PALB2 connects BRCA1 and BRCA2 in the G2/M checkpoint response

Abstract

The G2/M checkpoint inhibits mitotic entry upon DNA damage, thereby preventing segregation of broken chromosomes and preserving genome stability. The tumor suppressor proteins BRCA1, PALB2 and BRCA2 constitute a BRCA1-PALB2-BRCA2 axis that is essential for homologous recombination (HR)-based DNA doublestrand break repair. Besides HR, BRCA1 has been implicated in both the initial activation and the maintenance of the G2/M checkpoint, while BRCA2 and PALB2 have been shown to be critical for its maintenance. Here we show that all three proteins can play a significant role in both checkpoint activation and checkpoint maintenance, depending on cell type and context, and that PALB2 links BRCA1 and BRCA2 in the checkpoint response. The BRCA1-PALB2 interaction can be important for checkpoint activation, whereas the PALB2-BRCA2 complex formation appears to be more critical for checkpoint maintenance. Interestingly, the function of PALB2 in checkpoint response appears to be independent of CHK1 and CHK2 phosphorylation. Following ionizing radiation, cells with disengaged BRCA1-PALB2 interaction show greatly increased chromosomal abnormalities due apparently to combined defects in HR and checkpoint control. These findings provide new insights into DNA damage checkpoint control and further underscore the critical importance of the proper cooperation of the BRCA and PALB2 proteins in genome maintenance.

Figure 1.

Effects of BRCA1, BRCA2 or PALB2 deficiencies on the G2/M checkpoint response in human and mouse tumor cells. (A) G2/M checkpoint responses in U2OS cells depleted of BRCA1, PALB2 and BRCA2. Cells were treated with control, BRCA1, PALB2 and BRCA2 siRNAs for 48 hr and then irradiated with 3 or 10 Gy of IR; cells were collected at indicated time points and their mitotic indexes were measured. Upper panel, schematic diagram of the timeline of the experiment; lower panel, relative mitotic indexes of the above cells at different time points after IR. (B) Representative western blots showing the depletion of BRCA1, PALB2 and BRCA2 proteins in A. (C,D) Relative mitotic indexes of the different mouse tumor cell lines before and after IR (3 Gy). The numbers of mitotic cells were normalized against that of unirradiated cells for each siRNA treatment or cell line. Values shown are the means of relative mitotic indexes from 2 (A and C) or 3 (D) independent experiments; error bars represent standard errors of the mean (SEMs).

Figure 2.

Role of p53 and MLH1 in BRCA- and PALB2-mediated G2/M checkpoint activation. (A) Isogenic p53 wt and null HCT116 cells were depleted of BRCA1, BRCA2 or PALB2, and checkpoint activation was analyzed 1 hr after 3 Gy of IR. Upper panels: representative immunoblots showing levels of the indicated proteins. Lower panels: relative mitotic indexes of each cell type and condition. (B) Isogenic HCT116 and HCT116:3–6 cells were treated and analyzed as in A. Data shown are means ± SEMs of the relative mitotic indexes from n>3 independent experiments. Statistical significance was calculated with one-way ANOVA. *, P<0.05; **, P<0.01.

Figure 3.

G2/M checkpoint defect of PALB2-deficient human fibroblasts and its rescue by re-expression of wt PALB2. (A) G2/M checkpoint activation in U2OS, FEN5280, EUFA1341, and EUFA1341 cells reconstituted with wt PALB2. Upper panel, representative western blots showing expression levels of PALB2, BRCA1 and BRCA2 in the cells; lower panel, relative mitotic indexes of the cells before and 1 hr after 3 Gy of IR. Data shown are means ± SEMs of the relative mitotic indexes from n>3 independent experiments. (B) Dose-dependent CHK1 and CHK2 phosphorylation in blank, empty vector-harboring and PALB2-reconstituted EUFA1341 cell lines. Cells were collected before or 1 hr after 3 or 10 Gy of IR; total and phosphorylated proteins were analyzed by western blotting. (C) Checkpoint maintenance in blank and reconstituted EUFA1341 cells. Cells were treated with 3 Gy of IR and mitotic cells were measured before and at 1, 2, 3 and 6 hr after IR. Data shown are means ± SEMs of the relative mitotic indexes from 3 independent experiments. (D) Kinetics of CHK1 and CHK2 phosphorylation in blank and reconstituted EUFA1341 cells. Cells were treated with 3 Gy of IR and collected at indicated time points; total and phosphorylated proteins were analyzed by western blotting. The lanes between dotted vertical lines were loaded in the wrong order, which were reversed back using Adobe Photoshop.

Figure 4.

Roles of BRCA1, BRCA2 and KEAP1 binding to PALB2 in G2/M checkpoint response. (A,B) PALB2, BRCA1 and BRCA2 expression levels (A) and checkpoint response (B) in EUFA1341 cells reconstituted with wt, BRCA1 binding mutant (L35P) or BRCA2 binding mutant (A1025R) of PALB2. (C,D) PALB2-BRCA1 interaction (C) and checkpoint response (D) in EUFA1341 cells reconstituted with wt or KEAP1 binding mutants (T92E and G93E) of PALB2. For B and D, data shown are means ± SEMs of the relative mitotic indexes from ≥3 independent experiments. Statistical significance was calculated with one-way ANOVA comparing the values of cells expressing PALB2-T92E and G93E to cells expressing the wt protein at the same time point. *, P<0.05.

Figure 5.

Defective checkpoint response and increased IR-induced chromosomal abnormalities in mouse cells with abrogated BRCA1-PALB2 interaction. (A,B) PALB2 expression levels (A) and G2/M checkpoint activation (B) in MEFs from wt (WT), heterozygous (HET) and homozygous (MUT) Palb2-cc6 knockin mutant mice. (C,D) CHK1 phosphorylation (C) and checkpoint response (D) in splenic B cells isolated from wt and homozygous mutant mice after 3 Gy of IR. Data shown are the means ± SEMs from 4 independent experiments. Statistical significance was calculated with one-way ANOVA. *, P<0.05; **, P<0.01, ***, P<0.001. (E) Chromosomal abnormalities in wt and Palb2 mutant B cells at indicated time points after 3 Gy of IR. Cells were arrested for 1 hr with colcemid prior to collection at each time point (for the 1 hr time point, colcemid was added 10–15 min after IR and incubated for 1 hr). CTB, chromatid break; CSB, chromosomal break. Data shown are the means ± standard deviations (STDVs) from 3 independent experiments. Statistical significance was calculated with two-tailed student’s t test. *, P<0.05; **, P<0.01, ***, P<0.001.