Results
Verification of the interaction of UVR8, COP1 and RUP2

In order to achieve artificial regulation of RUP2 protein expression, we introduced the RUP2 coding gene into the plasmid Tet-on, and obtained a tetracycline-induced RUP2 protein expression plasmid of pRP_TRE_RUP2_Flag.

Figure 1A:. The final plasmid graph of pRP_TRE_RUP2_Flag
Figure 1B:. The sequencing result of pRP_TRE_RUP2_Flag

The plasmid was transfected into 293t cell line, and the tetracycline concentration gradient was set for 48 h after the protein was completely expressed, and Western blot was performed. The results and statistics are as follows:

Figure 2: Westernblot results of each tetracycline concentration gradient are shown as fluorescence intensity statistics

In the previous pre-experiment, we verified RUP2 expression by Western blot and obtained the optimal concentration of 10 μg/ml for tetracycline induction by setting the concentration gradient. Plasmids pIRESN2_GFP_UVR8, pSNV2_CHER_NLS_COP1 and pRP_TRE_RUP2_Flag were simultaneously transfected into 293t cells. Transfection experiments were performed according to the following timeline, and the slides were observed under a confocal microscope after sealing.

Figure 3: Flow chart of operation after transfection

Cells were plated using cell climbing slices, and then three plasmids, numbered 1, 2, and 3, were co-transfected into the cells. After cultivation under conditions with and without tetracycline, observations were made using a confocal microscope and images were captured. A field of view was found using a 20x magnification. Subsequently, find a field with more representative cells in different channels, and a multi-channel film was used to prepare for cell counting.

Table 1: Grouping of cells per well and treatment of cells
Figure 4: 20x confocal microscope multichannel combination of four groups of cells A:Confocal images of the first group of cells. B:Confocal images of the second group of cells C:Confocal images of the third group of cells. D:Confocal images of the forth group of cells

We established a criterion for cell fluorescence. We counted the cells with cytoplasmic green fluorescence and no or weak nuclear green fluorescence. After observation, we counted the above cells in multiple fields of group 1, group 2 and group 3 and performed statistical analysis ,Results were as follows:

Table 2: Statistics of the number and proportion of cells with dark nuclear fluorescence
Figure 5: Statistical chat of nuclear fluorescence status

Transfection of pIRESN2_GFP_UVR8, pSNV2_CHER_NLS_COP1 and pRP_TRE_RUP2_Flag plasmids, without UV irradiation, found that the proportion of cells with dark green nuclei was 27.78% on average, after UV irradiation, the proportion decreased to about 6.91%, and the expression of rup2 was applied after UV irradiation. When the concentration of tetracycline was 10μg/ml, the proportion returned to about 25.99%. At a tetracycline concentration of 5μg/ml, this proportion returned to about 15.22%, and the following figure shows a statistical plot of these data.

In addition, we also implemented a time-lapse imaging approach to elucidate the subcellular dynamics and distribution of GFP-tagged UVR-8 in response to UV-B irradiation. A cohort of cells was randomly chosen for continuous observation, with digital snapshots taken at regular intervals post-irradiation. These visual records were meticulously curated to present a selection of illustrative images that encapsulate the temporal progression of GFP fluorescence within the cellular context. Utilizing ImageJ software for the quantitative analysis of cellular nuclear fluorescence intensity from microscopic images, our data show a statistically significant difference in fluorescence brightness before and after UV-B exposure. Together, our results revealed a significant enhancement in the nuclear GFP signal following UV-B exposure, suggesting a translocation of UVR-8 into the nucleus upon UV-B stimulation. This translocation is hypothesized to be a pivotal step in the initiation of the UV-B signaling cascade. The dynamic monitoring of cellular behavior provided a temporal resolution that underscored the real-time impact of UV-B on UVR-8 nuclear import, thereby reinforcing the validity of our experimental system in dissecting the mechanistic underpinnings of UV-B signaling.

Figure 6: Microscopic Images of GFP Fluorescence Distribution at Different Times And Fluorescence Intensity Measurement. A: Microscopic Images of GFP Fluorescence Distribution at Different Times. B: Images of Fluorescence Distribution Before and After UV-B Irradiation. C: Fluorescence intensity measurement in nuclei before and after UV-B treatment

In summary, we used Western blot to validate the RUP2 protein expression and set up a concentration gradient to obtain the optimal concentration of tetracycline. Then we transfected plasmids pIRESN2_GFP_UVR8, pSNV2_CHER_NLS_COP1 and pRP_TRE_RUP2_Flag into cell lines. After UV irradiation, we observed that a significant portion of the green fluorescence, which was originally in the cytoplasm, had entered the cell nucleus. Over time, co-localization of red and green fluorescence was observed within the nucleus, confirming that in mammalian cells, UV light can indeed induce an interaction between UVR8 and COP1. Upon induction of RUP2 expression with tetracycline, the number of cells with dark nuclei increased again, indicating that RUP2 can dissociate the interaction between UVR8 and COP1 and transport UVR8 out of the nucleus. In summary, we have verified that in mammalian cells, UV light can induce an interaction between UVR8 and COP1, while RUP2 can break the interaction of UVR8 and COP1, successfully validating the interplay of UVR8, COP1, and RUP2.

Verification the activation of the UV-responsive gene switch (qualitative validation)

Having confirmed that UVR8 dissociates into monomers upon UV-B activation and enters the nucleus, we aimed to link UVR8 with transcriptional regulatory factors to harness UV-B for gene expression activation. We constructed the following experimental system:

Figure 1. Schematic diagram of the UV-B activated GFP reporting system

We utilized GFP as a reporter gene to verify the feasibility of activating gene expression with UV-B. We conducted transfection experiments in HEK293T cells, followed by exposure to UV-B radiation 24 hours post-transfection. The outcomes were assessed using an inverted fluorescence microscope and a plate reader, with measurements taken 24 hours post-UV-B exposure.

In our experimental setup, a co-transfection approach was employed with the reporter gene plasmid in conjunction with the UVR8-VP64 plasmid and the COP1-NLS-Gal4 plasmid to form the experimental group. We established three control groups: one with solely the reporter gene plasmid, another with the reporter gene plasmid combined with the UVR8-VP64 plasmid, and a third with the reporter gene plasmid combined with the COP1-NLS-Gal4 plasmid. This strategy was designed to conclusively establish that the transcription activation mechanism is reliant on the synergistic action of all gene components, rather than the individual contribution of any single component. Furthermore, we included control groups to compare the effects of UV-B irradiation with those of non-irradiated conditions, thereby highlighting the regulatory role of UV-B within the system. A schematic representation of the transfection experimental design for both the experimental and control groups is depicted in the following figure 2:

Figure 2. Schematic diagram of the transfection experimental design for the experimental group cell experiments

To obtain the results, we observed GFP fluorescence under 488nm excitation and 560nm emission using an inverted fluorescence microscope. We found that only the experimental group co-transfected with all three plasmids and irradiated with UV-B showed significant fluorescence, while all other groups showed no significant fluorescence. Some control groups, such as the group with the target gene plasmid + UVR8-VP64 plasmid and irradiated with UV-B, showed a small amount of fluorescence, which we consider to be gene expression leakage within an acceptable range. Images are as follows:

Figure 3 10x microscopic images of cells transfected with different plasmids with and without UV-B irradiation treatment. For cells transfected with each plasmid combination, three fields of view are selected for observation. A. Fluorescence micrographs of cells transfected with PDL6_CMV_GFP, pSNV2_VP64_NLS_coplat and pIRESN2_Gal4_UVR8at. B. Fluorescence micrographs of cells transfected with PDL6_CMV_GFP, and pIRESN2_Gal4_UVR8at. C. Fluorescence micrographs of cells transfected with PDL6_CMV_GFP, and pSNV2_VP64_NLS_coplat. D. Fluorescence micrographs of cells transfected with PDL6_CMV_GFP.

In the previous pre-experiment, we verified RUP2 expression by Western blot and obtained the optimal concentration of 10 μg/ml for tetracycline induction by setting the concentration gradient. Plasmids pIRESN2_GFP_UVR8, pSNV2_CHER_NLS_COP1 and pRP_TRE_RUP2_Flag were simultaneously transfected into 293t cells. Transfection experiments were performed according to the following timeline, and the slides were observed under a confocal microscope after sealing.

Additionally, to ensure more reliable results, we selected a field of cells and took photographs of the fluorescence channel, bright field channel, and phase contrast channel separately. By comparing the three images, it can be observed that the GFP fluorescence is located in living cells. This confirms that the observed GFP fluorescence is the result of cellular protein expression, rather than fluorescence caused by errors such as cellular debris.

Figure 4 20x microscopic images of different channels of the same group of cells before and after UV-B irradiation. From left to right, they are the fluorescence channel image, bright field channel image, and phase contrast channel image, respectively.

For statistical data, we counted the cells with GFP fluorescence. After observation, we counted the cells in multiple areas of cells transfected with different plasmid combinations, calculated the number of fluorescent cells per unit area, and conducted a statistical analysis. The results are as follows:

Figure 5 Unit Area Average Fluorescent Cell Count Chart

In the results involving the transfection of PDL6_CMV_GFP, pSNV2_VP64_NLS_coplat, and pIRESN2_Gal4_UVR8at plasmids, we observed a significant enhancement in the number of fluorescent cells per unit area upon exposure to UV-B irradiation. Specifically, in the absence of UV-B irradiation, the average count of fluorescent cells was 2.83 per unit area. However, this number increased dramatically to 44.33 upon irradiation, indicating a substantial impact of UV-B on transfection efficiency. Conversely, when cells were co-transfected with two gene bricks or transfected with a single gene brick, the average number of luminescent cells per unit area remained below or equal to 2, irrespective of UV-B treatment. This suggests that the presence of UV-B is a critical factor in enhancing fluorescence, particularly when all three plasmids are used in combination.

In summary, in this part of the results, we constructed and demonstrated the feasibility of the UV-B-activated reporter gene transcription system using a GFP reporter gene. Our study establishes a correlation between UV-B irradiation and the initiation of gene expression by demonstrating the pivotal role of "UVR8 nuclear entry." Furthermore, the successful expression of GFP under specific conditions confirms the system's capability to discern between environments with and without UV-B exposure. This robust evidence underscores the viability of employing UV-B as a trigger for gene expression, paving the way for potential applications in genetic regulation and biotechnological advancements.

Quantitative Characterization of UV-B-Induced Gene Expression and Construction of a Negative Feedback Regulation System for Modulating Expression Levels

Having linked UVR8 with transcriptional regulatory factors to harness UV-B for gene expression activation, we aimed to make quantitative characterization of UV-B-Induced gene expression and construct a negative feedback regulation system to modulate the expression level of reporter gene. We constructed the following experimental systems, and in this part, we go from qualitative analysis to quantitation and optimization:

Figure 1. Schematic illustrations of three experimental systems

In the first system, which has already illustrated in result 2, we utilized GFP as a reporter gene to verify the feasibility of activating gene expression with UV-B.

Here, in the second & third systems, we sought to quantitatively characterize the strength of UV-B-activated gene expression using the Luciferase reporter gene and employed a novel element, RUP2, to construct a negative feedback system, controlling the expression of the reporter gene within an appropriate range.

The specific experimental design, as well as the results and statistical data, are as follows:

Figure 2. Schematic diagram of the timeline for the cell experiment process

We performed transfections in HEK293T cells, irradiated with UV-B 24 hours post-transfection, and obtained results using an inverted fluorescence microscope and a plate reader 24 hours after UV-B irradiation.

In terms of experimental design, we used a co-transfection group with the reporter gene plasmid, UVR8-VP64 plasmid, and COP1-NLS-Gal4 plasmid as the experimental group. We set up three control groups containing only the reporter gene plasmid, the reporter gene plasmid + UVR8-VP64 plasmid, and the reporter gene plasmid + COP1-NLS-Gal4 plasmid. This design was intended to rigorously demonstrate that the transcription activation system requires the coordinated function of all gene components, rather than the action of individual components. Additionally, we set up controls between irradiated and non-irradiated UV-B to illustrate the regulatory function of UV-B in the system. The schematic diagram of the transfection experimental design for the experimental and control groups is shown below:

Figure 3. Schematic diagram of the transfection experimental design for the control group cell experiments

For the experiments of systems 2 and 3, we lysed the cells according to the protocol and added firefly luciferase detection reagent, observing chemiluminescence using a plate reader. The statistical results are shown in the figure below:

Firstly, in order to quantitatively characterize the impact of UV-B irradiation on the transcriptional expression of the reporter gene, we conducted two parallel experiments. One set of experiments was subjected to UV-B irradiation, while the other served as a control. After the same duration, we lysed the cells and added a luciferin substrate to measure the activity of luciferase.

Figure 4. Result of Luciferase Reporter Gene Assay With Or Without UV-B Exposure

As illustrated in the figure 7, the impact of UV-B irradiation on the expression of the luciferase reporter gene in the complete system containing three genetic elements is significant. After exposure to UV-B, the expression of the reporter gene increased to nearly five times that of the original, indicating that the gene system we designed is capable of sensitively responding to UV-B input and using the expression of the reporter gene as an output. In other groups containing only two genetic elements or just the reporter gene plasmid, the presence or absence of UV-B does not significantly affect the expression of the reporter gene. This demonstrates that each element in the system is indispensable, and also solidifies the experimental results through the control.

However, we observed that the expression of the target gene was excessively strong after UV-B irradiation, which does not align with the requirement for stable and moderate expression of XPC in practical application scenarios. To further optimize the system, we incorporated the P2A-RUP2 element into the genetic components, constructing a negative feedback regulation system to control the expression level of the reporter gene at a reasonable level under UV-B irradiation.

Figure 5. Schematic illustrations of negative feedback regulation system construction using RUP2

combination under UV-B irradiation, while the right side represents the performance of the gene combination after the addition of the P2A-RUP2 negative feedback regulation system under UV-B irradiation. It can be observed that the incorporation of the P2A-RUP2 negative feedback regulation system has brought the expression of the reporter gene under control.

Figure 6. Luciferase Reporter Gene Assay Analysis of Different Gene Bricks Groups

However, there are issues present, such as the reporter gene expression levels not significantly differing with or without the UVR8-VP64 element. Upon analyzing this issue, we believe that RUP2 competes too strongly with COP1, to the extent that UVR8 can hardly bind with COP1 to enhance transcription initiation after entering the nucleus. To address this issue, we subsequently introduced rare codons into RUP2 in the hope of reducing its expression level, weakening the negative feedback regulation effect, and allowing the reporter gene to express at an appropriate level without being excessively high.

Those Luciferase Reporter Gene Assays provide a quantitative measure of the transcriptional activity of different gene regulatory elements and is a valuable tool for assessing the functionality of gene constructs in synthetic biology applications. It can be seen that we successfully quantitatively determined the activation of reporter gene expression by UV-B and verified that our designed P2A-RUP2 negative feedback regulation system can stably control the expression of the target gene to a reasonable level.

Furthermore, to demonstrate the safety of the components used in our designed system for use in mammals, we conducted a CCK-8 cell viability assay. The results shown in the figure below indicate that none of the components involved in our system exhibit significant toxicity. This illustrates the wisdom of applying plant system components to mammalian cells—a strategy that employs orthogonal systems with a high probability of non-interference. Although this is only a preliminary argument at the cellular experimental level, it provides some support for safety. This safety also lays a foundational premise for our subsequent application of the system in supplying XPC to human epidermal cells.

Figure 7. Assessment of System Component Toxicity in Mammalian Cells Using the CCK-8 Assay

In summary, in this part of the results, we quantitatively determined the strength of reporter gene transcription and designed elements to optimize the system, enabling the expression of the target gene to be controlled at a reasonable level. This lays the foundation for applying this expression system to the controllable and stable expression of XPC.

Conclusion

In conclusion, we have verified that in mammalian cells, UV light can induce an interaction between UVR8 and COP1, while RUP2 can break the interaction of UVR8 and COP1, successfully validating the interplay of UVR8, COP1, and RUP2.

Next, we constructed and demonstrated the feasibility of the UV-B-activated reporter gene transcription system using a GFP reporter gene. Our study establishes a correlation between UV-B irradiation and the initiation of gene expression. Furthermore, the successful expression of GFP under specific conditions confirms the system's capability to discern between environments with and without UV-B exposure. This robust evidence underscores the viability of employing UV-B as a trigger for gene expression.

Then, we quantitatively determined the strength of reporter gene transcription and designed elements to optimize the system, enabling the expression of the target gene to be controlled at a reasonable level.

In addition, the pDL6_5*UAS_hCMVmin_XPC_P2A_RUP2 plasmid has been successfully constructed. In subsequent experiments, we will verify the regulation of the XPC expression construct. Furthermore, the plasmid of pRP_TRE_RUP2_Flag containing rare codons has been successfully constructed. In subsequent experiments, we are going to verify the effect of different quantities of rare codons on the expression level of RUP2. Additionally, we can apply the regulatory effect of rare codons on RUP2 to the plasmid that contains both XPC and RUP2, in order to precisely control the concentration of XPC.