The N- and C-terminal regions of RBP-J interact with the ankyrin repeats of Notch1 RAMIC to activate transcription

Abstract

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.

Identification of Two Binding Regions for the Suppressor of Hairless Protein within the Intracellular Domain of Drosophila Notch

“…The PPD binding site is bipartite, with its two elements redundant in most experimental contexts, explaining why it has previously been difficult to identify by mutation or deletion. The previously described requirement for the ANK repeats in CSL-dependent Notch signaling (15,25,36) is consistent with our data showing that the presence of the ANK repeats greatly increases the effectiveness of Su(H) binding to PPD. Perhaps ANK holds PPD in a conformation that is favorable for Su(H) binding.…”

“…It has been suggested that Su(H) may interact in vitro with the ankyrin repeats of Drosophila Notch (24), and indeed, in contrast to the RAM domain, the ANK repeats are essential for all CSL-dependent Notch functions in all studied organisms. However, the binding of CSL proteins to the ANK region seems to be very much weaker than the binding to the RAM domain (25) and has not even been detectable in all studies. Moreover, no CSL binding site has been specifically mapped to this part of the protein.…”

“…Co-localization of transfected Notch and Su(H) in the nucleus of S2 cells was also dependent on the integrity of the ANK repeats. Surprisingly, however, Tamura et al (19) reported compelling evidence based on yeast two-hybrid experiments and GST pull-downs showing that CSL bound strongly within the RAM domain of Notch but only weakly, if at all, with an extended region including the ANK repeats (25). These data of Tamura et al (19) were also difficult to reconcile with earlier genetic data in flies showing a strong gain of function phenotype of Drosophila Notch intracellular domains missing the RAM Su(H) binding domain (22,23).…”

Notch signaling augments the canonical Wnt pathway to specify the size of the otic placode

“…In complementary experiments, we examined Notch1 mutants, in which a substantial portion of the Notch1 gene is deleted [amino acids 1056[amino acids -2049[amino acids (Conlon et al, 1995]. This deletion encompasses RAM and Ankyrin repeats required for RBPJκ signaling (Conlon et al, 1995;Fortini and Artavanis-Tsakonas, 1994;Kurooka et al, 1998a;Kurooka et al, 1998b;Lamar et al, 2001;Nam et al, 2003;Tani et al, 2001). We confirmed that posterior hindbrain patterning was normal in Notch1 mutants by assaying for Hoxb1, Fgf3 and Krox20 expression (see Fig.…”

The lytic switch protein of KSHV activates gene expression via functional interaction with RBP-Jκ (CSL), the target of the Notch signaling pathway

“…(For review, see Weinmaster 2000; Panin and Irvine 1998). This fragment is transported to the nucleus via its two NLS elements, where it can then interact with RBP-J (CSL) via its N-terminal RAM domain and adjacent ankyrin-like repeats (Tani et al 2001). This interaction is thought to occur primarily via binding to the central (repressive) domain of RBP-J (Hsieh and Hayward 1995), one of the two regions we have found to mediate interaction with RTA (Fig.…”