Letters
Trends in Biotechnology February 2015, Vol. 33, No. 2
Special Issue: Manifesting Synthetic Biology
UVB-based optogenetic tools Arash Kianianmomeni Department of Cellular and Developmental Biology of Plants, University of Bielefeld, Universita¨tsstraße 25, D-33615 Bielefeld, Germany
Plant and algae photoreceptors that are sensitive to blue and red wavelengths have recently paved the way for the development of optogenetic tools [1]. These tools allow the manipulation and control of cellular signaling pathways with great specificity and fine spatial and temporal precision. However, the development of new light-based strategies in cellular signaling research requires the discovery and engineering of light-sensitive modules with modified absorptive properties and improved kinetic features. The recently characterized UVR8 photoreceptor from the vascular plant Arabidopsis represents a new type of photoreceptor with maximal absorption at UVB wavelengths. UVR8 is a homodimer that dissociates into monomers on UVB exposure, and this dissociation is mediated by specific tryptophans that act as chromophores [2–4]. Subsequently, the UVR8 monomer interacts with its downstream signaling component CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) to regulate gene expression [5]. Advantageous features of UVR8, such as constitutive formation of photolabile homodimers, light-induced interaction with COP1, rapid reversal kinetics, and no need for an exogenous chromophore, make this module promising for synthetic biology applications. In addition, the activation wavelength allows for multicolor imaging in combination with commonly used fluorescent proteins. Engineered UVR8 has been used to control protein–protein interactions involved in protein secretion, nuclear import, chromatin targeting, and gene expression with UV light [6–8]. To control steps in protein secretion with UVB light, photoswitches were designed based on UVB-induced monomerization of UVR8 [6]. To control protein association with the plasma membrane, membrane-localized dimers were formed that could release one of the monomers after UVB exposure. One monomer of UVR8 was fused to mCherry (mCh) and one monomer to a membranelocalizable GFP containing a C-terminal CaaX prenylation motif for targeting to the plasma membrane. After dissociation of the UVR8 dimer in response to UVB exposure, mCh was released into the cytosol (Figure 1A). To control protein trafficking through the endoplasmic reticulum (ER), tandem copies of UVR8 were fused to the secreted VSVG protein, thus promoting the formation of oligomers that are efficiently trapped in the ER. Disruption of these Corresponding author: Kianianmomeni, A. ([email protected]) Keywords: light-sensitive modules; synthetic biology; protein–protein interaction; light-inducible gene expression technology. 0167-7799/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibtech.2014.06.004
oligomers with UVB light allowed forward trafficking through the secretory pathway (Figure 1B) from the ER to the Golgi apparatus and ultimately to the plasma membrane [6]. To demonstrate control of protein translocation to the nucleus, UVR8 was fused to GFP and COP1 was fused to a nuclear localization sequence (NLS) and mCh [7]. UVR8 homodimer formation prevents its interaction with COP1. Subsequent exposure to UVB induced UVR8 monomerization and allowed interaction with COP1, resulting in translocation of monomeric UVR8 to the nucleus (Figure 1C). This approach was extended by fusing histone H2B N-terminally to UVR8, allowing recruitment of COP1 to chromatin in response to UVB light (Figure 1D). In addition, a UVB-induced system of gene expression was built by fusing the NF-kB transcriptional activation domain (AD) to UVR8 and fusing the GAL4 DNA-binding domain (GAL4-BD) to COP1 [7]. UVB-induced UVR8 interaction with COP1 brings together AD and GAL4-BD to activate reporter gene expression from the GAL4 promoter (Figure 1E). Although light-induced transcriptional systems have been engineered before [9–11], combining blue- and redlight-inducible gene expression systems with a UVB-inducible system can allow multichromatic fine-tuning of gene expression in a spatiotemporal fashion [8].The UVB-inducible system here was built by fusing UVR8 to the macrolide repressor E (REP-E) and COP1 to the transactivation domain VP16 [8]. UVB light liberates UVR8 to recruit COP1-VP16, leading to activation of reporter gene expression (Figure 1F). The ability to regulate gene expression could be further extended to obtain more favorable optical control of gene transcription, for example by combining photoswitchable domains with TALE DNAbinding domains [12]. UVB-based photoswitchable tools can be used for precise and specific control of diverse functions in living cells by induction of light-triggered translocation of signaling molecules or protein–protein interactions, allowing finetuned regulation of multiple signaling pathways. Contribution of UVR8 to the optogenetic toolkit offers new potential for the design of multiplexed systems composed of various light switches without overlapping absorption spectra. Such systems could be used for simultaneous control of multiple cellular processes, pathways, and networks using light of different colors. However, to go a step further, more efforts toward application in living organisms or to large-scale bioreactors for cell culture would be necessary to support potential use in biotechnology. In particular, additional work in combination with 59
Letters
Trends in Biotechnology February 2015, Vol. 33, No. 2
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SEAP TRENDS in Biotechnology
Figure 1. Application of the UVB photoreceptor UVR8 in synthetic biology. (A) Light-induced dissociation of UVR8-fused proteins [6]. (B) Disruption of VSVG-UVR8 oligomers following UVB illumination allows forward trafficking through the secretory pathway [6]. (C) UVB-induced interaction between UVR8 and COP1 leads to subsequent translocation of UVR8 from the cytoplasm to the nucleus [7]. (D) Recruitment of COP1 to chromatin in response to UVB light by fusing UVR8 to H2B [7]. (E) UVBinducible gene expression system that increases luciferase activity in response to UVB [7]. (F) UVB-inducible gene expression system that results in activation of secreted alkaline phosphatase (SEAP) [8]. Abbreviations: mCh, mCherry; NLS, nuclear localization signal; AD, transcription activation domain of NF-kB; GAL4-BD, GAL4 DNA-binding domain; Fluc, firefly luciferase; VP16, herpes simplex-derived transactivation domain; REP-E, macrolide repressor E; SEAP, human placental secreted alkaline phosphatase; (etr)8, octameric E-responsive operator site. Panels were adapted, with permission, from the references indicated.
structure-based engineering of UVR8, especially with respect to UVB toxicity in mammalian cells (e.g., by engineering new proteins that require smaller doses of UVB for monomerization), is certainly required to broaden the application of this light-sensitive module in basic research and biomedical science. In light of current advances and growing diversity, future applications of optogenetic tools for precise and spatial control of signaling pathways in 60
complex biological systems, without the need for chemical additives, seem to be more favorable in comparison to chemical systems. Acknowledgments I apologize to colleagues whose work I was unable to cite because of space constraints. My work is supported by the German Research Foundation (DFG).
Letters References 1 Toettcher, J.E. et al. (2011) The promise of optogenetics in cell biology: interrogating molecular circuits in space and time. Nat. Methods 8, 35–38 2 Rizzini, L. et al. (2011) Perception of UV-B by the Arabidopsis UVR8 protein. Science 332, 103–106 3 Wu, D. et al. (2012) Structural basis of ultraviolet-B perception by UVR8. Nature 484, 214–219 4 Christie, J.M. et al. (2012) Plant UVR8 photoreceptor senses UV-B by tryptophan-mediated disruption of cross-dimer salt bridges. Science 335, 1492–1496 5 Favory, J.J. et al. (2009) Interaction of COP1 and UVR8 regulates UVB-induced photomorphogenesis and stress acclimation in Arabidopsis. EMBO J. 28, 591–601
Trends in Biotechnology February 2015, Vol. 33, No. 2
6 Chen, D. et al. (2013) A light-triggered protein secretion system. J. Cell Biol. 201, 631–640 7 Crefcoeur, R.P. et al. (2013) Ultraviolet-B-mediated induction of protein– protein interactions in mammalian cells. Nat. Commun. 4, 1779 8 Muller, K. et al. (2013) Multi-chromatic control of mammalian gene expression and signaling. Nucleic Acids Res. 41, e124 9 Kennedy, M.J. et al. (2010) Rapid blue-light-mediated induction of protein interactions in living cells. Nat. Methods 7, 973–975 10 Shimizu-Sato, S. et al. (2002) A light-switchable gene promoter system. Nat. Biotechnol. 20, 1041–1044 11 Muller, K. and Weber, W. (2013) Optogenetic tools for mammalian systems. Mol. Biosyst. 9, 596–608 12 Konermann, S. et al. (2013) Optical control of mammalian endogenous transcription and epigenetic states. Nature 500, 472–476
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Trends in Biotechnology February 2015, Vol. 33, No. 2
Special Issue: Manifesting Synthetic Biology
UVB-based optogenetic tools Arash Kianianmomeni Department of Cellular and Developmental Biology of Plants, University of Bielefeld, Universita¨tsstraße 25, D-33615 Bielefeld, Germany
Plant and algae photoreceptors that are sensitive to blue and red wavelengths have recently paved the way for the development of optogenetic tools [1]. These tools allow the manipulation and control of cellular signaling pathways with great specificity and fine spatial and temporal precision. However, the development of new light-based strategies in cellular signaling research requires the discovery and engineering of light-sensitive modules with modified absorptive properties and improved kinetic features. The recently characterized UVR8 photoreceptor from the vascular plant Arabidopsis represents a new type of photoreceptor with maximal absorption at UVB wavelengths. UVR8 is a homodimer that dissociates into monomers on UVB exposure, and this dissociation is mediated by specific tryptophans that act as chromophores [2–4]. Subsequently, the UVR8 monomer interacts with its downstream signaling component CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) to regulate gene expression [5]. Advantageous features of UVR8, such as constitutive formation of photolabile homodimers, light-induced interaction with COP1, rapid reversal kinetics, and no need for an exogenous chromophore, make this module promising for synthetic biology applications. In addition, the activation wavelength allows for multicolor imaging in combination with commonly used fluorescent proteins. Engineered UVR8 has been used to control protein–protein interactions involved in protein secretion, nuclear import, chromatin targeting, and gene expression with UV light [6–8]. To control steps in protein secretion with UVB light, photoswitches were designed based on UVB-induced monomerization of UVR8 [6]. To control protein association with the plasma membrane, membrane-localized dimers were formed that could release one of the monomers after UVB exposure. One monomer of UVR8 was fused to mCherry (mCh) and one monomer to a membranelocalizable GFP containing a C-terminal CaaX prenylation motif for targeting to the plasma membrane. After dissociation of the UVR8 dimer in response to UVB exposure, mCh was released into the cytosol (Figure 1A). To control protein trafficking through the endoplasmic reticulum (ER), tandem copies of UVR8 were fused to the secreted VSVG protein, thus promoting the formation of oligomers that are efficiently trapped in the ER. Disruption of these Corresponding author: Kianianmomeni, A. ([email protected]) Keywords: light-sensitive modules; synthetic biology; protein–protein interaction; light-inducible gene expression technology. 0167-7799/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibtech.2014.06.004
oligomers with UVB light allowed forward trafficking through the secretory pathway (Figure 1B) from the ER to the Golgi apparatus and ultimately to the plasma membrane [6]. To demonstrate control of protein translocation to the nucleus, UVR8 was fused to GFP and COP1 was fused to a nuclear localization sequence (NLS) and mCh [7]. UVR8 homodimer formation prevents its interaction with COP1. Subsequent exposure to UVB induced UVR8 monomerization and allowed interaction with COP1, resulting in translocation of monomeric UVR8 to the nucleus (Figure 1C). This approach was extended by fusing histone H2B N-terminally to UVR8, allowing recruitment of COP1 to chromatin in response to UVB light (Figure 1D). In addition, a UVB-induced system of gene expression was built by fusing the NF-kB transcriptional activation domain (AD) to UVR8 and fusing the GAL4 DNA-binding domain (GAL4-BD) to COP1 [7]. UVB-induced UVR8 interaction with COP1 brings together AD and GAL4-BD to activate reporter gene expression from the GAL4 promoter (Figure 1E). Although light-induced transcriptional systems have been engineered before [9–11], combining blue- and redlight-inducible gene expression systems with a UVB-inducible system can allow multichromatic fine-tuning of gene expression in a spatiotemporal fashion [8].The UVB-inducible system here was built by fusing UVR8 to the macrolide repressor E (REP-E) and COP1 to the transactivation domain VP16 [8]. UVB light liberates UVR8 to recruit COP1-VP16, leading to activation of reporter gene expression (Figure 1F). The ability to regulate gene expression could be further extended to obtain more favorable optical control of gene transcription, for example by combining photoswitchable domains with TALE DNAbinding domains [12]. UVB-based photoswitchable tools can be used for precise and specific control of diverse functions in living cells by induction of light-triggered translocation of signaling molecules or protein–protein interactions, allowing finetuned regulation of multiple signaling pathways. Contribution of UVR8 to the optogenetic toolkit offers new potential for the design of multiplexed systems composed of various light switches without overlapping absorption spectra. Such systems could be used for simultaneous control of multiple cellular processes, pathways, and networks using light of different colors. However, to go a step further, more efforts toward application in living organisms or to large-scale bioreactors for cell culture would be necessary to support potential use in biotechnology. In particular, additional work in combination with 59
Letters
Trends in Biotechnology February 2015, Vol. 33, No. 2
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SEAP TRENDS in Biotechnology
Figure 1. Application of the UVB photoreceptor UVR8 in synthetic biology. (A) Light-induced dissociation of UVR8-fused proteins [6]. (B) Disruption of VSVG-UVR8 oligomers following UVB illumination allows forward trafficking through the secretory pathway [6]. (C) UVB-induced interaction between UVR8 and COP1 leads to subsequent translocation of UVR8 from the cytoplasm to the nucleus [7]. (D) Recruitment of COP1 to chromatin in response to UVB light by fusing UVR8 to H2B [7]. (E) UVBinducible gene expression system that increases luciferase activity in response to UVB [7]. (F) UVB-inducible gene expression system that results in activation of secreted alkaline phosphatase (SEAP) [8]. Abbreviations: mCh, mCherry; NLS, nuclear localization signal; AD, transcription activation domain of NF-kB; GAL4-BD, GAL4 DNA-binding domain; Fluc, firefly luciferase; VP16, herpes simplex-derived transactivation domain; REP-E, macrolide repressor E; SEAP, human placental secreted alkaline phosphatase; (etr)8, octameric E-responsive operator site. Panels were adapted, with permission, from the references indicated.
structure-based engineering of UVR8, especially with respect to UVB toxicity in mammalian cells (e.g., by engineering new proteins that require smaller doses of UVB for monomerization), is certainly required to broaden the application of this light-sensitive module in basic research and biomedical science. In light of current advances and growing diversity, future applications of optogenetic tools for precise and spatial control of signaling pathways in 60
complex biological systems, without the need for chemical additives, seem to be more favorable in comparison to chemical systems. Acknowledgments I apologize to colleagues whose work I was unable to cite because of space constraints. My work is supported by the German Research Foundation (DFG).
Letters References 1 Toettcher, J.E. et al. (2011) The promise of optogenetics in cell biology: interrogating molecular circuits in space and time. Nat. Methods 8, 35–38 2 Rizzini, L. et al. (2011) Perception of UV-B by the Arabidopsis UVR8 protein. Science 332, 103–106 3 Wu, D. et al. (2012) Structural basis of ultraviolet-B perception by UVR8. Nature 484, 214–219 4 Christie, J.M. et al. (2012) Plant UVR8 photoreceptor senses UV-B by tryptophan-mediated disruption of cross-dimer salt bridges. Science 335, 1492–1496 5 Favory, J.J. et al. (2009) Interaction of COP1 and UVR8 regulates UVB-induced photomorphogenesis and stress acclimation in Arabidopsis. EMBO J. 28, 591–601
Trends in Biotechnology February 2015, Vol. 33, No. 2
6 Chen, D. et al. (2013) A light-triggered protein secretion system. J. Cell Biol. 201, 631–640 7 Crefcoeur, R.P. et al. (2013) Ultraviolet-B-mediated induction of protein– protein interactions in mammalian cells. Nat. Commun. 4, 1779 8 Muller, K. et al. (2013) Multi-chromatic control of mammalian gene expression and signaling. Nucleic Acids Res. 41, e124 9 Kennedy, M.J. et al. (2010) Rapid blue-light-mediated induction of protein interactions in living cells. Nat. Methods 7, 973–975 10 Shimizu-Sato, S. et al. (2002) A light-switchable gene promoter system. Nat. Biotechnol. 20, 1041–1044 11 Muller, K. and Weber, W. (2013) Optogenetic tools for mammalian systems. Mol. Biosyst. 9, 596–608 12 Konermann, S. et al. (2013) Optical control of mammalian endogenous transcription and epigenetic states. Nature 500, 472–476
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