Tani S, Kurooka H, Aoki T, Hashimoto N, Honjo T
Department of Medical Chemistry and Department of Neurosurgery, Graduate School of Medicine, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan.
Nucleic Acids Res. 2001 Mar 15;29(6):1373-80. doi: 10.1093/nar/29.6.1373.
The evolutionarily-conserved DNA-binding protein RBP-J directly interacts with the RAM domain and the ankyrin (ANK) repeats of the Notch intracellular region (RAMIC), and activates transcription of downstream target genes that regulate cell differentiation. In vitro binding assays demonstrate that the truncated N- and C-terminal regions of RBP-J bind to the ANK repeats but not to the RAM domain. Using an OT11 mouse cell line, in which the RBP-J locus is disrupted, we showed that RBP-J constructs mutated in the N- and C-terminal regions were defective in their transcriptional activation induced by either RAMIC or IC (the Notch intracellular region without the RAM domain) although they had normal levels of binding activity to DNA and the RAM domain. The studies using chimeric molecules between RBP-J and its homolog RBP-L showed that the N- and C-terminal regions of RBP-J conferred the IC- as well as RAMIC-induced transactivation potential on RBP-L, which binds to the same DNA sequence as RBP-J but fails to interact with RAMIC. Taken together, these results indicate that the interactions between the N- and C-terminal regions of RBP-J and the ANK repeats of RAMIC are important for transactivation of RBP-J by RAMIC.
进化上保守的DNA结合蛋白RBP-J直接与Notch细胞内区域（RAMIC）的RAM结构域和锚蛋白（ANK）重复序列相互作用，并激活调节细胞分化的下游靶基因的转录。体外结合试验表明，RBP-J截短的N端和C端区域与ANK重复序列结合，但不与RAM结构域结合。使用RBP-J基因座被破坏的OT11小鼠细胞系，我们发现N端和C端区域发生突变的RBP-J构建体，尽管它们对DNA和RAM结构域具有正常水平的结合活性，但在由RAMIC或IC（没有RAM结构域的Notch细胞内区域）诱导的转录激活方面存在缺陷。使用RBP-J与其同源物RBP-L之间的嵌合分子进行的研究表明，RBP-J的N端和C端区域赋予RBP-L IC以及RAMIC诱导的反式激活潜力，RBP-L与RBP-J结合相同的DNA序列，但不能与RAMIC相互作用。综上所述，这些结果表明RBP-J的N端和C端区域与RAMIC的ANK重复序列之间的相互作用对于RAMIC对RBP-J的反式激活很重要。
