Abstract
Sox9 is a master transcription factor for chondrogenesis, which is essential for chondrocyte proliferation, differentiation, and maintenance. Sox9 activity is regulated by multiple layers, including post-translational modifications, such as SUMOylation. A detection method for visualizing the SUMOylation in live cells is required to fully understand the role of Sox9 SUMOylation. In this study, we generated a quantitative reporter for Sox9 SUMOylation that is based on the NanoBiT system. The simultaneous expression of Sox9 and SUMO1 constructs that are conjugated with NanoBiT fragments in HEK293T cells induced luciferase activity in SUMOylation target residue of Sox9-dependent manner. Furthermore, the reporter signal could be detected from both cell lysates and live cells. The signal level of our reporter responded to the co-expression of SUMOylation or deSUMOylation enzymes by several fold, showing dynamic potency of the reporter. The reporter was active in multiple cell types, including ATDC5 cells, which have chondrogenic potential. Finally, using this reporter, we revealed a extracellular signal conditions that can increase the amount of SUMOylated Sox9. In summary, we generated a novel reporter that was capable of quantitatively visualizing the Sox9-SUMOylation level in live cells. This reporter will be useful for understanding the dynamism of Sox9 regulation during chondrogenesis.
Highlights
Sox9 is a transcription factor that is crucial for chondrogenesis [1]
We cloned fusion constructs for SUMO1/Sox9 and NanoBiT fragments (i.e., LgBiT or SmBiT)
These constructs were introduced into HEK293T cells in all possible combinations, in order to examine whether or not the NanoBiT fusion Sox9 and SUMO1 protein could be conjugated by the endogenous SUMOylation machinery in the cells
Summary
Sox is a transcription factor that is crucial for chondrogenesis [1]. Sox promotes the proliferation, differentiation, and maintenance of chondrocytes by regulating its target genes. No specific antibodies against SUMOylated protein have been reported, and SUMOylated proteins are usually detected by Western blotting, based on the shift in the molecular weight or by the combination of immunoprecipitation and Western blotting [15,18] Since these methods are laborious, non-quantitative, and cannot be applied to live cells, a novel approach is required to detect dynamic changes in protein SUMOylation in live cells. These fragments—known as LgBiT and SmBiT—have a low affinity for each other, so they only exert luciferase activity when their combined proteins interact [19] This reporter is often used for assessing non-covalent protein interactions, but it can be applied to detect protein PTMs [20]. We used this system to generate a quantitative reporter for Sox SUMOylation that could be applied to live cells (Figure 1A)
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