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HEMATOPOIESIS
Regulated interaction of the Fanconi anemia protein, FANCD2, with chromatin
Rocio Montes de Oca, Paul R. Andreassen, Steven P. Margossian, Richard C. Gregory, Toshiyasu Taniguchi, XiaoZhe Wang,
Scott Houghtaling, Markus Grompe, and Alan D. D’Andrea
DNA damage activates the monoubiquitination of the Fanconi anemia (FA) protein, FANCD2, resulting in the assembly
of FANCD2 nuclear foci. In the current
study, we characterize structural features
of FANCD2 required for this intranuclear
translocation. We have previously identified 2 normal mRNA splice variants of
FANCD2, one containing exon 44 sequence at the 3ⴕ end (FANCD2-44) and
one containing exon 43 sequence
(FANCD2-43). The 2 predicted FANCD2
proteins differ in their carboxy terminal
24 amino acids. In stably transfected
FANCD2ⴚ/ⴚ fibroblasts, FANCD2-44 and
FANCD2-43 proteins were monoubiquiti-
nated on K561. Only FANCD2-44 corrected the mitomycin C (MMC) sensitivity
of the transfected cells. We find that monoubiquitinated FANCD2-44 was translocated from the soluble nuclear compartment into chromatin. A mutant form of
FANCD2-44 (FANCD2-K561R) was not
monoubiquitinated and failed to bind
chromatin. A truncated FANCD2 protein
(Exon44-T), lacking the carboxy terminal
24 amino acids encoded by exon 44 but
retaining K561, and another mutant
FANCD2 protein, with a single amino acid
substitution at a conserved residue within
the C-terminal 24 amino acids (D1428A),
were monoubiquitinated. Both mutants
were targeted to chromatin but failed to
correct MMC sensitivity. Taken together,
our results indicate that monoubiquitination of FANCD2 regulates chromatin binding and that D1428 within the carboxy
terminal acidic sequence encoded by
exon 44 is independently required for
functional complementation of FA-D2
cells. We hypothesize that the carboxy
terminus of FANCD2-44 plays a critical
role in sensing or repairing DNA damage.
(Blood. 2005;105:1003-1009)
© 2005 by The American Society of Hematology
Introduction
Fanconi anemia (FA) is a rare autosomal recessive cancer susceptibility
syndrome characterized by developmental abnormalities, progressive
bone marrow failure, and cellular hypersensitivity to DNA cross-linking
agents.1 Eleven FA complementation groups have been identified (A, B,
C, D1, D2, E, F, G, I, J, and L)2,3 and 8 FA genes have been cloned.2,4,5
The FANCD1 gene is identical to the breast cancer susceptibility gene,
BRCA2.6 The 8 encoded FA proteins (A, C, D1, D2, E, F, G, L)
cooperate in a common cellular pathway, the FA/BRCA pathway.7
In this pathway, 6 of the FA proteins (A, C, E, F, G, L)8,9 bind
in a constitutive nuclear protein complex (the FA complex). In
response to DNA damage10 or during the S phase of the cell
cycle,11 the FA complex promotes the monoubiquitination of the
downstream FANCD2 protein. This event requires a molecular
interaction between the FANCE and FANCD2 proteins.12,13
Monoubiquitination of FANCD2 is required for targeting of
FANCD2 into nuclear foci containing BRCA1, FANCD1/
BRCA2, and RAD51.11 These subnuclear foci may be sites of
homologous recombination-mediated DNA repair, given the
known roles of BRCA1, BRCA2, and RAD51 in this process.14,15 Disruption of the FA/BRCA pathway results in the
characteristic cellular and clinical features of FA, including
hypersensitivity to DNA cross-linking agents.16
A critical regulatory event in the FA/BRCA pathway is the
monoubiquitination of FANCD2 on Lysine 561.10 Analysis of
FANCD2 monoubiquitination provides a rapid diagnostic screen
for the integrity of the FA/BRCA pathway.17 In addition, FANCD2
undergoes an ionizing radiation (IR)–inducible, ataxia telangiectasia (ATM)–dependent phosphorylation on Serine 222.18 Phosphorylation of this serine is required for the establishment of an
intra–S-phase checkpoint response but is not required for FANCD2
monoubiquitination, FANCD2 targeting to foci, or FANCD2mediated DNA repair.
Little is known about the regulation or functional outcome of
FANCD2 monoubiquitination. First, the newly cloned FANCL
protein has a plant homeodomain (PHD) domain with E3 ubiquitin
ligase activity, although its ubiquitination of FANCD2 has not been
demonstrated in vitro.2 Interestingly, BRCA1, an 1863–amino acid
tumor suppressor protein with an amino terminal E3 RING (Really
interesting new gene) finger ubiquitin ligase domain,19 is required
for efficient FANCD2 activation following DNA damage, although
BRCA1-deficient cells still exhibit some FANCD2 monoubiquitination.20 BRCA1 and/or its heterodimeric E3 ligase partner, BRCA1associated ring domain 1 (BARD1),21 may therefore contribute
directly or indirectly to FANCD2 monoubiquitination. Second, the
From the Department of Radiation Oncology, Dana-Farber Cancer Institute,
Harvard Medical School, Boston, MA; Division of Hematology/Oncology,
Children’s Hospital of Boston, Boston, MA; and Department of Molecular and
Medical Genetics, Oregon Health Sciences University, Portland, OR.
the Amy Clare Potter Fund. T.T. is a Scholar Fellow of the American Society of
Hematology. R.C.G. is supported by the American Cancer Society.
Submitted November 21, 2003; accepted September 9, 2004. Prepublished
online as Blood First Edition Paper, September 28, 2004; DOI 10.1182/blood2003-11-3997.
Supported by National Institutes of Health grants RO1HL52725,
RO1DK43889, P01DK50654, and PO1HL54785 (A.D.D.). P.R.A. is a Special
Fellow of the Leukemia and Lymphoma Society. S.P.M. is supported in part by
BLOOD, 1 FEBRUARY 2005 䡠 VOLUME 105, NUMBER 3
Reprints: Alan D. D’Andrea, Dana-Farber Cancer Institute, Department of
Radiation Oncology, Harvard Medical School, 44 Binney St, Boston, MA 02115;
e-mail: [email protected].
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2005 by The American Society of Hematology
1003
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1004
MONTES DE OCA et al
monoubiquitinated isoform of FANCD2 (FANCD2-L) accumulates in discrete nuclear foci in damaged cells,10 suggesting that it is
actively transported to these structures. Accordingly, these foci
may contain a specific receptor for FANCD2-L or its ubiquitin
moiety. Third, following DNA repair or during the mitotic phase of
the cell cycle, FANCD2-L is deubiquitinated, suggesting a reversible and more complex mechanism of regulation.
The nucleus is organized into an integrated structure in which
chromatin is associated with a nonhistone scaffold termed the
nuclear matrix.22 Various aspects of nucleic acid metabolism,
including DNA replication, transcription, and the repair of UVinduced thymidine dimers, require an interaction between chromatin and the nuclear matrix. A previous study indicates that FA
proteins required for FANCD2 monoubiquitination are found in the
chromatin,23 and we recently reported that monoubiquitinated
FANCD2 interacts with the breast cancer susceptibility gene
product BRCA2/FANCD1 in chromatin.24
In the current study, we examined structural features of
FANCD2 required for monoubiquitination, nuclear foci assembly,
and functional activity in the FA/BRCA pathway. We identified a
conserved 24–amino acid carboxy terminal region of FANCD2
encoded by exon 44. This region is predicted to form a coiled-coil
structure and it is required for FANCD2 functional activity
downstream in the FA/BRCA pathway but not for monoubiquitination of FANCD2. Further, monoubiquitination, but not the Cterminal region of the protein, is required for FANCD2 localization
to chromatin.
Materials and methods
Cell culture
Epstein-Barr virus (EBV)–transformed lymphoblasts and adherent cells
(HeLa and PD20F lines) were maintained in RPMI and Dulbecco modified
Eagle medium (DMEM) media, respectively, supplemented with 15%
heat-inactivated fetal calf serum (FCS). Cells were grown in a humidified
5% CO2-containing atmosphere at 37°C. The FA-G lymphoblast line,
EUFA316, was provided by Dr Hans Joenje (Department of Clinical
Genetics and Human Genetics, VU University Medical Center, Amsterdam,
The Netherlands).4 The simian virus 40 (SV40)–transformed FA-D2
fibroblast, PD20F, expressing wild-type FANCD2 or FANCD2-K561R or
transfected with pMMP vector, has previously been described.5
BLOOD, 1 FEBRUARY 2005 䡠 VOLUME 105, NUMBER 3
first-strand cDNA using Superscript II RNase H(-) Reverse Transcriptase
(Invitrogen) and oligo(dT)15 (Invitrogen) as instructed by the manufacturer. Negative control reactions without reverse transcriptase were also
performed. The shared upstream primer for both exon 43 and exon 44
variants of FANCD2 was DF3862 (5⬘-CATCCTGTTCTGCATGTATG-3⬘).
The specific downstream primer for exon 43 variant was DR4462 (5⬘TCGTTGTTTCTTCTGGGTCC-3⬘) and the specific downstream primer
for exon 44 variant was DSR4360 (5⬘-GGGTCTAATCAGAGTCATCA3⬘). An aliquot (0.4 ␮L) of cDNA (40 ng RNA equivalent) was placed in 40
␮L of 1 ⫻ PCR buffer supplied with Taq polymerase, 200 ␮M of each
deoxyribonucleotide triphosphate (dNTP), 0.2 ␮M of each primer (DF3862,
DR4462, and DSR4360), and 2 U of AmpliTaq polymerase (Roche,
Indianapolis, IN). PCR was run for 30 cycles and each cycle constituted
denaturation (45 seconds at 94°C, first cycle 4 minutes 45 seconds),
annealing (1 minute at 55°C), and extension (1 minute at 72°C, last cycle 8
minutes). The PCR reaction (10 ␮L) was subjected to electrophoresis on a
1.2% agarose gel containing ethidium bromide. The expected sizes of the
PCR products are as follows: exon 43 form, 601 base pair (bp); exon 44
form, 499 bp.
MMC sensitivity assay
The mitomycin C (MMC) sensitivity assay was performed essentially as
previously described.26 Cells were passaged into 12-well plates at 6 ⫻ 103
cells per well. The following morning, a series of concentrations of MMC
was added and cells were cultured for 5 days. Cells were then washed with
phosphate-buffered saline (PBS) and fixed 10 minutes with 10% (vol/vol)
methanol/10% (vol/vol) acetic acid at room temperature. Cells were then
stained 10 minutes with 1% (wt/vol) crystal violet (Sigma-Aldrich, St.
Louis, MO) in methanol. The plates were washed in an excess volume of
dH2O, and absorbed dye was released by incubation with agitation for 1
hour at room temperature in methanol containing 0.1% sodium dodecyl
sulfate (SDS). Dye containing solution was then transferred to 96-well
microtiter plates, and dilutions (1:2) were prepared. The optical density
(OD) at 595 nm was then assayed in a microplate reader (BioRad model
3550; Hercules, CA). Results were normalized based upon the untreated
samples (0 ng/mL MMC).
Cell cycle synchronization
HeLa cells were synchronized by the double thymidine block method as
previously described, with minor modifications.27 Briefly, cells were treated
with 2 mM thymidine for 18 hours, thymidine-free media for 10 hours, and
additionally with 2 mM thymidine for 18 hours to arrest the cell cycle at
the G1/S boundary. Cells were washed twice with PBS, released in
DMEM ⫹15% FCS, and analyzed at various time intervals.
Retroviral infection of FA cell lines
Preparation of cellular fractions for immunoblotting
The indicated pMMP constructs were transfected with Fugene 6 by
lipofection into 293-GPG producer cells (human embryonic kidney cells)
expressing the vesicular stomatitis virus-G (VSV-G) envelope protein.25
Retroviral supernatants were collected on day 5 following lipofection, as
previously described.8 Retroviral supernatants contained 4.6 ⫻ 106 infectious units/mL, as estimated by Southern blot analysis of infected NIH-3T3
cells (data not shown).
FA fibroblasts were infected with the various pMMP supernatants by a
6- to 8-hour incubation in the presence of 8 ␮g/mL polybrene.8 Infected
cells were washed free of viral supernatant and resuspended in growth
media. After 48 hours, cells were transferred to media containing puromycin (1 ␮g/mL). Surviving cells were grown under continuous selection
in puromycin.
Cells were grown on 15-cm culture dishes. Cells were either left untreated
or were exposed to MMC (170 ng/mL) or IR (15 Gy), trypsinized 12 hours
later, and collected by centrifugation. Cells were washed once with cold
PBS and aliquoted equally into 4 eppendorf tubes and collected by
centrifugation in a Sorvall RT 6000D centrifuge (Kendro, Asheville, NC) at
4°C for 3 minutes at 300 g (1200 rpm). One pellet, representing the
whole-cell pellet (P1), was frozen in liquid nitrogen. Remaining pellets
were resuspended in cold buffer A (10 mM PIPES [piperazine diethanesulfonic acid], pH 7; 100 mM NaCl; 3 mM MgCl2; 1 mM EGTA [ethyleneglycotetraacetic acid]; 300 mM sucrose; 0.5 mM Na3VO4; 50 mM NaF; 10
␮g/mL aprotinin; 10 ␮g/mL leupeptin; 10 ␮g/mL pepstatin A; and 1 mM
PMSF [phenylmethylsulfonyl fluoride]) containing 0.5% Triton X-100 and
incubated at room temperature for 2 minutes to permeabilize cells. The
suspension was then recentrifuged at 4°C for 3 minutes at 300 g, and the
supernatant (S2), representing cytosol and nucleosol, was collected and
frozen. The pellet was washed with cold buffer A. One pellet, representing
detergent-insoluble nuclei (P2), was frozen. Nuclei were then digested with
RNase-free DNase I (200 U/mL) in buffer A for 30 minutes at room
temperature. The residual pellet was collected by centrifugation as described above. The supernatant (S3) was frozen, the pellet was washed with
RNA extraction and reverse transcription–polymerase chain
reaction (RT-PCR)
Total RNAs were extracted using Trizol reagent (Invitrogen, Carlsbad, CA),
measured by optic density, and stored at ⫺80°C. An aliquot (2 ␮g) of total
RNA was placed in 20 ␮L of room temperature reaction and converted to
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BLOOD, 1 FEBRUARY 2005 䡠 VOLUME 105, NUMBER 3
REGULATED INTERACTION OF FANCD2 WITH CHROMATIN
1005
cold buffer A, and one residual pellet (P3) was frozen. The remaining
residual pellet was extracted with cold buffer A containing 0.25 M
ammonium sulfate for 5 minutes at room temperature to extract remaining
chromatin. The residual pellet (P4), representing nuclear matrix, was
collected by centrifugation at 1877 g for 3 minutes and the supernatant (S4),
containing chromatin as identified by the presence of histones, was frozen.
The pellet was frozen following a single wash with cold buffer A and
centrifugation at 800 g for 3 minutes. Cell-equivalent volumes from each
cell fraction were separated by SDS–polyacrylamide gel electrophoresis
(PAGE) and immunoblotted for FANCD2, histone H4 (chromatin marker),
and lamin B (nuclear matrix marker).
Nuclear fractionation for immunofluorescence microscopy
HeLa cells were grown for 36 hours on glass coverslips (12-mm diameter)
coated with 0.1 mg/mL poly-L-lysine to promote adhesion. Cells were then
treated with 15 Gy IR for 12 hours. Fixation was at room temperature with
2% paraformaldehyde in PBS. Cells were fixed either prior to any
fractionation (equivalent of P1), following permeabilization for 2 minutes
at room temperature with buffer A containing 0.5% Triton X-100 (equivalent of P2), or following permeabilization and sequential treatment with 200
U/mL DNase I for 30 minutes in buffer A and then 250 mM ammonium
sulfate for 5 minutes in buffer A (equivalent of P4). Following fixation, cells
were washed with PBS. Cells that had not been previously permeabilized
were treated 3 minutes with 0.2% Triton X-100 in PBS. Incubation with
primary and secondary antibodies and washes were as previously described.10 The nuclear matrix was detected with a goat antibody to lamin B
(M-20; Santa Cruz Biotechnology, Santa Cruz, CA).
Immunofluorescence microscopy
Immunofluorescence microscopy of human fibroblasts was performed as
previously described,10 with modifications for fractionation as described in
the preceding section. In brief, cells were pre-permeabilized with 0.25%
Triton X-100 in PBS for 1 minute on ice and then fixed with 4%
paraformaldehyde in PBS for 15 minutes, followed by permeabilization
with 0.5% Triton X-100 in PBS for 1 minute. For immunofluorescence
microscopy, fixed cells were incubated with specific primary antibodies at
the appropriate dilution in 3% bovine serum albumin-PBS for 1 hour at
room temperature (RT). Phosphorylated histone H2AX (Ser 139) was
detected with rabbit polyclonal antibodies (Trevigen, Gaithersburg, MD)
diluted 200-fold. For colocalization with H2AX, FANCD2 was detected
with mouse monoclonal antibody (FI-17; Santa Cruz Biotechnology)
diluted 200-fold. Cells were subsequently washed 3 times in PBS and
incubated for 1 hour 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 Laboratories). Images
were acquired using an Axioplan 2 imaging microscope (Carl Zeiss,
Thornhill, NY), at 100 ⫻/1.4 NA (Figure 3) or 60 ⫻/1.4 NA (Figure 5),
with a Hamamatsu Orca CCD camera (Bridgewater, NJ). Images were
processed with Openlab software (Improvision, Lexington, MA).
Results
Two splice variants of FANCD2
We previously identified 2 normal mRNA splice variants of
FANCD2 that differ in their 3⬘ coding sequence (GenBank accession no. AF273251), as shown schematically in Figure 1A. One
splice variant (FANCD2-43), containing exon 43 sequence, is
predicted to encode a FANCD2 protein of 1471 amino acids (157
kDa; Figure 1A). The second splice variant (FANCD2-44), containing exon 44 sequence, is predicted to encode a FANCD2 protein of
1451 amino acids (155 kDa). Both splice variants contain the
Figure 1. Expression of 2 splice variants of the FANCD2 gene. (A) Schematic
representation of 2 splice variants of FANCD2. A 24–amino acid (aa) sequence,
containing 50% acidic residues, is encoded by exon 44 sequence. (B) Detection of
FANCD2-44 and FANCD2-43 mRNAs by RT-PCR in various FA cell lines. (C)
Detection of FANCD2-44 and FANCD2-43 mRNAs by RT-PCR throughout the cell
cycle or following exposure to MMC. The indicated primer pairs (A) were used to
amplify the alternatively spliced mRNAs. The mRNA was prepared from either
uncorrected lymphoblasts from the indicated FA subtypes, from cells in different
stages of the cell cycle, or following exposure to MMC. Ub indicates ubiquitin.
codon in exon 19 encoding lysine 561, the site of FANCD2
monoubiquitination.10 A cDNA corresponding to FANCD2-44 has
previously been described and is known to functionally complement the MMC sensitivity of transfected FA-D2 cells.5 FANCD2-44
contains a unique 24–amino acid stretch at its extreme C-terminus.
We confirmed the expression of these splice variants in human
cell lines by RT-PCR (Figure 1B-C). Although this analysis is
semiquantitative, the ratio of the 2 mRNAs was approximately 1:10
(exon 43–exon 44 variant). This ratio was constant, regardless of
the cell line examined (Figure 1B), the phase of the cell cycle, or
whether cells were exposed to MMC (Figure 1C). Northern blot
analysis confirmed the expression of these 2 major mRNA splice
variants in multiple tissues (data not shown), suggesting that these
variants have ubiquitous expression.
Expression of wild-type and mutant forms of FANCD2 in
FA-D2 fibroblasts
To determine the functional significance of the carboxy terminal
sequence of FANCD2, we generated a series of cDNAs encoding
the FANCD2 splice variants and mutant FANCD2 proteins (Figure
2A). The carboxy terminus of the FANCD2-44 protein is highly
conserved between human and rodent (Mus musculus and Rattus
norvegicus; Figure 2B) and may therefore have an important
cellular function. This sequence is highly acidic, containing 12
acidic residues (E or D) of 24 residues (50%) in the human
sequence. This includes an amino acid stretch (1427-1430) that is
conserved in humans and rodents, which we will refer to as the
“EDGE” region for the single-letter amino acid code of the
corresponding address. A highly conserved aspartic acid residue in
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1006
BLOOD, 1 FEBRUARY 2005 䡠 VOLUME 105, NUMBER 3
MONTES DE OCA et al
Figure 2. Generation of cDNAs encoding normal splice variants and mutant
forms of the FANCD2 protein. (A) Schematic series of FANCD2 proteins. (B)
Alignment of amino acids encoded by exon 44 sequence derived from Homo sapiens,
M. musculus, and R. norvegicus. The sequence is highly acidic and the D1428
residue is conserved.
the EDGE sequence (D1428) is conserved in the species listed
(Figure 2B).
We generated the Exon44-T mutant of FANCD2, truncated at
the carboxy terminus and lacking the EDGE region (Figure 2A).
Another construct, the mutant EDGE variant, contains the exon 44
sequence–encoded carboxy terminus but the conserved aspartic
acid residue at position 1428 was converted to alanine (D1428A).
Another mutant, FANCD2-K561R, has previously been described.10 This mutation disrupts the monoubiquitination of the
FANCD2 protein, its targeting to nuclear foci, and its ability to
functionally complement FA-D2 cells.
We next expressed the wild-type or mutant FANCD2 proteins in
the FANCD2(⫺/⫺) fibroblast line, PD20F, which lacks endogenous FANCD2 protein (Figure 3). Previous studies have shown that
FANCD2-44 is normally expressed as 2 isoforms, the unubiquitinated isoform (FANCD2-S) and the monoubiquitinated isoform
(FANCD2-L).10 Following DNA damage10 or during S phase of the
cell cycle,11 FANCD2 was monoubiquitinated. Like the wild-type
FANCD2 protein, the Exon44-T and mutant EDGE proteins were
monoubiquitinated in response to IR (Figure 3A, compare lanes 6,
8, and 10). The Exon44-T protein exhibited a slightly faster
electrophoretic mobility (see bottom bands in Figure 3A lanes 7
and 8), consistent with the removal of 24 amino acids from its
carboxy terminus. The FANCD2-43 variant was also monoubiquitinated on K561 (Table 1; data not shown). Taken together, these
results indicate that the carboxy terminus of FANCD2 is not
required for monoubiquitination.
Monoubiquitination of wild-type FANCD2-44 on K561
results in targeting to subnuclear foci.10 Similarly, monoubiquitinated Exon44-T and mutant EDGE were targeted to foci (Figure
3B; Table 1), indicating that the carboxy terminal region of
FANCD2-44 is not required for this intranuclear translocation.
In contrast, the K561R mutant failed to assemble in foci. In
addition, we demonstrate for the first time that wild-type
FANCD2 colocalizes with the phosphorylated histone H2AX
(Ser139), which is associated with DNA damage foci sites,28,29
following IR (Figure 3C). H2AX does not form prominent foci
in randomly cycling HeLa cells.
Figure 3. FANCD2 carboxy terminal mutants are monoubiquitinated following
ionizing radiation. (A) FA-D2 fibroblasts were stably transduced with either
hemagglutinin (HA)–pMMP (empty vector), HA-FANCD2 (wild type), HA–
Exon44-T, or HA–mutant-EDGE, as indicated. Cells were irradiated with 15 Gy, as
indicated, and whole-cell lysates were immunoblotted with anti-FANCD2 antisera.
(B) Immunofluorescence of the transduced cells 8 hours after cellular exposure to
IR, using an anti-FANCD2 antiserum. (C) Colocalization of FANCD2 and phosphorylated histone H2AX in DNA damaged-inducible foci. HeLa cells were either
untreated (top) or exposed to IR (15 Gy, 15 hours) (bottom), as indicated. HeLa
cells were double-stained with monoclonal anti-FANCD2 (FI-17; green) and
polyclonal anti–histone ␥-H2AX (Ser139; red). Merges of corresponding antiFANCD2 and anti–histone ␥-H2AX images are shown. Original magnifications
⫻ 630 (B-C).
The carboxy terminus of FANCD2-44 is required for functional
complementation of FA-D2 cells
We next examined the FANCD2 variants for their ability to
functionally complement the MMC hypersensitivity of FA-D2
cells (Figure 4). Wild-type FANCD2-44 corrected the MMC
sensitivity of transduced FA-D2 cells, as measured by a
colorimetric assay of cellular viability,5,10,26 consistent with
previously reported results.5 Interestingly, the FANCD2-43
variant, the Exon44-T mutant, and the mutant EDGE protein,
D1428A, failed to correct MMC hypersensitivity (Figure 4;
Table 1). Taken together, these results indicate that the carboxy
terminal 24 amino acids of FANCD2-44 are required for MMC
resistance.
Table 1. Characterization of splice variants and mutant forms
of the FANCD2 protein compared with wild-type FANCD2
Monoubiquitin
on K561
Detected
in
chromatin
Forms
nuclear
foci
Corrects
MMC
sensitivity
FANCD2-43
⫹
ND
⫹
⫺
FANCD2-44
⫹*
⫹
⫹*
⫹*
⫹
⫹
⫹
⫺
Mutant EDGE, D1428A
⫹
⫹
⫹
⫺
K561R
⫺*
⫺
⫺*
⫺*
Exon44-T
ND indicates not determined.
*Data reported in Garcia-Higuera et al.10
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BLOOD, 1 FEBRUARY 2005 䡠 VOLUME 105, NUMBER 3
Figure 4. The carboxy terminal region of FANCD2 is required for MMC
resistance. Stably transduced PD20 (FA-D2) fibroblasts, expressing the indicated
FANCD2 variants and mutant proteins, were grown in the presence of the indicated
concentrations of MMC. Cell survival was assessed by a colorimetric assay using
crystal violet. Data points represent the average of 3 experiments, with standard
deviation.
Monoubiquitination of FANCD2 is required for chromatin
binding
We next determined whether monoubiquitination of FANCD2 targets
the protein to discrete nuclear compartments (Figure 5). Nuclear
proteins from HeLa cells were fractionated into soluble nuclear,
chromatin, or nuclear matrix components (Figure 5A), and fractions
were immunoblotted for FANCD2 (Figure 5B). The unubiquitinated
FANCD2 protein fractionated with soluble nuclear proteins (Figure 5B
S2 fraction). The FANCD2-L (monoubiquitinated) isoform fractionated
with chromatin (Figure 5B S4 fraction). Neither FANCD2-S nor
FANCD2-L was detected in the nuclear matrix fraction (P4), a fraction
that contains lamin B (Figure 5B lane 7). These results suggest that
monoubiquitination of FANCD2 is required for chromatin targeting.
The localization of FANCD2-L to chromatin was consistent
with results from immunofluorescence microscopy (Figure 5C).
REGULATED INTERACTION OF FANCD2 WITH CHROMATIN
1007
Irradiated HeLa cells were examined by anti-FANCD2 immunofluorescence, either with or without permeablization with Triton
X-100. Triton X-100 removed background nuclear fluorescence but
failed to remove FANCD2 foci (P2), consistent with the presence
of FANCD2-L in the foci. Further, exposure of cells to DNase I and
ammonium sulfate resulted in loss of the FANCD2-L immunofluorescence signal but failed to extract lamin B (P4). Taken together,
these results confirm that the monoubiquitinated isoform of
FANCD2 resides in the chromatin fraction (S4) and that this
fraction correlates with FANCD2 observed in the foci. Thus,
FANCD2 foci that form following exposure to IR represent
monoubiquitinated FANCD2 (FANCD2-L) in chromatin.
FA-D2 fibroblasts, expressing either wild-type or mutant
FANCD2 polypeptides, were next examined by cell fractionation
(Figure 6). Cells were exposed to MMC, resulting in expression of
approximately equal levels of unubiquitinated (FANCD2-S) and
monoubiquitinated (FANCD2-L) isoforms in the residual pellet
following detergent permeabilization (lane 1). FANCD2-K561R
was not monoubiquitinated and was expressed only as FANCD2-S,
which was present in residual amounts following detergent permeabilization (Figure 6 lane 1). The unubiquitinated FANCD2-K561R
mutant was not detected in the chromatin fraction (Figure 6 lane 4).
The monoubiquitinated isoforms of FANCD2-44, Exon44-T, and
mutant EDGE were localized to the chromatin fraction (Figure 6
lane 4), whereas unubiquitinated FANCD2 (FANCD2-S) was
present in residual amounts prior to extraction of chromatin with
ammonium sulfate (Figure 6 lanes 1-3). The chromatin fraction
(S4) also contained histone H4. Again, neither FANCD2-L nor
FANCD2-S protein was detected in the nuclear matrix fraction
containing lamin B (Figure 6 lane 5).
Discussion
In the current study, we compared the function of the C-terminal
acidic region of FANCD2 that differs significantly in 2 endogenous
splice variants of FANCD2. Only one of these splice variants,
FANCD2-44, encodes a functional protein capable of correcting
Figure 5. Monoubiquitinated FANCD2 is preferentially retained in the chromatin fraction. (A) Protocol for nuclear fractionation of transduced FA-D2 fibroblasts.
Cytoplasm and nucleoplasm were extracted by permeabilization with detergent, and resulting nuclei were nuclease-digested and extracted with NH2SO4. Supernatants (S) and
pellets (P) were analyzed for FANCD2, histone H4 (chromatin marker), and lamin B (nuclear matrix marker) by Western blot. (B) Monoubiquitinated FANCD2 is preferentially
retained in detergent-insoluble nuclei (P2) and is subsequently extracted with chromatin (S4). (C) FANCD2 nuclear foci are extracted with the chromatin fraction. HeLa cells
were grown on coverslips and exposed to IR (15 Gy). After 12 hours, cells were prepared for immunofluorescence microscopy with an antibody to either FANCD2 or lamin B, as
indicated. Immunofluorescence microscopy was performed on either unpermeabilized (whole) cells (equivalent to P1 fraction), cells permeabilized with Triton X-100
(equivalent to P2), or cells permeabilized and extracted with DNase I and ammonium sulfate (equivalent to P4 fraction). FANCD2 and lamin B images are from different
representative cells. Original magnification ⫻ 630.
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1008
MONTES DE OCA et al
Figure 6. Monoubiquitination is required for targeting of FANCD2 to the
chromatin fraction. PD-20Fs (FA-D2 fibroblasts) were stably transduced with
retroviral constructs encoding HA-FANCD2-44 (wild type), HA–Exon44-T, HA–
mutant EDGE, or HA–FANCD2-K561R. Cells were pretreated with MMC to increase
the level of FANCD2-L to approximately 50% of total cellular FANCD2. Cells were
then fractionated as described in Figure 5A, and fractions were immunoblotted with
antisera to FANCD2, histone H4, or lamin B. Fractions are shown beginning with the
detergent-insoluble pellet following permeabilization (P2).
the FA/BRCA pathway in FA-D2 cells (Figure 4; Table 1). The
other variant, FANCD2-43, may have a different cellular function,
not identified in the current study. Since FANCD2-44 contains a
unique, conserved 24–amino acid sequence at the carboxy terminus, we focused our attention on this sequence. Several conclusions
are possible. First, the carboxy terminal region of FANCD2 is not
required for FANCD2 monoubiquitination on K561. Second,
monoubiquitination of FANCD2 is required for accumulation of
FANCD2 into chromatin. Third, the carboxy terminal 24 amino
acids of FANCD2-44 is required for the function of FANCD2 in the
FA/BRCA pathway (Figure 7).
The carboxy terminal 24 amino acids of FANCD2-44, containing
the EDGE region, act independently from FANCD2 monoubiquitination at K561. Truncation or point mutation of this C-terminal region did
not alter the efficiency of FANCD2 monoubiquitination, its targeting to
foci, or its distribution to chromatin. By contrast, monoubiquitination at
K561 is required both for FANCD2 foci formation10 and FANCD2
fractionation with chromatin24 (Figure 6).
Perhaps local sequence determinants, such as amino acids
adjacent to K561, control the sequence-specific monoubiquitination of FANCD2. For instance, FANCD2 contains several conserved serine residues in its primary sequence adjacent to K561.
One or more of these serine residues may become phosphorylated
in vivo, leading to FANCD2 monoubiquitination. Analogously,
protein polyubiquitination at an internal lysine residue is often
preceded by nearby serine phosphoryation.30
Monoubiquitination may alter the conformation of FANCD2, thereby
revealing a chromatin binding motif. Alternatively, a monoubiquitin
receptor in the chromatin, perhaps assembled at sites of damaged
chromatin, may mediate FANCD2-L accumulation. Ubiquitin binding
proteins may contain a ubiquitin-associated (UBA) domain31 or some
BLOOD, 1 FEBRUARY 2005 䡠 VOLUME 105, NUMBER 3
other ubiquitin binding activity.32 Other examples of protein monoubiquitination in protein sorting are known.32 The Ste2p protein in yeast is
internalized from the cell surface after monoubiquitination of its
cytoplasmic tail.33,34 Also, PCNA is monoubiquitinated following cellular exposure to DNA damage and perhaps targeted to sites of DNA
repair.35 Monoubiquitination may therefore play a more general role in
the targeting of DNA repair proteins to sites of damaged chromatin.
Increasing evidence suggests a role of chromatin modification in
DNA repair. H2AX, which assembles in nucleosomes,36 is phosphorylated in response to double-strand breaks and is believed to recruit other
DNA damage response machinery to these sites.28,29 Also, chromatin
assembly, mediated by modification of histones by either acetylation or
methylation, is required for DNA repair following intrastrand crosslinks or double-strand breaks.37,38 Besides FANCD2, other chromatinassociated proteins, including the MRE11/NBS1/RAD50 complex, play
a role in the DNA damage response.39,40
The finding that nucleotide excision repair (NER) requires
chromatin relaxation involving acetyltransferase activity41 is of
particular interest in the context of our finding that activated
FANCD2 is chromatin associated. Along with homologous recombination, nucleotide excision repair pathways are required for the
repair of DNA interstrand cross-links.42,43 FA cells are specifically
sensitive to DNA interstrand cross-linkers, such as MMC.
According to the model (Figure 7), DNA damage activates the
monoubiquitination of FANCD2 by an E3 ligase, which is probably
the FANCL subunit of the FA complex.44 Monoubiquitinated
FANCD2 subsequently binds to chromatin.24 There it may interact
with other protein components of the DNA repair response or with
damaged DNA itself in a manner that requires the C-terminal
24–amino acid EDGE region. In the current study, we show that
DNA damage activates the colocalization of FANCD2-Ub with
Figure 7. Schematic model indicating the downstream function of the FANCD2
protein in the FA/BRCA pathway. Following cellular exposure to DNA damaging
agents (IR or MMC, for example), FANCD2 is monoubiquitinated in an FA complex–
dependent manner. Monoubiquitinated FANCD2 is then targeted to the chromatin,
perhaps by a chromatin targeting factor. The carboxy terminal EDGE region of
FANCD2, required for its functional activity (resistance to MMC), may then interact
with other downstream proteins involved in DNA repair. Also, the monoubiquitin
moiety of FANCD2 may also interact with other chromatin factors(s) containing a
ubiquitin binding domain.
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BLOOD, 1 FEBRUARY 2005 䡠 VOLUME 105, NUMBER 3
REGULATED INTERACTION OF FANCD2 WITH CHROMATIN
histone ␥-H2AX foci. Recent studies indicate that monoubiquitinated FANCD2 may also interact with either ATM,18 the FANCD1/
BRCA2 protein,6 or the MRE11/NBS1/RAD50 complex.45 Other
proteins involved in the DNA repair response, such as MDC146,47
and 53BP1,48,49 may also have interactions with FANCD2. Whether
the EDGE region of FANCD2 binds directly to any of these
chromatin components remains to be determined.
Since FA cells are hypersensitive to DNA cross-linking agents, it will
be important to determine whether the EDGE region plays a specific
role in the DNA cross-link response. While both monoubiquitination of
FANCD2 and the C-terminal EDGE region are required for a normal
1009
cellular response to MMC, the observation that monoubiquitination
occurs in FANCD2 polypeptides lacking the EDGE region suggests that
the EDGE region may be involved in sensing or repairing damaged
DNA after FANCD2 is targeted into chromatin by monoubiquitination.
These 2 elements of FANCD2 function could therefore act by interaction with different sets of proteins.
Acknowledgment
We thank H. Joenje for EUFA316 cells.
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2005 105: 1003-1009
doi:10.1182/blood-2003-11-3997 originally published online
September 28, 2004
Regulated interaction of the Fanconi anemia protein, FANCD2, with
chromatin
Rocio Montes de Oca, Paul R. Andreassen, Steven P. Margossian, Richard C. Gregory, Toshiyasu
Taniguchi, XiaoZhe Wang, Scott Houghtaling, Markus Grompe and Alan D. D'Andrea
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