(A) HeLa cells were incubated with 0.5 μM aphidicolin or carrier alone for 24 h and then processed for ChIP using either irrelevant IgG or BMI1-specific antibodies. PCR was performed with two different sets of primers specific for the FRA-3B locus. (B) HeLa cells were mock treated or treated with 0.5 μM aphidicolin for 24 h and then subjected to ChIP using antibodies to BMI1 (blue bars) or pH2AX (red bars). Quantitative real-time PCR was performed with sets of primers that covered ∼18 kb of the FRA-3B locus, as shown. The relative fold increase in signal between untreated cells and aphidicolin-treated cells is shown for each primer pair for the ChIP using BMI1 antibody (blue bars) or pH2AX antibody (red bars) shown in the upper panel. The lower panel shows the relative occupancy of BMI1 and pH2AX at the positive control locus (HOXA3) and at a nonfragile site (MBS85) in both untreated and aphidicolin-treated cells. The data shown are means of at least three experiments ± the SEM. (C) A scheme of the ZFN strategy to create specific DNA DSBs is shown in the upper panel. HeLa cells were transfected with mRNA encoding a ZFN targeting the AAVS1 site or mock transfected and processed for ChIP using antibodies to pH2AX, BMI1, CBX2, or UbH2A or an irrelevant IgG and primers to either the flanking region of AAVS1, to MBS85 site on chromosome 19, or to the HOXA3 locus. The ChIP signal was quantitated by RT-PCR, and the lower panel shows the relative fold enrichment for each condition. Each point is the mean of at least three experiments ± the SEM.

(A) Bmi1^+/+; Ink4a^−/−; Arf^−/− MEFs, Bmi1^−/−; Ink4a^−/− Arf^−/− MEFs, H2AX^−/− MEFs, or H2AX^−/− MEFs expressing S139A mutant H2AX were treated with laser scissors and, after 30 min, processed for IF using antibody to BMI1 and either 53BP1 or pH2AX as indicated. A bar graph shows the percentages of cells with laser scissors-induced damage showing colocalization of BMI1 with 53BP1 in each MEF strain. The data plotted are the mean ± the SEM of three separate experiments. (B) In the top two panels, HeLa cells were treated with either 0 or 0.5 μM ATMi and subjected to laser scissors and, after 30 min, processed for IF using antibodies to BMI1 or 53BP1. In the middle two panels, Seckel cells or Rnf8^−/− MEFs were treated with laser scissors and processed for IF using the antibodies indicated. In the bottom two panels, HeLa cells were treated with either control siRNA or ATR-specific siRNA and then treated with lasers scissors and processed for IF using antibodies to BMI1 and 53BP1. Western blots showing the effect of siATR on ATR protein levels are shown below the panels. Bar graph shows quantitation of percentage of cells that had clear colocalization of BMI1 with 53BP1 or pH2AX. The data are plotted as means ± the SEM of at least three separate experiments.

(A) Loss of BMI1 leads to accumulation of cells in G₂/M associated with DNA damage-associated checkpoint activation. In the top panels, wild-type MEFs or Bmi1^−/−; Ink4a^−/− Arf^−/− MEFs were harvested, stained with propidium iodide, and analyzed by FACS for DNA content. Plots of the FACS analysis are shown in the left panels, and quantitation of the percentages in G₂/M is shown in the bar graph. In the bottom panels, HeLa cells were treated either with control siRNA or BMI1-specific siRNA and then processed for DNA content-based cell cycle analysis as described. The percentages in G₂/M are plotted. The data shown are means ± the SEM of three separate experiments. In the inset, a Western blots shows the expression of BMI1 and tubulin in cells treated with control or specific siRNA. (B) HeLa cells were treated either with control siRNA or BMI1-specific siRNA. Protein extracts were then subjected to Western blotting with antibodies for BMI1, phoshpo-CHK2-S19 (pCHK2), total CHK2 (CHK2), pH2AX, or tubulin. (C) Control siRNA- or BMI1 siRNA-treated HeLa cells were exposed to various doses of IR, and colony formation was assayed, plotted, and normalized to that of untreated cells. (D) U20S cells contain an integrated tandem GFP reporter of HR was treated with control siRNA, BMI1-specific siRNA, or BMI1-specific siRNA plus wt-BMI1. The cells were then transfected with an I-SceI-expressing vector. Cells transfected with an empty vector are also shown as a negative control. After 24 h, the percentages of cells expressing GFP were measured by flow cytometry. Each data point represents the mean ± the SEM of three separate experiments. Western blots showing the expression of BMI1 in control cells, BMI1 siRNA-treated cells, and BMI1 siRNA-treated cells reconstituted with wt-BMI1 are shown to the right.

BMI1 and CBX2 are recruited to sites of DNA damage. (A) HeLa cells were subjected to laser scissors-induced DNA damage and, after 30 min, localization of pH2AX and either BMI1 (top panel) or CBX2 (bottom panel) was assayed by IF. The percentage of cells with colocalization of pH2AX with BMI1 or CBX2 is indicated (right panel). (B) The top panels show HeLa cells treated with either 0 Gy of IR (−IR) or 10 Gy of IR (+IR). After 8 h, the localization of BMI1 and pH2AX was assayed by IF. In the bottom panels, HeLa cells were treated with either no or 3 mM HU for 2 h, and localization of BMI1 and 53BP1 was assayed by IF. In the right panel, the mean numbers of BMI1 foci colocalizing with pH2AX foci per cell are plotted for each condition. The data shown are means ± the standard error of the mean (SEM) from at least three separate experiments.

(A) Diagrams of wt-Bmi1 and mutant Bmi1 constructs are shown. (B) Empty vector or vectors encoding wt-Bmi1, Δ-RF Bmi1, or Δ-HT Bmi1 were introduced into Bmi1^−/−; Ink4a^−/− Arf^−/− MEFs. Western blots with an antibody that can recognize both wild-type and mutant constructs are shown. (C) The cells were then treated with laser scissors and processed for IF analysis using antibodies to Bmi1 and pH2AX. A bar graph shows the percentages of cells with a laser scissors-induced region of pH2AX that had colocalizing regions of Bmi1 staining in each condition. Only cells with both vector expression and evidence of laser scissors damage were assessed. The data are plotted as means ± the SEM of three separate experiments.

(A) Bmi1^+/+; Ink4a^−/−; Arf^−/− MEFs, Bmi1^−/−; Ink4a^−/− Arf^−/− MEFs, Bmi1^−/−; Ink4a^−/− Arf^−/− MEFs reconstituted with wt-BMI1, or H2AX^−/− MEFs were treated with laser scissors and, after 30 min, processed for IF using antibody to Ub-H2A-K119 and either 53BP1 or pH2AX as indicated. (B) Bmi1^+/+; Ink4a^−/−; Arf^−/− MEFs or Bmi1^−/−; Ink4a^−/− Arf^−/− MEFs were treated with 10 Gy of IR or mock treated and, after 30 min, chromatin extracts were prepared and processed for Western blotting with antibody to Ub-H2AK119 or antibody to histone H2A. (C) Bmi1^−/−; Ink4A^−/− Arf^−/− MEFs were treated with laser scissors and, after 30 min, processed for IF with antibodies to polyubiquitin (FK2) or RAP80 and 53BP1 as indicated. (D) Bar graphs show the percentages of the indicated cell types with laser scissors-induced regions of pH2AX or 53BP1 that also show colocalization with Ub-H2A-K119, FK1, or RAP80. (E) Bmi1^+/+; Ink4a^−/− Arf^−/− MEFs, Bmi1^−/−; Ink4a^−/− Arf^−/− MEFs, or Rnf8^−/− MEFs were treated with laser scissors and, after 30 min, processed for IF using antibodies to RING1B and either pH2AX or 53BP1 as indicated. The percentages of cells with laser scissors-induced damage showing colocalization of RING1B with pH2AX or 53BP1 for each MEF strain is shown in the bar graph. All data are plotted as means ± the SEM of at least three independent experiments.

Model showing the role for Bmi1 in the DNA damage response pathway. See the text for details.