A light-switchable gene promoter system

Abstract

Regulatable transgene systems providing easily controlled, conditional induction or repression of expression are indispensable tools in biomedical and agricultural research and biotechnology. Several such systems have been developed for eukaryotes. Most of these rely on the administration of either exogenous chemicals or heat shock. Despite the general success of many of these systems, the potential for problems, such as toxic, unintended, or pleiotropic effects of the inducing chemical or treatment, can impose limitations on their use. We have developed a promoter system that can be induced, rapidly and reversibly, by short pulses of light. This system is based on the known red light-induced binding of the plant photoreceptor phytochrome to the protein PIF3 and the reversal of this binding by far-red light. We show here that yeast cells expressing two chimeric proteins, a phytochrome-GAL4-DNA-binding-domain fusion and a PIF3-GAL4-activation-domain fusion, are induced by red light to express selectable or "scorable" marker genes containing promoters with a GAL4 DNA-binding site, and that this induction is rapidly abrogated by subsequent far-red light. We further show that the extent of induction can be controlled precisely by titration of the number of photons delivered to the cells by the light pulse. Thus, this system has the potential to provide rapid, noninvasive, switchable control of the expression of a desired gene to a preselected level in any suitable cell by simple exposure to a light signal.

Phytochromes and Phytochrome Interacting Factors

“…2); however, the functional importance of this interaction has yet to be determined (Fujimori et al, 2004). Interestingly, the APB domain of PIF6 and PIF3 along with the N-terminal domain of phyB have been used to develop a light-switchable gene regulatory module for studying cellular signaling pathways in metazoans (Shimizu-Sato et al, 2002;Levskaya et al, 2009;Toettcher et al, 2011aToettcher et al, , 2011bToettcher et al, , 2013.…”

Genetically encoded photoswitching of actin assembly through the Cdc42-WASP-Arp2/3 complex pathway

“…Recent studies have demonstrated that holo-phytochrome B (holo-PhyB) can be heterologously produced in E. coli through coexpression with two enzymes, heme oxygenase and bilin reductase, that together generate P⌽B from heme (15,16). Similarly, holo-PhyB can be generated in yeast through growth of strains expressing the apo-protein on media containing the phycocyanobilin chromophore (PCB, a close analog of P⌽B) isolated from blue-green algae (17,18). Together, these properties of phytochrome make it an ideal candidate for engineering a reversible, photoswitchable signaling system that combines elements of speed, spatial precision, and reversibility to control the location and timing of protein function.…”

The use of oscillatory signals in the study of genetic networks

“…The light switch is based on a molecule (phytochrome in ref. 5) that is synthesized in darkness in the Q1 form. When the Q1 form absorbs a red light photon (wavelength 664 nm) it is transformed into the form Q2.…”