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Polarized displacement by transcription activator-like effectors for regulatory circuits

Nature Chemical Biologyvolume 15pages8087 (2019) | Download Citation


The interplay between DNA-binding proteins plays an important role in transcriptional regulation and could increase the precision and complexity of designed regulatory circuits. Here we show that a transcription activator-like effector (TALE) can displace another TALE protein from DNA in a highly polarized manner, displacing only the 3′- but not 5′-bound overlapping or adjacent TALE. We propose that the polarized displacement by TALEs is based on its multipartite nature of binding to DNA. The polarized TALE displacement provides strategies for the specific regulation of gene expression, for construction of all two-input Boolean genetic logic circuits based on the robust propagation of the displacement across multiple neighboring sites, for displacement of zinc finger-based transcription factors and for suppression of Cas9–gRNA-mediated genome cleavage, enriching the synthetic biology toolbox and contributing to the understanding of the underlying principles of the facilitated displacement.

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The data that support the findings of this study are available from the corresponding author upon reasonable request.

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  1. 1.

    Khalil, A. S. et al. A synthetic biology framework for programming eukaryotic transcription functions. Cell 150, 647–658 (2012).

  2. 2.

    Weber, W. & Fussenegger, M. Engineering of synthetic mammalian gene networks. Chem. Biol. 16, 287–297 (2009).

  3. 3.

    Gaber, R. et al. Designable DNA-binding domains enable construction of logic circuits in mammalian cells. Nat. Chem. Biol. 10, 203–208 (2014).

  4. 4.

    Kiani, S. et al. CRISPR transcriptional repression devices and layered circuits in mammalian cells. Nat. Methods 11, 723–726 (2014).

  5. 5.

    Lebar, T. & Jerala, R. Benchmarking of TALE- and CRISPR/dCas9-based transcriptional regulators in mammalian cells for the construction of synthetic genetic circuits. ACS Synth. Biol. 5, 1050–1058 (2016).

  6. 6.

    Qi, L. S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173–1183 (2013).

  7. 7.

    Kleinjan, D. A., Wardrope, C., Nga Sou, S. & Rosser, S. J. Drug-tunable multidimensional synthetic gene control using inducible degron-tagged dCas9 effectors. Nat. Commun. 8, 1191 (2017).

  8. 8.

    Green, A. A., Silver, P. A., Collins, J. J. & Yin, P. Toehold switches: de-novo-designed regulators of gene expression. Cell 159, 925–939 (2014).

  9. 9.

    Weinberg, B. H. et al. Large-scale design of robust genetic circuits with multiple inputs and outputs for mammalian cells. Nat. Biotechnol. 35, 453–462 (2017).

  10. 10.

    Siuti, P., Yazbek, J. & Lu, T. K. Synthetic circuits integrating logic and memory in living cells. Nat. Biotechnol. 31, 448–452 (2013).

  11. 11.

    Moscou, M. J. & Bogdanove, A. J. A simple cipher governs DNA recognition by TAL effectors. Science 326, 1501 (2009).

  12. 12.

    Fu, Y. et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat. Biotechnol. 31, 822–826 (2013).

  13. 13.

    Huntley, S. et al. A comprehensive catalog of human KRAB-associated zinc finger genes: insights into the evolutionary history of a large family of transcriptional repressors. Genome Res. 16, 669–677 (2006).

  14. 14.

    Zhang, F. et al. Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Nat. Biotechnol. 29, 149–153 (2011).

  15. 15.

    Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

  16. 16.

    Boch, J. et al. Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326, 1509–1512 (2009).

  17. 17.

    Li, Y. et al. Modular construction of mammalian gene circuits using TALE transcriptional repressors. Nat. Chem. Biol. 11, 207–213 (2015).

  18. 18.

    Lebar, T. et al. A bistable genetic switch based on designable DNA-binding domains. Nat. Commun. 5, 5007 (2014).

  19. 19.

    Lonzarić, J., Lebar, T., Majerle, A., Manček-Keber, M. & Jerala, R. Locked and proteolysis-based transcription activator-like effector (TALE) regulation. Nucleic Acids Res. 44, 1471–1481 (2016).

  20. 20.

    Gao, X. et al. Comparison of TALE designer transcription factors and the CRISPR/dCas9 in regulation of gene expression by targeting enhancers. Nucleic Acids Res. 42, e155 (2014).

  21. 21.

    Lienert, F. et al. Two- and three-input TALE-based AND logic computation in embryonic stem cells. Nucleic Acids Res. 41, 9967–9975 (2013).

  22. 22.

    Deng, D. et al. Structural basis for sequence-specific recognition of DNA by TAL effectors. Science 35, 720–723 (2012).

  23. 23.

    Mak, A. N.-S., Bradley, P., Cernadas, R. A., Bogdanove, A. J. & Stoddard, B. L. The crystal structure of TAL effector PthXo1 bound to its DNA target. Science 335, 716–719 (2012).

  24. 24.

    Gao, H., Wu, X., Chai, J. & Han, Z. Crystal structure of a TALE protein reveals an extended N-terminal DNA binding region. Cell Res. 22, 1716–1720 (2012).

  25. 25.

    Cuculis, L., Abil, Z., Zhao, H. & Schroeder, C. M. Direct observation of TALE protein dynamics reveals a two-state search mechanism. Nat. Commun. 6, 7277 (2015).

  26. 26.

    Meckler, J. F. et al. Quantitative analysis of TALE-DNA interactions suggests polarity effects. Nucleic Acids Res. 41, 4118–4128 (2013).

  27. 27.

    Werner, J. & Gossen, M. Modes of TAL effector-mediated repression. Nucleic Acids Res. 42, 13061–13073 (2014).

  28. 28.

    Kramer, B. P., Fischer, C. & Fussenegger, M. BioLogic gates enable logical transcription control in mammalian cells. Biotechnol. Bioeng. 87, 478–484 (2004).

  29. 29.

    Mussolino, C. et al. A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity. Nucleic Acids Res. 39, 9283–9293 (2011).

  30. 30.

    Chavez, A. et al. Highly efficient Cas9-mediated transcriptional programming. Nat. Methods 12, 326–328 (2015).

  31. 31.

    Kamar, R. I. et al. Facilitated dissociation of transcription factors from single DNA binding sites. Proc. Natl. Acad. Sci. USA 114, E3251–E3257 (2017).

  32. 32.

    Tsai, M.-Y., Zhang, B., Zheng, W. & Wolynes, P. G. Molecular mechanism of facilitated dissociation of Fis protein from DNA. J. Am. Chem. Soc. 138, 13497–13500 (2016).

  33. 33.

    Hadizadeh, N., Johnson, R. C. & Marko, J. F. Facilitated dissociation of a nucleoid protein from the bacterial chromosome. J. Bacteriol. 198, 1735–1742 (2016).

  34. 34.

    Giuntoli, R. D. et al. DNA-segment-facilitated dissociation of Fis and NHP6A from DNA detected via single-molecule mechanical response. J. Mol. Biol. 427, 3123–3136 (2015).

  35. 35.

    Sing, C. E., Olvera, M., Cruz, D. & Marko, J. F. Multiple-binding-site mechanism explains concentration-dependent unbinding rates of DNA-binding proteins. Nucleic Acids Res. 42, 3783–3791 (2014).

  36. 36.

    Cocco, S., Marko, J. F. & Monasson, R. Stochastic ratchet mechanisms for replacement of proteins bound to DNA. Phys. Rev. Lett. 112, 238101 (2014).

  37. 37.

    Åberg, C., Duderstadt, K. E. & van Oijen, A. M. Stability versus exchange: a paradox in DNA replication. Nucleic Acids Res. 44, 4846–4854 (2016).

  38. 38.

    Thiel, G., Lietz, M. & Hohl, M. How mammalian transcriptional repressors work. Eur. J. Biochem. 271, 2855–2862 (2004).

  39. 39.

    Groner, A. C. et al. KRAB-zinc finger proteins and KAP1 can mediate long-range transcriptional repression through heterochromatin spreading. PLoS Genet. 6, e1000869 (2010).

  40. 40.

    Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).

  41. 41.

    Mekler, V., Minakhin, L. & Severinov, K. Mechanism of duplex DNA destabilization by RNA-guided Cas9 nuclease during target interrogation. Proc. Natl. Acad. Sci. USA 114, 5443–5448 (2017).

  42. 42.

    Gilbert, L. A. et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442–451 (2013).

  43. 43.

    Perez-Pinera, P. et al. RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Nat. Methods 10, 973–976 (2013).

  44. 44.

    Maeder, M. L. et al. CRISPR RNA-guided activation of endogenous human genes. Nat. Methods 10, 977–979 (2013).

  45. 45.

    Hinz, J. M., Laughery, M. F. & Wyrick, J. J. Nucleosomes Inhibit Cas9 Endonuclease Activity in Vitro. Biochemistry 54, 7063–7066 (2015).

  46. 46.

    Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).

  47. 47.

    Guschin, D. Y. et al. A rapid and general assay for monitoring endogenous gene modification. Methods Mol. Biol. 649, 247–256 (2010).

  48. 48.

    Pettersen, E. F. et al. UCSF Chimera--a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

  49. 49.

    Deng, D., Yan, C., Wu, J., Pan, X. & Yan, N. Revisiting the TALE repeat. Protein Cell 5, 297–306 (2014).

  50. 50.

    Webb, B. & Sali, A. Comparative protein structure modeling using MODELLER. Curr. Protoc. Protein Sci. 2016, 2.9.1–2.9.37 (2016).

  51. 51.

    Krivov, G. G., Shapovalov, M. V. & Dunbrack, R. L. Jr. Improved prediction of protein side-chain conformations with SCWRL4. Proteins 77, 778–795 (2009).

  52. 52.

    Langlois, C. et al. NMR structure of the complex between the Tfb1 subunit of TFIIH and the activation domain of VP16: structural similarities between VP16 and p53. J. Am. Chem. Soc. 130, 10596–10604 (2008).

  53. 53.

    Hoops, S. et al. COPASI--a COmplex PAthway SImulator. Bioinformatics 22, 3067–3074 (2006).

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This research was supported by grants from the Slovenian Research Agency (J1-6740, P4-0176) and in part by EU structural funds assigned to the EN-FIST Centre of Excellence. T.L. is partially supported by the UNESCO-L’OREAL national fellowship “For Women in Science”. We thank M. Ptashne (Memorial Sloan Kettering Cancer Center, New York City, USA) for the plasmid containing the VP16 domain and O. Griesbeck (Max Planck Institute of Neurobiology, Münich, Germany) for the plasmid with the mCitrine fluorescent protein. We thank K. Ivičak Kocjan and J. Mazej for help in initial experiments on overlapping target sites and R. Krese for help with cloning and experiments on TALE displacement-based logic gates. We are grateful to D. Lainšček for his help and insightful discussions.

Author information


  1. Department of Synthetic Biology and Immunology, National Institute of Chemistry, Ljubljana, Slovenia

    • Tina Lebar
    • , Anže Verbič
    • , Ajasja Ljubetič
    •  & Roman Jerala
  2. EN-FIST Centre of Excellence, Ljubljana, Slovenia

    • Tina Lebar
    •  & Roman Jerala


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T.L. and A.V. prepared the plasmid constructs and conducted the experiments. T.L. and R.J. designed the mechanistic model, and T.L. performed the simulations. A.L. generated the docking and molecular models. T.L. and R.J. designed the experiments and wrote the manuscript. R.J. conceived the study.

Competing interests

The authors filed a patent application on TALE displacement-based inhibition of binding of DNA-binding proteins to DNA.

Corresponding author

Correspondence to Roman Jerala.

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