Light-controlled Gene Expression Research Articles

Development of label-free light-controlled gene expression technologies using mid-IR and terahertz light.

Gene expression is a fundamental process that regulates diverse biological activities across all life stages. Given its vital role, there is an urgent need to develop innovative methodologies to effectively control gene expression. Light-controlled gene expression is considered a favorable approach because of its ability to provide precise spatiotemporal control. However, current light-controlled technologies rely on photosensitive molecular tags, making their practical use challenging. In this study, we review current technologies for light-controlled gene expression and propose the development of label-free light-controlled technologies using mid-infrared (mid-IR) and terahertz light.

PCSboost: A Multi-Model Machine Learning Framework for Key Fragments Selection of Channelrhodopsins Achieving Optogenetics

Optogenetics combines optical and genetic methods to modulate light-controlled gene expression, protein localization, signal transduction and protein interactions to achieve precise control of specific neuronal activity, with the advantages of low tissue damage, high spatial and temporal resolution, and genetic specificity. It provides a cutting-edge approach to establishing a causal relationship between brain activity and behaviors associated with health and disease. Channelrhodopsin (ChR) functions as a photogenic activator for the control of neurons. As a result, ChR and its variants are more widely used in the realization of optogenetics. To enable effective optogenetics, we propose a novel multi-model machine learning framework, i.e., PCSboost, to accurately assist key fragments selection of ChRs segments that realize optogenetics from protein sequence structure and information dataset. We investigate the key regions of the ChR variant protein fragments that impact photocurrent properties of interest and automatically screen important fragments that realize optogenetics. To address the issue of the dataset containing a limited quantity of data but a high feature dimension, we employ principal component analysis (PCA) to reduce the dimensionality of the data and perform feature extraction, followed by the XGBoost model to classify the ChRs based on their kinetics, photocurrent and spectral properties. Simultaneously, we employ the SHAP interpretability analysis to perform an interpretability analysis of the ChR variant protein for pointwise, characteristic similarities to identify key regions of the protein fragment structure that contribute to the regulation of photocurrent intensity, photocurrent wavelength sensitivity and nonkinetic properties. Experimental findings demonstrate that our proposed PCSboost approach can speed up genetic and protein engineering investigations, simplify the screening of important protein fragment sections, and potentially be used to advance research in the areas of optogenetics, genetic engineering and protein engineering.

Enhancement of Vivid-based photo-activatable Gal4 transcription factor in mammalian cells.

The Gal4/UAS system is a versatile tool to manipulate exogenous gene expression of cells spatially and temporally in many model organisms. Many variations of light-controllable Gal4/UAS system are now available, following the development of photo-activatable (PA) molecular switches and integration of these tools. However, many PA-Gal4 transcription factors have undesired background transcription activities even in dark conditions, and this severely attenuates reliable light-controlled gene expression. Therefore, it is important to develop reliable PA-Gal4 transcription factors with robust light-induced gene expression and limited background activity. By optimization of synthetic PA-Gal4 transcription factors, we have validated configurations of Gal4 DNA biding domain, transcription activation domain and blue light-dependent dimer formation molecule Vivid (VVD), and applied types of transcription activation domains to develop a new PA-Gal4 transcription factor we have named eGAV (enhanced Gal4-VVD transcription factor). Background activity of eGAV in dark conditions was significantly lower than that of hGAVPO, a commonly used PA-Gal4 transcription factor, and maximum light-induced gene expression levels were also improved. Light-controlled gene expression was verified in cultured HEK293T cells with plasmid-transient transfections, and in mouse EpH4 cells with lentivirus vector-mediated transduction. Furthermore, light-controlled eGAV-mediated transcription was confirmed in transfected neural stem cells and progenitors in developing and adult mouse brain and chick spinal cord, and in adult mouse hepatocytes, demonstrating that eGAV can be applied to a wide range of experimental systems and model organisms.Key words: optogenetics, Gal4/UAS system, transcription, gene expression, Vivid.

Recent Synthetic Biology Approaches for Temperature- and Light-Controlled Gene Expression in Bacterial Hosts.

The expression of genes of interest (GOI) can be initiated by providing external stimuli such as temperature shifts and light irradiation. The application of thermal or light stimuli triggers structural changes in stimuli-sensitive biomolecules within the cell, thereby inducing or repressing gene expression. Over the past two decades, several groups have reported genetic circuits that use natural or engineered stimuli-sensitive modules to manipulate gene expression. Here, we summarize versatile strategies of thermosensors and light-driven systems for the conditional expression of GOI in bacterial hosts.

Nuclear Localization Signals for Optimization of Genetically Encoded Tools in Neurons.

Nuclear transport in neurons differs from that in non-neuronal cells. Here we developed a non-opsin optogenetic tool (OT) for the nuclear export of a protein of interest induced by near-infrared (NIR) light. In darkness, nuclear import reverses the OT action. We used this tool for comparative analysis of nuclear transport dynamics mediated by nuclear localization signals (NLSs) with different importin specificities. We found that widely used KPNA2-binding NLSs, such as Myc and SV40, are suboptimal in neurons. We identified uncommon NLSs mediating fast nuclear import and demonstrated that the performance of the OT for nuclear export can be adjusted by varying NLSs. Using these NLSs, we optimized the NIR OT for light-controlled gene expression for lower background and higher contrast in neurons. The selected NLSs binding importins abundant in neurons could improve performance of genetically encoded tools in these cells, including OTs and gene-editing tools.

Optochemical Control of Bacterial Gene Expression: Novel Photocaged Compounds for Different Promoter Systems.

Photocaged compounds are applied for implementing precise, optochemical control of gene expression in bacteria. To broaden the scope of UV‐light‐responsive inducer molecules, six photocaged carbohydrates were synthesized and photochemically characterized, with the absorption exhibiting a red‐shift. Their differing linkage through ether, carbonate, and carbamate bonds revealed that carbonate and carbamate bonds are convenient. Subsequently, those compounds were successfully applied in vivo for controlling gene expression in E. coli via blue light illumination. Furthermore, benzoate‐based expression systems were subjected to light control by establishing a novel photocaged salicylic acid derivative. Besides its synthesis and in vitro characterization, we demonstrate the challenging choice of a suitable promoter system for light‐controlled gene expression in E. coli. We illustrate various bottlenecks during both photocaged inducer synthesis and in vivo application and possibilities to overcome them. These findings pave the way towards novel caged inducer‐dependent systems for wavelength‐selective gene expression.

Modular and Molecular Optimization of a LOV (Light-Oxygen-Voltage)-Based Optogenetic Switch in Yeast.

Optogenetic switches allow light-controlled gene expression with reversible and spatiotemporal resolution. In Saccharomyces cerevisiae, optogenetic tools hold great potential for a variety of metabolic engineering and biotechnology applications. In this work, we report on the modular optimization of the fungal light–oxygen–voltage (FUN-LOV) system, an optogenetic switch based on photoreceptors from the fungus Neurospora crassa. We also describe new switch variants obtained by replacing the Gal4 DNA-binding domain (DBD) of FUN-LOV with nine different DBDs from yeast transcription factors of the zinc cluster family. Among the tested modules, the variant carrying the Hap1p DBD, which we call “HAP-LOV”, displayed higher levels of luciferase expression upon induction compared to FUN-LOV. Further, the combination of the Hap1p DBD with either p65 or VP16 activation domains also resulted in higher levels of reporter expression compared to the original switch. Finally, we assessed the effects of the plasmid copy number and promoter strength controlling the expression of the FUN-LOV and HAP-LOV components, and observed that when low-copy plasmids and strong promoters were used, a stronger response was achieved in both systems. Altogether, we describe a new set of blue-light optogenetic switches carrying different protein modules, which expands the available suite of optogenetic tools in yeast and can additionally be applied to other systems.

Fungal Light-Oxygen-Voltage Domains for Optogenetic Control of Gene Expression and Flocculation in Yeast.

ABSTRACTOptogenetic switches permit accurate control of gene expression upon light stimulation. These synthetic switches have become a powerful tool for gene regulation, allowing modulation of customized phenotypes, overcoming the obstacles of chemical inducers, and replacing their use by an inexpensive resource: light. In this work, we implemented FUN-LOV, an optogenetic switch based on the photon-regulated interaction of WC-1 and VVD, two LOV (light-oxygen-voltage) blue-light photoreceptors from the fungus Neurospora crassa. When tested in yeast, FUN-LOV yields light-controlled gene expression with exquisite temporal resolution and a broad dynamic range of over 1,300-fold, as measured by a luciferase reporter. We also tested the FUN-LOV switch for heterologous protein expression in Saccharomyces cerevisiae, where Western blot analysis confirmed strong induction upon light stimulation, surpassing by 2.5 times the levels achieved with a classic GAL4/galactose chemical-inducible system. Additionally, we utilized FUN-LOV to control the ability of yeast cells to flocculate. Light-controlled expression of the flocculin-encoding gene FLO1, by the FUN-LOV switch, yielded flocculation in light (FIL), whereas the light-controlled expression of the corepressor TUP1 provided flocculation in darkness (FID). Altogether, the results reveal the potential of the FUN-LOV optogenetic switch to control two biotechnologically relevant phenotypes such as heterologous protein expression and flocculation, paving the road for the engineering of new yeast strains for industrial applications. Importantly, FUN-LOV’s ability to accurately manipulate gene expression, with a high temporal dynamic range, can be exploited in the analysis of diverse biological processes in various organisms.

Light-controlled gene expression in yeast using photocaged Cu2+

The manipulation of cellular function, such as the regulation of gene expression, is of great interest to many biotechnological applications and often achieved by the addition of small effector molecules. By combining effector molecules with photolabile protecting groups that mask their biological activity until they are activated by light, precise, yet minimally invasive, photocontrol is enabled. However, applications of this trendsetting technology are limited by the small number of established caged compound-based expression systems. Supported by computational chemistry, we used the versatile photolabile chelator DMNP-EDTA, long-established in neurobiology for photolytic Ca2+ release, to control Cu2+ release upon specific UV-A irradiation. This permits light-mediated control over the widely used Cu2+-inducible pCUP1 promoter from S. cerevisiae and thus constitutes the first example of a caged metal ion to regulate recombinant gene expression. We screened our novel DMNP-EDTA-Cu system for best induction time and expression level of eYFP with a high-throughput online monitoring system equipped with an LED array for individual illumination of every single well. Thereby, we realized a minimally invasive, easy-to-control, parallel and automated optical expression regulation via caged Cu2+ allowing temporal and quantitative control as a beneficial alternative to conventional induction via pipetting CuCl2 as effector molecule.

Optogenetic switches for light-controlled gene expression in yeast.

Light is increasingly recognized as an efficient means of controlling diverse biological processes with high spatiotemporal resolution. Optogenetic switches are molecular devices for regulating light-controlled gene expression, protein localization, signal transduction and protein-protein interactions. Such molecular components have been mainly developed through the use of photoreceptors, which upon light stimulation undergo conformational changes passing to an active state. The current repertoires of optogenetic switches include red, blue and UV-B light photoreceptors and have been implemented in a broad spectrum of biological platforms. In this review, we revisit different optogenetic switches that have been used in diverse biological platforms, with emphasis on those used for light-controlled gene expression in the budding yeast Saccharomyces cerevisiae. The implementation of these switches overcomes the use of traditional chemical inducers, allowing precise control of gene expression at lower costs, without leaving chemical traces, and positively impacting the production of high-value metabolites and heterologous proteins. Additionally, we highlight the potential of utilizing this technology beyond laboratory strains, by optimizing it for use in yeasts tamed for industrial processes. Finally, we discuss how fungal photoreceptors could serve as a source of biological parts for the development of novel optogenetic switches with improved characteristics. Although optogenetic tools have had a strong impact on basic research, their use in applied sciences is still undervalued. Therefore, the invitation for the future is to utilize this technology in biotechnological and industrial settings.

Light-Controlled Cell Factories: Employing Photocaged Isopropyl-β-d-Thiogalactopyranoside for Light-Mediated Optimization of lac Promoter-Based Gene Expression and (+)-Valencene Biosynthesis in Corynebacterium glutamicum.

Optogenetic tools are light-responsive modules that allow for a simple triggering of cellular functions with unprecedented spatiotemporal resolution and in a noninvasive fashion. Specifically, light-controlled gene expression exhibits an enormous potential for various synthetic bio(techno)logical purposes. Before our study, poor inducibility, together with phenotypic heterogeneity, was reported for the IPTG-mediated induction of lac-based gene expression in Corynebacterium glutamicum By applying photocaged IPTG as a synthetic inducer, however, these drawbacks could be almost completely abolished. Especially for increasing numbers of parallelized expression cultures, noninvasive and spatiotemporal light induction qualifies for a precise, homogeneous, and thus higher-order control to fully automatize or optimize future biotechnological applications.

Mesoporous silica-coated upconversion nanocrystals for near infrared light-triggered control of gene expression in zebrafish.

To develop a platform technology for photoactivation of gene expression in deep tissues. Upconversion nanoparticles (UCNs) were synthesized from rare earth elements like Ytterbium, Yttrium and Thulium. The nanoparticles were then further coated with a layer of mesoporous silica and loaded with photomorpholinos or photocaged plasmids and tested in zebrafish. The UCNs were activated using safe near-infrared (NIR) light which in turn produced UV light locally to enable photoactivation in deep tissues. Light-controlled gene knockdown was demonstrated in an in vivo model, namely zebrafish. UCNs loaded with photomorpholinos were used to knockdown a gene - ntl, which is essential for notochord formation and mesoderm patterning in zebrafish using NIR light. UCN-mediated light-controlled gene expression was also achieved by expressing GFP in tumor cells transplanted into adult zebrafish by irradiating the fish with NIR light. Apart from the delivery and control of genes, the UCNs were also used as imaging agents to image both zebrafish embryos and adult zebrafish. enabled excellent background-free, fluorescent imaging of both embryos and adult zebrafish. This technique of controlling gene expression/knockdown through NIR using UCNs is a game changer in the field of genetic manipulation and has the potential of being an excellent, safe and easy to implement tool for developmental biologists to investigate the role of specific genes in development. However, this technique is not restricted to be used only in zebrafish and can be extended for use in other animal models and even for clinical use, in various gene therapy applications.

Optogenetics for gene expression in mammalian cells.

Molecular switches that are controlled by chemicals have evolved as central research instruments in mammalian cell biology. However, these tools are limited in terms of their spatiotemporal resolution due to freely diffusing inducers. These limitations have recently been addressed by the development of optogenetic, genetically encoded, and light-responsive tools that can be controlled with the unprecedented spatiotemporal precision of light. In this article, we first provide a brief overview of currently available optogenetic tools that have been designed to control diverse cellular processes. Then, we focus on recent developments in light-controlled gene expression technologies and provide the reader with a guideline for choosing the most suitable gene expression system.

Near-infrared-light-based nano-platform boosts endosomal escape and controls gene knockdown in vivo.

Current nanoparticle-based gene delivery techniques face two major limitations, namely, endosomal degradation and poor cytosolic release of the nanoparticles and nonspecificity of treatment. These limitations can be overcome with certain light-based techniques, such as photochemical internalization to enable endosomal escape of the delivered nanoparticles and light-controlled gene expression to overcome the nonspecific effects. However, these techniques require UV/visible light, which is either phototoxic and/or has low tissue penetration capabilities, thus preventing their use in deep tissues in a clinical setting. In an effort to overcome these barriers, we have successfully demonstrated a light-based gene delivery system that significantly boosts cytosolic gene delivery, with precise control over gene expression and the potential for use in nonsuperficial tissues. Core-shell fluorescent upconversion nanoparticles excited by highly penetrating near-infrared radiation and emitting simultaneously in the ultraviolet and visible ranges were synthesized and used as remote nanotransducers to simultaneously activate endosomal escape and gene knockdown. Gene knockdown using photomorpholinos was enhanced as much as 30% in vitro compared to the control without endosomal escape facilitation. A similar trend was seen in vivo in a murine melanoma model, demonstrating the enormous clinical potential of this system.

Preparation and affinity-based purification of caged linear DNA for light-controlled gene expression in mammalian cells

A facile and useful method for preparing caged DNAs was developed. The method includes a caging reaction of a linear dsDNA having a minimal sequence of protein expression with Bio-Bhc-diazo and affinity separation of the caged DNA. Effective suppression and photo-mediated restoration of protein expression were demonstrated.

BiotecVisions 2013, February

BiotecVisions 2013, February

CUE1: A Mesophyll Cell-Specific Positive Regulator of Light-Controlled Gene Expression in Arabidopsis

CUE1: A Mesophyll Cell-Specific Positive Regulator of Light-Controlled Gene Expression in Arabidopsis

CUE1: A Mesophyll Cell-Specific Positive Regulator of Light-Controlled Gene Expression in Arabidopsis.

Light plays a key role in the development and physiology of plants. One of the most profound effects of light on plant development is the derepression of expression of an array of light-responsive genes, including the genes encoding the chlorophyll a/b binding proteins (CAB) of photosystem II. To understand the mechanism by which light signals nuclear gene expression, we developed a genetic selection to identify mutants with reduced CAB transcription. Here, we describe a new Arabidopsis locus, CUE1 (for CAB underexpressed). Mutations at this locus result in defects in expression of several light-regulated genes, specifically in mesophyll but not in bundle-associated or epidermis cells. Reduced accumulation of CAB and other photosynthesis-related mRNAs in the mesophyll was correlated with defects in chloroplast development in these cells, resulting in a reticulate pattern with veins greener than the interveinal regions of leaves. Moreover, chalcone synthase mRNA, although known to be regulated by both phytochrome and a blue light receptor, accumulated normally in the leaf epidermis. Dark basal levels of CAB expression were unaffected in etiolated cue1 seedlings; however, induction of CAB transcription by pulses of red and blue light was reduced, suggesting that CUE1 acts downstream from both phytochrome and blue light photoreceptors. CUE1 appears to play a role in the primary derepression of mesophyll-specific gene expression in response to light, because cue1 mutants are severely deficient at establishing photoautotrophic growth. Based on this characterization, we propose that CUE1 is a cell-specific positive regulator linking light and intrinsic developmental programs in Arabidopsis leaf mesophyll cells.