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NBS1 interacts with Notch signaling in neuronal homeostasis

Zhou, Zhong-Wei, Kirtay, Murat, Schneble, Nadine, Yakoub, George, Ding, Mingmei, Rüdiger, Tina, Siniuk, Kanstantsin, Lu, Ruiqing, Jiang, Yi-Nan, Li, Tang-Liang, Kaether, Christoph, Barzilai, Ari, Wang, Zhao-Qi·2020·Nucleic Acids Research
DOI 10.1093/nar/gkaa716PMC PMC7641754
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Abstract

NBS1 is a critical component of the MRN (MRE11/RAD50/NBS1) complex, which regulates ATM- and ATR-mediated DNA damage response (DDR) pathways. Mutations in NBS1 cause the human genomic instability syndrome Nijmegen Breakage Syndrome (NBS), of which neuronal deficits, including microcephaly and intellectual disability, are classical hallmarks. Given its function in the DDR to ensure proper proliferation and prevent death of replicating cells, NBS1 is essential for life. Here we show that, unexpectedly, Nbs1 deletion is dispensable for postmitotic neurons, but compromises their arborization and migration due to dysregulated Notch signaling. We find that Nbs1 interacts with NICD-RBPJ, the effector of Notch signaling, and inhibits Notch activity. Genetic ablation or pharmaceutical inhibition of Notch signaling rescues the maturation and migration defects of Nbs1-deficient neurons in vitro and in vivo. Upregulation of Notch by Nbs1 deletion is independent of the key DDR downstream effector p53 and inactivation of each MRN component produces a different pattern of Notch activity and distinct neuronal defects. These data indicate that neuronal defects and aberrant Notch activity in Nbs1-deficient cells are unlikely to be a direct consequence of loss of MRN-mediated DDR function. This study discloses a novel function of NBS1 in crosstalk with the Notch pathway in neuron development.

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Figures

Figure 1. The deletion of Nbs1 inhibits neurite outgrowth. (A) In vitro cultured neurons (8dpo) of control (Ctr) or Nbs1 inducible deletion (Nbs1-iKO) genotypes (see Supplementary Figure S2a) were stained with the dendritic marker MAP2. Nbs1-iKO: Nbs1-CER (Nbs1f/f-CreERT2), with 4-OHT treatment. Controls include all genotypes without 4-OHT treatment or Nb1+/+ or Nbs1f/+ with 4-OHT treatment. (B, C) The number (B) and the average length (C) of neurites per neuron were quantified from the indicated number of neurons (n) from four animals of each genotype. n, the number of neurons analyzed. Data are mean ± SEM. Welch's t-test (for B) or Mann–Whitney test (for C) was used for statistical analysis. **P < 0.01, ***P < 0.001. (D) GFP+ cells after IUE at E15.5 (see Supplementary Figure S3a) in layers II/III of the cortex of postnatal days 26 (P26) mice are shown. (E) The number of primary neurites per neuron was quantified from three to four animals of each treatment. N: number of animals analyzed. The number of neurons analyzed is depicted within the bar. Data are mean ± SEM. Welch's t-test was used for statistical analysis. ***P < 0.001. (F) The whole cross sections of P16 brains after IUE at E15.5 were imaged by confocal microscopy and the GFP signal in selected areas (i, ii) are shown enlarged on the right. (G) The average GFP intensity in control (shLuc) and Nbs1 knockdown (shNbs1) brain in area (ii) of the contralateral cortex were quantified. Three to five sections from each of three animals were analyzed for each condition. Data are presented as Mean ± SEM. The contralateral cortex region was equally divided into ten segments for statistical analysis by two-way ANOVA with Holm-Sidak's multiple comparison test. *P < 0.05.
Figure 1. The deletion of Nbs1 inhibits neurite outgrowth. (A) In vitro cultured neurons (8dpo) of control (Ctr) or Nbs1 inducible deletion (Nbs1-iKO) genotypes (see Supplementary Figure S2a) were stained with the dendritic marker MAP2. Nbs1-iKO: Nbs1-CER (Nbs1f/f-CreERT2), with 4-OHT treatment. Controls include all genotypes without 4-OHT treatment or Nb1+/+ or Nbs1f/+ with 4-OHT treatment. (B, C) The number (B) and the average length (C) of neurites per neuron were quantified from the indicated number of neurons (n) from four animals of each genotype. n, the number of neurons analyzed. Data are mean ± SEM. Welch's t-test (for B) or Mann–Whitney test (for C) was used for statistical analysis. **P < 0.01, ***P < 0.001. (D) GFP+ cells after IUE at E15.5 (see Supplementary Figure S3a) in layers II/III of the cortex of postnatal days 26 (P26) mice are shown. (E) The number of primary neurites per neuron was quantified from three to four animals of each treatment. N: number of animals analyzed. The number of neurons analyzed is depicted within the bar. Data are mean ± SEM. Welch's t-test was used for statistical analysis. ***P < 0.001. (F) The whole cross sections of P16 brains after IUE at E15.5 were imaged by confocal microscopy and the GFP signal in selected areas (i, ii) are shown enlarged on the right. (G) The average GFP intensity in control (shLuc) and Nbs1 knockdown (shNbs1) brain in area (ii) of the contralateral cortex were quantified. Three to five sections from each of three animals were analyzed for each condition. Data are presented as Mean ± SEM. The contralateral cortex region was equally divided into ten segments for statistical analysis by two-way ANOVA with Holm-Sidak's multiple comparison test. *P < 0.05.CC BY-NC · PubMed Central
Figure 2. Nbs1 deficiency inhibits neuronal migration. (A) Brain sections of P16 mice electroporated with indicating GFP-shRNA vectors at E15.5 are shown. GFP-shLuc targeting luciferase is control and GFP-shNbs1#1 or shNbs1#2 targets Nbs1. (B) The brain cortex from (A) was equally divided into ten segments and the percentage of GFP+ cells from each segment quantified based on three to five sections from each of three (N) animals of each condition (right). Data are Mean ± SEM. Two-way ANOVA with Holm-Sidak's multiple comparison test was used for statistical analysis. *P < 0.05; **P < 0.01; ***P < 0.001; n.s. = not significant. (C) Cortical sections were imaged after staining with CUX1, a marker for recently born neurons. The right panels are enlargements of the indicated areas of shNbs1 images. Arrows indicate GFP+CUX1+ cells. (D) The layers V and VI of the cortex (C) are shown after staining with CUX1 and early-born neuronal marker Ctip2. Arrowheads indicate GFP+CUX1+Ctip2– cells. (E) In vitro transwell migration of Neuo2A cells after co-transfection with shLuc or shNbs1 together with GFP-empty vector (EV) or GFP-shNbs1-resistant Nbs1 (rsNbs1) vector. The migratory activity was analyzed 24 h after plating on the membrane. Data are Mean ± SEM of minimum three independent experiments. Statistical analysis was performed using Two-way ANOVA followed by Tukey's post-hoc test. ***P < 0.001. (F) Transwell migration assay of primary neurons from E17.5 cortex of embryos that were electroporated with indicated shRNA expression vectors at E15.5. The migratory activity was calculated as GFP+, or GFP and NeuN double-positive (GFP+NeuN+) cells among the number of DAPI positive cells counted (n). Data are mean ± SEM of at least three embryos. Statistical analysis was performed using two-way ANOVA followed by Tukey's post-hoc test. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 2. Nbs1 deficiency inhibits neuronal migration. (A) Brain sections of P16 mice electroporated with indicating GFP-shRNA vectors at E15.5 are shown. GFP-shLuc targeting luciferase is control and GFP-shNbs1#1 or shNbs1#2 targets Nbs1. (B) The brain cortex from (A) was equally divided into ten segments and the percentage of GFP+ cells from each segment quantified based on three to five sections from each of three (N) animals of each condition (right). Data are Mean ± SEM. Two-way ANOVA with Holm-Sidak's multiple comparison test was used for statistical analysis. *P < 0.05; **P < 0.01; ***P < 0.001; n.s. = not significant. (C) Cortical sections were imaged after staining with CUX1, a marker for recently born neurons. The right panels are enlargements of the indicated areas of shNbs1 images. Arrows indicate GFP+CUX1+ cells. (D) The layers V and VI of the cortex (C) are shown after staining with CUX1 and early-born neuronal marker Ctip2. Arrowheads indicate GFP+CUX1+Ctip2– cells. (E) In vitro transwell migration of Neuo2A cells after co-transfection with shLuc or shNbs1 together with GFP-empty vector (EV) or GFP-shNbs1-resistant Nbs1 (rsNbs1) vector. The migratory activity was analyzed 24 h after plating on the membrane. Data are Mean ± SEM of minimum three independent experiments. Statistical analysis was performed using Two-way ANOVA followed by Tukey's post-hoc test. ***P < 0.001. (F) Transwell migration assay of primary neurons from E17.5 cortex of embryos that were electroporated with indicated shRNA expression vectors at E15.5. The migratory activity was calculated as GFP+, or GFP and NeuN double-positive (GFP+NeuN+) cells among the number of DAPI positive cells counted (n). Data are mean ± SEM of at least three embryos. Statistical analysis was performed using two-way ANOVA followed by Tukey's post-hoc test. *P < 0.05, **P < 0.01, ***P < 0.001.CC BY-NC · PubMed Central
Figure 3. The deletion of Nbs1 elevates Notch signaling. (A, B) Real-Time-quantitative-PCR (qPCR) analyses of Notch receptors and their target genes after Nbs1 deletion. (A) RNA was isolated from cerebellar neurons of P26 Nbs1-CNSΔ and control mice. Data are Mean ± SEM from two repeat experiments of 2–3 animals of each genotype. (B) RNA was from one Nbs1+/f-CreERT2 (+/F) and two independent Nbs1f/f-CreERT2 (F/F) MEF cell lines after treatment with or without 4OHT for 4 days and analyzed at 8dpo. Data are mean ± SEM of 2–3 independent experiments. Two-way ANOVA followed by uncorrected Fisher's LSD test was used for statistical analysis. *P < 0.05; **P < 0.01; ***P < 0.001; n.s.: not significant; UD: undetectable, ND: not detected. (C, D) Western blot analysis of Notch protein in primary neurons (C) or in 8dpo MEFs (D) with the indicated antibodies. The Notch1 antibody detects NICD at ∼110 kDa. Lamin B is a loading control. Data are mean ± SD of the relative protein level after quantifying two animals of each genotype (C), or two repeats of two independent cell lines of each genotype (D). (E) Western blot analysis of cell fractionations prepared from Nbs1-iKO MEFs (8dpo) using the indicated antibodies. The Notch1 antibody detects NICD (∼110 kDa). Histone 3 and β-Tubulin were used to control the nuclear and cytoplasmic fractions, respectively. WCE: whole-cell extract; S1: soluble cytoplasmic fraction; S2: soluble nuclear fraction; P2: chromatin-bound fraction. +/F: Nbs1+/f-CreERT2, heterozygous of Nbs1 alleles; F/F: Nbs1f/f-CreERT2, homozygous of Nbs1 floxed alleles. The mean ± SEM of the relative protein level is from four independent experiments (right panel). Two-way ANOVA followed by uncorrected Fisher's LSD test was used for statistical analysis. *P < 0.05; **P < 0.01; ***P < 0.001. (F) Notch activity in Nbs1-deleted MEF cells. Control or Nbs1-iKO MEFs were co-transfected with the luciferase reporters together with empty vector (EV) or shNotch1 at 7dpo. Dual-luciferase assay was carried out 24 h after transfection. Data are mean ± SEM of three independent experiments. Two-way ANOVA followed by Tukey's post-hoc test was used for statistical analysis. ***P < 0.001.
Figure 3. The deletion of Nbs1 elevates Notch signaling. (A, B) Real-Time-quantitative-PCR (qPCR) analyses of Notch receptors and their target genes after Nbs1 deletion. (A) RNA was isolated from cerebellar neurons of P26 Nbs1-CNSΔ and control mice. Data are Mean ± SEM from two repeat experiments of 2–3 animals of each genotype. (B) RNA was from one Nbs1+/f-CreERT2 (+/F) and two independent Nbs1f/f-CreERT2 (F/F) MEF cell lines after treatment with or without 4OHT for 4 days and analyzed at 8dpo. Data are mean ± SEM of 2–3 independent experiments. Two-way ANOVA followed by uncorrected Fisher's LSD test was used for statistical analysis. *P < 0.05; **P < 0.01; ***P < 0.001; n.s.: not significant; UD: undetectable, ND: not detected. (C, D) Western blot analysis of Notch protein in primary neurons (C) or in 8dpo MEFs (D) with the indicated antibodies. The Notch1 antibody detects NICD at ∼110 kDa. Lamin B is a loading control. Data are mean ± SD of the relative protein level after quantifying two animals of each genotype (C), or two repeats of two independent cell lines of each genotype (D). (E) Western blot analysis of cell fractionations prepared from Nbs1-iKO MEFs (8dpo) using the indicated antibodies. The Notch1 antibody detects NICD (∼110 kDa). Histone 3 and β-Tubulin were used to control the nuclear and cytoplasmic fractions, respectively. WCE: whole-cell extract; S1: soluble cytoplasmic fraction; S2: soluble nuclear fraction; P2: chromatin-bound fraction. +/F: Nbs1+/f-CreERT2, heterozygous of Nbs1 alleles; F/F: Nbs1f/f-CreERT2, homozygous of Nbs1 floxed alleles. The mean ± SEM of the relative protein level is from four independent experiments (right panel). Two-way ANOVA followed by uncorrected Fisher's LSD test was used for statistical analysis. *P < 0.05; **P < 0.01; ***P < 0.001. (F) Notch activity in Nbs1-deleted MEF cells. Control or Nbs1-iKO MEFs were co-transfected with the luciferase reporters together with empty vector (EV) or shNotch1 at 7dpo. Dual-luciferase assay was carried out 24 h after transfection. Data are mean ± SEM of three independent experiments. Two-way ANOVA followed by Tukey's post-hoc test was used for statistical analysis. ***P < 0.001.CC BY-NC · PubMed Central
Figure 4. Inhibition of Notch signaling rescues the migration and neurite defects of Nbs1-deficient neural cells. (A, B) Blockage of Notch signaling by inhibitors (10μM of DAPT or L685, 458) (A) or shRNA targeting Notch1 (B) improves migration deficiency of N2A cells caused by shNbs1 knockdown. The migratory activity was analyzed 24 h after drug treatment or shRNA transfection. Data are Mean ± SEM from four independent experiments. Two-way ANOVA followed by Tukey's post-hoc test was used for statistical analysis. *P < 0.05; **P < 0.01; ***P < 0.001. (C) Rescue of the neuronal migration in vivo. Brain cortical sections from P16 mice after IUE with indicated plasmids at E15.5 were imaged by confocal microscopy. The distribution of GFP+ cells in indicated regions of the cortex was quantified (right). The mean ± SEM of the percentage of GFP+ cells in indicated regions from three to five animals is shown (right panel). N: number of mice analyzed. Two-way ANOVA followed by Tukey's post-hoc test was used for statistical analysis within marked corresponding layer group. *P < 0.05; ** or ##P < 0.01; *** or ### or $$$P < 0.001; n.s.: not significant. (D) Rescue of the neurite outgrowth in vivo. Brain cortical sections from P26 mice after IUE with indicated plasmids at E15.5 were imaged and GFP+ neurons in layers II/III of the cortex are presented (up penal). The mean ± SEM were quantified from indicated animals of each treatment. N: number of mice analyzed; n: number of neurons analyzed. One-way ANOVA followed by Dunnett's multiple comparisons test was used for statistical analysis. *P < 0.05; **P < 0.01; ***P < 0.001. (E) Inhibition of Notch signaling by DAPT ameliorates the arborization defects of Nbs1-iKO neurons. Primary neurons were isolated from two Nbs1+/f-CreERT2 (+/F) and two Nbs1f/f-CreERT2 (F/F) E15.5 embryonic brains and cultured with 4-OHT for four days. Cells were then treated with or without 10 μM of DAPT for another 4 days before analysis at 8dpo. The number of primary neurites (marked by MAP2) per neuron from 41∼131 neurons (from at least two animals) are shown (right panel). The Mean ± SEM is shown. One-way ANOVA followed by Dunnett's multiple comparisons test was used for statistical analysis. *P < 0.05; **P < 0.01; ***P < 0.001. (F) Inhibition of Notch activity corrects neurite outgrowth defects of shNbs1 knockdown N2A. N2A cells were transfected with Tomato-tagged shRNA against luciferase (shLuc) or Nbs1 (shNbs1). Cells were induced to neuronal differentiation by 2.5 mM cAMP in the presence or absence of Notch inhibitors (10 μM of DAPT or L685, 458) 12 h after transfection. Cells were imaged 24 h after differentiation induction and represented imaged (left). Quantification of the total length of neurites and the areas of cell clusters was acquired by the IncuCyte3 system. Arrows mark the neurites. Data are presented as mean ± SEM of the relative neurite length (dividing total length by areas of cell cluster) from four experiments (right panel). Two-way ANOVA followed by Tukey's post-hoc test was used for statistical analysis. ***P < 0.001.
Figure 4. Inhibition of Notch signaling rescues the migration and neurite defects of Nbs1-deficient neural cells. (A, B) Blockage of Notch signaling by inhibitors (10μM of DAPT or L685, 458) (A) or shRNA targeting Notch1 (B) improves migration deficiency of N2A cells caused by shNbs1 knockdown. The migratory activity was analyzed 24 h after drug treatment or shRNA transfection. Data are Mean ± SEM from four independent experiments. Two-way ANOVA followed by Tukey's post-hoc test was used for statistical analysis. *P < 0.05; **P < 0.01; ***P < 0.001. (C) Rescue of the neuronal migration in vivo. Brain cortical sections from P16 mice after IUE with indicated plasmids at E15.5 were imaged by confocal microscopy. The distribution of GFP+ cells in indicated regions of the cortex was quantified (right). The mean ± SEM of the percentage of GFP+ cells in indicated regions from three to five animals is shown (right panel). N: number of mice analyzed. Two-way ANOVA followed by Tukey's post-hoc test was used for statistical analysis within marked corresponding layer group. *P < 0.05; ** or ##P < 0.01; *** or ### or $$$P < 0.001; n.s.: not significant. (D) Rescue of the neurite outgrowth in vivo. Brain cortical sections from P26 mice after IUE with indicated plasmids at E15.5 were imaged and GFP+ neurons in layers II/III of the cortex are presented (up penal). The mean ± SEM were quantified from indicated animals of each treatment. N: number of mice analyzed; n: number of neurons analyzed. One-way ANOVA followed by Dunnett's multiple comparisons test was used for statistical analysis. *P < 0.05; **P < 0.01; ***P < 0.001. (E) Inhibition of Notch signaling by DAPT ameliorates the arborization defects of Nbs1-iKO neurons. Primary neurons were isolated from two Nbs1+/f-CreERT2 (+/F) and two Nbs1f/f-CreERT2 (F/F) E15.5 embryonic brains and cultured with 4-OHT for four days. Cells were then treated with or without 10 μM of DAPT for another 4 days before analysis at 8dpo. The number of primary neurites (marked by MAP2) per neuron from 41∼131 neurons (from at least two animals) are shown (right panel). The Mean ± SEM is shown. One-way ANOVA followed by Dunnett's multiple comparisons test was used for statistical analysis. *P < 0.05; **P < 0.01; ***P < 0.001. (F) Inhibition of Notch activity corrects neurite outgrowth defects of shNbs1 knockdown N2A. N2A cells were transfected with Tomato-tagged shRNA against luciferase (shLuc) or Nbs1 (shNbs1). Cells were induced to neuronal differentiation by 2.5 mM cAMP in the presence or absence of Notch inhibitors (10 μM of DAPT or L685, 458) 12 h after transfection. Cells were imaged 24 h after differentiation induction and represented imaged (left). Quantification of the total length of neurites and the areas of cell clusters was acquired by the IncuCyte3 system. Arrows mark the neurites. Data are presented as mean ± SEM of the relative neurite length (dividing total length by areas of cell cluster) from four experiments (right panel). Two-way ANOVA followed by Tukey's post-hoc test was used for statistical analysis. ***P < 0.001.CC BY-NC · PubMed Central
Figure 5. Distinct effects of MRN and DNA damage on Notch activity and neuronal development. (A) The effect of the MRN complex on Notch activity. The luciferase reporters were co-transfected with empty (EV) or indicated shRNA expression vectors into wild-type MEF cells. Dual-luciferase assay was carried out 24 h after transfection. Data are mean ± SEM of the relative activity from seven independent experiments. One-way ANOVA with Holm–Sidak's multiple comparison test was used for statistical analysis. *P < 0.05; **P < 0.01; n.s.: not significant. (B) Neuron migration after knockdown of the MRN complex. Cortical sections of P16 mice after IUE at E15.5 with indicated shRNA expression vectors are shown. High magnification of immunostained sections with neuron markers (NeuN, CUX1, Ctip2) are shown under respective images. Arrows mark the GFP+NeuN+ or GFP+CUX1+ cells. (C) The percentage of GFP+ cells in the indicated regions from 4∼7 sections of three animals of each treatment is shown (right panel). Layers of the cortex are marked I to VI in (B). Data are presented as mean ± SEM. Two-way ANOVA followed by Tukey's post-hoc test was used for statistical analysis within marked corresponding layer group. *P < 0.05; **P < 0.01; ***P < 0.001; n.s.: not significant. (D) Western blot analysis of Nbs1-iKO MEFs at 8dpo, (see Supplementary Figure S2a) in the p53+/+ or p53−/− background using the indicated antibodies. The Notch1 antibody detects NICD (∼110 kDa). Actin serves as a loading control. The mean ± SEM of the relative protein level from four independent experiments is shown (right panel). Two-way ANOVA followed by uncorrected Fisher's LSD test was used for statistical analysis. *P < 0.05; **P < 0.01; ***P < 0.001. (E) Notch activity analysis was carried out using MEF cells transfected with the luciferase reporter, 24 h before treatment with different kinase inhibitor (1.6 μM ATRi, 1.0 μM DNA-PKi, 5 mM caffeine, 10 μM ATMi). Cells were exposed to 10 Gy of IR 1 h after drug incubation. Dual-luciferase assay was carried out 1 h after IR treatment. Data are mean ± SEM of the relative activity from three independent experiments. One-way ANOVA with Holm-Sidak's multiple comparison test was used for statistical analysis. *P < 0.05; **P < 0.01; ***P < 0.001. (F) ChIP quantitative real-time PCR detection of binding of NICD to the Hes5 promoter after MEF cells were treated with 10 Gy of IR. The Notch1 antibody was used for the ChIP assay and rabbit IgG was used as a negative control. All data shown as fold increase of binding activity after normalization first to the input, then the ratio of NICD to IgG in IR untreated control samples, were used to calculate the fold change. The mean ± SEM of fold changes from three experiments is shown. Welch's t-test was used for statistical analysis. *P < 0.05.
Figure 5. Distinct effects of MRN and DNA damage on Notch activity and neuronal development. (A) The effect of the MRN complex on Notch activity. The luciferase reporters were co-transfected with empty (EV) or indicated shRNA expression vectors into wild-type MEF cells. Dual-luciferase assay was carried out 24 h after transfection. Data are mean ± SEM of the relative activity from seven independent experiments. One-way ANOVA with Holm–Sidak's multiple comparison test was used for statistical analysis. *P < 0.05; **P < 0.01; n.s.: not significant. (B) Neuron migration after knockdown of the MRN complex. Cortical sections of P16 mice after IUE at E15.5 with indicated shRNA expression vectors are shown. High magnification of immunostained sections with neuron markers (NeuN, CUX1, Ctip2) are shown under respective images. Arrows mark the GFP+NeuN+ or GFP+CUX1+ cells. (C) The percentage of GFP+ cells in the indicated regions from 4∼7 sections of three animals of each treatment is shown (right panel). Layers of the cortex are marked I to VI in (B). Data are presented as mean ± SEM. Two-way ANOVA followed by Tukey's post-hoc test was used for statistical analysis within marked corresponding layer group. *P < 0.05; **P < 0.01; ***P < 0.001; n.s.: not significant. (D) Western blot analysis of Nbs1-iKO MEFs at 8dpo, (see Supplementary Figure S2a) in the p53+/+ or p53−/− background using the indicated antibodies. The Notch1 antibody detects NICD (∼110 kDa). Actin serves as a loading control. The mean ± SEM of the relative protein level from four independent experiments is shown (right panel). Two-way ANOVA followed by uncorrected Fisher's LSD test was used for statistical analysis. *P < 0.05; **P < 0.01; ***P < 0.001. (E) Notch activity analysis was carried out using MEF cells transfected with the luciferase reporter, 24 h before treatment with different kinase inhibitor (1.6 μM ATRi, 1.0 μM DNA-PKi, 5 mM caffeine, 10 μM ATMi). Cells were exposed to 10 Gy of IR 1 h after drug incubation. Dual-luciferase assay was carried out 1 h after IR treatment. Data are mean ± SEM of the relative activity from three independent experiments. One-way ANOVA with Holm-Sidak's multiple comparison test was used for statistical analysis. *P < 0.05; **P < 0.01; ***P < 0.001. (F) ChIP quantitative real-time PCR detection of binding of NICD to the Hes5 promoter after MEF cells were treated with 10 Gy of IR. The Notch1 antibody was used for the ChIP assay and rabbit IgG was used as a negative control. All data shown as fold increase of binding activity after normalization first to the input, then the ratio of NICD to IgG in IR untreated control samples, were used to calculate the fold change. The mean ± SEM of fold changes from three experiments is shown. Welch's t-test was used for statistical analysis. *P < 0.05.CC BY-NC · PubMed Central
Figure 6. NBS1 interacts with NICD-RBPJ and suppresses NICD activity. (A) Prediction of inter-relationship between NBS1 and NICD. Co-evolution analysis was carried out by using amino acid sequences of NBS1 and NICD of Notch1 from different species. The potential of functional interaction between NBS1 and different domains of NICD (left panel), or between NICD and different domains of NBS1 (right panel) were scored. FHA: forkhead-associated domain; BRCT: BRCA1 C-Terminus domain; RAM: RBPJ-associated molecule domain; ANK: ankyrin repeats domain; TAD: transactivation domain; PEST: proline (P), glutamic acid (E), serine (S), and threonine (T) rich domain; BRCT: BRCA1 C-terminal domain; C’: C-terminus. (B) Co-immunoprecipitation (Co-IP) analysis of endogenous interaction between Nbs1 and NICD or RBPJ in HeLa cells. Antibodies used for IP and Immunoblotting (IB) are indicated. The Notch1 antibody detects NICD (∼110 kDa). Representative images of four experiments are shown. (C) Identification of the binding domains between Nbs1 and NICD by Co-IP. Schematic structures of functional domains of Nbs1 and NICD (upper panel) are shown. HEK293 cells were co-transfected with Flag-tagged full length or truncated mutants of Nbs1 together with GFP-tagged NICD. IP (IP:Flag) samples were used for immunoblot analysis with indicated antibodies (lower panel). FL: full length; Δ24–330: deletion of N-terminal amino acids 24–330; Δ331–670: deletion of mid-part amino acids 331–670; Δ671–752: deletion of C-terminal amino acids 671–752. Representative images from four experiments are shown. (D) mCherry-tagged RBPJ was co-transfected with Flag-Nbs1 or GFP-NICD into U2OS cells and a Co-IP assay was performed using indicated antibodies. Representative images from two experiments are shown. (E) Notch activity assay of wildtype (Ctr) or Nbs1-iKO MEF cells (7dpo) after co-transfection with the luciferase reporter and the GFP-tagged empty vector (EV) or the full length Nbs1FL (FL) or N-terminal deletion mutant Nbs1Δ24–330 (Δ24–330) vector. Reporter activity was measured 24 hr after transfection. Data are from three independent experiments. Data are Mean ± SEM of relative Notch activity from three independent experiments. Two-way ANOVA followed by Tukey's post-hoc test was used for statistical analysis. **P < 0.01; ***P < 0.001, n.s.: not significant. (F) Transwell migration assay of N2A cells after co-transfection with Tomato-tagged shRNA targeting luciferase (shLuc) or Nbs1 (shNbs1), together with GFP-empty vector (EV) or a GFP-shNbs1-resistant Nbs1-N terminus (Nbs1-N) plasmid. The migratory activity was analyzed 24 hr after plating cells on the membrane. Data are mean ± SEM of the relative migration activity from five independent experiments. Two-way ANOVA followed by Tukey's post-hoc test was used for statistical analysis. **P < 0.01; ***P < 0.001.
Figure 6. NBS1 interacts with NICD-RBPJ and suppresses NICD activity. (A) Prediction of inter-relationship between NBS1 and NICD. Co-evolution analysis was carried out by using amino acid sequences of NBS1 and NICD of Notch1 from different species. The potential of functional interaction between NBS1 and different domains of NICD (left panel), or between NICD and different domains of NBS1 (right panel) were scored. FHA: forkhead-associated domain; BRCT: BRCA1 C-Terminus domain; RAM: RBPJ-associated molecule domain; ANK: ankyrin repeats domain; TAD: transactivation domain; PEST: proline (P), glutamic acid (E), serine (S), and threonine (T) rich domain; BRCT: BRCA1 C-terminal domain; C’: C-terminus. (B) Co-immunoprecipitation (Co-IP) analysis of endogenous interaction between Nbs1 and NICD or RBPJ in HeLa cells. Antibodies used for IP and Immunoblotting (IB) are indicated. The Notch1 antibody detects NICD (∼110 kDa). Representative images of four experiments are shown. (C) Identification of the binding domains between Nbs1 and NICD by Co-IP. Schematic structures of functional domains of Nbs1 and NICD (upper panel) are shown. HEK293 cells were co-transfected with Flag-tagged full length or truncated mutants of Nbs1 together with GFP-tagged NICD. IP (IP:Flag) samples were used for immunoblot analysis with indicated antibodies (lower panel). FL: full length; Δ24–330: deletion of N-terminal amino acids 24–330; Δ331–670: deletion of mid-part amino acids 331–670; Δ671–752: deletion of C-terminal amino acids 671–752. Representative images from four experiments are shown. (D) mCherry-tagged RBPJ was co-transfected with Flag-Nbs1 or GFP-NICD into U2OS cells and a Co-IP assay was performed using indicated antibodies. Representative images from two experiments are shown. (E) Notch activity assay of wildtype (Ctr) or Nbs1-iKO MEF cells (7dpo) after co-transfection with the luciferase reporter and the GFP-tagged empty vector (EV) or the full length Nbs1FL (FL) or N-terminal deletion mutant Nbs1Δ24–330 (Δ24–330) vector. Reporter activity was measured 24 hr after transfection. Data are from three independent experiments. Data are Mean ± SEM of relative Notch activity from three independent experiments. Two-way ANOVA followed by Tukey's post-hoc test was used for statistical analysis. **P < 0.01; ***P < 0.001, n.s.: not significant. (F) Transwell migration assay of N2A cells after co-transfection with Tomato-tagged shRNA targeting luciferase (shLuc) or Nbs1 (shNbs1), together with GFP-empty vector (EV) or a GFP-shNbs1-resistant Nbs1-N terminus (Nbs1-N) plasmid. The migratory activity was analyzed 24 hr after plating cells on the membrane. Data are mean ± SEM of the relative migration activity from five independent experiments. Two-way ANOVA followed by Tukey's post-hoc test was used for statistical analysis. **P < 0.01; ***P < 0.001.CC BY-NC · PubMed Central

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Metadata

Authors
Zhou, Z.-W., Kirtay, M., Schneble, N., Yakoub, G., Ding, M., Rüdiger, T., Siniuk, K., Lu, R., Jiang, Y.-N., Li, T.-L., Kaether, C., Barzilai, A., & Wang, Z.-Q.
Affiliations
  • Leibniz Institute on Aging – Fritz Lipmann Institute (FLI), Jena, Germany
  • School of Medicine (Shenzhen), Sun Yat-Sen University, Guangzhou, China
  • Department of Neurobiology, George S. Wise Faculty of Life Sciences and Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel
ORCID
Venue
Nucleic Acids Research
Publisher
Oxford University Press (OUP)
Published
2020-10-03
Volume
48
Issue
19
Pages
10924-10939
ISSN
  • 0305-1048
  • 1362-4962
DOI
10.1093/nar/gkaa716
PMC
PMC7641754
Cited by
20
References
5
Language
en
Licence
creativecommons.org/licenses/by-nc/4.0/ (vor)
Funders
  • DFG (WA2627/1-1, WA2627/5-1)
  • Leibniz Association (SAW2014, SAW2015)
  • German-Israel Foundation (I-1307-418.13/2015)
  • Natural Science Foundation of Guangdong Province (2019A1515010881)

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