Pan Xin, Li Huiyan, Zhang Peijing, Jin Baofeng, Man Jianghong, Tian Lifeng, Su Guiying, Zhao Jie, Li Weihua, Liu Hui, Gong Weili, Zhou Tao, Zhang Xuemin
National Center of Biomedical Analysis, Beijing 100850, PR China.
Biochem Biophys Res Commun. 2006 Jun 9;344(3):727-34. doi: 10.1016/j.bbrc.2006.03.194. Epub 2006 Apr 17.
SOX4 is a member of SOX transcriptional factor family that is crucial for many cellular processes. In this study, a yeast two-hybrid screening of human mammary cDNA library identified human ubiquitin-conjugating enzyme 9 (hUbc9) that interacted with SOX4. This interaction was confirmed by GST pull-down in vitro and co-immunoprecipitation assays in vivo. Deletion mapping demonstrated that HMG-box domain of SOX4 is required to mediate the interaction with Ubc9 in yeast. Furthermore, confocal microscopy showed that Ubc9 co-localized with SOX4 in the nucleus. Luciferase assays found that Ubc9 specifically repressed SOX4 transcriptional activity in 293T cells. We further demonstrated that Ubc9 could functionally repress the transcriptional activity of endogenous SOX4 induced by progesterone in T47D cells. The C93S mutant of Ubc9, which abrogates SUMO-1 conjugation activity, did not abolish the ability to repress SOX4 activity. It shows that Ubc9 interacts with SOX4 and represses its transcriptional activity independent of its SUMO-1-conjugating activity.
SOX4是SOX转录因子家族的成员，对许多细胞过程至关重要。在本研究中，通过酵母双杂交筛选人乳腺cDNA文库，鉴定出与SOX4相互作用的人泛素结合酶9（hUbc9）。这种相互作用通过体外GST下拉实验和体内共免疫沉淀实验得到证实。缺失作图表明，SOX4的HMG盒结构域是介导酵母中与Ubc9相互作用所必需的。此外，共聚焦显微镜显示Ubc9与SOX4在细胞核中共定位。荧光素酶实验发现，Ubc9在293T细胞中特异性抑制SOX4的转录活性。我们进一步证明，Ubc9在功能上可以抑制T47D细胞中孕酮诱导的内源性SOX4的转录活性。Ubc9的C93S突变体消除了SUMO-1缀合活性，但并未消除其抑制SOX4活性的能力。这表明Ubc9与SOX4相互作用并抑制其转录活性，且不依赖于其SUMO-1缀合活性。
