Functional analysis of SUMO modification in nuclear architecture and transcription regulation

在细胞核内，基因转录、复制、修复等许多重要事件在复杂的调控下发生。核因子在细胞核内不是随机或均匀分布的，而是高度动态地划分到几个区域，这使得许多反应在细胞核内快速有效。本项目的目的是阐明SUMOylation调节细胞核的机制。我设计并进行了以下实验。RanBP2的SUMOylation在核结构域的形成中起作用。通过原位SUMO酰化实验，可以看到SUMO酰化活性的亚细胞定位，我发现RanBP2在核孔复合物上发挥SUMO E3活性。通过siRNA敲低RanBP2，核孔复合物的SUMOylation活性丧失，同时核结构域发生了一些变化，如PML小体数量的减少和核斑点的异常形式。提出了一种新的模型，其中蛋白质从细胞质转运到细胞核时，在核孔复合体上被RanBP2溶解。核斑的作用及其形成机制。核斑包含转录、RNA加工、转运等多种因子，参与基因表达调控。我分析了核斑异常形成的RanBP2敲低细胞中基因表达模式的变化和核斑成分的定位。该研究提出了建立核结构的第一个分子可处理途径之一。综上所述，我揭示了SUMOylation在核结构域形成中的重要作用，并为了解细胞核的功能和动力学做出了贡献。

In the cell nucleus, many important events including gene transcription, replication, repairs occur under complicated regulations. Nuclear factors are not randomly or evenly distributed within the nucleus, rather they are highly and dynamically compartmentalized into several domains, which enables many reactions to be rapid and efficient in the nucleus. Goal of this project is to elucidate the mechanism of how SUMOylation regulates the nucleus. I designed and performed following experiments.SUMOylation by RanBP2 plays a role in nuclear domain formations.With in situ SUMOylation assay that visualizes sub-cellular localization of SUMOylation activity, I showed that RanBP2 exerts SUMO E3 activity at the nuclear pore complex. By knockdown of RanBP2 by siRNA, SUMOylation activity at the nuclear pore complex was lost, together with several changes in nuclear domains, such as reduction of numbers of PML bodies and aberrant forms of nuclear speckles. A novel model was suggested, in which proteins are SUMOlyated at the nuclear pore complex by RanBP2, when they are transported from cytoplasm to the nucleus.Function of nuclear speckle and the mechanism for its formation.Nuclear speckle contains many factors for transcription, RNA processing, transport and so on and thus it is involved in gene expression regulation. I analyzed changes in gene expression patterns and localizations of nuclear speckle components in RanBP2 knockdown cells in which nuclear speckles are aberrantly formed. The study presents one of the first molecularly tractable pathways for establishing nuclear architecture. In summary, I revealed significant role of SUMOylation in nuclear domain formations, and made contributions to understand function and dynamics of the cell nucleus.