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Near-infrared optogenetic pair for protein regulation and spectral multiplexing

Nature Chemical Biology volume 13, pages 633–639 (2017)Cite this article

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Abstract

Multifunctional optogenetic systems are in high demand for use in basic and biomedical research. Near-infrared-light-inducible binding of bacterial phytochrome BphP1 to its natural PpsR2 partner is beneficial for simultaneous use with blue-light-activatable tools. However, applications of the BphP1–PpsR2 pair are limited by the large size, multidomain structure and oligomeric behavior of PpsR2. Here, we engineered a single-domain BphP1 binding partner, Q-PAS1, which is three-fold smaller and lacks oligomerization. We exploited a helix–PAS fold of Q-PAS1 to develop several near-infrared-light-controllable transcription regulation systems, enabling either 40-fold activation or inhibition. The light-induced BphP1–Q-PAS1 interaction allowed modification of the chromatin epigenetic state. Multiplexing the BphP1–Q-PAS1 pair with a blue-light-activatable LOV-domain-based system demonstrated their negligible spectral crosstalk. By integrating the Q-PAS1 and LOV domains in a single optogenetic tool, we achieved tridirectional protein targeting, independently controlled by near-infrared and blue light, thus demonstrating the superiority of Q-PAS1 for spectral multiplexing and engineering of multicomponent systems.

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Figure 1: Characterization of BphP1 interaction with PpsR2 deletion mutants in vitro.
Figure 2: Transcription inhibition via light-induced relocalization approach.
Figure 3: Light-induced dissociation of chimeric transcription factor.
Figure 4: Light-controlled activation of reporter expression.
Figure 5: Spectral multiplexing of the BphP1–Q-PAS1 system with blue-light-activatable tools.

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References

  1. Deisseroth, K. Optogenetics. Nat. Methods 8, 26–29 (2011).

    Article  CAS  Google Scholar 

  2. Shcherbakova, D.M., Shemetov, A.A., Kaberniuk, A.A. & Verkhusha, V.V. Natural photoreceptors as a source of fluorescent proteins, biosensors, and optogenetic tools. Annu. Rev. Biochem. 84, 519–550 (2015).

    Article  CAS  Google Scholar 

  3. Wang, X., Chen, X. & Yang, Y. Spatiotemporal control of gene expression by a light-switchable transgene system. Nat. Methods 9, 266–269 (2012).

    Article  CAS  Google Scholar 

  4. Strickland, D. et al. TULIPs: tunable, light-controlled interacting protein tags for cell biology. Nat. Methods 9, 379–384 (2012).

    Article  CAS  Google Scholar 

  5. Niopek, D. et al. Engineering light-inducible nuclear localization signals for precise spatiotemporal control of protein dynamics in living cells. Nat. Commun. 5, 4404 (2014).

    Article  CAS  Google Scholar 

  6. Yumerefendi, H. et al. Control of protein activity and cell fate specification via light-mediated nuclear translocation. PLoS One 10, e0128443 (2015).

    Article  Google Scholar 

  7. Niopek, D., Wehler, P., Roensch, J., Eils, R. & Di Ventura, B. Optogenetic control of nuclear protein export. Nat. Commun. 7, 10624 (2016).

    Article  CAS  Google Scholar 

  8. Kennedy, M.J. et al. Rapid blue-light-mediated induction of protein interactions in living cells. Nat. Methods 7, 973–975 (2010).

    Article  CAS  Google Scholar 

  9. Taslimi, A. et al. An optimized optogenetic clustering tool for probing protein interaction and function. Nat. Commun. 5, 4925 (2014).

    Article  CAS  Google Scholar 

  10. Wang, H. et al. LOVTRAP: an optogenetic system for photoinduced protein dissociation. Nat. Methods 13, 755–758 (2016).

    Article  CAS  Google Scholar 

  11. Guntas, G. et al. Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins. Proc. Natl. Acad. Sci. USA 112, 112–117 (2015).

    Article  CAS  Google Scholar 

  12. Taslimi, A. et al. Optimized second-generation CRY2-CIB dimerizers and photoactivatable Cre recombinase. Nat. Chem. Biol. 12, 425–430 (2016).

    Article  CAS  Google Scholar 

  13. Bhoo, S.H., Davis, S.J., Walker, J., Karniol, B. & Vierstra, R.D. Bacteriophytochromes are photochromic histidine kinases using a biliverdin chromophore. Nature 414, 776–779 (2001).

    Article  CAS  Google Scholar 

  14. Toettcher, J.E., Weiner, O.D. & Lim, W.A. Using optogenetics to interrogate the dynamic control of signal transmission by the Ras/Erk module. Cell 155, 1422–1434 (2013).

    Article  CAS  Google Scholar 

  15. Buckley, C.E. et al. Reversible optogenetic control of subcellular protein localization in a live vertebrate embryo. Dev. Cell 36, 117–126 (2016).

    Article  CAS  Google Scholar 

  16. Ulijasz, A.T. & Vierstra, R.D. Phytochrome structure and photochemistry: recent advances toward a complete molecular picture. Curr. Opin. Plant Biol. 14, 498–506 (2011).

    Article  CAS  Google Scholar 

  17. Piatkevich, K.D., Subach, F.V. & Verkhusha, V.V. Engineering of bacterial phytochromes for near-infrared imaging, sensing, and light-control in mammals. Chem. Soc. Rev. 42, 3441–3452 (2013).

    Article  CAS  Google Scholar 

  18. Shcherbakova, D.M., Baloban, M. & Verkhusha, V.V. Near-infrared fluorescent proteins engineered from bacterial phytochromes. Curr. Opin. Chem. Biol. 27, 52–63 (2015).

    Article  CAS  Google Scholar 

  19. Shcherbakova, D.M. & Verkhusha, V.V. Near-infrared fluorescent proteins for multicolor in vivo imaging. Nat. Methods 10, 751–754 (2013).

    Article  CAS  Google Scholar 

  20. Kaberniuk, A.A., Shemetov, A.A. & Verkhusha, V.V. A bacterial phytochrome-based optogenetic system controllable with near-infrared light. Nat. Methods 13, 591–597 (2016).

    Article  CAS  Google Scholar 

  21. Bellini, D. & Papiz, M.Z. Structure of a bacteriophytochrome and light-stimulated protomer swapping with a gene repressor. Structure 20, 1436–1446 (2012).

    Article  CAS  Google Scholar 

  22. Braatsch, S., Johnson, J.A., Noll, K. & Beatty, J.T. The O2-responsive repressor PpsR2 but not PpsR1 transduces a light signal sensed by the BphP1 phytochrome in Rhodopseudomonas palustris CGA009. FEMS Microbiol. Lett. 272, 60–64 (2007).

    Article  CAS  Google Scholar 

  23. Heintz, U., Meinhart, A. & Winkler, A. Multi-PAS domain-mediated protein oligomerization of PpsR from Rhodobacter sphaeroides. Acta Crystallogr. D Biol. Crystallogr. 70, 863–876 (2014).

    Article  CAS  Google Scholar 

  24. Winkler, A. et al. A ternary AppA-PpsR-DNA complex mediates light regulation of photosynthesis-related gene expression. Nat. Struct. Mol. Biol. 20, 859–867 (2013).

    Article  CAS  Google Scholar 

  25. Mallo, M. Controlled gene activation and inactivation in the mouse. Front. Biosci. 11, 313–327 (2006).

    Article  CAS  Google Scholar 

  26. Bacchus, W., Aubel, D. & Fussenegger, M. Biomedically relevant circuit-design strategies in mammalian synthetic biology. Mol. Syst. Biol. 9, 691 (2013).

    Article  CAS  Google Scholar 

  27. Whiteside, S.T. & Goodbourn, S. Signal transduction and nuclear targeting: regulation of transcription factor activity by subcellular localisation. J. Cell Sci. 104, 949–955 (1993).

    CAS  PubMed  Google Scholar 

  28. Hong, M. et al. Structural basis for dimerization in DNA recognition by Gal4. Structure 16, 1019–1026 (2008).

    Article  CAS  Google Scholar 

  29. Delorenzi, M. & Speed, T. An HMM model for coiled-coil domains and a comparison with PSSM-based predictions. Bioinformatics 18, 617–625 (2002).

    Article  CAS  Google Scholar 

  30. Gautier, R., Douguet, D., Antonny, B. & Drin, G. HELIQUEST: a web server to screen sequences with specific alpha-helical properties. Bioinformatics 24, 2101–2102 (2008).

    Article  CAS  Google Scholar 

  31. Laherty, C.D. et al. Histone deacetylases associated with the mSin3 corepressor mediate mad transcriptional repression. Cell 89, 349–356 (1997).

    Article  CAS  Google Scholar 

  32. Xie, M., Haellman, V. & Fussenegger, M. Synthetic biology-application-oriented cell engineering. Curr. Opin. Biotechnol. 40, 139–148 (2016).

    Article  CAS  Google Scholar 

  33. Blancafort, P., Segal, D.J. & Barbas, C.F. III. Designing transcription factor architectures for drug discovery. Mol. Pharmacol. 66, 1361–1371 (2004).

    Article  CAS  Google Scholar 

  34. Ayer, D.E., Laherty, C.D., Lawrence, Q.A., Armstrong, A.P. & Eisenman, R.N. Mad proteins contain a dominant transcription repression domain. Mol. Cell. Biol. 16, 5772–5781 (1996).

    Article  CAS  Google Scholar 

  35. Burcin, M.M., Schiedner, G., Kochanek, S., Tsai, S.Y. & O'Malley, B.W. Adenovirus-mediated regulable target gene expression in vivo. Proc. Natl. Acad. Sci. USA 96, 355–360 (1999).

    Article  CAS  Google Scholar 

  36. Ornitz, D.M., Moreadith, R.W. & Leder, P. Binary system for regulating transgene expression in mice: targeting int-2 gene expression with yeast GAL4/UAS control elements. Proc. Natl. Acad. Sci. USA 88, 698–702 (1991).

    Article  CAS  Google Scholar 

  37. Dong, Z., Peng, J. & Guo, S. Stable gene silencing in zebrafish with spatiotemporally targetable RNA interference. Genetics 193, 1065–1071 (2013).

    Article  CAS  Google Scholar 

  38. Lewandoski, M. Conditional control of gene expression in the mouse. Nat. Rev. Genet. 2, 743–755 (2001).

    Article  CAS  Google Scholar 

  39. Anantharaman, V., Balaji, S. & Aravind, L. The signaling helix: a common functional theme in diverse signaling proteins. Biol. Direct 1, 25 (2006).

    Article  Google Scholar 

  40. Yumerefendi, H. et al. Light-induced nuclear export reveals rapid dynamics of epigenetic modifications. Nat. Chem. Biol. 12, 399–401 (2016).

    Article  CAS  Google Scholar 

  41. Arnoys, E.J. & Wang, J.L. Dual localization: proteins in extracellular and intracellular compartments. Acta Histochem. 109, 89–110 (2007).

    Article  CAS  Google Scholar 

  42. Ausländer, S. & Fussenegger, M. Engineering gene circuits for mammalian cell-based applications. Cold Spring Harb. Perspect. Biol. 8, a023895 (2016).

    Article  Google Scholar 

  43. Müller, K. et al. Multi-chromatic control of mammalian gene expression and signaling. Nucleic Acids Res. 41, e124 (2013).

    Article  Google Scholar 

  44. Webb, B. & Sali, A. Protein structure modeling with MODELLER. Methods Mol. Biol. 1137, 1–15 (2014).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  46. Piatkevich, K.D., Subach, F.V. & Verkhusha, V.V. Far-red light photoactivatable near-infrared fluorescent proteins engineered from a bacterial phytochrome. Nat. Commun. 4, 2153 (2013).

    Article  Google Scholar 

  47. Alland, L. et al. Identification of mammalian Sds3 as an integral component of the Sin3/histone deacetylase corepressor complex. Mol. Cell. Biol. 22, 2743–2750 (2002).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank M. Papiz (Liverpool University, UK), E. Giraud (Institute for Research and Development, France), W. Weber (University of Freiburg, Germany) and F. Zhang (Broad Institute of MIT and Harvard, USA) for plasmids, and A. Leopold (University of Helsinki), A. Kaberniuk and A. Shemetov (both from Albert Einstein College of Medicine) for useful suggestions. We thank Biomedicum Imaging Unit, AAV Gene Transfer and Cell Therapy and Biomedicum Flow Cytometry core facilities staff (University of Helsinki) for the technical assistance. This work was supported by grants GM105997, GM108579 and NS099573 from the US National Institutes of Health, ERC-2013-ADG-340233 from the EU FP7 program, and 263371 and 266992 from the Academy of Finland.

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Authors and Affiliations

  1. Department of Biochemistry and Developmental Biology, Faculty of Medicine, University of Helsinki, Helsinki, Finland

    Taras A Redchuk, Evgeniya S Omelina, Konstantin G Chernov & Vladislav V Verkhusha

  2. Department of Anatomy and Structural Biology and Gruss Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, New York, USA

    Vladislav V Verkhusha

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Contributions

T.A.R. and K.G.C. characterized the proteins in vitro, and T.A.R., E.S.O. and K.G.C. analyzed the optogenetic pair in mammalian cells. V.V.V. planned and directed the project and together with T.A.R., E.S.O. and K.G.C. designed the experiments, analyzed the data and wrote the manuscript.

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Correspondence to Vladislav V Verkhusha.

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Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–3 and Supplementary Figures 1–18 (PDF 2816 kb)

Supplementary Video 1

Sequential light-induced relocalization of the iRIS tool from a cytoplasm to the nucleus and to the plasma membrane in a single cell after the illumination periods (either 5 min with 460 nm of 1 mW cm−2 or 5 min with 740 nm of 1 mW cm−2 light) followed by the dark relaxation periods. (AVI 33424 kb)

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Redchuk, T., Omelina, E., Chernov, K. et al. Near-infrared optogenetic pair for protein regulation and spectral multiplexing. Nat Chem Biol 13, 633–639 (2017). https://doi.org/10.1038/nchembio.2343

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