The N- and C-terminal regions of RBP-J interact with the ankyrin repeats of Notch1 RAMIC to activate transcription.
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.
Department of Medical Chemistry and Department of Neurosurgery, Graduate School of Medicine, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan.
S
S
Tani
H
H
Kurooka
T
T
Aoki
N
N
Hashimoto
T
T
Honjo
D000818:Animals; D017089:Ankyrin Repeat; D001665:Binding Sites; D001667:Binding, Competitive; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009687:Nuclear Proteins; D011485:Protein Binding; D051881:Receptor, Notch1; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors; D015533:Transcriptional Activation
D004268:DNA-Binding Proteins; D008565:Membrane Proteins; D009687:Nuclear Proteins; C484553:Rbpsuhl protein, mouse; D051881:Receptor, Notch1; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors