Journal: PLoS Genetics

Article Title: Tankyrases Promote Homologous Recombination and Check Point Activation in Response to DSBs

doi: 10.1371/journal.pgen.1005791

Figure Lengend Snippet: (A) TNKS tethering to the lacO array leads to MERIT40 and RAP80 binding in a PARP activity independent manner even in the absence of DSBs. U2OS17 cells were transfected with GFP-lacR, GFP-lacR-TNKS1 or the PARP activity mutant version: GFP-lacR-TNKS1mut. ISce-I was co-transfected to induce pure DSBs at the lacO array. Immunofluorescence staining was performed to visualize the localization pattern of MERIT40 or RAP80 in the cells. The values represent the results of three independent experiments with SD (N = 100). On the right representative confocal microscopy pictures are shown. (B) The recruitment of MERIT40 and RAP80 is affected in TNKS1/2 knock down cells. U2OS17 cells were transfected with the indicated siRNAs. After depletion, cells were transfected with mCherry-lacR and ISce-I where indicated. Immunofluorescence staining was performed to visualize Merit40 or RAP80 in the cells. Relative values compared to the control are the results of three independent experiments with SD (N = 100 cells in each condition). (C) TNKS depleted cells are deficient for RAP80 foci formation. U2OS cells were transfected with siRNAs as shown and treated with NCS 48 hours later. The frequency of foci-positive cells was determined in three independent experiments (N = 100) and is shown as relative to the control (D) MDC1-mediated TNKS recruitment is necessary for efficient binding of the BRCA1 complex to DSBs in vivo . U2OS17 cells were transfected with mCherry-lacR-MDC1 wt or its TBD mutant version and ISce-I. Percent of cells harboring MERIT40 or RAP80 signal on the array was determined after immunofuorescence staining. Results from three independent experiments are shown with SD (N = 100) as relative frequencies compared to control. (E) MERIT40 stabilizes BRCA1 on the TNKS-bound chromatin. Control or MERIT40 depleted U2OS17 cells were transfected with GFP-lacR, GFP-lacR-TNKS1, GFP-lacR-TNKS1mut and ISce-I. Percent of cells harboring BRCA1 signal on the array was determined. Results from three independent experiments are shown with SD (N = 100).

Article Snippet: The following antibodies were used during our study: Anti-lacR (gift from Dr A. Belmont), anti-flag (F7425, Sigma), anti-flag M2 (F3165, Sigma), anti-Myc (clone 9E10), anti- γ-H2AX (ab22551, abcam), anti-H2AX (ab11175, abcam) anti-53BP1 (NB100-304, Novus), anti-RAD51 (PC130, Calbiochem), anti-BRCA1 (OP92 and OP93, Calbiochem), anti-MERIT40 (gift from Dr J. Chen), anti-histone H3 S10P (51TA2H12, Active Motif), anti-MDC1 (MDC1-50, Sigma), anti-TNKS (ab13587, abcam), anti-HA (11867423001, Roche), anti-ATM (Novus NB100-104), anti-DNA-PKcs (Millipore 04–1024), anti-tubulin (Sigma-Aldrich T5168), anti-Merit40 (A302-516A Bethyl), anti RAP80 (A300-763A Bethyl), anti-CtIP (Clone 14–1 Active motif), anti-phospho-RPA32 S4/8 (A300-245A, Bethyl).

Techniques: Binding Assay, Activity Assay, Transfection, Mutagenesis, Immunofluorescence, Staining, Confocal Microscopy, Knockdown, Control, In Vivo

Journal: PLoS Genetics

Article Title: Tankyrases Promote Homologous Recombination and Check Point Activation in Response to DSBs

doi: 10.1371/journal.pgen.1005791

Figure Lengend Snippet: (A) RNF8 mediated ubiquitination is dispensable for BRCA1A complex’s loading onto the chromatin. U2OS17 cells were transfected with siSCR control or siRNF8, and GFP-lacR, GFP-lacR-TNKS1 or its PARP activity mutant version. The frequency of MERIT40, RAP80 and BRCA1 signal on the lacO array was analyzed after immunofluorescence staining with the corresponding antibodies. Results of three independent experiments are shown with SD (N = 100). (B) RAP80 is recruited to TNKS1 bound chromatin in an ubiquitin-binding independent manner. U2OS17 cells were transfected with GFP-lacR or GFP-lacR-TNKS1 together with mCherry-RAP80 (wt or UIM mutant). Pure DSBs were inflicted with the cotransfection of ISce-I. Colocalization frequency of the GFP and mCherry signal is shown from three independent experiments (N = 100) with SD. (C) TNKS depleted cells show RAP80 foci-forming deficiency at later timepoints after DSB induction. U2OS cells were transfected with the indicated siRNAs, treated with NCS and fixed at the indicated timepoints. Results from three independent experiments are shown with SD (N = 100). (D) Knockdown of TNKS1/2 leads to G2/M checkpoint escape and elevated number of mitotic cells after DSB induction compared to the control. U2OS cells were transfected with the indicated siRNAs. 48 hours later cells were treated with Phleomycin and released for 6 hours. Mitotic cells were detected by an anti- histone H3 S10P fluorescence staining and their frequency determined. Values are represented on a Whisker box plot as relative to the control from three independent experiments. (E) Schematic action of TNKSs in DSB repair is shown.

Article Snippet: The following antibodies were used during our study: Anti-lacR (gift from Dr A. Belmont), anti-flag (F7425, Sigma), anti-flag M2 (F3165, Sigma), anti-Myc (clone 9E10), anti- γ-H2AX (ab22551, abcam), anti-H2AX (ab11175, abcam) anti-53BP1 (NB100-304, Novus), anti-RAD51 (PC130, Calbiochem), anti-BRCA1 (OP92 and OP93, Calbiochem), anti-MERIT40 (gift from Dr J. Chen), anti-histone H3 S10P (51TA2H12, Active Motif), anti-MDC1 (MDC1-50, Sigma), anti-TNKS (ab13587, abcam), anti-HA (11867423001, Roche), anti-ATM (Novus NB100-104), anti-DNA-PKcs (Millipore 04–1024), anti-tubulin (Sigma-Aldrich T5168), anti-Merit40 (A302-516A Bethyl), anti RAP80 (A300-763A Bethyl), anti-CtIP (Clone 14–1 Active motif), anti-phospho-RPA32 S4/8 (A300-245A, Bethyl).

Techniques: Ubiquitin Proteomics, Transfection, Control, Activity Assay, Mutagenesis, Immunofluorescence, Staining, Binding Assay, Cotransfection, Knockdown, Fluorescence, Whisker Assay

Journal: Cell

Article Title: TRIP12 and UBR5 suppress spreading of chromatin ubiquitylation at damaged chromosomes.

doi: 10.1016/j.cell.2012.06.039

Figure Lengend Snippet: Figure 6. Deregulation of TRIP12 and UBR5 Alters the Dynamics of DNA Repair (A) U-2-OS cells were transfected with the indi- cated siRNA for 72 hr and irradiated. WCE were analyzed by immunoblotting. (B) U-2-OS cells were irradiated (2 Gy), and 1 hr later, they were immunostained with an antibody to RPA (the FITC channel); nuclear DNA was coun- terstained by DAPI. Cell-cycle distribution was determined for each individual cell by quantifying the total DAPI and chromatin-bound RPA intensity per nucleus. (C) U-2-OS cells were transfected with the indi- cated siRNAs, irradiated, immunostained with antibodies to RPA and RAD51, sorted according to cell cycle as in (B), and subjected to an automated analysis of RAD51 focus formation. At least 500 cells were analyzed for each condition; represen- tative images are shown. (D) U-2-OS cells were transfected with siRNAs for 72 hr and irradiated. Where indicated, cells were treated for 2 hr with ATM or DNA-PK inhibitors prior to irradiation. WCE were analyzed by immuno- blotting (left). In parallel, cells were treated with the indicated siRNAs and inhibitors as indicated, irra- diated (2 Gy), and after 4 hr, were subjected to an automated single cell analysis for the number of MDC1 nuclear foci (right). (E) U-2-OS cells were treated with the indicated siRNAs and inhibitors, irradiated, and analyzed as in (D). Scale bar, 10 mm. See also Figure S7.

Article Snippet: Rabbit polyclonal antibodies included the following: RNF168 (Stewart et al., 2009; provided by Daniel Durocher), RNF8 (rabbit antiserum, provided by Xiaochun Yu), 53BP1 (sc-22760, Santa Cruz), MDC1 (ab11171, Abcam), TRIP12 (A301-814A, Bethyl), UBR5/EDD (A300-573A, Bethyl), PARP1 (sc-7150, Santa Cruz), RPA32 phospho Ser33 (A300-246A, Bethyl), RPA32-phospho S4/S8 (A300245A, Bethyl), CHK1 (sc-8404, Santa Cruz), CHK1 phospho S345 (#2341, Cell Signaling), pCHK1-S317 (#2344, Cell Signaling), KAP1 (A300-274A, Bethyl), KAP1 phospho S824 (ab70369, Abcam), Herc2 (Bekker-Jensen et al., 2010), Smc1 (ab9262, Abcam), Rap80 (A300-763A, Bethyl), RAD51 (sc-8394, Santa Cruz), H2AX (NB100-638 Novus Biologicals).

Techniques: Transfection, Irradiation, Western Blot, Single-cell Analysis

Journal: Cell

Article Title: TRIP12 and UBR5 suppress spreading of chromatin ubiquitylation at damaged chromosomes.

doi: 10.1016/j.cell.2012.06.039

Figure Lengend Snippet: Figure 1. Identification of TRIP12 and UBR5 as Regulators of Chromatin Ubiquitylation at Spontaneous DNA Lesions (A) A schematic depiction of the screening procedure combining siRNA arrays spanning the human ubiquitinome and a readout based on monitoring the increase of the GFP-RNF168- associated fluorescence at the G1 nuclear bodies. (B) A scatterplot of Z-scores derived from the screen shown in (A). Insets show representative images from manual re-examination of nuclear body expansion in U-2-OS/GFP-RNF168 cells treated with the indicated siRNAs; henceforth, siRNA is abbreviated as si, Control as Con, and TRIP12 and UBR5 as T12 and U5, respectively. (C) List of genes whose knockdown by two inde- pendent siRNA increased the expansion of GFP- 53BP1-decorated nuclear bodies. Scale bars, 10 mm. See also Table S1.

Article Snippet: Rabbit polyclonal antibodies included the following: RNF168 (Stewart et al., 2009; provided by Daniel Durocher), RNF8 (rabbit antiserum, provided by Xiaochun Yu), 53BP1 (sc-22760, Santa Cruz), MDC1 (ab11171, Abcam), TRIP12 (A301-814A, Bethyl), UBR5/EDD (A300-573A, Bethyl), PARP1 (sc-7150, Santa Cruz), RPA32 phospho Ser33 (A300-246A, Bethyl), RPA32-phospho S4/S8 (A300245A, Bethyl), CHK1 (sc-8404, Santa Cruz), CHK1 phospho S345 (#2341, Cell Signaling), pCHK1-S317 (#2344, Cell Signaling), KAP1 (A300-274A, Bethyl), KAP1 phospho S824 (ab70369, Abcam), Herc2 (Bekker-Jensen et al., 2010), Smc1 (ab9262, Abcam), Rap80 (A300-763A, Bethyl), RAD51 (sc-8394, Santa Cruz), H2AX (NB100-638 Novus Biologicals).

Techniques: Derivative Assay, Control, Knockdown

Journal: Cell

Article Title: TRIP12 and UBR5 suppress spreading of chromatin ubiquitylation at damaged chromosomes.

doi: 10.1016/j.cell.2012.06.039

Figure Lengend Snippet: Figure 2. Expansion of the Ubiquitylated Nuclear Domains in TRIP12- and UBR5- Depleted Cells Is Accompanied by Reduced Transcription (A) U-2-OS cells were transfected with the indi- cated siRNAs for 72 hr and immunostained with the FK2 antibody against conjugated ubiquitin. Numbers indicate the fraction of nuclei with enlarged ubiquitin-decorated nuclear bodies (larger than 80 pixels). For each condition, a randomly selected cohort of at least 500 cells was analyzed. One pixel, 0.2 3 0.2 mm. (B) U-2-OS cells were transfected with the indi- cated siRNAs for 72 hr and immunostained with antibodies to 53BP1 and ubiquitylated H2A. (C) U-2-OS cells were transfected with siRNAs as in (B) and immunostained with antibodies to 53BP1 and RNF168. (D) U-2-OS cells were transfected with the indi- cated siRNAs for 72 hr, incubated in the presence of 5-ethinyl uridin (5-EU) for the last 1 hr, and immunostained with antibodies to 53BP1. The 5-EU incorporation to nascent mRNA was devel- oped with Click-iT chemistry. The fluorescence intensity profiles were quantified along a line drawn through the center of the nuclear bodies. Insets show larger magnification of nuclear bodies; graphs integrate data from ten cells for each condition. Error bars represent SD. Scale bars, 10 mm. See also Figure S1.

Article Snippet: Rabbit polyclonal antibodies included the following: RNF168 (Stewart et al., 2009; provided by Daniel Durocher), RNF8 (rabbit antiserum, provided by Xiaochun Yu), 53BP1 (sc-22760, Santa Cruz), MDC1 (ab11171, Abcam), TRIP12 (A301-814A, Bethyl), UBR5/EDD (A300-573A, Bethyl), PARP1 (sc-7150, Santa Cruz), RPA32 phospho Ser33 (A300-246A, Bethyl), RPA32-phospho S4/S8 (A300245A, Bethyl), CHK1 (sc-8404, Santa Cruz), CHK1 phospho S345 (#2341, Cell Signaling), pCHK1-S317 (#2344, Cell Signaling), KAP1 (A300-274A, Bethyl), KAP1 phospho S824 (ab70369, Abcam), Herc2 (Bekker-Jensen et al., 2010), Smc1 (ab9262, Abcam), Rap80 (A300-763A, Bethyl), RAD51 (sc-8394, Santa Cruz), H2AX (NB100-638 Novus Biologicals).

Techniques: Transfection, Ubiquitin Proteomics, Incubation

Journal: Cell

Article Title: TRIP12 and UBR5 suppress spreading of chromatin ubiquitylation at damaged chromosomes.

doi: 10.1016/j.cell.2012.06.039

Figure Lengend Snippet: Figure 3. Catalytic Activities of TRIP12 and UBR5 Regulate Ubiquitin Spreading, but Not Chromatin Decondensation, after DNA Damage (A) U-2-OS-TetO cells harboring 240 TetO repeats next to the I-SceI restriction site were transfected with the indicated siRNAs, induced to cut the I-SceI site, and immunostained with antibodies to 53BP1; the locus with integrated TetO repeats was visualized by transient transfection of TetR-GFP. (B) U-2-OS cells were transfected with the indicated siRNAs for 72 hr and immunostained with the indicated antibodies. (C) Total fluorescence intensities of 53BP1 nuclear bodies were plotted against 53BP1 nuclear body areas. Coefficient of determination of a linear regression (R2) and the slope of the linear regression are provided for each condition. The fraction of 53BP1 nuclear bodies with an area greater than 100 pixels is indicated. For each condition, a randomly selected cohort of at least 300 cells was analyzed. One pixel, 0.2 3 0.2 mm. (D) U-2-OS cells were transfected with the indicated siRNAs for 72 hr, exposed to IR (0.25 Gy), and 45 min later, were immunostained with an antibody to 53BP1. A randomly selected cohort of 500 cells was analyzed; numbers indicate total fluorescence intensity per 53BP1 focus.

Article Snippet: Rabbit polyclonal antibodies included the following: RNF168 (Stewart et al., 2009; provided by Daniel Durocher), RNF8 (rabbit antiserum, provided by Xiaochun Yu), 53BP1 (sc-22760, Santa Cruz), MDC1 (ab11171, Abcam), TRIP12 (A301-814A, Bethyl), UBR5/EDD (A300-573A, Bethyl), PARP1 (sc-7150, Santa Cruz), RPA32 phospho Ser33 (A300-246A, Bethyl), RPA32-phospho S4/S8 (A300245A, Bethyl), CHK1 (sc-8404, Santa Cruz), CHK1 phospho S345 (#2341, Cell Signaling), pCHK1-S317 (#2344, Cell Signaling), KAP1 (A300-274A, Bethyl), KAP1 phospho S824 (ab70369, Abcam), Herc2 (Bekker-Jensen et al., 2010), Smc1 (ab9262, Abcam), Rap80 (A300-763A, Bethyl), RAD51 (sc-8394, Santa Cruz), H2AX (NB100-638 Novus Biologicals).

Techniques: Ubiquitin Proteomics, Transfection

Journal: Cell

Article Title: TRIP12 and UBR5 suppress spreading of chromatin ubiquitylation at damaged chromosomes.

doi: 10.1016/j.cell.2012.06.039

Figure Lengend Snippet: Figure 5. RNF168 Abundance Is Rate Limiting for 53BP1 Focus Formation (A) U-2-OS cells were transfected with siRNAs for 72 hr and exposed to increasing doses of IR as indicated, and after 1 hr, they were immunostained with an antibody to 53BP1. Wide-field images were acquired from randomly selected fields. The graph shows the total fluorescence intensity per 53BP1-decorated nuclear focus. Representative images for each condition are shown. Scale bar, 10 mm. (B) U-2-OS cells were transfected with siRNAs for 72 hr and irradiated as indicated, and 1 hr later, they were immunostained with an antibody to 53BP1. Wide-field images of 53BP1 were ac- quired from randomly selected fields. The graph shows the average sum of total fluorescence intensity associated with 53BP1 nuclear foci normalized to the total fluorescence intensity of 53BP1 per nucleus. (C) U-2-OS were transfected with control siRNA and irradiated as indicated, and 1 hr later, they were immunostained with antibodies to 53BP1 and g-H2AX. Wide-field images were acquired from randomly selected fields and analyzed for the number of nuclear foci per nucleus. Representa- tive images are shown. (D) U-2-OS cells were transfected with the TRIP12 and UBR5 siRNAs and analyzed as in (C). (E) U-2-OS cells expressing GFP-RNF168 or naive U-2-OS cells were irradiated and analyzed for 53BP1-associated fluorescence as in (A). (F) U-2-OS cells stably expressing GFP-RNF168, U-2-OS cell line induced to express RNF168 shRNA for 72 hr (used here as a negative control for suppression of 53BP1 focus formation), or naive U-2-OS cells were irradiated and analyzed for 53BP1 foci number. For each condition in (A)–(F), a randomly selected cohort of at least 500 cells was analyzed. Scale bars, 10 mm. See also Figure S6.

Article Snippet: Rabbit polyclonal antibodies included the following: RNF168 (Stewart et al., 2009; provided by Daniel Durocher), RNF8 (rabbit antiserum, provided by Xiaochun Yu), 53BP1 (sc-22760, Santa Cruz), MDC1 (ab11171, Abcam), TRIP12 (A301-814A, Bethyl), UBR5/EDD (A300-573A, Bethyl), PARP1 (sc-7150, Santa Cruz), RPA32 phospho Ser33 (A300-246A, Bethyl), RPA32-phospho S4/S8 (A300245A, Bethyl), CHK1 (sc-8404, Santa Cruz), CHK1 phospho S345 (#2341, Cell Signaling), pCHK1-S317 (#2344, Cell Signaling), KAP1 (A300-274A, Bethyl), KAP1 phospho S824 (ab70369, Abcam), Herc2 (Bekker-Jensen et al., 2010), Smc1 (ab9262, Abcam), Rap80 (A300-763A, Bethyl), RAD51 (sc-8394, Santa Cruz), H2AX (NB100-638 Novus Biologicals).

Techniques: Transfection, Irradiation, Control, Expressing, Stable Transfection, shRNA, Negative Control

Journal: Cell

Article Title: TRIP12 and UBR5 suppress spreading of chromatin ubiquitylation at damaged chromosomes.

doi: 10.1016/j.cell.2012.06.039

Figure Lengend Snippet: Figure 7. Mechanisms Determining RNF168 Turnover Influence Survival after DNA Damage and Can Be Deregulated in Human Cancer (A) H1299 dA3-1 cells were transfected with indi- cated siRNAs for 24 hr, followed by transfection with a plasmid expressing I-SceI for 72 hr, and then they were harvested. Where indicated, DNA- PKcs inhibitor NU7026 was added to cells at the time of I-SceI transfection. The proportion of GFP- positive cells (as a measure of NHEJ activity) was determined by flow cytometry. Data represent the mean from three experiments. (B) U-2-OS/DR-GFP cells were transfected with siRNAs as indicated and then cotransfected with expression plasmids for I-SceI and RFP for 48 hr. Flow cytometry analysis of GFP/RFP ratio was used to measure HR efficiency. Data represent the mean of five experiments. (C) MCA survival assay. U-2-OS cells expressing GFP were cotransfected with TRIP12 and UBR5 siRNAs for 48 hr and mixed in a 1:1 ratio with a U-2- OS cell line expressing mCherry transfected with control siRNA. Where indicated, U-2-OS/YFP- RNF168 induced to express the transgene by addition of Doxycycline (Dox) were mixed with the U-2-OS/mCherry cells and processed as above. After an additional 24 hr, cells were irradiated (5 Gy) and were analyzed 72 hr later by flow cytometry. The survival was determined as the GFP/mCherry ratio. The data represent a summary from three experiments. Error bars in (A)–(C) represent SD. (D) Paraffin sections from the indicated tumor types were immunostained with antibodies to 53BP1 or RNF168. Insets show larger magnifica- tions of the marked fields; arrows point to nuclear bodies. The HPV status of the depicted tumors is indicated. (E) Model of TRIP12/UBR5-mediated suppression of chromatin ubiquitylation after DNA breakage. Left, TRIP12 and UBR5 determine the nuclear pool of RNF168. This pool is under full control by the upstream regulators and is available to ubiq- uitylate a limited fraction of the DSB-flanking chromatin. Right, in the absence of TRIP12 and UBR5, a fraction of hyperaccumulated RNF168 escapes upstream regulators and triggers spreading of H2A and H2AX ubiquitylation far away from the initial DNA lesions. Scale bar, 10 mm.

Article Snippet: Rabbit polyclonal antibodies included the following: RNF168 (Stewart et al., 2009; provided by Daniel Durocher), RNF8 (rabbit antiserum, provided by Xiaochun Yu), 53BP1 (sc-22760, Santa Cruz), MDC1 (ab11171, Abcam), TRIP12 (A301-814A, Bethyl), UBR5/EDD (A300-573A, Bethyl), PARP1 (sc-7150, Santa Cruz), RPA32 phospho Ser33 (A300-246A, Bethyl), RPA32-phospho S4/S8 (A300245A, Bethyl), CHK1 (sc-8404, Santa Cruz), CHK1 phospho S345 (#2341, Cell Signaling), pCHK1-S317 (#2344, Cell Signaling), KAP1 (A300-274A, Bethyl), KAP1 phospho S824 (ab70369, Abcam), Herc2 (Bekker-Jensen et al., 2010), Smc1 (ab9262, Abcam), Rap80 (A300-763A, Bethyl), RAD51 (sc-8394, Santa Cruz), H2AX (NB100-638 Novus Biologicals).

Techniques: Transfection, Plasmid Preparation, Expressing, Activity Assay, Cytometry, Flow Cytometry, Clonogenic Cell Survival Assay, Control, Irradiation