53BP1 and USP28 mediate p53 activation and G1 arrest after centrosome loss or extended mitotic duration - PubMed (original) (raw)
53BP1 and USP28 mediate p53 activation and G1 arrest after centrosome loss or extended mitotic duration
Franz Meitinger et al. J Cell Biol. 2016.
Abstract
In normal human cells, centrosome loss induced by centrinone-a specific centrosome duplication inhibitor-leads to irreversible, p53-dependent G1 arrest by an unknown mechanism. A genome-wide CRISPR/Cas9 screen for centrinone resistance identified genes encoding the p53-binding protein 53BP1, the deubiquitinase USP28, and the ubiquitin ligase TRIM37. Deletion of TP53BP1, USP28, or TRIM37 prevented p53 elevation in response to centrosome loss but did not affect cytokinesis failure-induced arrest or p53 elevation after doxorubicin-induced DNA damage. Deletion of TP53BP1 and USP28, but not TRIM37, prevented growth arrest in response to prolonged mitotic duration. TRIM37 knockout cells formed ectopic centrosomal-component foci that suppressed mitotic defects associated with centrosome loss. TP53BP1 and USP28 knockouts exhibited compromised proliferation after centrosome removal, suggesting that centrosome-independent proliferation is not conferred solely by the inability to sense centrosome loss. Thus, analysis of centrinone resistance identified a 53BP1-USP28 module as critical for communicating mitotic challenges to the p53 circuit and TRIM37 as an enforcer of the singularity of centrosome assembly.
© 2016 Meitinger et al.
Figures
Figure 1.
Genome-wide CRISPR/Cas9 screen for genes involved in activating p53 upon centrosome loss. (A, top) Immunofluorescence images of RPE1 cells, stained for DNA (red) and the centrosomal protein Cep192 (green), after treatment with DMSO or centrinone for 5 d. Bar, 10 µm. (bottom) Schematic highlighting the two classes of genes that would be identified in a centrinone-resistance screen. (B) Summary of the screen designed to identify genes that activate p53 in response to centrosome loss. (C) Table summarizing the results of two independent screens. All 15 colonies had one of the three listed genes deleted; no colony had more than one. (D) Schematic of the three proteins 53BP1, USP28, and TRIM37, identified by the screen.
Figure 2.
Loss of TP53BP1, USP28, or TRIM37 suppresses p53 elevation and proliferation arrest triggered by centrosome loss. (A, top) Outline of the procedure used to generate RPE1 knockouts. (A, bottom) Immunoblots of extracts from control (Ctrl) and knockout RPE1 lines. Bands corresponding to each protein (arrowheads) and nonspecific bands (asterisks) are indicated. α-Tubulin serves as a loading control. (B) Outline of cell proliferation analysis and assessment of p53 and p21 levels after acute treatment with centrinone or Mdm2i. (C) Graphs plotting the results of passaging assays monitoring the growth of control and knockout RPE1 cell lines after addition at day 0 of DMSO (vehicle), centrinone, or Mdm2i. (D) Immunoblots probed with the indicated antibodies after addition of Mdm2i (left) or centrinone (right). α-Tubulin (α-tub) serves as a loading control. (E) Immunofluorescence analysis of Cep192 and p53 after 5-d centrinone treatment. Representative images (left) and graph (right) plotting the distributions of nuclear p53 fluorescence for one of three experiments (for quantification of the other two experiments, see
Fig. S2 E
). Graph shows 5–95% box-and-whiskers plots. Bar, 10 µm.
Figure 3.
53BP1 and USP28, but not TRIM37, are essential for activating p53 in response to prolonged mitotic duration. (A) Analysis of p53 and p21 levels after induction of DNA damage with doxorubicin; schematic describes experimental protocol and GAPDH serves as a loading control. (B) Analysis of cytokinesis failure–induced division arrest; schematic describes experimental protocol. Red dots show results from two independent experiments. Immunoblot confirms efficient p53 depletion. (C) Analysis of extended mitotic duration–induced division arrest; schematic describes experimental protocol. Vertical bars represent individual daughter cells. Bar height shows the time the mother cells spent in mitosis, and bar color indicates whether they arrested (red) or divided (gray). Black dashed line marks the mitotic duration cutoff in control RPE1 cells, after which resulting daughter cells arrest in G1. (D) Schematic shows two possible models for how centrosome loss might trigger p53 activation, either directly (left) or indirectly through successive prolonged mitoses (right).
Figure 4.
TRIM37Δ cells form foci containing centrosomal markers. (A) Immunofluorescence images after 5-d treatment with DMSO (top row) or centrinone (bottom row). Cells were stained for DNA (red) and with antibodies to the centrosomal protein Cep192 (green; insets 2.5× magnified). (B) Immunofluorescence images of interphase TRIM37Δ cells stained for DNA (red) and with antibodies to the indicated centrosomal proteins (green) after 5-d treatment with DMSO (top) or centrinone (bottom). Images are representative, and each marker was equivalently scaled for the two conditions. Yellow arrow points to bright ectopic Plk4 focus; blue arrow points to centrioles. (C) Immunofluorescence images of a microtubule regrowth experiment. Control RPE1 (left) and TRIM37Δ (middle) cells were pretreated for 5 d with DMSO (top) or centrinone (bottom) followed by 2-h treatment with nocodazole to depolymerize microtubules. Microtubules were allowed to grow for 4 min after nocodazole washout before fixation. Graph shows quantification of microtubule regrowth foci. (D) Immunofluorescence images of mitotic TRIM37Δ cells stained for DNA (red) and with antibodies to the indicated centrosomal proteins (green) after 5-d treatment with DMSO (top) or centrinone (bottom). Each marker was equivalently scaled for the two conditions. Yellow arrow points to bright ectopic Plk4 focus; blue arrows point to centrioles. Bars, 10 µm.
Figure 5.
Knockout of TRIM37, but not TP53BP1 or USP28, suppresses mitotic defects in centrosomeless cells. (A) Selected images from timelapse series of representative DMSO-treated control RPE1 cells and centrinone-treated TP53BP1Δ, USP28Δ, and TRIM37Δ mutant cells, acquired as outlined in the schematic. (B) Graph plotting the distribution of mitotic phenotypes observed for each condition along with representative images. (C) Graph plotting mitotic duration. Individual cell values (red triangles) are shown along with the mean and SD (black bars) for each condition. NEBD, nuclear envelope breakdown. (D) Graphs plotting the results of passaging assays monitoring the growth of control and knockout RPE1 cell lines after acute addition at day 0 of DMSO (vehicle) or centrinone. (E) Representative phase-contrast images of fields of DMSO-treated control RPE1 and knockout mutant cells after prolonged (>20 d) treatment with centrinone. Bars: (A and B) 10 µm; (E) 100 µm.
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