Embed Size (px)
Citation preview
MOLECULAR AND CELLULAR BIOLOGY, July 2004, p. 5850–5862 Vol. 24, No. 130270-7306/04/$08.00�0 DOI: 10.1128/MCB.24.13.5850–5862.2004Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Functional Interaction of Monoubiquitinated FANCD2 andBRCA2/FANCD1 in Chromatin
XiaoZhe Wang, Paul R. Andreassen, and Alan D. D’Andrea*Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School,
Boston, Massachusetts 02115
Received 16 November 2003 /Returned for modification 22 January 2004 /Accepted 7 April 2004
Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with at least 11 complemen-tation groups (A, B, C, D1, D2, E, F, G, I, J, and L), and eight FA genes have been cloned. The FANCD1 geneis identical to the breast cancer susceptibility gene, BRCA2. The FA proteins cooperate in a common pathway,but the function of BRCA2/FANCD1 in this pathway remains unknown. Here we show that monoubiquitinationof FANCD2, which is activated by DNA damage, is required for targeting of FANCD2 to chromatin, where itinteracts with BRCA2. FANCD2-Ub then promotes BRCA2 loading into a chromatin complex. FANCD2�/�
cells are deficient in the assembly of DNA damage-inducible BRCA2 foci and in chromatin loading of BRCA2.Functional complementation with the FANCD2 cDNA restores BRCA2 foci and its chromatin loading followingDNA damage. BRCA2�/� cells expressing a carboxy-terminal truncated BRCA2 protein form IR-inducibleBRCA2 and FANCD2 foci, but these foci fail to colocalize. Functional complementation of these cells withwild-type BRCA2 restores the interaction of BRCA2 and FANCD2. The C terminus of BRCA2 is thereforerequired for the functional interaction of BRCA2 and FANCD2 in chromatin. Taken together, our resultsdemonstrate that monoubiquitination of FANCD2, which is regulated by the FA pathway, promotes BRCA2loading into chromatin complexes. These complexes appear to be required for normal homology-directed DNArepair.
Fanconi anemia (FA) is an autosomal recessive diseasecharacterized by cancer susceptibility and cellular hypersensi-tivity to DNA cross-linking agents, such as mitomycin C(MMC) and cisplatin. Eight FA genes have been cloned, cor-responding to FA subtypes A, C, BRCA2/D1, D2, E, F, G, andL. The encoded FA proteins cooperate in a common path-way—the FA/BRCA pathway (6, 15). Six of the FA proteins(A, C, E, F, G, and L) assemble in a multisubunit nuclearcomplex (8, 18, 19), required for the activation (monoubiquiti-nation) of the downstream FANCD2 protein (9). Whether theE3 ubiquitin ligase, BRCA1, also participates in the mono-ubiquitination of FANCD2 remains unclear (28). The acti-vated FANCD2 protein is subsequently targeted to nuclearfoci (9).
Whether BRCA2 participates with other FA proteins in thispathway has remained uncertain. On the one hand,BRCA2�/� patients share most of the clinical and cellularphenotypic features of other FA subtypes, suggesting a com-mon pathway (13). On the other hand, BRCA2-deficient cellshave a more severe defect in homologous recombination repair(21, 30), suggesting that BRCA2 may have functions indepen-dent of the FA pathway. Also, BRCA2�/� patients generallyhave a more severe clinical phenotype, with earlier onset ofcancer (11). Although two-hybrid studies suggest that BRCA2may interact with other FA proteins, such as FANCG (14),direct biochemical evidence linking BRCA2 to other FA pro-teins is lacking.
Unlike other FA proteins, BRCA2 has a well-defined role inhomology-directed DNA repair (HDR). BRCA2 binds to sin-gle- and double-stranded DNA (31), interacts directly withRAD51 (4, 17), modulates RAD51 activity in vitro (7), andregulates the level of HDR (21, 27). BRCA2 is also requiredfor the stabilization of stalled DNA replication forks (16), aprocess that could be related to the role of BRCA2 in HDR.Evidence of a biochemical interaction between BRCA2 andanother FA protein may link the pathway to the BRCA2-mediated process of homologous recombination.
In the present study, we show that monoubiquitinatedFANCD2 assembles with BRCA2 and another FA protein,FANCE, in a functional chromatin complex. DNA damage-inducible monoubiquitination of FANCD2 is required for itstargeting to chromatin, and monoubiquitinated FANCD2 thenpromotes the assembly of ionizing radiation (IR)-inducibleBRCA2 foci and chromatin loading of BRCA2. Moreover, thecarboxy terminus of BRCA2 is required for the interaction ofBRCA2 and FANCD2. An FA patient-derived mutant form ofBRCA2 lacking amino acids 3226 to 3418 fails to colocalize orcoimmunoprecipitate with FANCD2. The presence of BRCA2and FANCD2 chromatin complexes suggests a link betweenthe FA pathway and the process of homologous recombinationDNA repair in mammalian cells.
MATERIALS AND METHODS
Cell culture. The simian virus 40-transformed fibroblasts, PD20 (FA-D2),EUFA423 (FA-D1), and GM6914 (FA-A), as well as HeLa and U2OS cells, weregrown in Dulbecco’s modified Eagle’s medium supplemented with 15% heat-inactivated fetal calf serum (FCS) in a humidified 5% CO2 incubator at 37°C.PD20 fibroblasts expressing either empty vector, full-length FANCD2 cDNA, orthe FANCD2(K561R) mutant were described previously (9). AT22IJE-T fibro-blasts (AT) expressing either empty vector or full-length ataxia telangiectasia
* Corresponding author. Mailing address: Dana-Farber Cancer In-stitute, Department of Pediatric Oncology, Harvard Medical School,44 Binney St., Boston, MA 02115. Phone: (617) 632-2112. Fax: (617)632-5757. E-mail: [email protected].
5850
Dow
nloa
ded
from
http
s://j
ourn
als.
asm
.org
/jour
nal/m
cb o
n 17
Feb
ruar
y 20
22 b
y 22
2.10
3.22
.67.
mutated (ATM) cDNA (AT � ATM) were described previously (33). EUFA423fibroblasts stably expressing empty vector or the hemagglutinin (HA) tag plasmidpcDNA3 HA-BRCA2 (EUFA423 � HA-BRCA2), and chromosome 13-trans-fected FA-D1 cells (EUFA423 � BRCA2) were described previously (13).BRCA2�/� CAPAN-1 cells were grown in Iscove’s modified Dulbecco’s mediumwith 15% heat-inactivated FCS. Epstein-Barr virus-transformed lymphoblastsPD7 (wild-type), HSC230 (FA-B), and EUFA130 (FA-E) were maintained inRPMI 1640 with 15% FCS. EUFA130 lymphoblasts expressing full-length HA-FANCE were described previously (23).
Generation of DNA damage. Gamma irradiation was delivered with a Gam-macell 40 apparatus (MDS Nordion, Ottawa, Canada). For MMC (Sigma Chem-ical) treatment, cells were continuously exposed to the drug for the indicatedtimes. MMC sensitivity assays of human fibroblasts and lymphoblasts have beendescribed previously (9).
Transient transfection. EUFA423 (FA-D1) cells were transfected with theplasmids pcDNA3 and pcDNA3 HA-BRCA2 (13), using FuGENE 6 (Roche),according to the manufacturer’s protocol.
Preparation of cellular fractions. Cells were grown on 15-cm-diameter culturedishes and were exposed to IR (15 Gy, 15 h) or MMC (170 ng/ml, 15 h). Cellswere trypsinized and washed with cold phosphate-buffered saline (PBS), ali-quoted equally into four Eppendorf tubes, and collected by centrifugation at1,200 rpm for 3 min at 4°C in a Sorvall RT 6000D centrifuge. One pellet,representing the whole-cell extracts (P1), was frozen in liquid N2. The remainingthree pellets were resuspended in cold buffer A (10 mM PIPES, pH 7, 100 mMNaCl, 1 mM EGTA, 300 mM sucrose, 0.5 mM sodium orthovanadate, 50 mMsodium fluoride, 10-�g/ml aprotinin, 10-�g/ml leupeptin, 10-�g/ml pepstatin A,1 mM phenylmethylsulfonyl fluoride [PMSF]) containing 0.5% Triton X-100 andwere incubated at room temperature (RT) for 2 min to permeabilize the cells.The suspension was then centrifuged as described above, and the supernatant(S2; detergent-soluble nuclear proteins) was collected and frozen. Pellets werewashed with cold buffer A. One pellet (P2; detergent-insoluble nuclear proteins)was frozen. Nuclei were then digested with RNase-free DNase I (200 U/ml)(Roche) in buffer A for 30 min at RT. The residual pellet was collected bycentrifugation as described above. The supernatant (S3; DNase I-sensitive pro-teins) from one tube was frozen, and all pellets were washed with cold buffer A.One residual pellet (P3; DNase I-resistant protein) was frozen. The remainingresidual pellet was extracted with cold buffer A containing 250 mM ammoniumsulfate for 5 min at RT to extract the remaining chromatin. The supernatant (S4;chromatin) was collected following centrifugation at 3,000 rpm for 3 min and wasthen frozen. Cell-equivalent volumes from each cell fraction were separated bysodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) andimmunoblotted with antibodies.
Immunoblotting. Cells were fractionated, and whole-cell extracts were sub-jected to 6% SDS-PAGE or 3 to 8% NuPAGE Tris-acetate (Invitrogen) gelelectrophoresis, transferred to nitrocellulose membranes, and subjected to West-ern blot analysis (9). The following antibodies were used: anti-BRCA2 (mono-clonal Ab-1 and polyclonal Ab-2) (Oncogene Research Products), anti-FANCD2(affinity-purified E35 polyclonal antibody and FI-17; Santa Cruz Biotech.), an-ti-HA (HA.11; Babcock), anti-histone H4 (Santa Cruz Biotech.), anti-FANCE (agenerous gift from Grover Bagby, Oregon Health and Science University, Port-land), and anti-ATM (Novus Biologicals).
Coimmunoprecipitation. For immunoprecipitation from whole-cell extracts,cells were lysed with lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mMEDTA, 0.5% NP-40) supplemented with protease inhibitors (1-�g/ml leupeptinand pepstatin, 2-�g/ml aprotinin, 1 mM PMSF) and phosphatase inhibitors (1mM sodium orthovanadate, 10 mM sodium fluoride). Immunoprecipitation ofprotein (6 mg) from whole-cell extracts or of chromatin proteins extracted asdescribed above was performed by using the monoclonal antibody to BRCA2(Ab-1), polyclonal antibody to BRCA2 (H-300) (Santa Cruz Biotech.), or poly-clonal antibodies to FANCD2 (E35). As a negative control, preimmune rabbitserum or normal mouse immunoglobulin G (IgG) was used. The lysates wereprecleared with protein A-Sepharose for 30 min at 4°C. Cleared extracts werethen incubated with antibodies overnight at 4°C before protein A-Sepharose wasadded for 2 to 4 h. The immunoprecipitated materials were washed extensivelywith lysis buffer and analyzed by 6% SDS-PAGE or 3 to 8% NuPAGE Tris-acetate gel electrophoresis, followed by immunoblot analysis with the indicatedantibodies and enhanced chemiluminescence detection with the ECL system.
Detection of monoubiquitinated FANCD2 in chromatin. HeLa cells weretransfected with a HA-tagged ubiquitin expression vector (pMT 123) using Fu-GENE 6 (Roche) as previously described (9). After transfection, cells weretreated with the indicated dose of IR and MMC and then were fractionated intoS2 (soluble nuclear proteins) and S4 (chromatin) fractions. These two fractionsfrom HeLa cells were subjected to immunoprecipitation with a polyclonal anti-
body (E35) to FANCD2. Immune complexes were run on 6% SDS-PAGEtransferred to nitrocellulose, and immunoblotted with anti-FANCD2 (FI-17) oranti-HA (HA.11) monoclonal antibodies.
Immunoprecipitation and phosphatase assay. To determine whether shifts ofBRCA2 mobility on immunoblots corresponded to protein phosphorylation,BRCA2 was immunoprecipitated with monoclonal antibody (Ab-1) and treatedwith alkaline phosphatase as previously described (9). Samples were separated by3 to 8% NuPAGE Tris-Acetate (Invitrogen) gel electrophoresis and immuno-blotted with polyclonal antibody to BRCA2 (Ab-2).
Immunofluorescence microscopy. Preparation of cells for immunofluores-cence microscopy was performed essentially as previously described (9). Cellswere prepermeabilized with 0.25% Triton X-100 in PBS for 1 min on ice andthen fixed with 4% paraformaldehyde in PBS for 15 min, followed by permeabi-lization with 0.5% Triton X-100 in PBS for 1 min. For immunofluorescencemicroscopy, fixed cells were incubated with specific primary antibodies at theappropriate dilution in 3% bovine serum albumin–PBS for 1 h at RT. FANCD2was detected with the affinity-purified E35 polyclonal antibody or monoclonalantibody FI-17. BRCA2 was detected with monoclonal antibody (Ab-1) or poly-clonal antibody (Ab-2). Anti-HA monoclonal antibody (HA.11) was used todetect overexpressed HA-tagged BRCA2; RAD51 was detected with anti-RAD51 monoclonal antibody (Oncogene Research Products). Cells were sub-sequently washed three times in PBS and incubated for 1 h at RT with species-specific fluorescein (Jackson Immunoresearch) or Texas red-conjugated(Amersham) secondary antibodies diluted in 3% bovine serum albumin in PBS.Cells were counterstained with 4�6-diamidine-2-phenylindole dihydrochloride(Roche) to visualize nuclei. Slides were mounted in Vectashield (Vector Labo-ratories). Images were acquired using an Axioplan 2 imaging microscope (CarlZeiss) equipped with a digital camera and processed by using Openlab software.
RDS assay. The radioresistant DNA synthesis (RDS) assay was performed asdescribed previously (25). Briefly, cells were plated in 60-mm-diameter plates,incubated with 10 nCi of [14C]thymidine for 24 h, and cultured in the absence oflabeling medium for 24 h. The cells were then treated in replicate with 0, 4, or8 Gy of IR. At 30 min following DNA damage, cells were incubated with2.5-�Ci/ml [3H]thymidine for 15 min. Cells were fixed, loaded onto glass fiberfilters, and counted as previously described (20).
RESULTS
DNA damage activates the colocalization, cofractionation,and coimmunoprecipitation of BRCA2 and FANCD2. In theabsence of DNA damage, BRCA2 and FANCD2 were ex-pressed diffusely in the nuclei of HeLa cells (Fig. 1A). IR at 2or 15 Gy, or MMC exposure, activated the assembly of BRCA2and FANCD2 foci, and these foci showed a �80% colocaliza-tion (Fig. 1A, merge). This strong colocalization of FANCD2and BRCA2 following IR or MMC treatment suggested thatthe proteins may be loaded together into chromatin complexes,perhaps at sites of DNA damage. Consistent with this notion,phosphorylated histone H2AX also colocalized with FANCD2and BRCA2 following exposure to IR or MMC (data notshown).
Previous studies have indicated that FANCD2 exists as twoisoforms: an unubiquitinated isoform (FANCD2-S) and a mono-ubiquitinated isoform (FANCD2-L) (9). Cellular exposure toIR, MMC, or other genotoxic agents results in the FA com-plex-dependent conversion of FANCD2-S to FANCD2-L (9).We next determined the cellular localization of FANCD2-Sand FANCD2-L by cellular fractionation. Nuclei from eitheruntreated or treated U2OS cells were fractionated into deter-gent-soluble (S2) and insoluble (P2) nuclear fractions andchromatin (S4) components, as outlined in Fig. 1B. Interest-ingly, FANCD2-S was nearly exclusively extracted with thesoluble nuclear fraction (Fig. 1C, lane 4), while the IR-induc-ible or MMC-inducible FANCD2-L isoform was selectivelyretained in the chromatin fraction (lane 8). In contrast, littleFANCD2-L protein was present in the chromatin fraction de-
VOL. 24, 2004 FANCD2-Ub INTERACTS WITH BRCA2 IN CHROMATIN 5851
Dow
nloa
ded
from
http
s://j
ourn
als.
asm
.org
/jour
nal/m
cb o
n 17
Feb
ruar
y 20
22 b
y 22
2.10
3.22
.67.
rived from untreated U2OS cells. These results suggest thatthe monoubiquitin tag of FANCD2-L may function as a chro-matin targeting signal.
We also determined the cellular localization of BRCA2 follow-ing DNA damage (Fig. 1C, anti-BRCA2 immunoblots). An in-
creased amount of BRCA2 was detected in the chromatin frac-tion (S4) following treatment with IR or MMC (lane 8), ascompared to that in untreated cells. A fraction of BRCA2 cofrac-tionated with chromatin-associated monoubiquitinated FANCD2(lane 8), suggesting that the proteins may interact in chromatin.
FIG. 1. Colocalization, cofractionation, and coimmunoprecipitation of monoubiquitinated FANCD2 and BRCA2 in chromatin. (A) Colocal-ization of FANCD2 and BRCA2 in DNA damage-inducible foci. HeLa cells were either untreated, exposed to IR (2 or 15 Gy), or treated withMMC (80 ng/ml), as indicated. HeLa cells were double stained with polyclonal anti-FANCD2 (E35) (red) and monoclonal anti-BRCA2 (Ab-1)(green) antibodies after the indicated time of treatment. Magnification, �630. (B) Protocol utilized for nuclear fractionation of cells. Cytoplasmand nucleoplasm were extracted by permeabilization with detergent, the resulting nuclei were DNase I digested, and chromatin was extracted withammonium sulfate (NH2SO4). (C) U2OS cells, either untreated or exposed to IR (15 Gy) or MMC (170 ng/ml), were fractionated 15 h after theinitiation of DNA damage. Supernatants (S) and pellets (P) were subjected to Western blot analysis with the indicated antibodies: E35 forFANCD2 and Ab-1 for BRCA2. The presence of chromatin in the S4 fraction was confirmed by blotting with anti-histone H4 antibody. (D) S2(soluble nuclear proteins) and S4 (chromatin) fractions of U2OS cells were subjected to immunoprecipitation with mouse monoclonal anti-BRCA2antibody (Ab-1) or a control mouse antibody (mIgG) and then immunoblotted with either polyclonal anti-BRCA2 (Ab-2) or anti-FANCD2 (E35)antibodies. Heavy chain IgG was used as a loading control. (E) HeLa cells were transfected with a cDNA encoding HA-ubiquitin, as indicated.After transfection, cells were treated with the indicated dose of IR or MMC. S2 (soluble nuclear proteins) and S4 (chromatin) fractions of HeLacells were immunoprecipitated (IP) with a polyclonal antibody (E35) to FANCD2, as indicated. Immune complexes were run on SDS-PAGE andimmunoblotted with anti-FANCD2 (FI-17) or anti-HA (HA.11) monoclonal antibodies. WCE, whole-cell extract.
5852 WANG ET AL. MOL. CELL. BIOL.
Dow
nloa
ded
from
http
s://j
ourn
als.
asm
.org
/jour
nal/m
cb o
n 17
Feb
ruar
y 20
22 b
y 22
2.10
3.22
.67.
To test for an interaction between BRCA2 and FANCD2,we next immunoprecipitated BRCA2 from the soluble nuclear(S2) and chromatin (S4) fractions derived from U2OS cells(Fig. 1D). Although more BRCA2 was detected in the S2fraction (lanes 3 and 4, anti-BRCA2 immunoblot), FANCD2-Sdid not coimmunoprecipitate with it. In contrast, FANCD2-Lin the S4 (chromatin) fraction selectively coimmunoprecipi-tated with BRCA2 (lanes 7 and 8), and this association wasenhanced by IR (compare lanes 7 and 8 of FANCD2-L). TheBRCA2 protein had a slightly slower electrophoretic mobilityin chromatin (i.e., increased mass) after IR treatment, suggest-ing that BRCA2 in chromatin is posttranslationally modifiedfollowing IR (Fig. 1D, compare lanes 7 and 8). A reciprocalcoimmunoprecipitation with an antibody to FANCD2, fol-lowed by an immunoblot with anti-BRCA2 antibodies, con-firmed the interaction of FANCD2-L and BRCA2 in the chro-matin (S4) fraction (see Fig. 7D, lanes 4).
To confirm that the FANCD2 protein in the S4 (chromatin)fraction is indeed monoubiquitinated, we transfected HeLa
cells with a cDNA encoding HA-ubiquitin, and endogenousFANCD2 was then immunoprecipitated (Fig. 1E). As ex-pected, IR and MMC treatment activated the monoubiquiti-nation (HA labeling) of FANCD2 (lanes 9 and 10, respec-tively). FANCD2 in the S2 (soluble nuclear proteins) fractionwas nonubiquitinated, as demonstrated by the absence of HA-ubiquitin in the FANCD2 immunoprecipitate (lanes 3 to 5).
Monoubiquitination of FANCD2 promotes the assembly ofIR-inducible BRCA2 foci. To determine the functional impor-tance of the interaction between BRCA2 and FANCD2, weexamined an MMC-sensitive patient-derived FANCD2�/� cellline (FA-D2), PD20F (Fig. 2). As expected, FANCD2 was notdetected in anti-BRCA2 immune complexes immunoprecipi-tated from whole-cell extracts of FA-D2 cells (Fig. 2A, lanes 3and 4). The interaction between BRCA2 and FANCD2 wasobserved after stable transfection with the FANCD2 cDNA(Fig. 2A, lanes 6 and 7).
PD20F (FA-D2) fibroblasts had no FANCD2 foci, as previ-ously described (9) (Fig. 2B). Although these cells did exhibit
FIG. 1—Continued.
VOL. 24, 2004 FANCD2-Ub INTERACTS WITH BRCA2 IN CHROMATIN 5853
Dow
nloa
ded
from
http
s://j
ourn
als.
asm
.org
/jour
nal/m
cb o
n 17
Feb
ruar
y 20
22 b
y 22
2.10
3.22
.67.
FIG. 2. Monoubiquitinated FANCD2 promotes IR-induced assembly of BRCA2 foci. (A) Whole-cell extracts of PD20 (FA-D2) fibroblastsstably expressing empty vector (PD20F � Vec) and PD20 fibroblasts corrected with FANCD2 (PD20F � FANCD2) were subjected toimmunoprecipitation with mouse monoclonal anti-BRCA2 (Ab-1) or a control mouse antibody (mIgG) and then immunoblotted with eitheranti-FANCD2 (E35) or anti-BRCA2 (Ab-2) antibodies. Heavy chain IgG was used as a loading control. (B) Formation of subnuclear FANCD2and BRCA2 foci in response to IR treatment. PD20 (FA-D2) fibroblasts, stably expressing empty vector alone (upper panel) or full-lengthFANCD2 cDNA (lower panel), were either untreated or treated with IR (15 Gy) and fixed 4 h later. Cells were double stained with polyclonalanti-FANCD2 (E35) (red) and monoclonal anti-BRCA2 (Ab-1) (green) antibodies and analyzed by immunofluorescence microscopy. Magnifi-cation, �630. (C) Quantification of BRCA2 foci. PD20 (FA-D2) fibroblasts stably expressing empty vector alone (PD20F � Vec), FANCD2(PD20F � FANCD2), or FANCD2-K561R mutant (PD20F � K561R), GM6914 (FA-A) fibroblasts and corrected GM6914 fibroblasts stablyexpressing FANCA (GM6914 � FANCA), EUFA130 (FA-E) lymphoblasts and corrected EUFA130 lymphoblasts (EUFA130 � HA-FANCE),and HeLa cells were either untreated or treated with IR (15 Gy) and fixed 4 h later. Cells with more than four distinct foci were counted as positive.A total of 200 cells/sample were analyzed. The values shown are the mean � standard deviation from three separate experiments.
5854 WANG ET AL. MOL. CELL. BIOL.
Dow
nloa
ded
from
http
s://j
ourn
als.
asm
.org
/jour
nal/m
cb o
n 17
Feb
ruar
y 20
22 b
y 22
2.10
3.22
.67.
BRCA2 foci (Fig. 2B), the percentage of cells with BRCA2foci did not increase upon cellular exposure to IR (Fig. 2C).Correction of these cells with the FANCD2 cDNA restored theformation of IR-inducible BRCA2 foci, as measured by thepercentage of cells with BRCA2 foci following IR treatment(Fig. 2C). A nonubiquitinated mutant of FANCD2 (FANCD2-K561R) (9) failed to complement the cells and failed to in-crease the percentage of cells with IR-activated BRCA2 foci(Fig. 2C and Table 1). FA-A (complementation group A) andFA-E fibroblasts also exhibited no increase in the percentageof cells with BRCA2 foci following IR. Functional complemen-tation of these cells, with either the FANCA or FANCEcDNA, respectively, restored FANCD2 monoubiquitinationand MMC resistance (Table 1) and the ability to assemblyIR-inducible BRCA2 foci (Fig. 2C). Taken together, theseresults indicate that the generation of monoubiquitinatedFANCD2 by the functional FA/BRCA pathway promotes theassembly of DNA damage-inducible BRCA2 foci.
The carboxy terminus of BRCA2 is required for the func-tional interaction of BRCA2 and FANCD2. To further appre-ciate the functional importance of BRCA2 and FANCD2 com-plexes, we examined a FA-D1 (BRCA2�/�) fibroblast line,EUFA423 (Fig. 3). These cells have two mutant alleles ofBRCA2 (13). The cells express a truncated BRCA2 protein,encoded by one of the mutant alleles (exon 27, 9900insA) (13),and this mutant protein lacks the carboxy-terminal 192 aminoacids of BRCA2 (Fig. 3A). The truncated BRCA2 protein canbe detected with anti-BRCA2 antibody (Ab-1) but not withAb-2. Uncorrected EUFA423 cells or EUFA423 cells stablytransfected with the full-length BRCA2 cDNA were examinedfor FANCD2 monoubiquitination (Fig. 3B). UncorrectedEUFA423 cells were hypersensitive to DNA damage (Table 1)and upregulated the monoubiquitination of FANCD2 at lowerdoses (5 Gy) (Fig. 3B, compare lanes 4 and 9) and shorter timepoints (4 h) after IR than corrected EUFA423 cells (Fig. 3B,compare lanes 16 and 21). The increase in FANCD2 mono-ubiquitination in EUFA423 cells probably results from thegreater accumulation of DNA damage in these DNA repair-deficient cells.
We next examined BRCA2 foci and FANCD2 foci in these
FA-D1 cells. Uncorrected EUFA423 cells exhibited IR-induc-ible BRCA2 foci (Fig. 3C) and an abnormally high percentageof cells with FANCD2 foci, both with and without IR treat-ment (Fig. 3D). The increase in the percentage of FA-D1 cellswith FANCD2 foci is consistent with the increased monoubiq-uitination of FANCD2 observed on immunoblots in Fig. 3B. Incontrast, BRCA2-corrected EUFA423 cells exhibited similarpercentages of cells with FANCD2 foci as HeLa cells (Fig.3D). Interestingly, based on counts, the FANCD2 foci inFA-D1 cells failed to colocalize with BRCA2 foci (Fig. 3E).Colocalization of BRCA2 and FANCD2 was restored in cor-rected EUFA423 cells (Fig. 3E). Importantly, using an anti-body to FANCD2, FANCD2-L and BRCA2 from the damagedchromatin of BRCA2-corrected EUFA423 cells coimmuno-precipitated (Fig. 3F, lane 8) but C-terminally truncatedBRCA2 from damaged chromatin of EUFA423 cells did notcoimmunoprecipitate with FANCD2-L (Fig. 3F, lane 7).Taken together, these results indicate that FA-D1 cells assem-ble elevated FANCD2 foci, but that the carboxy-terminal 192amino acids of BRCA2 are required for the functional inter-action of BRCA2 and FANCD2.
FA-D1 and FA-D2 cells are defective in the assembly ofIR-inducible RAD51 foci. Previous studies have indicated thatBRCA2 binds RAD51 and that BRCA2-deficient cell lines aredefective in RAD51 focus formation (4, 17, 32). We thereforeexamined IR-induced RAD51 foci in FA-D1 and FA-D2 cells(Fig. 4). EUFA423 (BRCA2-deficient) cells were defective inIR-inducible RAD51 focus assembly, as demonstrated by adecreased percentage of cells with RAD51 foci, either with orwithout IR treatment, relative to HeLa cells or correctedEUFA423 cells (Fig. 4A). Similarly, a much lower percentageof PD20 (FANCD2-deficient) cells had IR-inducible RAD51foci, either with or without exposure to IR, than PD20 cellscorrected by the expression of wild-type FANCD2. PD20 cellsexpressing the FANCD2-K561R mutant were also defective inthe assembly of IR-inducible RAD51 foci (Fig. 4B). Takentogether, these results suggest that at least one function of theFA/BRCA pathway is to assemble DNA damage-inducibleRAD51 foci.
TABLE 1. Characterization of FA and corrected FA cell lines
Cell line/plasmid FA group MMC sensitivityaIR induction of focib: FANCD2/BRCA2 complex
in chromatinBRCA2 FANCD2
FibroblastsHeLa Wild type R 1 1 �GM6914 A S � � �GM6914 � FANCA A R 1 1 �PD20 � Vec D2 S � � �PD20 � FANCD2 D2 R 1 1 �PD20 � K561R D2 S � � �EUFA423 D1 S 1c 111 �EUFA423 � BRCA2 D1 R 1 1 �
LymphoblastsEUFA130 E S � � �EUFA130 � HA-FANCE E R 1 1 �
a R, resistant; S, sensitive.b1, increase; �, no change.c The C terminus-truncated BRCA2 in EUFA423 (FA-D1) cells was present in both cytoplasm and nucleus.
VOL. 24, 2004 FANCD2-Ub INTERACTS WITH BRCA2 IN CHROMATIN 5855
Dow
nloa
ded
from
http
s://j
ourn
als.
asm
.org
/jour
nal/m
cb o
n 17
Feb
ruar
y 20
22 b
y 22
2.10
3.22
.67.
FIG. 3. FANCD2 and BRCA2 foci fail to associate in FA-D1 (BRCA2�/�) cells. (A) Schematic diagram of human BRCA2 protein, indicatingmutations in EUFA 423 (FA-D1) cells. The positions of epitopes for the anti-BRCA2 antibodies Ab-1 and Ab-2 are shown. (B) EUFA423 (FA-D1)fibroblasts and corrected cells stably transfected with human chromosome 13 (EUFA423 � BRCA2) were exposed to IR, either at different doses(left) or for different periods of time (right). Western blotting was performed with anti-BRCA2 (Ab-1 or Ab-2) or anti-FANCD2 (E35) antibodies.(C) Immunofluorescent localization of BRCA2 and FANCD2 following treatment with IR (15 Gy) was examined in EUFA423 (FA-D1)fibroblasts, EUFA423 fibroblasts transiently transfected with pcDNA3 HA-BRCA2 (EUFA423 � HA-BRCA2), and EUFA 423 fibroblasts stablytransfected with human chromosome 13 (EUFA423 � BRCA2). Cells were double stained with the indicated antibodies and analyzed byimmunofluorescence microscopy. Magnification, �630. (D and E) Quantification of the percentage of cells with FANCD2 foci (D) and thepercentage of cells with colocalization of FANCD2 and BRCA2 foci (E) in HeLa cells, EUFA423 fibroblasts, EUFA423 fibroblasts transientlyexpressing HA-BRCA2 (EUFA423 � HA-BRCA2), and EUFA423 fibroblasts stably expressing human chromosome 13 (EUFA423 � BRCA2).Cells were either untreated or treated with IR (15 Gy) and fixed 4 h later for immunofluorescence microscopy. Cells with more than four distinctfoci were counted as positive. Two hundred and 100 cells/sample were analyzed in panels D and E, respectively. The values shown are the mean� standard deviation from three separate experiments. (F) S2 (soluble nuclear proteins) and S4 (chromatin) fractions of irradiated EUFA423 cellsand EUFA423 cells stably expressing human chromosome 13 (EUFA423 � BRCA2) were subjected to immunoprecipitation with rabbit polyclonalanti-FANCD2 antibody (E35) or a control nonimmunized rabbit serum (Pre-imm) and then immunoblotted with either monoclonal anti-BRCA2(Ab-1) or anti-FANCD2 (FI-17) antibodies. Heavy chain IgG was used as a loading control. WCE, whole-cell extract.
5856
Dow
nloa
ded
from
http
s://j
ourn
als.
asm
.org
/jour
nal/m
cb o
n 17
Feb
ruar
y 20
22 b
y 22
2.10
3.22
.67.
Monoubiquitination of FANCD2 promotes IR-inducible ac-cumulation of BRCA2 in chromatin. We next fractionated cellsfrom multiple FA subtypes (Fig. 5) and analyzed the recruit-ment of BRCA2 and FANCD2-L to chromatin. In U2OS cells,which have a functional FA pathway, IR activated the recruit-ment of monoubiquitinated FANCD2 and BRCA2 to chroma-tin (Fig. 5A, compare lanes 4 and 6). In FA-A cells and FA-Bcells, no monoubiquitinated FANCD2 was observed (lanes 10and 21), and there was no increase of BRCA2 in chromatin(S4) fractions following IR (compare lanes 8 and 10 and 19 and21 for FA-A and FA-B cells, respectively).
FA-D2 cells were also defective in IR-inducible loading ofBRCA2 onto chromatin (Fig. 5B, compare lanes 4 and 6).
Wild-type FANCD2 (PD20F � HA-FANCD2), but not thenonubiquitinated mutant of FANCD2 (PD20F � HA-K561R),corrected IR-inducible BRCA2 chromatin loading (comparelanes 8 and 10 and 12 and 14 for PD20 cells expressing wild-type HA-FANCD2 and the FANCD2-K561R mutant HA-K561R, respectively). These results confirm that monoubiquiti-nated FANCD2 promotes the loading of BRCA2 ontodamaged chromatin. Failure to load BRCA2 onto damagedchromatin is a common feature of all FA subtypes examined inthis study (FA subtypes A, B, D1, D2, and E) and was consis-tently seen in different experiments in this study.
Even though FANCD2 is strongly monoubiquitinated inFA-D1 cells and efficiently transported to chromatin (Fig. 5A,
FIG. 3—Continued.
FIG. 4. FA-D1 and FA-D2 cells are defective in the assembly of IR-inducible RAD51 foci. (A) Quantification of RAD51 foci in EUFA423(FA-D1) fibroblasts, EUFA423 fibroblasts stably transfected with human chromosome 13 (EUFA423 � BRCA2), and HeLa cells. Cells were eitheruntreated or treated with IR (2 or 15 Gy) and fixed 15 h later. Cells were stained with monoclonal anti-RAD51 antibody and analyzed byimmunofluorescence microscopy. Cells with more than four distinct foci were counted as positive. Two hundred cells/sample were analyzed. Thevalues shown are the mean � standard deviation from three separate experiments. (B) Quantification of RAD51 foci in PD20 (FA-D2) fibroblastsstably expressing empty vector alone (PD20F � Vec), full-length FANCD2 cDNA (PD20F � FANCD2), or the FANCD2 K561R mutant (PD20F� K561R), and HeLa cells. Cells were either untreated or treated with IR (2 or 15 Gy) and fixed 8 h later. Immunofluorescence microscopy andcounts were performed as described above for panel A.
VOL. 24, 2004 FANCD2-Ub INTERACTS WITH BRCA2 IN CHROMATIN 5857
Dow
nloa
ded
from
http
s://j
ourn
als.
asm
.org
/jour
nal/m
cb o
n 17
Feb
ruar
y 20
22 b
y 22
2.10
3.22
.67.
compare lanes 12 and 14), these cells exhibited decreasedcolocalization of BRCA2 and FANCD2 foci (Fig. 3E) andwere deficient in the interaction of monoubiquitinatedFANCD2 and BRCA2 in chromatin (Fig. 3F). Therefore, themonoubiquitination and targeting of FANCD2 onto chromatinoccur independently of the carboxy terminus of BRCA2. Thecarboxy terminus of BRCA2 is required, however, for the ef-ficient interaction of BRCA2 and FANCD2 in chromatin.
IR activates the ATM-dependent phosphorylation ofBRCA2, resulting in activation of an intra-S-phase checkpoint.BRCA2 has multiple SQ phosphorylation consensus sites andmay therefore be a direct substrate of ATM or ATR (1). Giventhat BRCA2 in chromatin migrates more slowly following IR(Fig. 1D, compare lanes 7 and 8), we tested whether thiselectrophoretic change represents an IR-dependent phosphor-ylation of chromatin-derived BRCA2 (Fig. 6A). Irradiationactivated an upward shift of BRCA2 in chromatin (S4) frac-
tions (lane 4), and in vitro phosphatase treatment reversed thisshift (lane 5). Taken together, these results indicate that IRactivates the phosphorylation of BRCA2 and that phosphory-lated BRCA2 accumulates in the chromatin of IR-damagedcells. To test whether ATM is involved in this process, weexamined ATM�/� fibroblasts or ATM cDNA-corrected cells(Fig. 6B). IR failed to activate BRCA2 phosphorylation inchromatin in AT (ATM�/�) cells (compare lanes 3 to 5),suggesting that ATM may be the kinase for this reaction.Functional complementation of these cells with the ATMcDNA restored the IR-dependent phosphorylation of BRCA2(Fig. 6B, compare lanes 6 to 8). Also, IR activated the associ-ation of ATM protein with the S4 (chromatin) fraction, con-sistent with previous studies (2).
The IR-activated, ATM-dependent phosphorylation of sev-eral proteins, including NBS1 (10), BRCA1 (5), and FANCD2(25), triggers an intra-S-phase checkpoint. Cells deficient in
FIG. 5. Monoubiquitination of FANCD2 promotes IR-inducible accumulation of BRCA2 in chromatin. (A) S2 (soluble nuclear proteins) andS4 (chromatin) fractions were prepared from U2OS and FA cells, which were either untreated or treated with IR (15 Gy, fractionated after 15 h).Fractions were subjected to Western blot analysis with anti-FANCD2 (E35) or anti-BRCA2 (Ab-1) antibodies. The extraction of chromatin in theS4 fraction was confirmed by blotting with anti-histone H4 antibody, as indicated. The FA cells are represented by GM6914 (FA-A) fibroblasts,EUFA423 (FA-D1) fibroblasts, and HSC230 (FA-B) lymphoblasts. (B) S2 (soluble nuclear proteins) and S4 (chromatin) fractions were preparedfrom PD20 (FA-D2) fibroblasts stably expressing empty vector alone (PD20F � HA-PMMP), HA-FANCD2 (PD20F � HA-FANCD2), orFANCD2-HA-K561R mutant (PD20F � HA-K561R), which were either untreated or treated with IR (15 Gy, fractionated after 6 h). Fractionswere subjected to Western blot analysis with anti-FANCD2 (E35) or anti-BRCA2 (Ab-1) antibodies. The extraction of chromatin in the S4 fractionwas confirmed by blotting with anti-histone H4 antibody.
5858 WANG ET AL. MOL. CELL. BIOL.
Dow
nloa
ded
from
http
s://j
ourn
als.
asm
.org
/jour
nal/m
cb o
n 17
Feb
ruar
y 20
22 b
y 22
2.10
3.22
.67.
any of these ATM substrates lack this checkpoint and exhibitRDS. Interestingly, EUFA423 (FA-D1) cells have a defect inthe intra-S checkpoint response (Fig. 6C), similar to the defectobserved in FA-D2 (PD20F) cells (25). Correction ofEUFA423 with wild-type BRCA2 restored the intra-S check-point, further indicating that BRCA2 is an ATM substrate thatparticipates in the intra-S checkpoint. We have previously re-ported that while the K561 mutant of FANCD2 is still func-tional for the intra-S checkpoint in PD20 cells, it is associatedwith sensitivity to MMC (25). Thus, the interaction betweenmonoubiquitinated FANCD2 and BRCA2 in chromatin ap-pears to be required for establishing the MMC resistance ofcells; however, this interaction is not required for establish-ment of the S-phase checkpoint.
Interaction of BRCA2 with other FA proteins. Recent stud-ies have indicated that the FANCD2 protein binds directly toFANCE, a subunit of the FA complex (A/C/E/F/G/L complex)(22). We next determined whether FANCE is present inBRCA2 and FANCD2 immune complexes (Fig. 7). An anti-
body to BRCA2 coimmunoprecipitated FANCE from eitheruntreated or DNA-damaged HeLa whole-cell extracts (Fig.7A). As expected, BRCA2 failed to coimmunoprecipitate withFANCE from whole-cell extracts of FA-E (FANCE deficient)lymphoblasts (Fig. 7B, lanes 1 and 2). Coimmunoprecipitationof BRCA2 and HA-FANCE was observed after functionalcomplementation with the HA-FANCE cDNA (Fig. 7B, lanes4 and 5). Moreover, HA-FANCE complementation of FA-Ecells promoted the monoubiquitination of FANCD2 and thestability of BRCA2 (Fig. 7C, lanes 8 to 12). BRCA2, FANCD2-Ub, and FANCE coimmunoprecipitated from S4 (chromatin)fractions (Fig. 7D). These results suggest that BRCA2 inter-acts, either directly or indirectly, with FANCE and perhapsother components of the FA pathway.
DISCUSSION
Interaction of BRCA2 and FANCD2 in a common DNArepair pathway. Our results demonstrate that BRCA2 is in-
FIG. 6. ATM is required for IR-activated phosphorylation of BRCA2 in chromatin. (A) BRCA2 immunoprecipitated (Ab-1) from chromatinfractions was treated either with or without �-phosphatase and phosphatase inhibitors, as indicated. Chromatin fractions were derived either fromuntreated U2OS cells or 15 h following IR treatment (15 Gy). The mobility shift of BRCA2 is shown in lanes 4 and 6. WCE, whole-cell extract.(B) BRCA2 derived from the chromatin fraction of corrected AT (AT � ATM) fibroblasts at time points after treatment with IR (15 Gy)undergoes a mobility shift, while BRCA2 from the chromatin fraction of AT (ATM�/�) fibroblasts does not. Chromatin fractions wereimmunoblotted with either anti-BRCA2 (Ab-1), anti-ATM, or anti-FANCD2 (E35) antibodies. (C) EUFA423 (FA-D1) and PD20 (FA-D2) cellsare defective in an IR-inducible S-phase checkpoint. RDS was assessed 30 min after delivery of IR to the indicated cell lines.
VOL. 24, 2004 FANCD2-Ub INTERACTS WITH BRCA2 IN CHROMATIN 5859
Dow
nloa
ded
from
http
s://j
ourn
als.
asm
.org
/jour
nal/m
cb o
n 17
Feb
ruar
y 20
22 b
y 22
2.10
3.22
.67.
FIG. 7. Interaction of FANCE and BRCA2. (A) Whole-cell ex-tracts (WCE) from HeLa cells, either untreated or exposed to IR (15Gy, harvested 15 h later) or MMC (40 ng/ml, harvested 24 h later),were subjected to immunoprecipitation with mouse monoclonal anti-BRCA2 antibody (Ab-1) or a control mouse antibody (mIgG). Heavychain IgG was used as a loading control. (B) Whole-cell extracts fromunirradiated or irradiated EUFA130 (FA-E), and EUFA130 cells sta-bly expressing HA-FANCE (EUFA130 � HA-FANCE), were sub-jected to immunoprecipitation with polyclonal anti-BRCA2 antibody(H-300) or a control rabbit antibody (rIgG) and then immunoblottedwith anti-BRCA2 (Ab-1) or anti-HA antibodies. Heavy chain IgG wasused as a loading control. (C) EUFA130 (FA-E) lymphoblasts andEUFA130 cells stably expressing HA-FANCE (EUFA130 � HA-FANCE) were either untreated or exposed to IR at different doses, asindicated, and harvested after 4 h. Western blotting was performedwith anti-BRCA2 (Ab-1), anti-FANCD2 (E35), or anti-HA antibodies.(D) Reciprocal coimmunoprecipitation of FANCD2 and BRCA2 withother antibodies. The indicated fractions (S2 and S4), prepared fromirradiated U2OS cells, were immunoprecipitated with antibody toFANCD2 (E35), FANCE, or a control nonimmunized rabbit serum(Pre-imm), and the immune complexes were immunoblotted with anti-BRCA2 (Ab-1) or anti-FANCD2 (FI-17) antibodies. Heavy chain IgGwas used as a loading control. (E) Schematic model of the ATM-BRCA2-FANCD2-mediated DNA damage response. IR activatesATM, resulting in the phosphorylation of BRCA2, FANCD2, andseveral other protein substrates. Activated BRCA2 is then recruited tochromatin by FANCD2, which is activated by monoubiquitination. Theinteraction of BRCA2 and FANCD2 requires both the C terminus ofBRCA2 and FANCD2 monoubiquitination. Following its loading ontochromatin, BRCA2 then functions downstream of monubiquitinatedFANCD2.
5860 WANG ET AL. MOL. CELL. BIOL.
Dow
nloa
ded
from
http
s://j
ourn
als.
asm
.org
/jour
nal/m
cb o
n 17
Feb
ruar
y 20
22 b
y 22
2.10
3.22
.67.
deed a protein component of the FA pathway and that itfunctions downstream in this pathway. BRCA2 colocalizes,cofractionates, and coimmunoprecipitates with monoubiquiti-nated FANCD2 from chromatin. According to one possiblemodel (Fig. 7E), DNA damage by IR activates the FA com-plex-dependent monoubiquitination of FANCD2, resulting inits targeting to chromatin. Monoubiquitinated FANCD2 inchromatin then promotes the loading of BRCA2 onto chro-matin. The carboxy-terminal 192 amino acids of BRCA2 arerequired for the functional interaction of BRCA2 andFANCD2 in nuclear foci. Whether FANCD2-L binds toBRCA2 directly, or indirectly via other protein intermediates,remains unknown. Recent studies using the two-hybrid systemsuggest a direct interaction between BRCA2 and FANCD2(C. Mathew, personal communication). Importantly, loss ofthe interaction between monoubiquitinated FANCD2 andBRCA2 in chromatin is a common feature of other FA sub-types. This defect correlates with the chromosome instabilityand abnormal homology-directed DNA repair of FA cells (30).
Our results are consistent with those of Bruun et al. (3),which suggest that BRCA2 is epistatic with other FA genes andfunctions downstream in the pathway. According to that study,disruption of BRCA2 expression by small inhibitory RNA didnot further increase MMC-induced chromosome breakage inFA-A cells.
While it has been recently reported that BRCA2 interactswith the FA protein, FANCG, by two-hybrid analysis (14), herewe present the first direct evidence of an intracellular interac-tion of endogenous levels of BRCA2 with an upstream FAprotein, FANCE, and with downstream monoubiquitinatedFANCD2. The interaction of FANCE with BRCA2 appears tobe independent of FANCD2, since it occurs in PD20 (FA-D2)cells lacking FANCD2 and in EUFA423 (FA-D1) fibroblasts(data not shown), in which FANCD2 does not bind to BRCA2(Fig. 3F). This may be of functional significance for BRCA2loading onto chromatin. FANCE binds to both FANCD2 andFANCC (22) and thus may link FANCD2 to the machinery formonoubiquitination. As such, the constitutive interaction ofFANCE with BRCA2 may facilitate the targeting of BRCA2 tochromatin. The finding that BRCA2, like FANCD2, is phos-phorylated in chromatin in an ATM-dependent manner mayalso be of significance to the regulation of BRCA2 loadingonto chromatin (Fig. 6). Whether ATM-phosphorylatedFANCD2 and BRCA2 cooperate in the S-phase checkpointresponse remains unknown.
Possible functions of FANCD2-L in homology-directed DNArepair. The function of the chromatin complex of monoubiq-uitinated FANCD2 (FANCD2-L) and BRCA2 remains un-known, but increasing evidence suggests that the complex mayplay a role in HDR. While the requirement for BRCA2 inHDR is well known (7, 17, 21, 31), the function of FANCD2-Lis less clear. FANCD2-L may be required for the proper pair-ing of homologous chromosomes (or homologous regions ofsister chromatids), thus allowing BRCA2 to unload RAD51 atappropriate sites (31). Consistent with this view, meiotic cellsisolated from Fancd2�/� mice exhibit increased chromosomemispairing (12). In support of such a role for FANCD2-L,FANCD2 colocalizes with RAD51 during S phase (24). Alter-natively, FANCD2-L may contribute to the efficiency or timingof repair, but not to the actual strand invasion or processing of
Holliday junctions. Accordingly, FA-G cells, which lack mo-noubiquitinated FANCD2, have only a mild defect in DNArepair (30) compared to the more profound eightfold deficitobserved in BRCA2�/� cells (21).
FA cells are specifically impaired in the repair of interstrandcross-links. Cross-link repair requires the generation of a dou-ble-strand break (DSB) intermediate and subsequent HDRactivity. The activated (monoubiquitinated) FANCD2 proteinis therefore likely to contribute to the sensing of the DNAcross-link or the processing of the cross-link to a DSB. Theinteraction of FANCD2-L with BRCA2/RAD51 complexessuggests that FANCD2-L may either (i) redirect the BRCA2/RAD51 complexes to specific sites of cross-link lesions or (ii)enable the BRCA2/RAD51 complexes to process and repairsuch lesions. How the chromatin-associated FANCD2-L en-ables BRCA2 to repair DNA cross-links remains a centralunanswered question in FA research.
Germ line or somatic disruption of the FA/BRCA pathway incancer. The interaction of the breast/ovarian cancer suscepti-bility gene BRCA2/FANCD1 in a common pathway with otherFANC genes suggests that inherited or acquired defects in thispathway may result in cancer in the general (non-FA) popu-lation. Several lines of evidence support this notion. First,epigenetic inactivation of FANCF (26) accounts for the chro-mosome instability and cisplatin hypersensitivity of a subset ofovarian tumors. Second, germ line mutations in BRCA2,FANCC, or FANCG are found in individuals with inheritedpancreatic cancer (29). Third, germ line disruption of the mu-rine Fancd2 gene results in mice with ovarian and breast epi-thelial cancers (12). We predict that inherited (germ line)mutations or polymorphisms in other FANC genes may ac-count for an increased cancer risk of individuals in the general(non-FA) population. The actual cancer risk and tumor spec-trum may depend on the presence of specific mutant FA al-leles.
ACKNOWLEDGMENTS
We thank M. Buchwald for the primary FA-B lymphoblast line,HSC230. We thank G. Bagby for the anti-FANCE antibody. We thankL. Moreau for chromosome breakage analysis.
This work was supported by National Institutes of Health grantsRO1HL52725, RO1DK43889, and PO1HL54785 (A.D.D.). P.R.A. is aSpecial Fellow of the Leukemia and Lymphoma Society.
REFERENCES
1. Abraham, R. T. 2001. Cell cycle checkpoint signaling through the ATM andATR kinases. Genes Dev. 15:2177–2196.
2. Andegeko, Y., L. Moyal, L. Mittelman, I. Tsarfaty, Y. Shiloh, and G. Rotman.2001. Nuclear retention of ATM at sites of DNA double strand breaks.J. Biol. Chem. 276:38224–38230.
3. Bruun, D., A. Folias, Y. M. Akkari, Y. Cox, S. Olson, and R. Moses. 2003.siRNA depletion of BRCA1, but not BRCA2, causes increased genomeinstability in Fanconi anemia cells. DNA Repair 2:1007–1013.
4. Chen, P. L., C. F. Chen, Y. Chen, J. Xiao, Z. D. Sharp, and W. H. Lee. 1998.The BRC repeats in BRCA2 are critical for RAD51 binding and resistanceto methyl methanesulfonate treatment. Proc. Natl. Acad. Sci. USA 95:5287–5292.
5. Cortez, D., Y. Wang, J. Qin, and S. J. Elledge. 1999. Requirement of ATM-dependent phosphorylation of brca1 in the DNA damage response to dou-ble-strand breaks. Science 286:1162–1166.
6. D’Andrea, A. D., and M. Grompe. 2003. The Fanconi anaemia/BRCA path-way. Nat. Rev. Cancer 3:23–34.
7. Davies, A. A., J. Y. Masson, M. J. McIlwraith, A. Z. Stasiak, A. Stasiak, A. R.Venkitaraman, and S. C. West. 2001. Role of BRCA2 in control of theRAD51 recombination and DNA repair protein. Mol. Cell 7:273–282.
8. Garcia-Higuera, I., Y. Kuang, D. Naf, J. Wasik, and A. D. D’Andrea. 1999.
VOL. 24, 2004 FANCD2-Ub INTERACTS WITH BRCA2 IN CHROMATIN 5861
Dow
nloa
ded
from
http
s://j
ourn
als.
asm
.org
/jour
nal/m
cb o
n 17
Feb
ruar
y 20
22 b
y 22
2.10
3.22
.67.
Fanconi anemia proteins FANCA, FANCC, and FANCG/XRCC9 interactin a functional nuclear complex. Mol. Cell. Biol. 19:4866–4873.
9. Garcia-Higuera, I., T. Taniguchi, S. Ganesan, M. S. Meyn, C. Timmers,J. Hejna, M. Grompe, and A. D. D’Andrea. 2001. Interaction of the Fanconianemia proteins and BRCA1 in a common pathway. Mol. Cell 7:249–262.
10. Gatei, M., D. Young, K. M. Cerosaletti, A. Desai-Mehta, K. Spring, S.Kozlov, M. F. Lavin, R. A. Gatti, P. Concannon, and K. Khanna. 2000.ATM-dependent phosphorylation of nibrin in response to radiation expo-sure. Nat. Genet. 25:115–119.
11. Hirsch, B., A. Shimamura, L. Moreau, S. Baldinger, M. Hag-Alshiekh, B.Bostrom, S. Sencer, A. D. D’Andrea. 2004. Association of biallelic BRCA2/FANCD1 mutations with spontaneous chromosomal instability and solidtumors of childhood. Blood 103:2554–2559.
12. Houghtaling, S., C. Timmers, M. Noll, M. J. Finegold, S. N. Jones, M. S.Meyn, and M. Grompe. 2003. Epithelial cancer in Fanconi anemia comple-mentation group D2 (Fancd2) knockout mice. Genes Dev. 17:2021–2035.
13. Howlett, N. G., T. Taniguchi, S. Olson, B. Cox, Q. Waisfisz, C. De Die-Smulders, N. Persky, M. Grompe, H. Joenje, G. Pals, H. Ikeda, E. A. Fox,and A. D. D’Andrea. 2002. Biallelic inactivation of BRCA2 in Fanconi ane-mia. Science 297:606–609.
14. Hussain, S., E. Witt, P. A. Huber, A. L. Medhurst, A. Ashworth, and C. G.Mathew. 2003. Direct interaction of the Fanconi anaemia protein FANCGwith BRCA2/FANCD1. Hum. Mol. Genet. 12:2503–2510.
15. Joenje, H., and K. J. Patel. 2001. The emerging genetic and molecular basisof Fanconi anaemia. Nat. Rev. Genet. 2:446–457.
16. Lomonosov, M., S. Anand, M. Sangrithi, R. Davies, A. R. Venkitaraman.2004. Stabilization of stalled DNA replication forks by the BRCA2 breastcancer susceptibility protein. Genes Dev. 17:3017–3022.
17. Marmorstein, L. Y., T. Ouchi, and S. A. Aaronson. 1998. The BRCA2 geneproduct functionally interacts with p53 and RAD51. Proc. Natl. Acad. Sci.USA 95:13869–13874.
18. Medhurst, A. L., P. A. Huber, Q. Waisfisz, J. P. de Winter, and C. G.Mathew. 2001. Direct interactions of the five known Fanconi anaemia pro-teins suggest a common functional pathway. Hum. Mol. Genet. 10:423–429.
19. Meetei, A. R., S. Sechi, M. Wallisch, D. Yang, M. K. Young, H. Joenje, M. E.Hoatlin, and W. Wang. 2003. A multiprotein nuclear complex connectsFanconi anemia and Bloom syndrome. Mol. Cell. Biol. 23:3417–3426.
20. Morgan, S. E., C. Lovly, T. K. Pandita, Y. Shiloh, and M. B. Kastan. 1997.Fragments of ATM which have dominant-negative or complementing activ-ity. Mol. Cell. Biol. 17:2020–2029.
21. Moynahan, M. E., A. J. Pierce, and M. Jasin. 2001. BRCA2 is required forhomology-directed repair of chromosomal breaks. Mol. Cell 7:263–272.
22. Pace, P., M. Johnson, W. M. Tan, G. Mosedale, C. Sng, M. Hoatlin, J. de
Winter, H. Joenje, F. Gergely, and K. J. Patel. 2002. FANCE: the linkbetween Fanconi anaemia complex assembly and activity. EMBO J. 21:3414–3423.
23. Taniguchi, T., and A. D. D’Andrea. 2002. The Fanconi anemia protein,FANCE, promotes the nuclear accumulation of FANCC. Blood 100:2457–2462.
24. Taniguchi, T., I. Garcia-Higuera, P. R. Andreassen, R. C. Gregory, M.Grompe, and A. D. D’Andrea. 2002. S-phase-specific interaction of the Fan-coni anemia protein, FANCD2, with BRCA1 and RAD51. Blood 100:2414–2420.
25. Taniguchi, T., I. Garcia-Higuera, B. Xu, P. R. Andreassen, R. C. Gregory,S. T. Kim, W. S. Lane, M. B. Kastan, and A. D. D’Andrea. 2002. Convergenceof the Fanconi anemia and ataxia telangiectasia signaling pathways. Cell109:459–472.
26. Taniguchi, T., M. Tischkowitz, N. Ameziane, S. V. Hodgson, C. G. Mathew,H. Joenje, S. C. Mok, and A. D. D’Andrea. 2003. Disruption of the Fanconianemia-BRCA pathway in cisplatin-sensitive ovarian tumors. Nat. Med.9:568–574.
27. Tutt, A., D. Bertwistle, J. Valentine, A. Gabriel, S. Swift, G. Ross, C. Griffin,J. Thacker, and A. Ashworth. 2001. Mutation in Brca2 stimulates error-prone homology-directed repair of DNA double-strand breaks occurringbetween repeated sequences. EMBO J. 20:4704–4716.
28. Vandenberg, C. J., F. Gergely, C. Y. Ong, P. Pace, D. L. Mallery, K. Hiom,and K. J. Patel. 2003. BRCA1-independent ubiquitination of FANCD2.Mol. Cell 12:247–254.
29. Van Der Heijden, M. S., C. J. Yeo, R. H. Hruban, and S. E. Kern. 2003.Fanconi anemia gene mutations in young-onset pancreatic cancer. CancerRes. 63:2585–2588.
30. Yamamoto, K., M. Ishiai, N. Matsushita, H. Arakawa, J. E. Lamerdin, J.-M.Buerstedde, M. Tanimoto, M. Harada, L. H. Thompson, and M. Takata.2003. Fanconi anemia FANCG protein in mitigating radiation- and enzyme-induced DNA double-strand breaks by homologous recombination in verte-brate cells. Mol. Cell. Biol. 23:5421–5430.
31. Yang, H., P. D. Jeffrey, J. Miller, E. Kinnucan, Y. Sun, N. H. Thoma, N.Zheng, P. L. Chen, W. H. Lee, and N. P. Pavletich. 2002. BRCA2 function inDNA binding and recombination from a BRCA2-DSS1-ssDNA structure.Science 297:1837–1848.
32. Yuan, S. S., S. Y. Lee, G. Chen, M. Song, G. E. Tomlinson, and E. Y. Lee.1999. BRCA2 is required for ionizing radiation-induced assembly of Rad51complex in vivo. Cancer Res. 59:3547–3551.
33. Ziv, Y., A. Bar-Shira, I. Pecker, P. Russell, T. J. Jorgensen, I. Tsarfati, andY. Shiloh. 1997. Recombinant ATM protein complements the cellular A-Tphenotype. Oncogene 15:159–167.
5862 WANG ET AL. MOL. CELL. BIOL.
Dow
nloa
ded
from
http
s://j
ourn
als.
asm
.org
/jour
nal/m
cb o
n 17
Feb
ruar
y 20
22 b
y 22
2.10
3.22
.67.
