Exploring plant-derived phytochrome chaperone proteins for light-switchable transcriptional regulation in mammals – Nature Communications

Plasmid construction

Comprehensive design and construction details for all expression vectors are provided in Supplementary Data 1. The DNA sequences for PTRC components are provided in Supplementary Table 1. Some plasmids were constructed using a MultiS One Step Cloning Kit (C113-01, Vazyme) according to the manufacturer’s instructions. All relevant genetic components have been confirmed by sequencing (Personalbio).

Cell culture and transfection

Human embryonic kidney cells (HEK293T cells, CRL-1573, ATCC), baby hamster kidney cells (BHK-21; CCL-10, ATCC), telomerase-immortalized human mesenchymal stem cells (hMSC-TERT; SCRC-4000, ATCC), B16-F10 melanoma cells (CRL-6475, ATCC), HEK293-derived Hana3A cells engineered for constitutive expression of RTP1, RTP2, REEP1 and Gαoλϕ⁵³, human cervical adenocarcinoma cells (HeLa; CCL-2, ATCC), human hepatocellular carcinoma cells (Huh7; TCHu182, Chinese Academy of Sciences), human Retinal Pigment Epithelial cell (ARPE-19; Chinese Academy of Sciences), and the mouse fibroblast NIH-3T3 cell line (CRL-1658, ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; C11995500BT, Gibco) supplemented with 10% (vol/vol) fetal bovine serum (FBS; 16000-044, Gibco) and 1% (vol/vol) penicillin/streptomycin solution (ST488-1/ST488-2, Beyotime). All cell lines were cultured at 37 °C in a humidified atmosphere containing 5% CO₂ and were regularly tested for the absence of mycoplasma and bacterial contamination.

HEK293T, hMSC-TERT, and Hana3A cells were transfected with an optimized polyethyleneimine (PEI)-based protocol. Unless explicitly indicated, all the cell experiments were performed as follows. Briefly, the cells were plated 6 × 10⁴ cells/well in a 24-well plate and cultured for 18 hours before transfection. Subsequently, cells were incubated for 6 hours with 50 µL of PEI and DNA mixture at a mass ratio of 3:1 (PEI, molecular weight 40,000, stock solution 1 mg/mL in double distilled water; 24765, Polysciences). For the transfection of B16-F10, HeLa, Huh7, ARPE-19, and NIH-3T3 cells, the cells were plated at 5 × 10⁴ cells/well in a 24-well plate and cultivated for 12 hours at the time of transfection using Lipofectamine 8000 transfection reagent (C0533FT, Beyotime) according to manufacturer’s protocol. For transfection, HEK293T cells were plated at 5 × 10⁴ cells/well in a 24-well plate and cultivated for 12 hours. At the time of transfection, INVI DNA Transfection Reagent (IV1214, Invigentech) was used according to the manufacturer’s protocol. Detailed transfection mixtures are provided in Supplementary Data 2 and 3.

Small molecule induction

Resveratrol (50 mM; R5010, Sigma-Aldrich) was purchased from Sigma-Aldrich, prepared in 50 mM stock solutions in 100% DMSO, stored at −20 °C, and diluted to different working concentrations using DMEM medium. Stocks (100×) of ABA (10 mM; 90769, Sigma-Aldrich) and RAPA (10 μM; A606203, Sangon Biotech) were prepared in 100% ethanol and stored at −20 °C. A 100× stock of GZV (1 mM; A173303, Adooq Bioscience), DNV (1 mM; RG7227, Adooq Bioscience), and PCB (500 μM; P14137, Frontier Scientific) were made in DMSO and stored at −20 °C. These molecules were added to cell cultures, so the final concentration was 1× at induction time. For mouse experiments, ABA (200 mg/kg) and PCB (5 mg/kg) were intraperitoneally injected after being dissolved into 100 μL PBS (A600100, Sangon Biotech).

Light illumination

For cell experiments, 24-well plates containing the samples were placed below a custom-designed LED array (4 × 6) emitting blue, red, or far-red light (465 nm, 660 nm, or 730 nm; Shenzhen Bested Opto-electronic, with each LED centered above a single well). The illumination intensity was set to 1 mW/cm² at 660 nm, 1 mW/cm² at 730 nm, and 1 mW/cm² at 465 nm, unless explicitly indicated. This illumination process was carried out after transfection in 37 °C humidified incubators. Plates not subjected to light treatment were wrapped in aluminum foil immediately after transfection. The light intensity was measured at a wavelength of 465 nm, 660 nm, or 730 nm using an optical power meter (Q8230, Advantest).

For mouse experiments, the devices and LED were sourced from Shenzhen Kiwi Lighting Co. Ltd. The LED beads had a power rating of 50 mW/cm², with available wavelengths of 465 nm or 660 nm. The light angle of the lamp was between 115 and 130°, which was narrowed to 60° upon the addition of a spotlight kit. The mice were exposed to illumination 8 hours post-injection, at an intensity of 5 mW/cm² (1 minute on, 5 minutes off, alternating) for 450 nm or at 10 mW/cm2 (1 minute on, 5 minutes off, alternating) for 660 nm, unless explicitly indicated.

SEAP assay

The quantification of human placental SEAP in cell culture medium was conducted as previously reported⁵⁴. Briefly, 120 μL of substrate solution [100 μL of 2×assay buffer containing 20 mM homoarginine (1483-01-8, Sangon Biotech), 1 mM MgCl₂, 21% (w/w) diethanolamine (pH 9.8), and 20 μL of substrate containing 120 mM p-nitro phenyl phosphate (333338-18-4, Sangon Biotech)] was added to 80 μL heat-inactivated (65 °C for 30 min) cell culture supernatant, and the light absorbance time course at 405 nm was measured using a Synergy H1 hybrid multimode microplate reader (BioTek Instruments) with Gen5 software (version 2.04). Unless explicitly indicated, SEAP production was detected 24 hours after transfection.

Fluorescence imaging

Fluorescence imaging of EGFP expression in cells was performed with an inverted fluorescence microscope (Olympus IX71, TH4-200, Olympus) equipped with an Olympus digital camera (Olympus DP71, Olympus) and a 495/535-nm (blue/green/red) excitation/emission filter set, and images were acquired with 480 nm (excitation) and 535 nm (emission) filters and analyzed using Image-Pro Express C software (version ipp6.0) for EGFP signal.

Luciferase reporter assay

Luciferase activity levels were measured using the Dual-Luciferase assay kit (RG005, Beyotime). Briefly, cell samples were treated with 200 μL cell lysis buffer per well of a 24-well plate for 5 minutes. Lysis supernatants were collected after centrifuged at 13,800 × g for 5 minutes. The mixture of 20 μL lysis supernatants and 20 μL luciferase substrate was added to the 96-well plate. The luminescence signal was detected using the Synergy H1 hybrid multi-mode microplate reader (BioTek Instruments).

qPCR analysis

Cells were harvested for total RNA isolation using an RNAiso Plus kit (9109, Takara). A total of 1 μg RNA was reverse transcribed into cDNA using a PrimeScript RT Reagent Kit (RR047, Takara). qPCR reactions were performed on the LightCycler 96 real-time PCR instrument (Roche) using the SYBR Premix Ex Taq (RR420, Takara) for detecting each target gene, and the 2^-ΔΔCt method was used to calculate relative gene expression. The gRNA sequences used in this study are listed in Supplementary Table 2, and the qPCR primers used in this study are listed in Supplementary Table 3.

Western blot analysis

Tissue samples were lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA pH 8.0, 1% NP-40) containing 1 mM phenylmethanesulfonyl fluoride (PMSF). The mixture was ground into homogenate on ice, and the supernatant was collected by centrifuging at 16,200 × g for 15 minutes at 4 °C. The protein concentration in samples was determined using a bicinchoninic acid assay kit (P0012S, Beyotime). Lysates were mixed with loading buffer and boiled for 10 minutes. An equal amount of proteins (30 µg) were run on a 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and then transferred onto a polyvinylidene fluoride (PVDF) membrane (IPVH00010, Millipore MA). The membrane was blocked with 5% nonfat milk in TBST buffer (50 mM Tris, 1.37 mM NaCl, 2.7 mM KCl, 0.05% Tween 20, pH 8.0) for 1 hour at room temperature. The membranes were then incubated with anti-tdTomato primary antibody (1:500; A00682, GenScript) or anti-GAPDH primary antibody (1:1000; AF1186, Beyotime) overnight at 4 °C. After washing three times with TBST buffer, the membrane was incubated with a secondary antibody (1:5000; Alexa Fluor790 Goat Anti-Rabbit, Jackson ImmunoResearch) for 1 hour at room temperature. After washing three times with TBST buffer, the membrane was visualized using a fluorescent Western blot imaging system (LI-COR Odyssey Clx).

PTRC-mediated gene expression dynamics

To evaluate PTRC-mediated gene expression, HEK293T cells transfected with PTRC-mediated transcriptional regulation system [5 ng pDQ521 (P_SV40-Gal4-FHL-pA), 300 ng pDQ362 (P_hCMV-ΔPhyA-2×ZIM3-pA), and 100 ng pYZ430 (5×UAS-P_TATA-SEAP-pA)] were illuminated with red light (660 nm, 1 mW/cm²) for different time periods (0-48 hours). SEAP production was quantified after illumination.

To evaluate exposure time-dependent transgene expression, HEK293T cells were transfected with a PTRC-mediated transcriptional regulation system. Twelve hours later, the transfected cells were illuminated with red light (660 nm, 1 mW/cm²) for different time periods (0-48 hours). SEAP production was quantified at 60 hours after transfection.

To evaluate illumination intensity-dependent gene expression, HEK293T cells transfected with a PTRC-mediated transcriptional regulation system were illuminated with red light (660 nm) at different light intensities (0–2 mW/cm²) for 48 hours, and then SEAP production was quantified.

To evaluate the long-term dynamics of light-responsive transcriptional regulation, the PTRC transcriptional regulation system was introduced into HEK293T cells using the INVI DNA Transfection Reagent. After transfection, the cells were either kept in darkness or exposed to red light (660 nm, 1 mW/cm²). The production of SEAP was quantified daily before the culture medium was replaced with fresh medium.

To evaluate the reversibility of the PTRC-mediated transcriptional regulation system, HEK293T cells transfected with this system were divided into two groups six hours after transfection. Cells in the ON-OFF-ON group were maintained in darkness for nine hours to activate transcription, followed by six hours of exposure to red light (660 nm, 1 mW/cm²) to deactivate it. Subsequently, these cells were exposed to far-red light (730 nm, 1 mW/cm²) for nine hours to reactivate transcription. Conversely, cells in the OFF-ON-OFF group were initially exposed to red light (660 nm, 1 mW/cm²) for six hours to deactivate transcription, then maintained in darkness for nine hours to activate it, and finally exposed again to red light (660 nm, 1 mW/cm²) for six hours to deactivate it. The relative mRNA levels of SEAP were quantified by qPCR following changes in the treatment conditions.

To determine the time required to completely remove the transcriptional activation effect, HEK293T cells transfected with the PTRC-mediated transcriptional regulation system were first maintained in darkness for 12 hours to induce transcriptional activation. Subsequently, they were divided into two groups. Cells in the Dark group continued to be kept in darkness, whereas those in the 660 nm group were exposed to red light (660 nm, 1 mW/cm²). The relative mRNA levels of SEAP were quantified using qPCR after exposure to red light for intervals of 0, 1, 2, 3, 4, 5, 6, and 12 hours.

Assessment of the activation/deactivation performance of the PTRCCIP-ABA system

HEK293T cells transfected with the PTRC_CIP-ABA system [100 ng pWY49 (P_hCMV-ABI-Gal4-pA), 100 ng pWY51 (P_hCMV-PYL1-FHL-pA), 100 ng pYZ430, and 200 ng pDQ362] were treated with 100 µM ABA in darkness for 12 hours before being divided into three groups. The first group was treated again with 100 µM ABA and kept in darkness (Dark + ABA). The second group was maintained in darkness without any additional ABA treatment (Dark – ABA), and the third group was exposed to red light (660 nm, 1 mW/cm²) without any further ABA treatment (660 nm – ABA). The relative mRNA levels of SEAP were quantified using qPCR at 0, 1, 2, and 3 hours.

Assessment of the activation/deactivation performance of the PTRC_DL system

HEK293T cells transfected with the PTRC_DL system [100 ng pDQ369 (P_hCMV-CIBN-Gal4-pA), 100 ng pDQ522 (P_hCMV-CRY2_PHR-FHL-pA), 100 ng pYZ450, and 200 ng pDQ362] were exposed to blue light (465 nm, 1 mW/cm²) for 12 hours before being divided into three groups. The first group continued to be exposed to blue light (465 nm−465 nm), the second group was kept in darkness (465 nm – Dark), and the third group was exposed to red light (465 nm−660 nm). The relative mRNA levels of SEAP were quantified using qPCR at intervals of 0, 1, 2, and 3 hours.

Animals

The experimental animals, including six-week-old C57BL/6 male mice and the transgenetic Cre-tdTomato reporter male mice (Gt (ROSA)26Sor^{tm14(CAG-tdTomato)Hze}) were obtained from the ECNU Laboratory Animal Centre. All the animals were kept on a standard alternating 12-hour light/12-hour darkness cycle and given a normal chow diet [6% fat and 18% protein (wt/wt)] and water.

FHY1/FHL-mediated DNA recombination in mice

For six-week-old C57BL/6 mice, two milliliters (10% of the body weight in grams) of Ringer’s solution (147 mM NaCl, 4 mM KCl, 1.13 mM CaCl₂) containing a total of 125 μg plasmids encoding DocS/Coh2-mediated split-Cre recombinase system [50 μg pDQ532 (P_hCMV-DocS-CreC-pA), 50 μg pDQ533 (P_hCMV-CreN-Coh2-pA), and 25 μg pXY185 (P_hCMV–loxp-STOP-loxp-Luciferase-pA)], or ΔFHY1/ΔFHL-mediated split-Cre recombinase system [50 μg pDQ683 (P_hCMV-ΔFHY1-CreC-pA), 50 μg pDQ661 (P_hCMV-CreN-ΔFHL-pA) and 25 μg pXY185] was hydrodynamically injected via tail vein injection. Negative control mice (Vehicle) were hydrodynamically injected with pXY185. At 24 hours after plasmid injection, luciferase reporter expression was measured using an IVIS Lumina II in vivo imaging system (IVIS, PerkinElmer). For 6-week-old transgenetic Cre-tdTomato reporter mice, two milliliters (10% of the body weight in grams) of Ringer’s solution containing a total of 200 μg plasmids encoding DocS/Coh2-mediated split-Cre recombinase system (100 μg pDQ532, 100 μg pDQ533), or ΔFHY1/ΔFHL-mediated split-Cre recombinase system (100 μg pDQ683, 100 μg pDQ661) was hydrodynamically injected via tail vein injection. Negative control mice (Vehicle) were hydrodynamically injected with pcDNA3.1(+). At seven days after plasmid injection, the mice were sacrificed, and the livers were isolated for fluorescence imaging, qPCR, Western blot, and histological analysis. The tdTomato signal from the isolated liver was detected using an IVIS equipped with tdTomato filter sets. The collected fluorescence emission signals were stored in epi-fluorescence units (radiance efficiency), and the total flux was calculated for ROI. All images were analyzed using the Living Image® 4.3.1 software.

PTRC-mediated transcriptional regulation in mice

Two milliliters (10% of the body weight in grams) of Ringer’s solution containing 248 μg plasmids [8 μg pDQ328 (P_hCMV-Gal4-FHL-pA), 160 μg pDQ362 and 80 μg pYZ450 (5×UAS-P_TATA-luciferase-pA)] were hydrodynamically injected into mice (male, six weeks old) via tail vein injection. Eight hours after plasmid injection, the mice were intraperitoneally injected with 5 mg/kg PCB and then exposed to red light (660 nm LED, 5 mW/cm², 1 minute on, 5 minutes off, alternating) for 16 hours. Negative control mice (Vehicle) were hydrodynamically injected with pYZ450. At 24 hours after plasmid injection, luciferase reporter expression was measured using an IVIS.

PTRC_dcas-mediated regulation of endogenous gene transcription in mice

Two milliliters (10% of the body weight in grams) of Ringer’s solution containing a total of 300 μg plasmids [100 μg pYZ561 (P_U6-sgRNA1_Ascl1::P_U6-sgRNA2_Ascl1::P_hCMV-dCas9-pA), 150 μg pDQ362 and 50 μg pDQ455 (P_hCMV-MS2-FUS-FHL-pA)] was hydrodynamically injected into mice (male, six weeks old) via tail vein injection. At 8 hours after plasmid injection, the mice were intraperitoneally injected with 5 mg/kg PCB and then exposed to red light (660 nm LED, 5 mW/cm², 1 minute on, 5 minutes off, alternating) for 16 hours. Negative control mice (Vehicle) were hydrodynamically injected with NTsgRNA. At 16 hours following illumination, the mice were sacrificed, and the livers were collected. RNA was extracted for qPCR analysis. The gRNA sequences used in this study are listed in Supplementary Table 2, and the qPCR primers used in this study are listed in Supplementary Table 3.

PTRC_CIP-mediated transcriptional regulation in mice

Two milliliters (10% of the body weight in grams) of Ringer’s solution containing 320 μg plasmids [80 μg pWY49 (P_hCMV-ABI-Gal4-pA), 40 μg pWY51 (P_hCMV-PYL1-FHL-pA), 40 μg pYZ450 and 160 μg pDQ362] were hydrodynamically injected into mice (6 weeks old) via tail vein injection. Eight hours after plasmid injection, the mice were intraperitoneally injected with 5 mg/kg PCB and 200 mg/kg ABA⁵⁵ and then exposed to red light (660 nm LED, 5 mW/cm², 1 minute on, 5 minutes off, alternating) for 16 hours. At 24 hours after plasmid injection, luciferase reporter expression was measured using an IVIS.

PTRC_DL-mediated transcriptional regulation in mice

Two milliliters (10% of the body weight in grams) of Ringer’s solution containing 320 μg plasmids [80 μg pDQ369, 40 μg pDQ522, 40 μg pYZ450, and 160 μg pDQ362] were hydrodynamically injected into mice (6 weeks old) via tail vein injection. Eight hours after plasmid injection, the mice were intraperitoneally injected with 5 mg/kg PCB and then exposed to red light (660 nm LED, 5 mW/cm², 1 minute on, 5 minutes off, alternating) or blue light (465 nm LED, 5 mW/cm², 1 minute on, 5 minutes off, alternating) as indicated. At 24 hours after plasmid injection, luciferase reporter expression was measured using an IVIS.

IVIS imaging

For in vivo imaging, each mouse was intraperitoneally injected with luciferin substrate solution (150 mg/kg; luc001, Shanghai Sciencelight Biology Science & Technology) intraperitoneally. Five minutes after the injection, bioluminescence images of the mice were captured using an IVIS. The bioluminescence images were then analyzed using Living Image software (version 4.3.1).

Liver histology imaging

Fresh livers were washed three times with cold PBS to remove impurities such as blood and then fixed in 4% (w/v) paraformaldehyde (PFA; 30525-89-4, Sangon Biotech) for 2 hours at 4 °C. Tissue blocks of ~1 cm³ were cut and embedded in an optimum cutting temperature compound (OCT; 03803389, Leica). Five µm thick liver sections were prepared using Cryostat Microtome (CM1950, Leica) and rinsed with PBS. Finally, samples were counterstained with 4’,6-diamidino-2-phenylindole (DAPI; 28718-90-3, Sigma) for 10 minutes. Endogenous gene tdTomato expression was observed on an inverted fluorescence microscope (DMI8, Leica).

Ethics

The experiments involving animals were approved by the East China Normal University (ECNU) Animal Care and Use Committee and in direct accordance with the Ministry of Science and Technology of the People’s Republic of China on Animal Care guidelines. The protocol (protocol ID: m20220412, m20220506) was approved by the ECNU Animal Care and Use Committee. All animals were euthanized after the experiments were terminated.

Statistical analysis

All in vitro data are expressed as the mean ± SD of three independent experiments (n = 3). For the animal experiments, each treatment group consisted of randomly selected mice (n = 4–6). The results are expressed as mean ± SEM. Statistical significance was analyzed by the Student’s t test. Neither animals nor samples were excluded from the study. Differences were considered statistically significant at p < 0.05 (*), very significant at p < 0.01 (**), and extremely significant at p < 0.001 (***). GraphPad Prism software version 6.0 was used for statistical analysis.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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• Source: https://www.nature.com/articles/s41467-024-49254-5