Fig 2: Su(H) and RBPJ AOS variants decrease DNA binding.A-B. Representative EMSAs with triplicate quantification shown on the right for purified (A) Drosophila Su(H) WT, E137V, and K132M proteins and (B) mouse Rbpj WT, E89G, and K195E proteins. P-values are reported for the 125 nM protein conditions and were determined by an ANOVA with Tukey Honest Significant Difference test ([***] P < 0.001, [**] P < 0.01, [*] P < 0.05 and [NS] not significant). WT Su(H) vs. Su(H) K132M P = 2.14E-6. WT Su(H) vs. E137V P = 9.9E-9. WT RBPJ vs. E89G P = 2.27E-8. WT RBPJ vs. K195E P = 6.42E-14. C-D. Representative thermograms showing the raw heat signal and nonlinear least squares fit to the integrated data for (C) Drosophila Su(H) and (D) mouse Rbpj variants binding to a 20mer oligo duplex containing the same RBPJ/Su(H) binding site used in the above EMSAs. Each ITC experiment was performed at 10°C with 20 injections. The mean dissociation constants (KD)and standard deviations from triplicate experiments are reported along with the p-value determined from a two-tailed T-test ([***] P < 0.001, [**] P < 0.01, [*] P < 0.05 and [NS] not significant). WT Su(H) vs. Su(H) K132M P = 2.22E-4. WT Su(H) vs. E137V P = 8.22E-4. WT RBPJ vs. E89G P = 4.06E-3. WT RBPJ vs. K195E P = 3.97E-3.

Fig 3: FBXO42 is a critical interactor of RBPJ and a positive regulator of the Notch pathway.(A) Interaction network of RBPJ. high-confidence interacting proteins (HCIPs) were grouped on the basis of their cellular functions as indicated by Gene Ontology (GO) analysis and a literature search. Ubiquitination (Ub)/ubiquitin-like (UbL)–related proteins, proteins that are potential positive and negative regulators, are indicated by different colored dots. Signaling pathways and proteins reported to be involved in Notch pathway regulation are indicated by different colored texts. NER, nuclear excision repair; BER, base excision repair; TH1/TH2, T helper 1/2 cells. (B and C) Expression of L3MBTL3 (B) and Notch target genes (C) was evaluated in wild-type (WT) and L3MBTL3 KO HEK293T cells. (D and E) Expression of FBXO42 (D) and Notch target genes (E) was evaluated in WT and FBXO42 KO HEK293T cells. (F and G) Green fluorescent protein (GFP) reporter (F) and luciferase assays (G) were performed to evaluate Notch signaling activities in WT and FBXO42 KO HEK293T cells. RBPJ-BS, RBPJ binding site. (H to J) Expression level of FBXO42 in Notch-related cancers as indicated was analyzed using The Cancer Genome Atlas (TCGA) datasets. (K and L) Expression level of FBXO42 in various cancer cell lines (K) and T-ALL cell lines (L) derived from the Cancer Cell Line Encyclopedia (https://sites.broadinstitute.org/ccle). (M and N) Expression correlation of FBXO42 and Notch pathway target genes in patients with DLBCL and acute myeloid leukemia (AML) was analyzed using TCGA dataset and UALCAN database. (B) to (G), n = 3. Quantitative data are presented as means ± SEM from three independent experiments. P values were calculated using two-tailed Student’s t tests. *P < 0.05, ***P < 0.001, and ****P < 0.0001. NS, not significant; TPM, transcript per million.

Fig 4: Mapping, mutagenesis and docking analysis of CSL/UPF1/Ku70/Ku80 interaction. a Co-IP analysis of HEK293T cells expressing MYC-tagged full length (FL) Ku70 and 1-257, 1-464, 1-573, or 257–609 domains plus/minus full length (FL) CSL with anti-MYC magnetic beads followed by immunoblotting with antibodies against the indicated proteins. b Co-IP analysis of HEK293T cells expressing MYC-tagged full length (FL) Ku70 or its 464–609 domain plus/minus full length (FL) CSL with anti-MYC magnetic beads followed by immunoblotting with antibodies against the indicated proteins. c Co-IP analysis of HEK293T cells expressing FLAG-tagged full length (FL) CSL and CSL BTD (166–334) domains plus/minus full length (FL) UPF1 with anti-FLAG magnetic beads followed by immunoblotting with antibodies against the indicated proteins. A second independent experiment is in Supplementary Fig. 9a. d Co-IP analysis of HEK293T cells expressing FLAG-tagged full length (FL) CSL and CSL point mutants (R192H, F235R, V237R, A258R, and Q307R) plus/minus full length (FL) Ku70 with anti-FLAG magnetic beads followed by immunoblotting with antibodies against the indicated proteins. Additional information on CSL point mutants is in Supplementary Fig. 9b. e Cartoon representation showing the docking complex between CSL (cyan)—Telomere DNA (orange)—Ku70 (magenta) and Ku80 (blue) using HDOCK server. Ku70 is shown to interact with CSL-BTD domain and bind to CSL bound telomere DNA through its SAP domain, while Ku80 binds indirectly through Ku70. f Close up view of the docking complex between CSL—Telomere DNA—Ku70 showing the α-helix of C-Ku domain (olive) interacting with CSL-BTD domain (cyan), and Ku70 SAP domain (magenta) interacting with telomere DNA (orange). The “hot spot” mutations that abrogate CSL-Ku70 interaction and Ku70-telomeric DNA association as in d are shown in pink (A258R and R192H). Additional non-interfering mutations are labeled in blue (F235R, V237R, and Q307R). g–j Telomeric binding assays with antibodies against the indicated proteins followed by qPCR with telomere- and alu-specific primers in HEK293T cells expressing CSL full length (WT) and point mutants (R192H, F235R, V237R, A258R, and Q307R). Non-immune IgGs were used for normalization. Bars represent mean ± SD

Fig 5: Combined Notch1/Notch2 and Trp53 deletion in Hes5+ cells leads to formation of forebrain tumors in adult mice. (A,B) Conditional deletion of Notch1, Notch2 and Trp53 or Rbpj and Trp53 in brain cells expressing the Notch target gene Hes5. Adult Hes5::CreERT2 mice carrying floxed Trp53 alleles and either floxed Notch1 and Notch2 alleles or floxed Rbpj alleles and a GFP Cre-reporter for lineage tracing were treated with tamoxifen (A) and were analyzed 10 months later (B). (C) Kaplan–Meier curves showing survival of Hes5::CreERT2 Trp53-/-, Trp53-/-Notch1-/-Notch2-/- and Trp53-/-Rbpj-/- mutant mice. (D) A Trp53-/-Notch1-/-Notch2-/- tumor in the forebrain. The GFP expression from the Rosa-CAG::GFP Cre-reporter indicates derivation from Hes5+ cells. LV, lateral ventricle. (E) Percentages of Trp53-/-, Trp53-/-Notch1-/-Notch2-/- and Trp53-/-Rbpj-/- mice that developed hyperplasia or tumors in the brain.