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Polarized displacement by transcription activator-like effectors for regulatory circuits

Abstract

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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Fig. 1: Polarized inhibition of transcription by TALE binding to overlapping and adjacent target sites.
Fig. 2: Benchmarking of displacement-based repression to other transcriptional inhibition strategies in mammalian cells.
Fig. 3: Chained TALE displacement.
Fig. 4: Implementation of the AND, OR, NAND and XOR Boolean logic functions based on TALE displacement.
Fig. 5: TALE displacement of other DNA-binding domains.
Fig. 6: TALE-mediated prevention of DNA cleavage by the Cas9 nuclease.

Data availability

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

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Acknowledgements

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.

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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.

Corresponding author

Correspondence to Roman Jerala.

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

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Lebar, T., Verbič, A., Ljubetič, A. et al. Polarized displacement by transcription activator-like effectors for regulatory circuits. Nat Chem Biol 15, 80–87 (2019). https://doi.org/10.1038/s41589-018-0163-8

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