Volume 50, Issue 4, Pages 565-576 (May 2013) Dynamic Methylation of Numb by Set8 Regulates Its Binding to p53 and Apoptosis  Gurpreet Kaur Dhami, Huadong Liu, Marek Galka, Courtney Voss, Ran Wei, Kimberly Muranko, Tomonori Kaneko, Sean P. Cregan, Lin Li, Shawn Shun-Cheng Li  Molecular Cell  Volume 50, Issue 4, Pages 565-576 (May 2013) DOI: 10.1016/j.molcel.2013.04.028 Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 1 Numb Binds to p53 in the Nucleus under Cellular Stress (A) The Numb and p53 interaction was enhanced by Doxo in a dose-dependent manner. MCF7 cells treated with 20 μM MG132 and the indicated concentration of Doxo were fractionated into the cytosolic (Cyto) and nuclear (Nuc) fractions prior to coIP and immunoblot (IB) to examine the Numb-p53 interaction. (B) Quantification of data in (A) to show the extent of Numb-p53 interaction in the nucleus normalized to the p53 level. (C) The Numb PTB domain is necessary for p53 binding. Flag-tagged Numb or a truncation mutant was expressed in MCF7 cells, and their binding to endogenous p53 was examined. (D) The Numb PTB domain is sufficient for binding p53. Bacterial expressed and purified GST-PTB domains were used to pull down p53 from the lysate of MCF7 cells treated with 0, 0.5, or 1.0 μM Doxo. (E) The DNA binding domain of p53 is necessary for Numb binding. Structures of p53 and the mutants used in the coIP experiment are as follows: gray, hashed, and black bars represent the transactivation, DNA-binding, and oligomeric domains, respectively. Flag-tagged p53 or a p53 truncation mutant was expressed in HeLa cells and assessed for binding to Numb by anti-Numb IP followed by anti-Flag IB. (F) The DNA-binding domain of p53 is sufficient for Numb binding. The indicated p53 mutants were expressed in HeLa cells and assayed for binding to Numb. (G) The DNA binding domain of p53 is required for apoptosis. MCF7 cells transfected with indicated proteins were treated with vehicle (DMSO) or Doxo for 24 hr and labeled with Annexin V and propidium iodide (PI) for apoptosis analysis by flow cytometry. Data representative of at least three independent experiments are shown in (B) and (G) (± SD, *p < 0.05; **p < 0.005). See also Figure S1. Molecular Cell 2013 50, 565-576DOI: (10.1016/j.molcel.2013.04.028) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 2 Numb Mediates Apoptosis through p53 and PUMA (A) A western blot showing the overexpression of Flag-Numb and/or knockdown of p53 by a specific siRNA, but not the scrambled control siRNA in MCF7 cells. (B) Flow-cytometric analysis of Annexin V-positive cells under the conditions specified. MCF7 cells expressing indicated siRNA were transfected with a Numb-expressing construct or the empty vector (Ctrl), treated with or without Doxo, stained for Annexin V, and sorted. (C) A bar graph of the data presented in (B) showing the percentage of apoptotic MCF7 cells (i.e., Annexin V-positive and PI-negative). Results represent mean ± SD from four independent experiments. **p < 0.005; *p < 0.05. (D) While RNA polymerase II was recruited to the GAPDH promoter, Numb and p53 were recruited to the PUMA promoter upon Doxo (1 μM) treatment. The IgG, RNA polymerase II, p53, or Numb-associated DNA fragments were immunoprecipitated, purified, and amplified by real-time PCR using primers for GAPDH and PUMA, respectively. Results represent mean ± SD from five different experiments. **p < 0.005; *p < 0.05. (E) Numb upregulated PUMA transcription. Shown is the ratio of PUMA/GAPDH mRNA in MCF7 cells transfected with the indicated constructs and treated or not with 1 μM Doxo. Results represent mean ± SD from three independent experiments. **p < 0.005. (F) The PUMA protein was upregulated by Numb. MCF7 cells expressing Numb or not were treated with or without Doxo and subjected to western blotting to determine the levels of PUMA, Numb (anti-Flag), p53, and β-tubulin using specific antibodies. (G) Depletion of endogenous Numb from MCF7 cells led to reduced PUMA expression. Numb was depleted by a specific shRNA, but not a control shRNA. Cells were treated with Doxo and immunoblotted for Numb, p53, PUMA, and β-tubulin, respectively. (H) Depletion of Numb conferred resistance to Doxo-induced apoptosis in MCF7 cells. Shown are the percentages of apoptotic cells expressing the control or Numb-shRNA before or after Doxo treatment. Results represent mean ± SD from four independent experiments. *p < 0.05. See also Figure S2. Molecular Cell 2013 50, 565-576DOI: (10.1016/j.molcel.2013.04.028) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 3 Dynamic Numb Methylation at Lys158 and Lys163 in Response to Doxorubicin Treatment (A) Extracted ion chromatograms (XIC) of four fragment ions detected in the MRM experiment designed to monitor the Numb_K158me2 peptide, AACLER(Kme2)QKREKECGVTATF (molecular mass M3+ = 766.0682) eluted at 22.66 min. (B) MS/MS spectrum of the Numb_158me2 peptide showing the key fragment ions indicative of lysine methylation. (C) Changes in the level of nuclear Numb_K158me2 before (black trace) and after (purple trace) Doxo treatment as measured by MRM-MS. The Numb_358–369 peptide (GQSSGAASPGLF, M2+ = 539.7618) was used as an internal control. (D) A bar graph showing the normalized (against the Numb_358–369 peptide), quantitative changes in the di- and trimethylated K158 and K163 in the cytosolic (S) versus the nuclear (N) fractions in the absence or presence of Doxo. (E) The structure of the Drosophila Numb PTB domain bound to a peptide based on PDB ID code 2NMB. The PTB domain is shown in ribbons, with the equivalent residues to human Numb K158 and K163 shown in green sticks. The bound peptide is shown in yellow sticks. See also Table S1 and Figure S3. Molecular Cell 2013 50, 565-576DOI: (10.1016/j.molcel.2013.04.028) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 4 Methylation of Numb on Residues K158 and K163 Negatively Regulates Its Interaction with p53 and Apoptosis (A) The Numb K158/163A and K158/163R mutants (Flag tagged) displayed diminished binding to p53. MCF7 cells expressing indicated proteins were subjected to anti-Flag IP followed by a p53 immunoblot. (B) The single K158A/R and the double mutants of Numb were defective in promoting Doxo-induced apoptosis in MCF7 cells. Shown are the percentages of apoptotic cells transfected with Flag-Numb or a mutant before and after Doxo treatment. Results are mean ± SD from three independent experiments. **p < 0.005. (C) The Numb K158A-containing mutants (Flag-tagged) exhibited diminished binding to p53 in U2OS cells. (D) Mutation of K158 compromised the ability of Numb to promote apoptosis of U2OS cells. The bar graph shows the Annexin V-positive cells under the indicated conditions. Results are mean ± SD from three independent experiments. **p < 0.005. (E) The function of Numb in regulating p53 ubiquitination and stability required K158 and K163. MCF7 cells containing the Numb-specific or scrambled siRNA were transfected with a plasmid encoding siRNA-resistant Numb (sr-Numb) or the K158/163R double mutant. Upon incubating with 20 μM MG132 for 4 hr, the cells were lysed for p53 IP followed by anti-ubiquitin and anti-p53 immunoblots. Also shown are the total Numb and p53 protein levels under the same conditions. See also Figure S4. Molecular Cell 2013 50, 565-576DOI: (10.1016/j.molcel.2013.04.028) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 5 Numb Undergoes Methylation at K158 and K163 in the Presence of Set8 (A) Numb is methylated by Set8. U2OS cells were transfected with Flag-Numb alone or together with HA-Set8 or GFP-SUV4-20h2. Numb was IPed with anti-Flag and immunoblotted with a panmethylation antibody Me1/2 (Abcam) to detect changes in Lys methylation. (B) Set8 methylates WT Numb, but not the K158/163A mutant. Numb or the double mutant was expressed in MCF7 cells together with HA-Set8 and immunoblotted for methylation using anti-Me1/2. (C) Interaction of endogenous Numb and Set8. Numb was IPed from MCF7 cells and immunoblotted for Set8. (D and E) Set8 was both a mono- and a dimethyltransferase. Set8 converted the Numb_K158 peptide substrate (biotin-AACLERKQKREKE) to both mono- (D) and dimethylated (E) forms in an in vitro methylation assay. Shown are MS/MS spectra of the corresponding peptides. (F) Set8 transcription was significantly repressed by Doxo. The mRNA level of Set8, as measured by qPCR, was compared for cells with or without Doxo treatment. Results are mean ± SD from three independent experiments. **p < 0.005. (G) Set8 was degraded in the presence of Doxo. A western blot shows the levels of Set8 in the presence and absence of Doxo and proteasome inhibitor MG132 in MCF7 cells. See also Table S2 and Figure S5. Molecular Cell 2013 50, 565-576DOI: (10.1016/j.molcel.2013.04.028) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 6 Methylation of Numb by Set8 Abolishes the Numb-p53 Interaction and Reduces Numb-Mediated Apoptosis (A) The interaction of Numb and p53 was diminished when Set8 was overexpressed in MCF7 cells. (B) Coexpression of Set8 with Numb markedly reduced the Numb-p53 interaction in U2OS cells. (C) Coexpression of Set8 with Numb decreased Numb-mediated apoptosis significantly. Results are mean ± SD from three independent experiments. **p < 0.005. (D) Depletion of endogenous Set8 by siRNA from MCF7 cells promoted Numb binding to p53. (E) Depletion of Set8 in U2OS cells by siRNA decreased Numb methylation and enhanced its binding to p53. Numb was IPed and immunoblotted for methylation (Me1/2) and for binding to p53, respectively. Also shown are the levels of Set8, p53, Numb, β-tubulin, H4 me1, and H4, respectively, in the U2OS cells transfected with the ctrl (left) and Set8-specific (right) siRNA. (F) Numb bound equally well to Flag-tagged p53 or the mutant K382R. (G) Bacterially expressed GST-p53 bound to Numb more proficiently in the Doxo-treated than the untreated U2OS cells. Numb methylation was markedly reduced in the presence of Doxo. GST showed no binding to Numb regardless of Doxo. Molecular Cell 2013 50, 565-576DOI: (10.1016/j.molcel.2013.04.028) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 7 Depletion of Set8 Sensitizes MCF7 Cells to Apoptosis, an Effect Dependent Entirely on Numb (A) Flow cytometry profiles of Annexin V-postive MCF7 cells transfected with control siRNA, with Set8 siRNA alone, or together with Numb siRNA before or after Doxo treatment. (B) A bar graph of the data presented in (A) showing the percentage of apoptotic MCF7 cells under the specified conditions. Results are mean ± SD from three independent experiments. **p < 0.005. (C) Western blots showing the efficiency of Numb and Set8 knockdown in MCF7 cells. (D) A model depicting the role of lysine methylation in the modulation of Numb-p53 interaction, p53 stability, and p53-mediated apoptosis. See also Figure S6. Molecular Cell 2013 50, 565-576DOI: (10.1016/j.molcel.2013.04.028) Copyright © 2013 Elsevier Inc. Terms and Conditions