PALB2 links BRCA1 and BRCA2 in the DNA-damage response

Abstract

BRCA1 and BRCA2 are often mutated in familial breast and ovarian cancer. Both tumor suppressors play key roles in the DNA-damage response. However, it remains unclear whether these two tumor suppressor function together in the same DNA-damage response pathway. Here, we show that BRCA1 associates with BRCA2 through PALB2/FANCN, a major binding partner of BRCA2. The interaction between BRCA1 and BRCA2 is abrogated in PALB2-deficient Fanconi anemia cells and in the cells depleted of PALB2 by small interfering RNA. Moreover, we show that BRCA1 promotes the concentration of PALB2 and BRCA2 at DNA-damage sites and the interaction between BRCA1 and PALB2 is important for the homologous recombination repair. Taken together, our results indicate that BRCA1 is an upstream regulator of BRCA2 in the DNA-damage response, and PALB2 is the linker between BRCA1 and BRCA2.

Figure 1. PALB2 Mediates the Interaction between BRCA1 and BRCA2

(A) Silver staining of affinity-purified PALB2 complexes. The nuclear extracts prepared from naive HeLa S3 cells or cells stably expressing PALB2-FLAG-HA were subjected to two rounds of affinity purification. Final elutes were resolved by SDS-PAGE and stained with silver. The protein bands were analyzed by mass spectrometry. Arrows indicate protein bands corresponding to BRCA1, BRCA2, and PALB2. (B) PALB2 interacts with BRCA1. 293T cell lysates were analyzed by immunoprecipitation (IP) and western blotting with PALB2 or BRCA1 antibodies respectively. Whole-cell lysates were blotted and shown as the input. An irrelevant IgG was used as the IP control. (C) The interaction between BRCA1 and PALB2 is DNA-damage independent. 293T cells were treated with 0 or 10 Gy IR. Extracts prepared from mock-treated or irradiated 293T cells were IPed with antibodies against PALB2 and BRCA1. The precipitates were separated by SDS-PAGE and immunoblotted with the indicated antibodies. A blot with anti-β-actin antibody was used as the protein loading control. (D) PALB2 siRNAs specifically downregulate PALB2. HeLa cells were treated with control or PALB2 siRNA. Cell lysates were examined by indicated antibodies. (E) Downregulation of PALB2 abrogates the association between BRCA1 and BRCA2. HeLa cells were treated with control or PALB2 siRNAs. Cell lysates were analyzed by immunoprecipitaion and western blotting with indicated antibodies. The amount of β-actin was used as the protein loading control. (F) Loss of BRCA1-BRCA2 interaction in PALB2-deficent Fanconi anemia cells. Whole-cell lysates of U2OS, FEN5280 (PALB2 wt), EUFA1341 (PALB2-deficent, FA-N), and the reconstituted EUFA1341-PALB2 cells were analyzed with indicated antibodies.

Figure 2. BRCA1 Impairs Focus Formation of PALB2 and BRCA2 at DNA-Damage Sites

(A) The DNA-damage-induced focus formation of PALB2 and BRCA2 was impaired in BRCA1-deficient cells. HCC1937 and HCC1937-BRCA1 cells were treated with 0 or 10 Gy of IR. Cells were stained with indicated antibodies. The percentage of foci positive cells was summarized. (B) BRCA1 siRNA specifically downregulates BRCA1. U2OS cells were treated with control siRNA or two different BRCA1 siRNA for 48 hr and the whole-cell lysates were analyzed by western blotting. (C) Downregulation of BRCA1 impairs the IR-induced focus formation of PALB2 and BRCA2. U2OS cells were treated with the same siRNAs as mentioned above for 45 hr and then treated with 10 Gy of IR. Cells were fixed 6 hr after IR and costained with indicated antibodies. The results shown were from BRCA1 siRNA #1. BRCA1 siRNA #2 produced similar effects (not shown).

Figure 3. The Coiled-Coil Domain of BRCA1 Interacts with PALB2 and Targets PALB2 and BRCA2 to DNA-Damage Sites

(A) The schematic diagrams of BRCA1 and its mutants. (B) The coiled-coil domain of BRCA1 interacts with PALB2. Wild-type BRCA1 and its internal deletion mutants were expressed in 293T cells. The interaction between PALB2 and the BRCA1 species were examined with indicated antibodies. (C) The coiled-coil domain of BRCA1 is important for targeting PALB2 and BRCA2 to DNA-damage sites. Wild-type BRCA1 or the BD5 mutant was expressed in HCC1937 cells. Cells were treated with 10 Gy of IR and then stained with indicated antibodies.

Figure 4. The Interaction between BRCA1 and PALB2 Is Critical for DNA-Damage-Induced Focus Formation of BRCA2 and HR Repair

(A) The N-terminal coiled-coil domain of PALB2 interacts with BRCA1. SBP-tagged mutant PALB2 was expressed in 293T cells. Cell lysates were analyzed by IP with BRCA1 antibody and western blotting with FLAG antibody. The expression level of exogenous PALB2 proteins was examined by western blotting with FLAG antibody. The schematic diagrams of PALB2 and its mutants were presented. (B) The N-terminal domain of PALB2 is essential for its translocation to DNA-damage sites. Wild-type PALB2 and the P1, P2, and P13 mutants were expressed in HeLa cells. Cells were treated with 10 Gy of IR and stained with indicated antibodies. Foci-positive cells were enumerated. (C) The N-terminal domain of PALB2 is important for HR repair. HR assay was performed as explained in Supplemental Experimental Procedures. The error bars were calculated from three independent experiments. Exogenous and endogenous PALB2 were analyzed by indicated antibodies.