Our project aims to develop a gene switch that responds to ultraviolet light and use this gene switch for the treatment of Xeroderma Pigmentosum Type C. Our project is mainly divided into three modules: First, we have verified the interaction of UVR8, COP1, and RUP2 in mammalian cells and their response to ultraviolet light. Second, we have successfully constructed a UV-responsive gene switch that activates the expression of target genes upon UV irradiation and demonstrated that RUP2 can dissociate the interaction between UVR8 and COP1, thereby stabilizing the expression level of the target gene to a certain extent. Third, through modeling, we have obtained results predicting the expression of the target protein after sequence optimization of RUP2 and successfully constructed plasmids with different numbers of rare codons added to the RUP2 gene, which will be validated through wet lab experiments.
</p>
...
...
@@ -106,10 +106,10 @@
</div>
<divclass="block">
<p>
GAL4 is a transcriptional activator derived from yeast, typically composed of two domains: the DNA-binding domain (BD) and the transcriptional activation domain (AD) (Struhl et al., 1995). It can recognize and bind to the upstream activation sequence (UAS), thereby activating the transcription of downstream genes. Our project utilizes the BD domain of GAL4 and the 5UAS sequence to facilitate protein localization through the binding of GAL4 to 5UAS. VP64 is a potent transcriptional activator that, when bound to the promoter, can activate gene transcription. The P2A peptide is a self-cleaving peptide that enables the independent translation of two genes located before and after the P2A sequence (Szymczak & Vignali, 1995)Szymczak.
GAL4 is a transcriptional activator derived from yeast which typically composed of two domains: the DNA-binding domain (BD) and the transcriptional activation domain (AD) (Struhl et al., 1995). It can recognize and bind to the upstream activation sequence (UAS), thereby activating the transcription of downstream genes. Our project utilizes the BD domain of GAL4 and the 5UAS sequence to facilitate protein localization through the binding of GAL4 to 5UAS. VP64 is a potent transcriptional activator that, when bound to the promoter, can activate gene transcription. The P2A peptide is a self-cleaving peptide that enables the independent translation of two genes located before and after the P2A sequence (Szymczak & Vignali, 1995).
</p>
<p>
We have fused UVR8 with VP64 and COP1 with BD domain of Gal4, added a nuclear localization sequence (NLS) behind COP1 to stabilize its location in the cell nucleus, which is more conducive to initiating gene expression. The target XPC gene is connected behind the 5UAS sequence and the CMV promoter, and the RUP2 is connected behind the XPC gene through the P2A sequence. Gal4 tightly binds to 5UAS within the nucleus, and when UV-B signal is present, UVR8 monomerizes and enters the nucleus to interact with COP1. Due to the localization effect of Gal4, the distance between VP64 and the CMV promoter is reduced, thereby activating the expression of the downstream XPC and RUP2 genes. The presence of P2A allows for the independent translation of XPC and RUP2. To ensure that the XPC protein reaches a certain concentration before RUP2 inhibits the interaction between UVR8 and COP1, we have optimized the RUP2 sequence by replacing the codons in the RUP2 sequence with synonymous codons that are rare. By adjusting the number of replacements, the expression level of XPC is brought close to that found in normal somatic cells. Due to the repeated interaction and disengagement of UVR8 and COP1, XPC protein is stably maintained at an appropriate concentration. When the UV-B signal is removed, the UVR8 dimer cannot monomerize, ultimately achieving complete dissociation of RUP2 from UVR8 and COP1, constructing a UV-responsive XPC gene switch.
We have fused UVR8 with VP64 and COP1 with the BD domain of Gal4 and added a nuclear localization sequence (NLS) behind COP1 to stabilize its location in the cell nucleus, which helps to initiate gene expression. The target XPC gene is connected behind the 5UAS sequence and the CMV promoter, and the RUP2 is connected behind the XPC gene through the P2A sequence. Gal4 tightly binds to 5UAS within the nucleus, and when UV-B signal is present, UVR8 monomerizes and enters the nucleus to interact with COP1. Due to the localization effect of Gal4, the distance between VP64 and the CMV promoter is reduced, thereby activating the expression of the downstream XPC and RUP2 genes. The presence of P2A allows for the independent translation of XPC and RUP2. To ensure that the XPC protein reaches a certain concentration before RUP2 inhibits the interaction between UVR8 and COP1, we have optimized the RUP2 sequence by replacing the codons in the RUP2 sequence with synonymous codons that are rare. By adjusting the number of replacements, the expression level of XPC is brought close to that found in normal somatic cells. Due to the repeated interaction and disengagement of UVR8 and COP1, XPC protein is stably maintained at an appropriate concentration. When the UV-B signal is removed, the UVR8 dimer cannot monomerize, ultimately achieving complete dissociation of RUP2 from UVR8 and COP1, constructing a UV-responsive XPC gene switch.
<figcaption> Fig.4: Schematic diagram of the UV-responsive XPC gene switch</figcaption>
...
...
@@ -119,7 +119,7 @@
</div>
<divclass="block">
<p>
Our project innovatively designed a gene therapy that responds to ultraviolet light, offering possibilities for the treatment of xeroderma pigmentosum. Meanwhile, this UV-responsive control system can be replaced with any target gene behind the CMV promoter, and it can also be combined with gene-editing systems such as CRISPR/Cas, providing solutions for various puzzles of skin genetic diseases, offering a more sensitive response and a high degree of spatial and temporal control.
Our project innovatively designed a gene therapy that responds to ultraviolet light, offering possibilities for the treatment of xeroderma pigmentosum. Meanwhile, this UV-responsive control system can be replaced with any target gene behind the CMV promoter, and it can also be combined with gene-editing systems such as CRISPR/Cas, providing solutions for various puzzles of skin genetic diseases, a more sensitive response, and a high degree of spatial and temporal control.
<divclass="title"id="Part 1 the ultraviolet-responsive interaction between UVR8 and COP1">
Part 1 the UV-responsive interaction between UVR8 and COP1
Part 1 The UV-responsive interaction between UVR8 and COP1
</div>
<divclass="block">
<p>In Arabidopsis, COP1 is an E3 ubiquitin ligase that targets HY5 for degradation. In absence of UV-B signals, UVR8, a photoreceptor protein, exists in the form of homodimers outside the nucleus. Upon UV-B exposure, UVR8 monomerizes and translocates into the nucleus, where it interacts with COP1 to regulate plant photomorphogenesis.</p>
