PALB2 functionally connects the breast cancer susceptibility proteins BRCA1 and BRCA2

Abstract

BRCA1 and BRCA2 are prominently associated with inherited breast and ovarian cancer. The encoded proteins function in DNA damage responses, but no functional link between BRCA1 and BRCA2 has been established. We show here that PALB2 physically and functionally connects BRCA1 and BRCA2 into a DNA damage response network that also includes the RAD51 recombinase. PALB2 directly binds BRCA1, as determined with bacterially expressed fragments of each protein. Furthermore, PALB2 independently interacts with BRCA1 and BRCA2 through its NH2 and COOH termini, respectively. Critically, two point mutants (L21P and L24P) of the PALB2 coiled-coil domain or an NH2-terminal deletion (Delta1-70) disrupt its interaction with BRCA1. We have reconstituted PALB2-deficient cells with PALB2Delta1-70, PALB2-L21P, or PALB2-L24P, or with COOH-terminally truncated PALB2 that is deficient for interaction with BRCA2. Using extracts from these cells, we find that PALB2 mediates the physical interaction of BRCA2 with a COOH-terminal fragment of BRCA1. Analysis of the assembly of foci in these cells by BRCA1, PALB2, BRCA2, and RAD51 suggests that BRCA1 recruits PALB2, which in turn organizes BRCA2 and RAD51. Resistance to mitomycin C and the repair of DNA double-strand breaks by homologous recombination require the interaction of PALB2 with both BRCA1 and BRCA2. These results suggest that BRCA1 and BRCA2 cooperate in DNA damage responses in a PALB2-dependent manner, and have important implications for the genesis of breast/ovarian cancer and for chemotherapy with DNA interstrand cross-linking agents.

FIGURE 1

PALB2 and BRCA1 coimmunoprecipitate and BRCA1 regulates PALB2 behavior. A. Levels of PALB2 and BRCA1 in extracts from undamaged MCF7, HeLa, or 293T cells are indicated by immunoblotting (input). These samples represent 2% of the amount of protein used for immunoprecipitation with anti-PALB2 or anti-BRCA1 antibodies. Preimmune serum for the PALB2 antibody was used as a control. B. Representative i mages of the assembly of PALB2 foci in HCC1937 cells, which are deficient for BRCA1, or their counterparts corrected by expression of BRCA1. Cells were fixed at 16 h following exposure to 15 Gy IR, and the position of nuclei was determined by counterstaining with 4′,6-diamidino-2-phenylindole (DAPI). C to E. Quantification of the assembly of PALB2 (C), BRCA2 (D), and RAD51 (E) foci in HCC1937 or their corrected counterparts. Cells were exposed to 15 Gy IR and were fixed after 16 h, or were left untreated (Untr.), and average values ± SD were determined from three independent counts of 150 or more cells each.

FIGURE 2

An NH₂-terminal domain of PALB2, distinct from the BRCA2-interacting domain of PALB2, mediates interaction with BRCA1 and assembly of PALB2 nuclear foci. A. Diagram of sets of PALB2 mutants used to examine the interaction of PALB2 with BRCA1. These included large NH₂- or COOH-terminal truncations (i), a series of smaller NH₂-terminal truncations (ii), and two different point mutants (*) within the NH₂-terminal coiled-coil domain of PALB2 (iii). Each PALB2 construct contained a Flag-HA epitope tag. In the case of NH₂-terminal deletion mutants, the epitope tags were at the COOH terminus of the protein. All other constructs contained NH₂-terminal epitope tags. FL, PALB2-full-length. B. Immunoprecipitation with anti-Flag antibodies was done for each experimental set. Antibodies against BRCA1, and BRCA2 or RAD51 in i, were used to determine whether the protein was present in immunoprecipitates. C. Diagram of amino acids 1 to 70 of PALB2. The predicted coiled-coil domain is underlined (amino acids 9–44); *, point mutants that were tested (L21P and L24P). D. Examples of PALB2 foci assembled by wild-type protein, PALB2/1–550, or PALB2-L21P following expression in 293T cells. Assembly of expressed PALB2 and its mutants, all of which contained an HA-Flag epitope tag, was detected with anti-HA antibodies. E. Quantification of the assembly of PALB2 foci, detected with anti-HA antibodies against the epitope tag for each set of mutants. Values for wild-type PALB2 and PALB2Δ1–70 are repeated in each data set for the purpose of comparison. Cells were fixed at 16 h following treatment with 15 Gy IR. Average values ± SD were determined from three independent counts of 150 or more cells each.

FIGURE 3

The interaction of PALB2 with BRCA1 is required for the assembly of PALB2, BRCA2, and RAD51 nuclear foci. A. Levels of stable expression of wild-type PALB2 (FL), PALB2ΔC, PALB2Δ1–70, PALB2-L21P, and PALB2-L24P with an HA-Flag epitope tag in EUFA1341 fibroblasts are indicated for each cell line (input). Coimmunoprecipitation of BRCA1, BRCA2, and RAD51 was determined by immunoblotting. Blots for input extracts represent 2.5% of the amount of protein used for immunoprecipitation. B. Examples of PALB2 foci assembled by full-length (wild-type) PALB2, PALB2ΔC, PALB2-L21P, or pMMP vector alone. In each case, the expressed protein contained HA-Flag epitope tags. PALB2 foci detected with anti-HA antibodies (red) and RAD51 foci detected with anti-RAD51 antibodies (green) are shown in a merged image. Colocalization is indicated by yellow foci, whereas red foci indicate the presence of PALB2 but not RAD51 foci. C. Quantification of the assembly of each form of PALB2 into nuclear foci (top), and of the assembly of RAD51 (center) or BRCA1 (bottom) into foci in EUFA1341 fibroblasts reconstituted with each form of PALB2. D. Quantification of the assembly of BRCA2 foci in U2OS cells exogenously expressing wild-type PALB2, PALB2ΔC, PALB2Δ1–70, PALB2-L21P, or PALB2-L24P that were depleted of endogenous PALB2 using a siRNA against its 3′-UTR. Controls trans-fected with a siRNA directed against GFP are indicated. Cells were analyzed at 4 d after transfection with the siRNA. Cells were fixed at 16 h following treatment with 15 Gy IR or were left untreated, and average values ± SD were determined from three independent counts of 150 or more cells each (C–D).

FIGURE 4

Direct binding of the coiled-coil domain of PALB2 to BRCA1 is required for the association of BRCA2 with BRCA1, for resistance to MMC, and for DSB-initiated HR. A. GST protein fused with the 1293–1863 fragment of BRCA1 was incubated with extracts from EUFA1341 fibroblasts exogenously expressing wild-type PALB2, PALB2ΔC, PALB2-L21P, or PALB2-L24P. Associated protein complexes were isolated on glutathione beads. The presence of BRCA2 or PALB2 in isolated protein complexes was determined by immunoblotting. GST-BRCA1-1293–1863 was detected with anti-GST antibodies. B. MBP, MBP/PALB2-1–70, MBP/PALB2-1–70(L21P), or MBP/PALB2-1–70(L24P), isolated using maltose beads (input), were incubated with GST or GST/ BRCA1-1293–1863 beads. Binding of each MBP fusion protein to GST or GST/BRCA1-1293–1863 was determined by immunoblotting with anti-MBP antibodies. The blot for input protein represents 10% of the amount of protein used for the pulldown assay. C. Survival of EUFA1341 fibroblasts stably expressing wild-type PALB2, PALB2-L21P, PALB2-L24P, or PALB2Δ1–70 with HA-Flag epitope tags was determined using a colorimetric assay following treatment with a range of concentrations of MMC (0–500 nmol/L). Relative survival for each cell line ± SD was normalized to the value for that cell line without treatment. D. Assay of HR in U2OS-DR cells stably expressing wild-type PALB2, PALB2ΔC, PALB2-L21P, or PALB2-L24P (with Flag-HA epitope tags), which were depleted of endogenous PALB2 using a siRNA against its 3′-UTR. Examples of dot plots demonstrating the level of GFP (inside box) versus phycoerytherin (PE) for cells expressing PALB2-L21P and I-SCEI. Cells were transfected with a control siRNA against LacZ or with a siRNA directed against the 3′-UTR of endogenous PALB2. At right is quantification of HR for each cell type, relative to controls for the corresponding cell line transfected with siLacZ instead of siPALB2–3′-UTR. The amount of GFP-positive cells ranged from 0% to 2% in each experiment. Columns, average from three independent experiments; bars, SD.

FIGURE 5

Proposed model for the role of PALB2 in linking BRCA1 with BRCA2 and RAD51 in a pathway of HR. We propose that BRCA1 binds to the NH₂ terminus of PALB2 following its own recruitment to sites of DNA damage. We suggest that the COOH terminus of positioned PALB2 then binds to BRCA2, which in turn regulates the assembly of the RAD51 nucleoprotein filament that initiates HR. In this manner, PALB2 links BRCA1, BRCA2, and RAD51 into a network that is required for HR. Our results suggest that BRCA1 and BRCA2 do not directly interact, but associate using PALB2 as an intermediary.