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An optogenetic gene expression system with rapid activation and deactivation kinetics

Nature Chemical Biology volume 10, pages 196–202 (2014)Cite this article

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Abstract

Optogenetic gene expression systems can control transcription with spatial and temporal detail unequaled with traditional inducible promoter systems. However, current eukaryotic light-gated transcription systems are limited by toxicity, dynamic range or slow activation and deactivation. Here we present an optogenetic gene expression system that addresses these shortcomings and demonstrate its broad utility. Our approach uses an engineered version of EL222, a bacterial light-oxygen-voltage protein that binds DNA when illuminated with blue light. The system has a large (>100-fold) dynamic range of protein expression, rapid activation (<10 s) and deactivation kinetics (<50 s) and a highly linear response to light. With this system, we achieve light-gated transcription in several mammalian cell lines and intact zebrafish embryos with minimal basal gene activation and toxicity. Our approach provides a powerful new tool for optogenetic control of gene expression in space and time.

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Figure 1: Model for the EL222-based light-inducible gene expression system.
Figure 2: Dose-dependent activation and photoreversibility of gene expression by VP-EL222.
Figure 3: Kinetic modeling of VP-EL222 activation.
Figure 4: Light-regulated gene expression of the splicing factor CELF2 using VP-EL222 in the T cell–derived JSL1 cell line.
Figure 5: VP-EL222 robustly activates reporter gene expression in the developing zebrafish embryo in a light-dependent manner.

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References

  1. Weber, W. & Fussenegger, M. Inducible product gene expression technology tailored to bioprocess engineering. Curr. Opin. Biotechnol. 18, 399–410 (2007).

    Article  CAS  Google Scholar 

  2. Weber, W. & Fussenegger, M. Emerging biomedical applications of synthetic biology. Nat. Rev. Genet. 13, 21–35 (2012).

    Article  CAS  Google Scholar 

  3. Briggs, W.R. & Spudich, J.L. Handbook of Photosensory Receptors (Wiley-VCH, 2005).

  4. Shimizu-Sato, S., Huq, E., Tepperman, J.M. & Quail, P.H. A light-switchable gene promoter system. Nat. Biotechnol. 20, 1041–1044 (2002).

    Article  CAS  Google Scholar 

  5. Levskaya, A. et al. Synthetic biology: engineering Escherichia coli to see light. Nature 438, 441–442 (2005).

    Article  CAS  Google Scholar 

  6. Yazawa, M., Sadaghiani, A.M., Hsueh, B. & Dolmetsch, R.E. Induction of protein-protein interactions in live cells using light. Nat. Biotechnol. 27, 941–945 (2009).

    Article  CAS  Google Scholar 

  7. 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 

  8. Ye, H., Daoud-El Baba, M., Peng, R.W. & Fussenegger, M. A synthetic optogenetic transcription device enhances blood-glucose homeostasis in mice. Science 332, 1565–1568 (2011).

    Article  CAS  Google Scholar 

  9. Ohlendorf, R., Vidavski, R.R., Eldar, A., Moffat, K. & Moglich, A. From dusk till dawn: one-plasmid systems for light-regulated gene expression. J. Mol. Biol. 416, 534–542 (2012).

    Article  CAS  Google Scholar 

  10. 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 

  11. Polstein, L.R. & Gersbach, C.A. Light-inducible spatiotemporal control of gene activation by customizable zinc finger transcription factors. J. Am. Chem. Soc. 134, 16480–16483 (2012).

    Article  CAS  Google Scholar 

  12. Liu, H., Gomez, G., Lin, S., Lin, S. & Lin, C. Optogenetic control of transcription in zebrafish. PLoS ONE 7, e50738 (2012).

    Article  CAS  Google Scholar 

  13. Nash, A.I. et al. Structural basis of photosensitivity in a bacterial light-oxygen-voltage/helix-turn-helix (LOV-HTH) DNA-binding protein. Proc. Natl. Acad. Sci. USA 108, 9449–9454 (2011).

    Article  CAS  Google Scholar 

  14. Huala, E. et al. Arabidopsis NPH1—a protein kinase with a putative redox-sensing domain. Science 278, 2120–2123 (1997).

    Article  CAS  Google Scholar 

  15. Rivera-Cancel, G., Motta-Mena, L.B. & Gardner, K.H. Identification of natural and artificial DNA substrates for light-activated LOV-HTH transcription factor EL222. Biochemistry 51, 10024–10034 (2012).

    Article  CAS  Google Scholar 

  16. Zoltowski, B.D., Motta-Mena, L.B. & Gardner, K.H. Blue light–induced dimerization of a bacterial LOV-HTH DNA-binding protein. Biochemistry 52, 6653–6661 (2013).

    Article  CAS  Google Scholar 

  17. Zoltowski, B.D., Nash, A.I. & Gardner, K.H. Variations in protein-flavin hydrogen bonding in a light, oxygen, voltage domain produce non-Arrhenius kinetics of adduct decay. Biochemistry 50, 8771–8779 (2011).

    Article  CAS  Google Scholar 

  18. Sadowski, I., Ma, J., Triezenberg, S. & Ptashne, M. GAL4–VP16 is an unusually potent transcriptional activator. Nature 335, 563–564 (1988).

    Article  CAS  Google Scholar 

  19. Sadowski, I., Bell, B., Broad, P. & Hollis, M. GAL4 fusion vectors for expression in yeast or mammalian cells. Gene 118, 137–141 (1992).

    Article  CAS  Google Scholar 

  20. Swanson, B., Fan, F. & Wood, K. in Promega Corporation Vol. 17 3–5 (Cell Notes, 2007).

  21. Chen, E., Swartz, T.E., Bogomolni, R.A. & Kliger, D.S.A. LOV story: the signaling state of the phot1 LOV2 photocycle involves chromophore-triggered protein structure relaxation, as probed by far-UV time-resolved optical rotatory dispersion spectroscopy. Biochemistry 46, 4619–4624 (2007).

    Article  CAS  Google Scholar 

  22. Kennis, J.T. et al. Primary reactions of the LOV2 domain of phototropin, a plant blue-light photoreceptor. Biochemistry 42, 3385–3392 (2003).

    Article  CAS  Google Scholar 

  23. Harper, S.M., Neil, L.C., Day, I.J., Hore, P.J. & Gardner, K.H. Conformational changes in a photosensory LOV domain monitored by time-resolved NMR spectroscopy. J. Am. Chem. Soc. 126, 3390–3391 (2004).

    Article  CAS  Google Scholar 

  24. Pan, Y.X., Chen, H. & Kilberg, M.S. Interaction of RNA-binding proteins HuR and AUF1 with the human ATF3 mRNA 3′-untranslated region regulates its amino acid limitation-induced stabilization. J. Biol. Chem. 280, 34609–34616 (2005).

    Article  CAS  Google Scholar 

  25. Qian, X. et al. Posttranscriptional regulation of IL-23 expression by IFN-γ through tristetraprolin. J. Immunol. 186, 6454–6464 (2011).

    Article  CAS  Google Scholar 

  26. Thompson, J.F., Hayes, L.S. & Lloyd, D.B. Modulation of firefly luciferase stability and impact on studies of gene regulation. Gene 103, 171–177 (1991).

    Article  CAS  Google Scholar 

  27. Larson, D.R., Zenklusen, D., Wu, B., Chao, J.A. & Singer, R.H. Real-time observation of transcription initiation and elongation on an endogenous yeast gene. Science 332, 475–478 (2011).

    Article  CAS  Google Scholar 

  28. Lynch, K.W. & Weiss, A. A model system for activation-induced alternative splicing of CD45 pre-mRNA in T cells implicates protein kinase C and Ras. Mol. Cell. Biol. 20, 70–80 (2000).

    Article  CAS  Google Scholar 

  29. Ip, J.Y. et al. Global analysis of alternative splicing during T-cell activation. RNA 13, 563–572 (2007).

    Article  CAS  Google Scholar 

  30. Martinez, N.M. et al. Alternative splicing networks regulated by signaling in human T cells. RNA 18, 1029–1040 (2012).

    Article  CAS  Google Scholar 

  31. Faustino, N.A. & Cooper, T.A. Identification of putative new splicing targets for ETR-3 using sequences identified by systematic evolution of ligands by exponential enrichment. Mol. Cell. Biol. 25, 879–887 (2005).

    Article  CAS  Google Scholar 

  32. Mallory, M.J. et al. Signal- and development-dependent alternative splicing of LEF1 in T cells is controlled by CELF2. Mol. Cell. Biol. 31, 2184–2195 (2011).

    Article  CAS  Google Scholar 

  33. Dembowski, J.A. & Grabowski, P.J. The CUGBP2 splicing factor regulates an ensemble of branchpoints from perimeter binding sites with implications for autoregulation. PLoS Genet. 5, e1000595 (2009).

    Article  Google Scholar 

  34. Yelon, D., Horne, S.A. & Stainier, D.Y. Restricted expression of cardiac myosin genes reveals regulated aspects of heart tube assembly in zebrafish. Dev. Biol. 214, 23–37 (1999).

    Article  CAS  Google Scholar 

  35. Harper, S.M., Neil, L.C. & Gardner, K.H. Structural basis of a phototropin light switch. Science 301, 1541–1544 (2003).

    Article  CAS  Google Scholar 

  36. Scheuermann, T.H. et al. Allosteric inhibition of hypoxia inducible factor-2 with small molecules. Nat. Chem. Biol. 9, 271–276 (2013).

    Article  CAS  Google Scholar 

  37. Mattis, J. et al. Principles for applying optogenetic tools derived from direct comparative analysis of microbial opsins. Nat. Methods 9, 159–172 (2012).

    Article  CAS  Google Scholar 

  38. Loew, R., Heinz, N., Hampf, M., Bujard, H. & Gossen, M. Improved Tet-responsive promoters with minimized background expression. BMC Biotechnol. 10, 81 (2010).

    Article  Google Scholar 

  39. Konermann, S. et al. Optical control of mammalian endogenous transcription and epigenetic states. Nature 500, 472–476 (2013).

    Article  CAS  Google Scholar 

  40. Krueger, M., Scholz, O., Wisshak, S. & Hillen, W. Engineered Tet repressors with recognition specificity for the tetO-4C5G operator variant. Gene 404, 93–100 (2007).

    Article  CAS  Google Scholar 

  41. Lynch, K.W. & Weiss, A.A. CD45 polymorphism associated with multiple sclerosis disrupts an exonic splicing silencer. J. Biol. Chem. 276, 24341–24347 (2001).

    Article  CAS  Google Scholar 

  42. Bookout, A.L. & Mangelsdorf, D.J. Quantitative real-time PCR protocol for analysis of nuclear receptor signaling pathways. Nucl. Recept. Signal. 1, e012 (2003).

    Article  Google Scholar 

  43. Schneider, C.A., Rasband, W.S. & Eliceiri, K.W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012).

    Article  CAS  Google Scholar 

  44. Westerfield, M. The Zebrafish Book. A Guide for the Laboratory Use of Zebrafish (Danio rerio) 5th edn. (The University of Oregon Press, 2007).

  45. Balciunas, D. et al. Harnessing a high cargo-capacity transposon for genetic applications in vertebrates. PLoS Genet. 2, e169 (2006).

    Article  Google Scholar 

  46. Huang, C.J., Tu, C.T., Hsiao, C.D., Hsieh, F.J. & Tsai, H.J. Germ-line transmission of a myocardium-specific GFP transgene reveals critical regulatory elements in the cardiac myosin light chain 2 promoter of zebrafish. Dev. Dyn. 228, 30–40 (2003).

    Article  CAS  Google Scholar 

  47. Maizel, A., von Wangenheim, D., Federici, F., Haseloff, J. & Stelzer, E.H. High-resolution live imaging of plant growth in near physiological bright conditions using light sheet fluorescence microscopy. Plant J. 68, 377–385 (2011).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was funded by grants from the US National Institutes of Health (R01 GM081875 and GM106239 to K.H.G.; R01 GM103383 to K.W.L.; R01 GM096164 to O.D.W.), Cancer Prevention and Research Institute of Texas (RP130312), Defense Advanced Research Projects Agency (Living Foundries HR0011-12-C-0068 to B. Chow (University of Pennsylvania), supporting S.G.) and the Robert A. Welch Foundation (I-1424 to K.H.G.). K.H.G. is the Virginia Lazenby O'Hara Chair in Biochemistry and W.W. Caruth Scholar in Biomedical Research. A.R. was supported by a National Science Foundation Graduate Research Fellowship. We thank S.L. McKnight and P.R. Potts (both at UT Southwestern Medical Center) for generously providing constructs.

Author information

Authors and Affiliations

  1. Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, Texas, USA

    Laura B Motta-Mena & Kevin H Gardner

  2. Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas, USA

    Laura B Motta-Mena & Kevin H Gardner

  3. Cardiovascular Research Institute, University of California–San Francisco, San Francisco, California, USA

    Anna Reade & Orion D Weiner

  4. Department of Biochemistry and Biophysics, University of California–San Francisco, San Francisco, California, USA

    Anna Reade & Orion D Weiner

  5. Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Michael J Mallory & Kristen W Lynch

  6. Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Spencer Glantz

  7. Structural Biology Initiative, Advanced Science Research Center, City University of New York, New York, New York, USA

    Kevin H Gardner

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  1. Laura B Motta-Mena
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Contributions

L.B.M.-M., A.R., O.D.W., K.W.L. and K.H.G. conceived and designed the experiments. L.B.M.-M., A.R. and M.J.M. performed the experiments. L.B.M.-M., A.R., M.J.M., O.D.W., K.W.L. and K.H.G. analyzed the data, with S.G. and K.H.G. generating the kinetic model. L.B.M.-M. and K.H.G. wrote the paper.

Corresponding author

Correspondence to Kevin H Gardner.

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Competing interests

L.B.M.M. and K.H.G. have filed US Patent Application PCT/US2012/065493 covering the method described in this paper.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–5, Supplementary Table 1 and Supplementary Notes 1 and 3. (PDF 2701 kb)

Supplementary Note 2

MATLAB code for kinetic model (DOC 28 kb)

Supplementary Video 1

Z-stack of 70% epiboly embryo showing mosaic expression of mCherry after illumination with blue light. (AVI 20581 kb)

Supplementary Video 2

Z-stack of 70% epiboly embryo showing no expression of mCherry under dark conditions. (AVI 24709 kb)

Supplementary Video 3

Localization of fluorescent mCherry in the heart of a developing zebrafish embryo at 24 h.p.f. (AVI 399 kb)

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Motta-Mena, L., Reade, A., Mallory, M. et al. An optogenetic gene expression system with rapid activation and deactivation kinetics. Nat Chem Biol 10, 196–202 (2014). https://doi.org/10.1038/nchembio.1430

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