. Clin Epigenetics. 2015;7(1):18.CrossRefPubMedPubMedCentralGoogle Scholar
  32. Kim CA, Berg JM. A 2.2 A resolution crystal structure of a designed zinc finger protein bound to DNA. Nat Struct Biol. 1996;3(11):940–5.CrossRefPubMedGoogle Scholar
  33. Kiss A, Weinhold E. Functional reassembly of split enzymes on-site: a novel approach for highly sequence-specific targeted DNA methylation. Chembiochem. 2008;9(3):351–3.CrossRefPubMedGoogle Scholar
  34. Kungulovski G, Jeltsch A. Epigenome editing: state of the art, concepts, and perspectives. Trends Genet. 2015;32(2):101–13.CrossRefPubMedGoogle Scholar
  35. Kungulovski G, Nunna S, Thomas M, Zanger UM, Reinhardt R, Jeltsch A. Targeted epigenome editing of an endogenous locus with chromatin modifiers is not stably maintained. Epigenetics Chromatin. 2015;8:12.CrossRefPubMedPubMedCentralGoogle Scholar
  36. Lara H, Wang Y, Beltran AS, Juarez-Moreno K, Yuan X, Kato S, et al. Targeting serous epithelial ovarian cancer with designer zinc finger transcription factors. J Biol Chem. 2012;287(35):29873–86.CrossRefPubMedPubMedCentralGoogle Scholar
  37. Ledford H. Targeted gene editing enters clinic. Nature. 2011;471(7336):16.CrossRefPubMedGoogle Scholar
  38. Ledford H. Epigenetics: the genome unwrapped. Nature. 2015;528(7580):S12–3.CrossRefPubMedGoogle Scholar
  39. Li F, Papworth M, Minczuk M, Rohde C, Zhang Y, Ragozin S, et al. Chimeric DNA methyltransferases target DNA methylation to specific DNA sequences and repress expression of target genes. Nucleic Acids Res. 2007;35(1):100–12.CrossRefPubMedGoogle Scholar
  40. Li H, Rauch T, Chen ZX, Szabo PE, Riggs AD, Pfeifer GP. The histone methyltransferase SETDB1 and the DNA methyltransferase DNMT3A interact directly and localize to promoters silenced in cancer cells. J Biol Chem. 2006;281(28):19489–500.CrossRefPubMedGoogle Scholar
  41. Li K, Pang J, Cheng H, Liu WP, Di JM, Xiao HJ, et al. Manipulation of prostate cancer metastasis by locus-specific modification of the CRMP4 promoter region using chimeric TALE DNA methyltransferase and demethylase. Oncotarget. 2015;6(12):10030–44.CrossRefPubMedPubMedCentralGoogle Scholar
  42. Lister R, Mukamel EA, Nery JR, Urich M, Puddifoot CA, Johnson ND, et al. Global epigenomic reconfiguration during mammalian brain development. Science. 2013;341(6146):1237905.CrossRefPubMedPubMedCentralGoogle Scholar
  43. Lister R, Pelizzola M, Dowen RH, Hawkins RD, Hon G, Tonti-Filippini J, et al. Human DNA methylomes at base resolution show widespread epigenomic differences. Nature. 2009;462(7271):315–22.CrossRefPubMedPubMedCentralGoogle Scholar
  44. Maeder ML, Angstman JF, Richardson ME, Linder SJ, Cascio VM, Tsai SQ, et al. Targeted DNA demethylation and activation of endogenous genes using programmable TALE-TET1 fusion proteins. Nat Biotechnol. 2013a;31(12):1137–42.CrossRefPubMedPubMedCentralGoogle Scholar
  45. Maeder ML, Linder SJ, Reyon D, Angstman JF, Fu Y, Sander JD, et al. Robust, synergistic regulation of human gene expression using TALE activators. Nat Methods. 2013b;10(3):243–5.CrossRefPubMedPubMedCentralGoogle Scholar
  46. McDonald JI, Celik H, Rois LE, Fishberger G, Fowler T, Rees R, et al. Reprogrammable CRISPR/Cas9-based system for inducing site-specific DNA methylation. Biol Open. 2016;5(6):866–74. (doi: 10.1242/bio.019067. PubMed PMID: 27170255; PubMed Central PMCID: PMC4920199).
  47. McNamara AR, Hurd PJ, Smith AE, Ford KG. Characterisation of site-biased DNA methyltransferases: specificity, affinity and subsite relationships. Nucleic Acids Res. 2002;30(17):3818–30.CrossRefPubMedPubMedCentralGoogle Scholar
  48. Miller JC, Tan S, Qiao G, Barlow KA, Wang J, Xia DF, et al. A TALE nuclease architecture for efficient genome editing. Nat Biotechnol. 2011;29(2):143–8.CrossRefPubMedGoogle Scholar
  49. Minczuk M, Papworth MA, Kolasinska P, Murphy MP, Klug A. Sequence-specific modification of mitochondrial DNA using a chimeric zinc finger methylase. Proc Natl Acad Sci U S A. 2006;103(52):19689–94.CrossRefPubMedPubMedCentralGoogle Scholar
  50. Mussolino C, Morbitzer R, Lutge F, Dannemann N, Lahaye T, Cathomen T. A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity. Nucleic Acids Res. 2011;39(21):9283–93.CrossRefPubMedPubMedCentralGoogle Scholar
  51. Nunna S, Reinhardt R, Ragozin S, Jeltsch A. Targeted methylation of the epithelial cell adhesion molecule (EpCAM) promoter to silence its expression in ovarian cancer cells. PLoS One. 2014;9(1):e87703.CrossRefPubMedPubMedCentralGoogle Scholar
  52. Pogribny IP, Pogribna M, Christman JK, James SJ. Single-site methylation within the p53 promoter region reduces gene expression in a reporter gene construct: possible in vivo relevance during tumorigenesis. Cancer Res. 2000;60(3):588–94.PubMedGoogle Scholar
  53. Raynal NJ, Si J, Taby RF, Gharibyan V, Ahmed S, Jelinek J, et al. DNA methylation does not stably lock gene expression but instead serves as a molecular mark for gene silencing memory. Cancer Res. 2012;72(5):1170–81.CrossRefPubMedPubMedCentralGoogle Scholar
  54. Reyon D, Tsai SQ, Khayter C, Foden JA, Sander JD, Joung JK. FLASH assembly of TALENs for high-throughput genome editing. Nat Biotechnol. 2012;30(5):460–5.CrossRefPubMedPubMedCentralGoogle Scholar
  55. Riggs AD. X inactivation, differentiation, and DNA methylation. Cytogenet Cell Genet. 1975;14(1):9–25.CrossRefPubMedGoogle Scholar
  56. Rivenbark AG, Stolzenburg S, Beltran AS, Yuan X, Rots MG, Strahl BD, et al. Epigenetic reprogramming of cancer cells via targeted DNA methylation. Epigenetics. 2012;7(4):350–60.CrossRefPubMedPubMedCentralGoogle Scholar
  57. Rots MG, Petersen-Mahrt SK. The 2012 IMB Conference: DNA demethylation, repair and beyond. Institute of Molecular Biology, Mainz, Germany, 18-21 October 2012. Epigenomics. 2013;5(1):25–8.CrossRefPubMedGoogle Scholar
  58. Rusk N. CRISPRs and epigenome editing. Nat Methods. 2014;11(1):28.CrossRefPubMedGoogle Scholar
  59. Sander JD, Joung JK. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol. 2014;32(4):347–55.CrossRefPubMedPubMedCentralGoogle Scholar
  60. Saxonov S, Berg P, Brutlag DL. A genome-wide analysis of CpG dinucleotides in the human genome distinguishes two distinct classes of promoters. Proc Natl Acad Sci U S A. 2006;103(5):1412–7.CrossRefPubMedPubMedCentralGoogle Scholar
  61. Sharif J, Muto M, Takebayashi S, Suetake I, Iwamatsu A, Endo TA, et al. The SRA protein Np95 mediates epigenetic inheritance by recruiting Dnmt1 to methylated DNA. Nature. 2007;450(7171):908–12.CrossRefPubMedGoogle Scholar
  62. Sharma S, Kelly TK, Jones PA. Epigenetics in cancer. Carcinogenesis. 2010;31(1):27–36.CrossRefPubMedGoogle Scholar
  63. Siddique AN, Nunna S, Rajavelu A, Zhang Y, Jurkowska RZ, Reinhardt R, et al. Targeted Methylation and Gene Silencing of VEGF-A in Human Cells by Using a Designed Dnmt3a-Dnmt3L Single-Chain Fusion Protein with Increased DNA Methylation Activity. J Mol Biol. 2012;425(3):479–91.CrossRefPubMedGoogle Scholar
  64. Siegfried Z, Eden S, Mendelsohn M, Feng X, Tsuberi BZ, Cedar H. DNA methylation represses transcription in vivo. Nat Genet. 1999;22(2):203–6.CrossRefPubMedGoogle Scholar
  65. Smith AE, Ford KG. Specific targeting of cytosine methylation to DNA sequences in vivo. Nucleic Acids Res. 2007;35(3):740–54.CrossRefPubMedGoogle Scholar
  66. Smith AE, Hurd PJ, Bannister AJ, Kouzarides T, Ford KG. Heritable gene repression through the action of a directed DNA methyltransferase at a chromosomal locus. J Biol Chem. 2008;283(15):9878–85.CrossRefPubMedGoogle Scholar
  67. Snowden AW, Gregory PD, Case CC, Pabo CO. Gene-specific targeting of H3K9 methylation is sufficient for initiating repression in vivo. Curr Biol. 2002;12(24):2159–66.CrossRefPubMedGoogle Scholar
  68. Song F, Smith JF, Kimura MT, Morrow AD, Matsuyama T, Nagase H, et al. Association of tissue-specific differentially methylated regions (TDMs) with differential gene expression. Proc Natl Acad Sci U S A. 2005;102(9):3336–41.CrossRefPubMedPubMedCentralGoogle Scholar
  69. Stein R, Razin A, Cedar H. In vitro methylation of the hamster adenine phosphoribosyltransferase gene inhibits its expression in mouse L cells. Proc Natl Acad Sci U S A. 1982;79(11):3418–22.CrossRefPubMedPubMedCentralGoogle Scholar
  70. Stolzenburg S, Beltran AS, Swift-Scanlan T, Rivenbark AG, Rashwan R, Blancafort P. Stable oncogenic silencing in vivo by programmable and targeted de novo DNA methylation in breast cancer. Oncogene. 2015;34(43):5427–35.CrossRefPubMedPubMedCentralGoogle Scholar
  71. van der Gun BT, Huisman C, Stolzenburg S, Kazemier HG, Ruiters MH, Blancafort P, et al. Bidirectional modulation of endogenous EpCAM expression to unravel its function in ovarian cancer. Br J Cancer. 2013;108(4):881–6.CrossRefPubMedPubMedCentralGoogle Scholar
  72. van der Gun BT, Maluszynska-Hoffman M, Kiss A, Arendzen AJ, Ruiters MH, McLaughlin PM, et al. Targeted DNA methylation by a DNA methyltransferase coupled to a triple helix forming oligonucleotide to down-regulate the epithelial cell adhesion molecule. Bioconjug Chem. 2010;21(7):1239–45. doi: 10.1021/bc1000388.
  73. van der Wijst MG, Huisman C, Mposhi A, Roelfes G, Rots MG. Targeting Nrf2 in healthy and malignant ovarian epithelial cells: protection versus promotion. Mol Oncol. 2015;9(7):1259–73.CrossRefPubMedGoogle Scholar
  74. Vandevenne M, Jacques DA, Artuz C, Nguyen CD, Kwan AH, Segal DJ, et al. New insights into DNA recognition by zinc fingers revealed by structural analysis of the oncoprotein ZNF217. J Biol Chem. 2013;288(15):10616–27.CrossRefPubMedPubMedCentralGoogle Scholar
  75. Vaquerizas JM, Kummerfeld SK, Teichmann SA, Luscombe NM. A census of human transcription factors: function, expression and evolution. Nat Rev Genet. 2009;10(4):252–63.CrossRefPubMedGoogle Scholar
  76. Vojta A, Dobrinić P, Tadić V, Bočkor L, Korać P, Julg B, et al. Repurposing the CRISPR-Cas9 system for targeted DNA methylation. Nucleic Acids Res. 2016;44(12):5615–28. (doi: 10.1093/nar/gkw159. Epub 2016 Mar 11; PMID: 26969735; PubMed Central PMCID: PMC4937303).
  77. Waddington CH. The epigenotype. 1942. Int J Epidemiol. 2012;41(1):10–3.CrossRefPubMedGoogle Scholar
  78. Xu GL, Bestor TH. Cytosine methylation targetted to pre-determined sequences. Nat Genet. 1997;17(4):376–8.CrossRefPubMedGoogle Scholar
  79. Xu X, Tao Y, Gao X, Zhang L, Li X, Zou W, et al. A CRISPR- based approach for targeted DNA demethylation. Cell Discov. 2016;3;2:16009. (doi: 10.1038/celldisc.2016.9. eCollection 2016. PubMed PMID: 27462456; PubMed Central PMCID: PMC4853773).

Copyright information

© Springer International Publishing Switzerland 2016

Authors and Affiliations

  • Sabine Stolzenburg
    • 1
  • Désirée Goubert
    • 1
  • Marianne G. Rots
    • 1
    Email author
  1. 1.Epigenetic Editing Research Group, Department of Pathology and Medical BiologyUniversity Medical Center Groningen, University of GroningenGroningenThe Netherlands

Personalised recommendations