Light inducible protein-protein interactions are powerful tools to manipulate biological processes. Genetically encoded light-gated proteins for controlling precise cellular behavior are a new and promising technology, called optogenetics. Here we exploited the blue light-induced transcription system in yeast and zebrafish, based on the blue light dependent interaction between two plant proteins, blue light photoreceptor Cryptochrome 2 (CRY2) and the bHLH transcription factor CIB1 (CRY-interacting bHLH 1). We demonstrate the utility of this system by inducing rapid transcription suppression and activation in zebrafish.

Funding: This work was supported by grants from the National Natural Science Foundation of China (number 31270285) and the start funding from Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, and also the United States National Institutes of Health (GM56265 to C.L.). The funders had no role in study design, data collection and manuscript.

Introduction

Using the combination of genetic and optical methods to control specific events in targeted cells or organisms has allowed recent development of optogenetics technology [1]. In comparison to other methods used to manipulate cellular functions and processes, optogenetics methods offer certain advantages, such as rapid delivery, lack of toxicity, and reversibility.

Light-dependent protein-protein interaction is a convenient approach in optogenetic control of cellular functions. For example, a phytochrome-dependent transcription regulatory system has been reported for the light control of gene expression in yeast. Phytochromes are plant red/far-red light photoreceptors that undergo red light-dependent physical interaction with the basic helix-loop-helix proteins PIFs (phytochrome interacting factors). Phytochromes and PIFs have been used as the dimerizer pair to make the red light controlled system, such as the light switchable transcription system in yeast [2]. In addition, other approaches, such as the light regulated protein translocation system have also been widely used [3]. Although the interaction between Phytochromes and PIF offers rapid stimulation and reversibility, the interaction requires a bilin cofactor which could not be found in lots of organisms, such as animals, including zebrafish. This deters the use of this system in other organisms that do not synthesize the bilin cofactor.

Cryptochromes (CRY) are photolyase-like photoreceptors that regulate growth and development in plants and the circadian clock in plants and animals. Plant cryptochromes are best studied in the reference plant Arabidopsis. Arabidopsis CRY1 and CRY2 mediate primarily blue light regulation of de-etiolation and photoperiodic control of flowering, respectively. The cryptochrome protein contains two domains, the N-terminal PHR (Photolyase-Homologous Region) domain of about 500 residues, and the C-terminal extension CCE (Cryptochrome C-terminal Extension, also called CCT) of various lengths. PHR is the chromophore-binding domain of cryptochromes that binds non-covalently to the chromophore flavin adenine dinucleotide (FAD) and possibly a second chromophore, 5,10-methenyltetrahydrofolate (MTHF) [4], [5], [6]. The CCE domain of Arabidopsis CRY1 and CRY2 which functions as an effector domain is approximately 180 and 110 residues in length, respectively. Arabidopsis CRY2 undergoes blue light-specific interaction with the bHLH protein CIB1 (CRY-interacting bHLH 1), which was isolated in a blue light-differentiated yeast-two-hybrid screen [7]. CIB1 is the first protein that interacts with CRY2 in a blue light specific manner in plant, and is a transcription factor whose activity is also blue light and CRY2 dependent.

The chromophore of cryptochromes, FAD, is synthesized in all organisms. The CRY2-CIB1 interaction can be triggered at a subsecond time scale, and is reversible within minutes [8], making it an attractive optogenetics system. A blue light triggered protein translocation system and a DNA recombination system in living cells were made based on the blue light triggered interaction of CIB1 and CRY2 [8]. Very recently, optogenetic control of phosphoinositide metabolism was also developed based on the CIB1 and CRY2 pair [9]. There are blue light inducible transcription systems in yeast and plants [7], [8], but so far no artificial light inducible transcription system has been reported in a vertebrate organism. Here we describe a blue light inducible transcription system in zebrafish.

PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Figure 1. Blue light-dependent transcription regulation in yeast. A. Schematic of split Gal4 modules expressed in yeast cells containing His or LacZ reporter genes under control of a galactose-inducible promoter. In the dark the reporter gene is off. To induce expression of the reporter gene, cells are exposed to blue light which triggers the interaction of CRY2 and CIB1, so that the reporter gene gets activated. B. Histidine auxotrophy assays showing blue light-dependent interaction between CRY2N-489 or 565 and CIB1 or CIB1 N171, and the lack of interaction between CRY2N-375 and CIB1 or CIB1 N171. Yeast cells containing plasmids encoding the indicated proteins were grown on medium in the presence (+) or absence (−) of histidine, under blue light (Blue, 30 µmolm−2 s−1) or in the dark (Dark) for 3 days. C. β-Gal assays of yeast cells expressing indicated proteins irradiated with different fluence rate of blue light (0 to 70 mmol m−2 s−1) for 60 minutes. D. Effect of dark treatment in reversing induction of gene expression by blue light treatment. Yeast cells co-transformed with BD-NCRY2 and AD-NCIB1 were grown in the dark first for 2 hour, then moved to blue light (20 mmol m–2 s–1 ) for 30,60,120,180 min, at every time point, yeast were either incubated further in the blue light for the periods indicated (B) or moved back to dark for the periods indicated (D). For example, when the yeast were treated with blue light for 30 min, samples were taken for the β-galactosidase assay, then the yeast cells were split into two part, one was kept in the blue light condition, while the other one was put into dark condition, 30 min later, samples were take from both the blue light treated and the dark kept yeast cells for the β-galactosidase assay. https://doi.org/10.1371/journal.pone.0050738.g001