Kurooka H, Kuroda K, Honjo T
Department of Medical Chemistry, Kyoto University Faculty of Medicine, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan.
Nucleic Acids Res. 1998 Dec 1;26(23):5448-55. doi: 10.1093/nar/26.23.5448.
The Notch intracellular region (RAMIC) interacts with a DNA binding protein RBP-J to activate transcription of genes that inhibit cell differentiation. The RAM domain and ankyrin (ANK) repeats of mouse Notch1 RAMIC were shown to be responsible for RBP-J binding and necessary for transactivation. The C-terminal portion of Notch1 RAMIC has also been suggested to be important for transactivation. Using GAL4 fusion constructs, we identified a novel transactivation domain (TAD) between the ANK repeats and the PEST sequence of mouse Notch1. The C-terminal half of mouse Notch2 RAMIC also exhibited TAD activity. Unexpectedly, the RBP-J chimeric protein with the Notch1 TAD failed to activate transcription but the activity was recovered by addition of either the RAM domain or ANK repeats. The results suggest that the activity of Notch1 TAD is repressed by fusion with RBP-J because of the presence of a RBP-J-associated co-repressor(s), which could be displaced by either the RAM domain or ANK repeats. Taken together, mouse Notch1 RAMIC can experimentally be separated into three functional domains: the RAM domain and ANK repeats for RBP-J binding and co-repressor displacement and the C-terminal TAD.
Notch细胞内区域（RAMIC）与DNA结合蛋白RBP-J相互作用，以激活抑制细胞分化的基因的转录。小鼠Notch1 RAMIC的RAM结构域和锚蛋白（ANK）重复序列被证明负责RBP-J结合，并且是反式激活所必需的。Notch1 RAMIC的C末端部分也被认为对反式激活很重要。使用GAL4融合构建体，我们在小鼠Notch1的ANK重复序列和PEST序列之间鉴定了一个新的反式激活结构域（TAD）。小鼠Notch2 RAMIC的C末端一半也表现出TAD活性。出乎意料的是，带有Notch1 TAD的RBP-J嵌合蛋白未能激活转录，但通过添加RAM结构域或ANK重复序列可恢复活性。结果表明，由于存在RBP-J相关的共抑制因子，Notch1 TAD与RBP-J融合后其活性受到抑制，而RAM结构域或ANK重复序列可取代该共抑制因子。综上所述，小鼠Notch1 RAMIC在实验上可分为三个功能结构域：用于RBP-J结合和共抑制因子置换的RAM结构域和ANK重复序列，以及C末端TAD。
