Journal: Genes & Development

Article Title: ZMYM3 regulates BRCA1 localization at damaged chromatin to promote DNA repair

doi: 10.1101/gad.292516.116

Figure Lengend Snippet: ZMYM3 antagonizes the BRCA1-A complex to promote HR. ( A ) ZMYM3 promotes HR and opposes RAP80 and ABRA1 inhibition of HR. DR-GFP reporter assays were performed after depletion or codepletion of the indicated proteins by siRNAs. Data represent mean ± SD. n = 3. ( B ) Confirmation of knockdown efficiency by Western blotting from experiments performed in A . ( C ) 53BP1 depletion rescues HR defects in ZMYM3-depleted cells. Experiments were performed as in A . ( D ) ZMYM3 knockout cells are sensitive to IR and PARP inhibitors compared with parental U2OS cells. Cells were challenged with IR or PARP inhibitor as indicated and were analyzed by colony formation assays. Data represent mean ± SD. n = 3. ( E ) Western blotting analysis of knockdown efficiency in cells transfected with BRCA1 siRNA. ( F ) Epistasis analysis of ZMYM3 and BRCA1. Wild-type and ZMYM3 knockout cells either alone or in combination with siBRCA1 were challenged with IR and PARP inhibitor followed by colony formation assays. ( G ) Chromosome aberration analyses in ZMYM3 knockout and BRCA1 knockdown cells. Experiments were performed as in E. ( H ) Complementation assay of ZMYM3 knockout cells. ZMYM3 knockout cells with empty vector or wild-type ZMYM3 were analyzed as in D . (*) P < 0.05; (**) P < 0.01; (***) P < 0.001 versus same treatment with control cells, Student's t -test.

Article Snippet: Primary antibodies used in this study were Flag M2 (Sigma, F1804), Myc (Santa Cruz Biotechnology, SC-40), H2AX (Millipore, 07-627), γH2AX (Millipore, 05-636), H2AZ (Cell Signaling, 2718S), macroH2A (Abcam, AB37264), H2A.Bbd (Millipore, 06-1319), ZMYM3 (Abcam, AB106626), ATM (Santa Cruz Biotechnology, SC-135663), pATM S1981 (Abcam, AB81292), β-tubulin (Abcam, AB6046), RAD51 (Abcam, AB88572), RAP80 (Bethyl Laboratories, A300-763A), ABRA1 (Abcam, AB139191), BRCA1 (Santa Cruz Biotechnology, SC-6954), HRP-linked anti-GST (Sigma, A7340), MBP (Abcam, AB9084), phospho-H3 S10 (Cell Signaling, 3377), 53BP1 (Novus Biologicals, NB100-304), RAD18 (Cell Signaling, 9040), RPA2 (Abcam, AB2175), pRPA2 S33 (Bethyl Laboratories, A300-246), pRPA2 S4/S8 (Bethyl Laboratories, A300-245), Chk1 (Santa Cruz Biotechnology, SC-8408), and pChk1 (Cell Signaling, 2348).

Techniques: Inhibition, Knockdown, Western Blot, Knock-Out, Transfection, Plasmid Preparation, Control

Journal: Cell death and differentiation

Article Title: 53BP1 represses mitotic catastrophe in syncytia elicited by the HIV-1 envelope.

doi: 10.1038/cdd.2009.159

Figure Lengend Snippet: Figure 1 Aggregation and phosphorylations of 53BP1 in karyogamic syncytia elicited by HIV-1 Env. (a–h) Syncytia, arising from the coculture of HeLa Env and HeLa CD4 cells for 48 h, were subjected to immunofluorescence stainings. Representative karyogamic syncytia stained with anti-53BP1, 53BP1S25P, 53BPS1778P, PML, ATMS1981P, g-H2AX antibodies and Hoechst 33342 are shown. Colocalization between 53BP1(a, d), 53BP1S25P (b, e, g) and 53BP1S1778P (c, f, h) with g-H2AX (a, b, c), PML (d, e, f) or ATMS1981P (g, h) are observed. (i) Kinetics of karyogamy (KG), of PML or of 53BP1 aggregation, of phosphorylations on serine 25 (53BP1S25P) or on serine 1778 of 53BP1 (53BP1S1778P), on serine 1981 of ATM (ATMS1981P) of H2AX, on serine 137 of H2AX (g-H2AX) and on nuclear apoptosis. (j) Quantification of PML or 53BP1 aggregation or phosphorylation of 53BP1 (53BP1S25P and 53BP1S1778P), ATM (ATMS1981P) and g-H2AX on single cells (SC), on prekaryogamic (pre-KG) and karyogamic (KG) syncytia and during apoptosis. Karyogamy and nuclear apoptosis are evaluated using Hoechst 33342 staining (X±S.D., n ¼ 3)

Article Snippet: Rabbit antibody against phospho-53BP1 (Novus Biologicals) and biotinylated goat anti-rabbit IgG were incubated with tissue sections.

Techniques: Staining, Phospho-proteomics

Journal: Cell death and differentiation

Article Title: 53BP1 represses mitotic catastrophe in syncytia elicited by the HIV-1 envelope.

doi: 10.1038/cdd.2009.159

Figure Lengend Snippet: Figure 3 ATM controls 53BP1 phosphorylations but not its aggregation. (a, b) 53BP1 aggregation occurs before the activation of PML, TopBP1 and ATM. HeLa CD4 and HeLa Env cells were transfected 48 h before fusion with siRNAs specific for PML, TopBP1 and ATM and then were cocultured for 36 h. Syncytia were then subjected to immunofluorescence microscopy for the detection of karyogamy (KG), apoptosis (nuclear chromatin condensation), g-H2AX foci or the aggregation of PML and 53BP1. Representative karyogamic syncytia are shown after staining with antibodies against PML and 53BP1 (a). Results are shown as means±S.D. of three independent experiments (b). (c) ATM-dependent 53BP1 phosphorylation. After 48 h transfection with specific siRNA against ATM, HeLa CD4 and HeLa Env cells were cocultured for 36 h and stained with specific antibodies against 53BP1S25P and 53BP1S1778P. The effect of ATM knockdown on 53BP1 phosphorylation was evaluated using immunofluorescence microscopy in four independent experiments (X±S.D.). (d, e) Partial inhibition of 53BP1S25P on syncytia obtained after HIV-1 infection of PHA/IL-2 lymphoblasts obtained from A-T patients. PHA/IL-2 lymphoblasts from controls or A-T patients (n ¼ 3) were infected by HIV-1, and the frequency of syncytia with 53BP1S25P þ

Article Snippet: Rabbit antibody against phospho-53BP1 (Novus Biologicals) and biotinylated goat anti-rabbit IgG were incubated with tissue sections.

Techniques: Activation Assay, Transfection, Microscopy, Staining, Phospho-proteomics, Knockdown, Inhibition, Infection

Journal: Cell death and differentiation

Article Title: 53BP1 represses mitotic catastrophe in syncytia elicited by the HIV-1 envelope.

doi: 10.1038/cdd.2009.159

Figure Lengend Snippet: Figure 4 53BP1 depletion induces syncytial cell death through an increase in mitoses. (a–c) HeLa CD4 and HeLa Env cells were separately transfected with 53BP1-specific siRNAs, and 48 h later the cells were cocultured for further 36 h, lysed and subjected to the immunodetection of 53BP1 and GAPDH (a). Representative karyogamic syncytia transfected during 36 h with specific siRNA against 53BP1 and stained with antibodies specific for 53BP1, 53BP1S1778P, PML, g-H2AX, cytochrome c (Cyto. c) or activated caspase-3 are shown (b). Note that 53BP1 depletion triggers cytochrome c release and chromatin condensation on Env-elicited syncytia. Effect of 53BP1 depletion on the pro-apoptotic signaling pathway induced by HIV-1 envelope and on mitosis (c). The frequency of syncytia showing nuclear aggregation of PML or 53BP1, phosphorylation events 53BP1S25P, 53BP1S1778P, ATMS1981P, g-H2AX, cytochrome c release or nuclear apoptosis (chromatin condensation) was assessed using immunofluorescence microscopy. Karyogamy (KG) and mitosis were determined using Hoechst 33342 staining (X±S.D., n ¼ 5)

Article Snippet: Rabbit antibody against phospho-53BP1 (Novus Biologicals) and biotinylated goat anti-rabbit IgG were incubated with tissue sections.

Techniques: Transfection, Immunodetection, Staining, Phospho-proteomics, Microscopy

Journal: Cell death and differentiation

Article Title: 53BP1 represses mitotic catastrophe in syncytia elicited by the HIV-1 envelope.

doi: 10.1038/cdd.2009.159

Figure Lengend Snippet: Figure 5 53BP1 knockdown induces mitotic catastrophe on HIV-1 Env-elicited syncytia. (a) Transient phosphorylation of 53BP1 on serine 1778 (53BP1S1778P) during normal mitosis. Detection of 53BP1S1778P during normal anaphase, normal telophase and normal cytokinesis using immunofluorescence microscopy. (b) Depletion of 53BP1 triggers abnormal mitosis and mitotic catastrophe of HIV-1 Env-elicited syncytia. HeLa CD4 and HeLa Env cells were separately transfected for 48 h with 53BP1- specific siRNAs. Syncytia arising from the coculture of these cells for 36 h were fixed and stained with specific antibody against a-tubulin and activated caspase-3 for immunofluorescence microscopy. Representative prekaryogamic and karyogamic syncytia, prophase, aberrant metaphase, telophase and mitotic catastrophe are shown. (c) Knockdown of 53BP1 leads to multipolar metaphases with multiple centrosomes. Cells were fixed and stained with antibody against g-tubulin for immunofluorescence microscopy. Representative aberrant metaphase revealing numerous centrosomes is shown. (d) Quantification of different mitotic stages and mitotic catastrophe induced by 53BP1 silencing was determined (X±S.D., n ¼ 3). (e) Inhibition of syncytial mitotic catastrophe by N9-isopropyl-olomoucine. HeLa CD4 and HeLa Env cells were separately transfected for 48 h with 53BP1-specific siRNAs, and syncytia arising from the coculture of cells for 36 h were then incubated with 10 mM of N9-isopropyl-olomoucine (N9-Olo). Cells were fixed and stained with specific antibodies against a-tubulin and activated caspase-3 for immunofluorescence microscopy. The effects of N9- isopropyl-olomoucine on mitotic catastrophe induced by 53BP1 knockdown were determined (X±S.D., n ¼ 3)

Article Snippet: Rabbit antibody against phospho-53BP1 (Novus Biologicals) and biotinylated goat anti-rabbit IgG were incubated with tissue sections.

Techniques: Knockdown, Phospho-proteomics, Microscopy, Transfection, Staining, Inhibition, Incubation

Journal: Cell death and differentiation

Article Title: 53BP1 represses mitotic catastrophe in syncytia elicited by the HIV-1 envelope.

doi: 10.1038/cdd.2009.159

Figure Lengend Snippet: Figure 6 53BP1 phosphorylation in vivo, in HIV-1-infected patients. (a) Phosphorylation of 53BP1 on serine 25 in syncytia detectable in frontal cortex from patients with HIV-1-associated encephalitis (HAE). 53BP1S25P phosphorylations were visualized using immunohistochemistry on histological sections from control patients or from untreated HIV-1 carriers with HAE but without opportunistic infection. The insert shows labeled giant multinuclear cells. The percentage of syncytia (X±S.E.M.) staining positively for 53BP1S25P amounted to 58±9, as determined for six different HIV-1 carriers. (b) 53BP1S25P phosphorylation in lymph nodes from controls and untreated HIV-1 carriers. The insert reveals bonafide labeled syncytia. (c) 53BP1S25P in PBMC derived from representative HIV-1-infected patients or healthy donors (Co.). Immunoperoxidase stainings are shown. (d, e) Correlation between 53BP1S25P, viral status and HAART treatment. PBMCs from untreated HIV-1-infected patients were subjected to 53BP1S25P staining and the values were plotted against the number of viral particles in the serum. The P-values refer to the correlation coefficients (d). A cohort of HIV-1- donors, untreated HIV-1 carriers and HAART-treated subjects with undetectable viremia were analyzed for 53BP1S25P (e)

Article Snippet: Rabbit antibody against phospho-53BP1 (Novus Biologicals) and biotinylated goat anti-rabbit IgG were incubated with tissue sections.

Techniques: Phospho-proteomics, In Vivo, Infection, Immunohistochemistry, Control, Labeling, Staining, Derivative Assay

Journal: Nature Communications

Article Title: Treacle controls the nucleolar response to rDNA breaks via TOPBP1 recruitment and ATR activation

doi: 10.1038/s41467-019-13981-x

Figure Lengend Snippet: a γH2AX, 53BP1, RPA2, RAD51 and BRCA1 localization in control depleted (siCtrl) and Treacle-depleted (siTreacle) U2OS cells 2 h after I-Ppo1 transfection. b Quantification of the experiment in a , γH2AX: siCtrl ( n = 177) versus siTreacle ( n = 200), 53BP1: siCtrl ( n = 261) versus siTreacle ( n = 239), RPA2: siCtrl ( n = 152) versus siTreacle ( n = 168) BRCA1: siCtrl ( n = 525) versus siTreacle ( n = 246) and RAD51: siCtrl ( n = 525) versus siTreacle ( n = 246) cells (all datapoints are shown, bar represents mean, error bars represent S.D.) c γH2AX, 53BP1, RPA2, RAD51 and BRCA1 localization in control depleted (siCtrl) and TOPBP1-depleted (siTOPBP1) U2OS cells 2 h after I-Ppo1 transfection. d Quantification of the experiment in c , γH2AX: siCtrl ( n = 403) versus siTOPBP1 ( n = 205), 53BP1: siCtrl ( n = 239) versus siTOPBP1 ( n = 256), RPA2: siCtrl ( n = 649) versus siTOPBP1 ( n = 359) BRCA1: siCtrl ( n = 228) versus siTOPBP1 ( n = 348) and RAD51: siCtrl ( n = 228) versus siTOPBP1 ( n = 348) cells (all datapoints are shown, bar represents mean, error bars represent S.D.). e Clonogenic surivival analysis of I-Ppo1-transfected control (siCtrl), Treacle-depleted (siTreacle) and TOPBP1-depleted (siTOPBP) U2OS cells (red bars represent the mean). f Clonogenic surivival analysis of I-Ppo1-transfected WT and NBS1DN U2OS cells (red bars represent the mean). g Model (see text for details). Source data are provided as a Source Data file.

Article Snippet: The following antibodies were used at the indicated dilutions: Treacle (rabbit, Sigma Life Science, HPA038237, 1/100), TOPBP1 (rabbit, Bethyl, A300-111A, 1/500), Nucleophosmin (mouse, Thermofisher, MA5-17141, 1/300), BRCA1 (mouse, sc-6454, Santa Cruz, 1/100), RAD51 (rabbit, Santa Cruz, sc-8349, 1/100), γH2AX (mouse, Millpore, 05-636, 1/500), MDC1 (mouse, Abcam, Ab50003, 1/300), Cyclin A (mouse, BD biosciences, 611269, 1/100), RPA2 (mouse, Abcam, Ab2175, 1/250), RPA2 pS4/S8 (rabbit, Bethyl A300-245, 1/400), 53BP1 (mouse, gift from T. Halazonetis, 1/20) NBS1 (rabbit, Novus, NB100-143, 1/200), MRE11 (mouse, GeneTex, GTX70212, 1/1000), UBF (mouse, Santa Cruz, sc-13121, 1/100), ATM (rabbit, Abcam, ab32420, 1/250), ATR (rabbit, Cell Signaling E1S3S, 1/100), V5 (mouse, Abcam, ab27671, 1/500).

Techniques: Control, Transfection

Journal: Nucleic Acids Research

Article Title: The COP9 signalosome is vital for timely repair of DNA double-strand breaks

doi: 10.1093/nar/gkv270

Figure Lengend Snippet: Elimination of the Atm phosphorylation site on murine endogenous Csn3 impairs the DSB response in mice. ( A ) Endogenous Csn3 is not phosphorylated on S410 in response to DSBs in various tissues of mice expressing a mutated protein with the S410A amino acid substitution. Ten-day-old mice were irradiated with 10 Gy of IR, and 1 h later tissues were collected and processed for immunoblotting analysis with the indicated antibodies. Phosphorylation of the Atm substrate Kap-1 on S824 served to monitor ATM activity. ( B ) Kaplan–Meier survival curve of 1-month old mice with various genotypes in the Cops3 locus encoding Csn3 following whole body irradiation with 6.5 Gy of IR. 10–20 animals of each genotype were irradiated. ( C ) Clonogenic survival of MEFs with different genotypes after treatment with various NCS doses. Cells expressing non-phosphorylatable Csn3 exhibit intermediate radiomimetic sensitivity between that of wild-type and that of Atm-deficient cells. ( D and E ). Lack of an Atm phosphorylation site on Csn3 slows the disappearance of DNA damage-induced γH2AX and 53BP1 nuclear foci. MEFs with the indicated version of endogenous Csn3 were irradiated with 1 Gy of IR, and subsequently fixed and stained with antibodies against γH2AX or 53BP1. Nuclear foci were counted in 150–250 cells per time point. ( F ) Similar analysis as in (D) and (E) using an antibody against Rad51. The diagram presents the fraction of cells which contained more than 15 nuclear foci. Experiments were carried out in triplicate, and one set of results is shown. Error bars represent SEM. Statistical analysis was based on Student's t test for γH2AX, 53BP1 and χ 2 for Rad51.

Article Snippet: Anti-53BP1 mouse monoclonal antibody was a generous gift from T. Halazonetis, anti-53BP1 polyclonal antibody was obtained from Novus Biologicals, LLC (Littleton CO, USA), anti-pS139-H2AX (γH2AX) polyclonal antibody and polyclonal antibodies against CSN subunits 1, 3 and 5 were obtained from Bethyl Laboratories, Inc. (Montgomery, TX, USA), anti-RNF168, anti-H2A, anti-pS139-H2AX monoclonal antibody and anti-BRCA1 antibodies were obtained from Merck Millipore (Darmstadt, Germany), anti-H4 was obtained from Abcam (Cambridge, MA, USA), anti-HSC70 monoclonal antibody was obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA), and anti-Cullin 1 antibody was obtained from Invitrogen Corporation (Camarillo, CA, USA).

Techniques: Phospho-proteomics, Expressing, Irradiation, Western Blot, Activity Assay, Staining