Abstract
Optogenetic switches are emerging molecular tools for studying cellular processes as they offer higher spatiotemporal and quantitative precision than classical, chemical-based switches. Light-controllable gene expression systems designed to upregulate protein expression levels meanwhile show performances superior to their chemical-based counterparts. However, systems to reduce protein levels with similar efficiency are lagging behind. Here, we present a novel two-component, blue light-responsive optogenetic OFF switch (‘Blue-OFF’), which enables a rapid and quantitative down-regulation of a protein upon illumination. Blue-OFF combines the first light responsive repressor KRAB-EL222 with the protein degradation module B-LID (blue light-inducible degradation domain) to simultaneously control gene expression and protein stability with a single wavelength. Blue-OFF thus outperforms current optogenetic systems for controlling protein levels. The system is described by a mathematical model which aids in the choice of experimental conditions such as light intensity and illumination regime to obtain the desired outcome. This approach represents an advancement of dual-controlled optogenetic systems in which multiple photosensory modules operate synergistically. As exemplified here for the control of apoptosis in mammalian cell culture, the approach opens up novel perspectives in fundamental research and applications such as tissue engineering.
Highlights
A common approach to study the function of a protein of interest in mammalian cells is to artificially manipulate its expression level
We envisioned that an optogenetic system that actively represses promoter activity and simultaneously targets protein stability would result in superior reduction of cellular protein levels in terms of rate and quantitative control
The optogenetic repressor module KRAB-EL222 was constructed by fusing a repressive KRAB domain, derived from the human kox-1 gene[19], and two nuclear localization signals (NLS) to the N-terminus of EL222
Summary
A common approach to study the function of a protein of interest in mammalian cells is to artificially manipulate its expression level. Chemical-based switches were used to manipulate expression levels by controlling transcription Such systems are based on transcriptional activators or repressors, which alter their conformation and their target DNA-binding affinity upon interaction with specific small molecules. None of the existing systems actively represses transcription which would contribute to an efficient and quantitative reduction of protein levels To address this limitation, we envisioned that an optogenetic system that actively represses promoter activity and simultaneously targets protein stability would result in superior reduction of cellular protein levels in terms of rate and quantitative control. We envisioned that an optogenetic system that actively represses promoter activity and simultaneously targets protein stability would result in superior reduction of cellular protein levels in terms of rate and quantitative control For this purpose, we have developed a novel dual-controlled optogenetic system (‘Blue-OFF’) that combines transcriptional repression with regulation of protein stability, upon illumination with a single wavelength. We demonstrate for the first time its use as light-inducible transcriptional repressor by fusing it to the KRAB transrepressor domain to inhibit transcription from a constitutive promoter[19]
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