Advertisement

Design and Application of 6mA-Specific Zinc-Finger Proteins for the Readout of DNA Methylation

  • Johannes A. H. Maier
  • Albert Jeltsch
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1867)

Abstract

Designed zinc-finger (ZnF) proteins can recognize AT base pairs by H-bonds in the major groove, which are disrupted, if the adenine base is methylated at the N6 position. Based on this principle, we have recently designed a ZnF protein, which does not bind to DNA, if its recognition site is methylated. In this review, we summarize the principles of the recognition of methylated DNA by proteins and describe the design steps starting with the initial bacterial two-hybrid screening of three-domain ZnF proteins that do not bind to CcrM methylated target sites, followed by their di- and tetramerization to improve binding affinity and specificity. One of the 6mA-specific ZnF proteins was used as repressor to generate a methylation-sensitive promoter/repressor system. This artificial promoter/repressor system was employed to regulate the expression of a CcrM DNA methyltransferase gene, thereby generating an epigenetic system with positive feedback, which can exist in two stable states, an off-state with unmethylated promoter, bound ZnF and repressed gene expression, and an on-state with methylated promoter and active gene expression. This system can memorize transient signals approaching bacterial cells and store the input in the form of DNA methylation patterns. More generally, the ability to bind to DNA in a methylation-dependent manner gives ZnF and TAL proteins an advantage over CRISPR/Cas as DNA-targeting device by allowing methylation-dependent genome or epigenome editing.

Key words

Zinc-finger protein DNA methylation Adenine-N6 methylation Protein design Bacterial gene expression Epigenetic circuit 

References

  1. 1.
    Allis CD, Jenuwein T (2016) The molecular hallmarks of epigenetic control. Nat Rev Genet 17:487–500CrossRefPubMedGoogle Scholar
  2. 2.
    Henikoff S, Greally JM (2016) Epigenetics, cellular memory and gene regulation. Curr Biol 26:R644–R648CrossRefPubMedGoogle Scholar
  3. 3.
    Jeltsch A (2002) Beyond Watson and Crick: DNA methylation and molecular enzymology of DNA methyltransferases. Chembiochem 3:274–293CrossRefPubMedGoogle Scholar
  4. 4.
    Schubeler D (2015) Function and information content of DNA methylation. Nature 517:321–326CrossRefPubMedGoogle Scholar
  5. 5.
    Ambrosi C, Manzo M, Baubec T (2017) Dynamics and context-dependent roles of DNA methylation. J Mol Biol 429:1459–1475CrossRefPubMedGoogle Scholar
  6. 6.
    O'Brown ZK, Greer EL (2016) N6-methyladenine: a conserved and dynamic DNA mark. Adv Exp Med Biol 945:213–246CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Bergman Y, Cedar H (2013) DNA methylation dynamics in health and disease. Nat Struct Mol Biol 20:274–281CrossRefPubMedGoogle Scholar
  8. 8.
    Baylin SB, Jones PA (2011) A decade of exploring the cancer epigenome—biological and translational implications. Nat Rev Cancer 11:726–734CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Jones PA (2012) Functions of DNA methylation: islands, start sites, gene bodies and beyond. Nat Rev Genet 13:484–492CrossRefPubMedGoogle Scholar
  10. 10.
    Wion D, Casadesus J (2006) N6-methyl-adenine: an epigenetic signal for DNA-protein interactions. Nat Rev Microbiol 4:183–192CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Marinus MG, Casadesus J (2009) Roles of DNA adenine methylation in host-pathogen interactions: mismatch repair, transcriptional regulation, and more. FEMS Microbiol Rev 33:488–503CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Casadesus J, Low DA (2013) Programmed heterogeneity: epigenetic mechanisms in bacteria. J Biol Chem 288:13929–13935CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Roberts RJ, Vincze T, Posfai J, Macelis D (2015) REBASE-a database for DNA restriction and modification: enzymes, genes and genomes. Nucleic Acids Res 43:D298–D299CrossRefPubMedGoogle Scholar
  14. 14.
    Barrangou R, Horvath P (2017) A decade of discovery: CRISPR functions and applications. Nat Microbiol 2:17092CrossRefPubMedGoogle Scholar
  15. 15.
    Gonzalez D, Collier J (2013) DNA methylation by CcrM activates the transcription of two genes required for the division of Caulobacter crescentus. Mol Microbiol 88:203–218CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Garvie CW, Wolberger C (2001) Recognition of specific DNA sequences. Mol Cell 8:937–946CrossRefPubMedGoogle Scholar
  17. 17.
    Seeman NC, Rosenberg JM, Rich A (1976) Sequence-specific recognition of double helical nucleic acids by proteins. Proc Natl Acad Sci U S A 73:804–808CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Travers AA (1989) DNA conformation and protein binding. Annu Rev Biochem 58:427–452CrossRefPubMedGoogle Scholar
  19. 19.
    Du Q, Luu PL, Stirzaker C, Clark SJ (2015) Methyl-CpG-binding domain proteins: readers of the epigenome. Epigenomics 7:1051–1073CrossRefPubMedGoogle Scholar
  20. 20.
    Shimbo T, Wade PA (2016) Proteins that read DNA methylation. Adv Exp Med Biol 945:303–320CrossRefPubMedGoogle Scholar
  21. 21.
    Jeltsch A (2008) Reading and writing DNA methylation. Nat Struct Mol Biol 15:1003–1004CrossRefPubMedGoogle Scholar
  22. 22.
    Meehan RR, Lewis JD, McKay S, Kleiner EL, Bird AP (1989) Identification of a mammalian protein that binds specifically to DNA containing methylated CpGs. Cell 58:499–507CrossRefPubMedGoogle Scholar
  23. 23.
    Hashimoto H, Zhang X, Vertino PM, Cheng X (2015) The mechanisms of generation, recognition, and erasure of DNA 5-methylcytosine and thymine oxidations. J Biol Chem 290:20723–20733CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Prokhortchouk A, Hendrich B, Jorgensen H, Ruzov A, Wilm M, Georgiev G, Bird A, Prokhortchouk E (2001) The p120 catenin partner Kaiso is a DNA methylation-dependent transcriptional repressor. Genes Dev 15:1613–1618CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Buck-Koehntop BA, Defossez PA (2013) On how mammalian transcription factors recognize methylated DNA. Epigenetics 8:131–137CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Liu Y, Toh H, Sasaki H, Zhang X, Cheng X (2012) An atomic model of Zfp57 recognition of CpG methylation within a specific DNA sequence. Genes Dev 26:2374–2379CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Liu Y, Olanrewaju YO, Zheng Y, Hashimoto H, Blumenthal RM, Zhang X, Cheng X (2014) Structural basis for Klf4 recognition of methylated DNA. Nucleic Acids Res 42:4859–4867CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Yin Y, Morgunova E, Jolma A, Kaasinen E, Sahu B, Khund-Sayeed S, Das PK, Kivioja T, Dave K, Zhong F, Nitta KR, Taipale M, Popov A, Ginno PA, Domcke S, Yan J, Schubeler D, Vinson C, Taipale J (2017) Impact of cytosine methylation on DNA binding specificities of human transcription factors. Science 356:eaaj2239CrossRefGoogle Scholar
  29. 29.
    Long HK, Blackledge NP, Klose RJ (2013) ZF-CxxC domain-containing proteins, CpG islands and the chromatin connection. Biochem Soc Trans 41:727–740CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Isalan M, Choo Y (2000) Engineered zinc finger proteins that respond to DNA modification by HaeIII and HhaI methyltransferase enzymes. J Mol Biol 295:471–477CrossRefPubMedGoogle Scholar
  31. 31.
    Deng D, Yin P, Yan C, Pan X, Gong X, Qi S, Xie T, Mahfouz M, Zhu JK, Yan N, Shi Y (2012) Recognition of methylated DNA by TAL effectors. Cell Res 22:1502–1504CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Maier JAH, Mohrle R, Jeltsch A (2017) Design of synthetic epigenetic circuits featuring memory effects and reversible switching based on DNA methylation. Nat Commun 8:15336CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Sander JD, Zaback P, Joung JK, Voytas DF, Dobbs D (2007) Zinc Finger Targeter (ZiFiT): an engineered zinc finger/target site design tool. Nucleic Acids Res 35:W599–W605CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Sander JD, Maeder ML, Reyon D, Voytas DF, Joung JK, Dobbs D (2010) ZiFiT (Zinc Finger Targeter): an updated zinc finger engineering tool. Nucleic Acids Res 38:W462–W468CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Wright DA, Thibodeau-Beganny S, Sander JD, Winfrey RJ, Hirsh AS, Eichtinger M, Fu F, Porteus MH, Dobbs D, Voytas DF, Joung JK (2006) Standardized reagents and protocols for engineering zinc finger nucleases by modular assembly. Nat Protoc 1:1637–1652CrossRefPubMedGoogle Scholar
  36. 36.
    O'Shea EK, Klemm JD, Kim PS, Alber T (1991) X-ray structure of the GCN4 leucine zipper, a two-stranded, parallel coiled coil. Science 254:539–544CrossRefPubMedGoogle Scholar
  37. 37.
    Harbury PB, Zhang T, Kim PS, Alber T (1993) A switch between two-, three-, and four-stranded coiled coils in GCN4 leucine zipper mutants. Science 262:1401–1407CrossRefPubMedGoogle Scholar
  38. 38.
    Oehler S, Eismann ER, Kramer H, Muller-Hill B (1990) The three operators of the lac operon cooperate in repression. EMBO J 9:973–979PubMedPubMedCentralCrossRefGoogle Scholar
  39. 39.
    Wolfe SA, Nekludova L, Pabo CO (2000) DNA recognition by Cys2His2 zinc finger proteins. Annu Rev Biophys Biomol Struct 29:183–212CrossRefGoogle Scholar
  40. 40.
    Pabo CO, Peisach E, Grant RA (2001) Design and selection of novel Cys2His2 zinc finger proteins. Annu Rev Biochem 70:313–340CrossRefPubMedGoogle Scholar
  41. 41.
    Segal DJ, Barbas CF 3rd (2001) Custom DNA-binding proteins come of age: polydactyl zinc-finger proteins. Curr Opin Biotechnol 12:632–637CrossRefPubMedGoogle Scholar
  42. 42.
    Beerli RR, Barbas CF 3rd (2002) Engineering polydactyl zinc-finger transcription factors. Nat Biotechnol 20:135–141CrossRefPubMedGoogle Scholar
  43. 43.
    Jamieson AC, Miller JC, Pabo CO (2003) Drug discovery with engineered zinc-finger proteins. Nat Rev Drug Discov 2:361–368CrossRefPubMedGoogle Scholar
  44. 44.
    Deltcheva E, Chylinski K, Sharma CM, Gonzales K, Chao Y, Pirzada ZA, Eckert MR, Vogel J, Charpentier E (2011) CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471:602–607CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821CrossRefGoogle Scholar
  46. 46.
    Sander JD, Joung JK (2014) CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol 32:347–355CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Wang H, La Russa M, Qi LS (2016) CRISPR/Cas9 in genome editing and beyond. Annu Rev Biochem 85:227–264CrossRefPubMedGoogle Scholar
  48. 48.
    Kungulovski G, Jeltsch A (2016) Epigenome editing: state of the art, concepts, and perspectives. Trends Genet 32:101–113CrossRefPubMedGoogle Scholar
  49. 49.
    Lee S, Doddapaneni K, Hogue A, McGhee L, Meyers S, Wu Z (2010) Solution structure of Gfi-1 zinc domain bound to consensus DNA. J Mol Biol 397:1055–1066CrossRefPubMedGoogle Scholar
  50. 50.
    Sera T, Uranga C (2002) Rational design of artificial zinc-finger proteins using a nondegenerate recognition code table. Biochemistry 41:7074–7081CrossRefPubMedGoogle Scholar

Copyright information

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

Authors and Affiliations

  1. 1.Department of Biochemistry, Institute of Biochemistry and Technical BiochemistryUniversity StuttgartStuttgartGermany

Personalised recommendations