Optogenetic regulation of transcription

Abstract

Optogenetics has become widely recognized for its success in real-time control of brain neurons by utilizing non-mammalian photosensitive proteins to open or close membrane channels. Here we review a less well known type of optogenetic constructs that employs photosensitive proteins to transduce the signal to regulate gene transcription, and its possible use in medicine. One of the problems with existing gene therapies is that they could remain active indefinitely while not allowing regulated transgene production on demand. Optogenetic regulation of transcription (ORT) could potentially be used to regulate the production of a biological drug in situ, by repeatedly applying light to the tissue, and inducing expression of therapeutic transgenes when needed. Red and near infrared wavelengths, which are capable of penetration into tissues, have potential for therapeutic applications. Existing ORT systems are reviewed herein with these considerations in mind.

Fig. 1

Tissue penetration spectrum. The range where light penetrates deepest in the tissue, also known as the absorption basin as the phototherapeutic window is located in the range of 750–1100 nm in the near infrared (NIR) part of the spectrum

Fig. 2

Blue light activated systems. a NFAT/Melanopsin system utilizes blue light. The cascade of signaling events opens TRPC to allow calcium ion influx, which, in turn, activates calcineurin that dephosphorylates NFAT and allows for its translocation into the nucleus and the expression of the transgene. b CIB/CRY2 systems are activated by blue light and deactivated by darkness. The figure shows how CRY2/CIB1 was used to induce activation of split Cre recombinase by reconstituting the enzyme through its dimerization. Cre is a site-specific recombinase that catalyzes the recombination between two LoxP to excise sequence between them. Cre enzyme was split into two parts: the N-terminal fragment of Cre fused to CRY2 and the C-terminal fragment of Cre fused to CIB1. Blue-light induced interaction between CRY2 and CIB1 lead to the reconstitution of Cre, which then catalyzes recombination at loxP sites. c In the absence of blue light, the split transcription FKF/GI factor Gal4 is inactive. In the presence of blue light, VP16 links an active domain to the binding domain region of a transcription factor Gal4 that regains its function and enables expression of the gene of interest. TRPC transient receptor protein channels; Gaq Gaq-type G protein; PLC phospholipase C; PKC protein kinase C; NFAT nuclear factor of activated T-cells; CRY2 Cryptochrome 2; CIB CRY2-interacting bHLH; CreC; Pol II polymerase II; goi gene of interest; FKF1 Flavin-binding Kelch repeat F-box; VP16 activation domain of transcription factor VP16; GI GIGANTEA protein; DBD DNA binding domain

Fig. 3

Green light inducible system. In this example of the green light inducible system, CcaS becomes phosphorylated upon illumination at 535 nm as green light activates the system. However, upon red light stimulation the cognate response regulator CcaR is dephosphorylated and the transgene expression is switched off. CcaS Cyanobacteriochrome, CcaR downstream cyanobacteriochrome regulator; pcpcG2 promoter of gene cpcG2 that is regulated by CcaR; cpcB 5′UTR sequence derived from the cpcB gene; ccaR ccaR gene

Fig. 4

Red light-activated systems. a Near-infrared light is detected by BphG1 which triggers the release of GMP and secondary messenger c-di-GMP. The c-di-GMP is recognized by STING and activates the phosphorylation of IRF3 by TBK1. IRF3 then translocates into the nucleus, binds to IRF3-specific operators and induces the IFN controlled promoters. b PhyB/PIF system is activated by 650 nm and deactivated by 750 nm. In this split system, PhyB component is bound to the membrane, while PIF3 is in the cytoplasm. Light activation results in the dimerization of PhyB/PIF and the translocation of PIF-bound protein of interest to the nucleus. c The system incorporates VP16 and the Tet operon with PhyB and PIF. In the TetR-PIF6 split transcription factor construct, the N-terminal fragment of nuclear-targeted PhyB is fused to the VP16 TA domain, and the N-terminal of PIF6—to the tetracycline repressor TetR. To control expression of the gene of interest, the TetR-specific operator, TetO, is inserted into its promoter. Illumination with 650 nm light leads to the reversible heterodimerization of PhyB with PIF6. Since PhyB is fused to TetR, heterodimerization brings VP16 in vicinity of TetO, initiating transcription of the gene of interest. After far-red (740 nm) light-induced conversion of PhyB into its inactive form, the dissociation of PhyB-PIF6 inhibits expression of the transgene. BphG1 Rhodobacter sphaeroides phytochrome BphG1; GMP Guanosine monophosphate; GTP Guanosine triphosphate; c-di-GMP cyclic diguanylate monophosphate; STING stimulator of interferon genes; IRF3 interferon regulatory factor 3; TBK1 tank-binding kinase 1; DGCL diguanylate cyclase; ER endoplasmic reticulum; PINF(ACD +) interferon promoter; Phy phytochrome; PIF phytochrome-interacting factor; TetO tetracycline operator; tetR tetracycline repressor; VP16 activation domain of transcription factor VP16; PIF16 phytochrome interacting factor 16; PhyBFR active FR form of phytochrome B; Pol II polymerase II; goi gene of interest