Laboratory of Animal Regeneration Systemology, Department of Life Sciences, School of Agriculture, Meiji University, Kanagawa 214-8571, Japan.
Department of Developmental Neurobiology, Institute of Development, Aging and Cancer, Tohoku University, 4-1 Seiryo, Aoba, Sendai, Miyagi 980-8575, Japan.
Int J Mol Sci. 2020 Feb 13;21(4):1274. doi: 10.3390/ijms21041274.
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.
Sox9 是软骨形成的主转录因子，对于软骨细胞的增殖、分化和维持至关重要。Sox9 的活性受到多个层面的调节，包括翻译后修饰，如 SUMO 化。为了充分了解 Sox9 SUMO 化的作用，需要有一种在活细胞中可视化 SUMO 化的检测方法。在这项研究中，我们基于 NanoBiT 系统生成了 Sox9 SUMO 化的定量报告器。在 HEK293T 细胞中同时表达 Sox9 和 SUMO1 构建体，这些构建体与 NanoBiT 片段缀合，以 Sox9 依赖性方式诱导荧光素酶活性。此外，该报告器的信号可以从细胞裂解物和活细胞中检测到。我们的报告器信号对 SUMO 化或去 SUMO 化酶的共表达响应了几倍，显示出报告器的动态效力。该报告器在多种细胞类型中具有活性，包括具有软骨形成潜力的 ATDC5 细胞。最后，使用该报告器，我们揭示了可以增加 SUMO 化 Sox9 量的细胞外信号条件。总之，我们生成了一种新型报告器，能够在活细胞中定量可视化 Sox9-SUMO 化水平。该报告器将有助于理解 Sox9 在软骨形成过程中的调节动态。
