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NEOPLASIA
The Fanconi anemia core complex associates with chromatin during S phase
Jun Mi and Gary M. Kupfer
Fanconi anemia (FA) is an autosomal
recessive disease marked by bone marrow failure, birth defects, and cancer. The
FA proteins FANCA, FANCC, FANCE,
FANCF, FANCG, and FANCL participate in
a core complex. We previously have
shown that several members of this complex bind to chromatin until mitosis and
that this binding increases after DNA damage. The purpose of the present study
was to determine the dynamics of complex movement between cytoplasm and
nuclear compartments. Fluorescent-tagged
versions of FANCA, FANCC, and FANCG
colocalize in cytoplasm and nucleus,
chiefly in chromatin. At the G1-S border,
the FA core complex exists as foci on
chromatin, progressively diffusing and
migrating to the nuclear periphery and
becoming completely excluded from condensed chromosomes by mitosis. Chromatin fiber analysis shows FA proteins
diffusely staining along chromatin fibers
during G1-S and S phase. Treatment with
the DNA cross-linker mitomycin C results
in a diffusion of foci and increased binding of complex proteins to chromatin,
as well as diffuse and increased complex binding to chromatin fibers. These
data are consistent with the idea that the
FA proteins function at the level of chromatin during S phase to regulate and
maintain genomic stability. (Blood. 2005;
105:759-766)
© 2005 by The American Society of Hematology
Introduction
Fanconi anemia (FA) is a genetic disease of cancer susceptibility
marked by congenital defects, bone marrow failure, and myeloid
leukemia.1-4 To date at least 11 complementation groups have been
defined5-7 and 8 genes have been cloned.8-17 However, the gene
products resemble no known proteins and have few identifiable
functional protein motifs.
Cells derived from patients with the disease exhibit characteristic hypersensitivity to DNA cross-linking agents and generalized
decreased survival.18-22 In addition, a well-described G2 phase cell
cycle delay has been described that is thought to be secondary to a
defective S or G2 checkpoint.23-25 Other studies have implicated
cytokine dysregulation, sensitivity to oxidative damage, and defects in DNA repair.23,26-29 However, no defined biochemical
mechanism for this hypersensitivity has been elucidated. Patient
and cellular phenotypes across all the complementation groups are
similar, suggesting an inter-relatedness or cooperativity between
the FA proteins.
This cooperativity has been borne out by work we have done in
showing binding of FANCA and FANCC in a core protein complex
in both nucleus and cytoplasm.30-32 Recent work has found the
FANCG, FANCE, FANCF, and FANCL proteins in the complex as
well.17,33-36 One group has shown that BRCA2/FANCD1 binds to
FANCG.37 A large complex is suggested by our recent work,38 and
formation of the core complex does not occur in any of the
complementation groups except the FA-D1, FA-D2, FA-I, and
FA-J groups.7
Our work has established that FA proteins bind to chromatin.39
Immunoblotting experiments revealed that the increased FA proteins were bound to chromatin after DNA damage and that the
complex underwent egress from the nucleus at mitosis. This
movement appears to be regulated, as the FANCG protein becomes
doubly phosphorylated at mitosis yet remains bound to the core
complex.39 Recent work has implicated FANCD2 in chromatin,
reinforcing the importance of the FA core complex, since it is
required for FANCD2 monoubiquitination.40,41
In this paper we take those observations further in an attempt to
better define at the level of chromatin (1) the dynamics of
movement of the complex by microscopy during the cell cycle, (2)
how the complex responds to DNA damage, and (3) the manner in
which the complex localizes to chromatin fibers. In order to look
exclusively at chromatin, we strip away soluble nuclear components and cytoplasmic proteins by in situ chromatin preparations.
In addition, we demonstrate true localization to chromatin fibers by
vertical extraction in a urea buffer, which causes release and
elongation of fibers. Our data suggest that the FA protein complex
achieves a focal presence on chromatin by G1-S, which is
accentuated during S phase, and that the increase in FA complex
caused by DNA damage is likely a manifestation of the S-phase
concentration of FA complex.
Materials and methods
Cell culture and cell line
Cells were grown at 37°C in a 5% CO2 incubator. FA fibroblast cell lines
were grown in Dulbecco modified Eagle medium (DMEM) supplemented
with 15% (vol/vol) fetal bovine serum (FBS). HeLa cells were grown in
DMEM with 10% FBS. FA-A primary mutant cells (GANO; gift of Hans
Joenje, Free University, The Netherlands) were grown in F12 nutrition
medium and 15% FBS. HSC536N (FA-C) and EUFA143 (FA-G) mutant
lymphoblasts were grown in RPMI 1640 and 15% FBS.
From the Departments of Microbiology and Pediatrics, the University of Virginia
Health System, University of Virginia, Charlottesville.
Reprints: Gary M. Kupfer, Box 441 Jordan Hall, University of Virginia,
Charlottesville, VA 22908; e-mail: [email protected].
Submitted January 6, 2004; accepted July 1, 2004. Prepublished online as
Blood First Edition Paper, July 15, 2004; DOI 10.1182/blood-2004-01-0001.
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.
The online version of this article contains a data supplement.
© 2005 by The American Society of Hematology
BLOOD, 15 JANUARY 2005 䡠 VOLUME 105, NUMBER 2
759
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MI and KUPFER
BLOOD, 15 JANUARY 2005 䡠 VOLUME 105, NUMBER 2
Plasmid constructs
2 ug/mL DAPI (4⬘,6-diamidino-2-phenylindole; Vector Laboratories, Burlingame, CA) after washing with cold PBS.39,42
760
The plasmid pECFP (cyano fluorescent protein)–FANCG was constructed
with the FANCG gene fragment from pMMP-FANCG through the BglII
and EcoRI site generated by polymerase chain reaction (PCR). The plasmid
pEGFP (green fluorescent protein)–FANCA was constructed with the
FANCA gene fragment from pcDNA3-Flag-FANCA through the HindIII
and EcoRI site generated by PCR. The plasmid pEYFP (yellow fluorescent
protein)–FANCC was constructed with the FANCC gene fragment from
pLXSN-FANCC through KpnI and BamHI site generated by PCR.
To generate plasmid single-point mutations, PCR was performed with
site-directed mutagenesis kit (Quickchange, Stratagene, La Jolla, CA).
pEGFP-FANCA (H1110P), pECFP-FANCG (G546R), and pEYFP-FANCC
(L554P) were constructed, respectively, by using following pairs of
primers: 5⬘-GCAGTTCTTCCCCTTGGTCA-3⬘ and 5⬘-GTTGACCAAGGGGAAGAACT-3⬘, 5⬘-GTGCCCACGTAATCGAGACA-3⬘ and
5⬘-CTCGATTACGTGGGCACATC-3⬘, 5⬘-TTAAAGAGCCGCGAACTCAAG-3⬘ and 5⬘-TTGAGTTCGCGGCTCTTTAAG-3⬘. Resulting
constructs were verified by DNA sequencing.
DNA transfection
Transfections were performed by lipofection per manufacturer’s protocol
(Lipofectamine; Gibco, Carlsbad, CA). For all transient and stable transfection, plasmid DNA was prepared by column purification (Qiagen, Valencia,
CA). 5 ⫻ 105 cells were placed on 60-mm petri dishes one day prior to
transfection and grown until the cells were 60% to 80% confluent. Plasmid
DNA was diluted in 100-␮L Opti-MEM medium (Gibco) and mixed with
plus reagent (Gibco); LipofectAMINE was diluted in 100 ␮L OPTI-MEM
medium (Gibco). These were gently mixed together 15 minutes after
incubation at room temperature, and incubated another 15 minutes at room
temperature. The final complex was placed on prewashed cells with
phosphate-buffered saline (PBS) and incubated for 5 hours.
Cell synchronization and drug treatment
HeLa cells were treated overnight with 2 mM thymidine, washed, released
into regular media, and treated again overnight with thymidine, as
previously described.39 Cells were then released for variable times or
incubated overnight in the presence of 1 ␮M nocodazole (Sigma, St
Louis, MO). Mitotic cells were collected the next morning by shaking
the plate and collecting the cells in suspension. Verification of cell cycle
state was achieved using FACScan (Becton Dickinson, San Jose, CA)
analysis after resuspension of 100 000 cells in 0.3% citrate, 0.01%
NP40, 100 ␮g/mL propidium iodide, and 10 ␮g/mL RNAse.39
Asynchronous cells or cells synchronized at G1-S border or S phase
were incubated with mitomycin (MMC) (0.1 ␮M) for 4 hours. For the time
course, asynchronous cells were incubated with MMC for 0 hours, 6 hours,
12 hours, and 24 hours. For transiently transfected HeLa cells, 48 hours
after transfection, cells were treated for 12 hours with MMC.
For sorting, 106 asynchronously growing HeLa cells transfected with
pEGFP-FANCA and pECFP-FANCG were collected and pelleted. The cells
were resuspended sequentially in 500 ␮L PBS and 500 ␮L 2% paraformaldehyde and incubated on ice for 1 hour. After pelleting, the cells were
washed and then incubated overnight at 4°C in 70% ethanol. The cells were
pelleted again and resuspended in 1 mL of 40 ␮g/mL propidium iodide and
100 ␮g/mL RNAse in PBS. This mix was incubated at 37°C for 30 minutes.
The cells were then sorted into G1, S, and G2-M groups on the FACS
instrument (Becton Dickinson, San Jose, CA).
In situ chromatin preparation
Cells grown on chamber slides were first washed in cold PBS. Soluble
proteins were removed by extraction in cytoskeletal buffer [CSK: 10 mM
PIPES (piperazine diethanesulfonic acid), pH 6.8/300 mM sucrose/100 mM
NaCl/3 mM MgCl2/1 mM EGTA (ethylene glycol tetraacetic acid)/RNAse
inhibitor (Gibco)/1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride] containing 0.5% Triton X-100 for 2 minutes at 4°C. The structure remaining was
extensively cross-linked by treatment with 4% formaldehyde in CSK for 40
minutes at 4°C. The slides were covered with mounting solution containing
Chromatin fiber preparation
Chromatin fibers were prepared according to the method of Blower et al.43
Cells were trypsinized 48 hours after transfection, washed twice with cold
PBS, then suspended in hypotonic solution (75 mM KCl in PBS) for 10
minutes. Cells adjusted to a density of 1 to 2 ⫻ 105 cells/mL were cytospun
onto charged slides (double funnels) at 800 rpm (150g) with high
acceleration for 4 minutes, 150 ␮L cells per slide. For attached/monolayer
cells, 1 ⫻ 105 cells/mL were used, and for suspension cultures, 2 ⫻ 105
cells/mL. After cytospin, slides were immersed in a Coplin jar filled with
lysis buffer [25 mM Tris (tris(hydroxymethyl)aminomethane) Cl, pH
7.5/0.5 M NaCl/1% Triton X-100/0.2 M urea for 3 to 10 minutes. The slide
was slowly lifted vertically from the lysis buffer, and the chromatin was
allowed to move down the slide. After fixation in 4% paraformaldehyde in
PBS-Tween20 (0.05%) for 20 minutes, the slides were covered with
mounting solution containing 2 ug/mL DAPI (4⬘6-diamidino-2-phenylindole 2HCl) (Vector Laboratories) after wash in PBS. For fluorescent
microscopy and analysis, inverted fluorescent microscope (Nikon, Melville,
NY) was used, with images captured by digital camera (Hamamatsu,
Hamamatsu City, Japan).
Chromatin preparation and immunoblotting
Chromatin extract was prepared and run on sodium dodecyl sulfate–
polyacrylamide gel electrophoresis (SDS-PAGE), as previously described,
essentially a modification of the above-described in situ preparation.39,42
Immunoblotting was conducted with anti-FANCG(N terminal), antiFANCA(N terminal), and Ku86 (Santa Cruz Biotechnology, Santa Cruz,
CA), respectively.39
Results
Fluorescent-tagged versions of FA proteins are functional
For the purpose of visualizing FANCA, FANCG, and FANCC by
direct fluorescent microscopy, we constructed FA cDNAs with
fluorescent tags: EGFP-FANCG, EYFP-FANCC, and ECFPFANCG. In order to determine if the fluorescent versions of the
FANCA, FANCC, and FANCG were functional, we transfected the
fluorescent and nonfluorescent tagged constructs into FA-A
(HSC72), FA-C (HSC536N), and FA-G (EUFA143) mutant cells,
respectively. After transfection the cells were analyzed for cytotoxicity against MMC. In Figure 1, each version of the FA genes was
able to restore normal MMC resistance in the corresponding
mutant line, indicating that the fluorescent tag did not interfere with
normal FA function. Thus, use of these constructs is valid in
experiments to explore the normal function of the FA proteins.
Tagged FANCA, FANCC, and FANCG localize to chromatin
In previous work we showed by immunoblotting that the endogenous FA core complex localized to chromatin. To determine if
tagged versions of the FA proteins localized to chromatin, we
doubly transfected HeLa cells with either EGFP-FANCA and
ECFP-FANCG or ECFP-FANCG and EYFP-FANCC. After 48
hours, we extracted the cells in situ to leave behind what we term a
“chromatin prep.” Direct microscopy of either whole cells or
chromatin prepared in situ revealed that FANCA, FANCC, and
FANCG all localized to the nucleus and chromatin and totally
colocalized (Figure 2A). Most expressing cells contained fluorescent protein in both cytoplasm and nucleus. No chromatin binding
was observed in control experiments for cells expressing only
EGFP, ECFP, or EYFP (Figure 2B). Transfection efficiency was
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BLOOD, 15 JANUARY 2005 䡠 VOLUME 105, NUMBER 2
Figure 1. Fluorescent-tagged versions of FA proteins are functional. Mutant
FA-G (EUFA143), FA-A (HSC72), and FA-C (HSC536N) cell lines were transfected
with fluorescent-tagged wild-type ECFP-FANCG, EGFP-FANCA, and EYFP-FANCC
cDNAs and the nonfluorescent tagged versions, respectively. The cells were treated
with the indicated doses of MMC for 5 days and analyzed by XTT (2,3-Bis[2-methoxy4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) assay. Each tagged version
restored MMC resistance to the level of the nontagged version.
estimated to be at least 50% in all cases, as determined by counting
fluorescent cells prior to extraction. In order to show that mutation
of the FA proteins could disrupt proper chromatin localization, we
prepared similarly tagged versions of FANCA (H1110P), FANCC
(L554P), and FANCG (G546R) by PCR-mediated mutagenesis.
Each of these mutants exist in nature in FA patients, and the
derivative proteins are expressed at near wild-type levels.8,44,45 The
FANCA (H1110P), FANCC (L554P), and FANCG (G546R) mutants all were cytoplasmic and were not visible upon chromatin
preparation (Figure 2C). Thus, patient-derived point mutations
resulted in abrogation of nuclear or chromatin localization, further
validating the use of our tagged constructs.
The FA proteins form foci at G1-S and during S phase
Our previous work did not show any marked change of FA protein
amount bound to chromatin during S phase, as compared to the
G1-S border.39 In order to show qualitative changes in the FA core
complex, we synchronized HeLa cells by double thymidine block,
then released into regular media. Chromatin preparations at the
FA AND CHROMATIN DURING S PHASE
761
G1-S border and during S phase revealed that the FA core complex
formed foci, which were not readily apparent in whole cells (Figure
3A). In all cases, FANCA and FANCG as well as FANCG and
FANCC colocalized. Interestingly, cells from G1-S to mitosis
contained predominately fluorescent FA proteins in the nucleus.
The whole cells in the micrographs were underexposed in order to
diminish cytoplasmic staining and increase visibility of the nucleus.
As the cells continued into G2, the foci appeared to diffuse and
migrate to the nuclear periphery by late G2 (Figures 3B-D). This
also was apparent in whole cells. Cells that became detached from
the plate after nocodazole arrest, assumed to be in mitosis, were
collected and cytospun onto slides. Examination of these cells
revealed complete exclusion from nuclei (prior to chromosome
condensation) in the whole cell as well in chromatin preps (Figure
3E). Once cells are released from G1-S block, approximately 10 to
12 hours later the cells are “shakable” from the surface of the plate
and represent mitotic cells. These cells were collected and allowed
to reattach to the slide surface for 4 hours. These cells, seen in
Figure 3F, had divided, but the FA proteins were still cytoplasmic.
These data are consistent with our previous work in which we
showed the FA complex is excluded from condensed chromosomes
at mitosis. In early G1 it is apparent that the complex is still
cytoplasmic. Representative FACS analyses from HeLa cells and
EGFP-FANCA/ECFP-FANCG are depicted in Figure 3G; no
differences in DNA histograms were seen in any of the transfectants and HeLa cells alone. Cell number at this stage was no
different in nontransfected and transfected cells.
In order to verify the phenomenon of protein shuttling in
asynchronously growing cells, we sorted by flow HeLa cells
cotransfected with GFP-FANCA and CFP-FANCG into G1, S, and
G2-M groups. Microscopy revealed that FANCA and FANCG were
cytoplasmic and colocalized in G1 and G2-M cells, while in S phase
cells they were predominately nuclear (Figure 3H). This is
consistent with the idea that FA proteins shuttle between nucleus
and cytoplasm during the cell cycle and that synchronization does
not affect this event.
To demonstrate that synchronization does not prevent cells from
proceeding to the next cell cycle, we collected HeLa cells
transfected with EGFP-FANCA and ECFP-FANCG and arrested at
mitosis. These cells were washed and then placed in fresh regular
media and observed under time-lapse photography. The film
shows that initially FANCA and FANCG are cytoplasmic. However, after 6 hours, FANCA and FANCG can be observed in the
nucleus, mostly at the periphery (Supplementary Video 1 [see the
Video link at the top of the online article on the Blood website]). FACS (fluorescence-activated cell scans) analysis demonstrates that the cells are at the G1-S border (Figure 3G). Time-lapse
photography taken of asynchronous cells demonstrates a similar
phenomenon: initially cytoplasmic proteins become nuclear after
2 hours of observation (Supplementary Video 2, also available on
the Blood website).
FA core complex localizes to chromatin fibers
Chromatin preparations afford the ability to see structures beyond
soluble nuclear material, yet chromatin still represents a densely
packed version of the genome. In order to visualize distinct
chromatin fibers, we extracted nuclei by vertical immersion in a
urea-based extraction buffer after cotransfection with EGFPFANCA and ECFP-FANCG. In the asynchronous cells, colocalizing FANCA and FANCG were seen in irregularly spaced foci
along the length of the fiber (Figure 4). In contrast, upon G1-S
and S-phase entry, the proteins could be seen slightly more
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MI and KUPFER
BLOOD, 15 JANUARY 2005 䡠 VOLUME 105, NUMBER 2
Figure 2. FANCA, FANCC, and FANCG colocalize in
chromatin. (A) HeLa cells were transfected with either
GFP-FANCA/CFP-FANCG or CFP-FANCG/YFP-FANCC.
Whole cells were fixed or chromatin preparations were
performed in situ. (B) HeLa cells were transfected with
EGFP, EYFP, or ECFP only–containing plasmid, and
whole cells and chromatin were prepared. No GFP, YFP,
or CFP signal was discernible on chromatin after preparation. Also, CFP was not visible through GFP filters, and
YFP and GFP were not visible through CFP filters. (C)
Point mutant versions of EGFP-FANCA(H1110P), EYFPFANCC(L554P), and ECFP-FANCG(G546R) were transfected into HeLa cells. Whole cells contained none of the
mutant proteins in the nucleus, and no mutant proteins
were visible at all after chromatin preparation.
prominently but more homogeneously spread along the fiber. At
G2, the proteins appeared to be more clumped. Strikingly and
consistent with our other data, a chromatin fiber prep performed
on mitotic cells revealed the complete absence of FA proteins,
while the DAPI-stained image showed predictably a heterochromatically staining fiber.
FA core complex diffuses after MMC treatment
Our previous work showed that increased FA proteins localized to
chromatin after MMC treatment. To demonstrate this phenomenon
in situ, we treated an asynchronous cell population with 0.1 ␮M
MMC and examined chromatin and whole cells at various time
points of MMC treatment. No obvious differences can be seen in
whole cells (Figure 5A). However, by 12 hours, foci seen in
untreated chromatin had become diffusely stained in MMC-treated
chromatin (Figure 5B). We analyzed a slide containing cells
similarly treated with MMC and performed chromatin fiber preparation. Increased and more-spread FA proteins stained the fibers
(Figure 5C), consistent with the in situ appearance in Figure 5B and
the immunoblotting data of our prior work.39
FA core complex localizes to chromatin in primary cells
Because HeLa cells are easily synchronized, we have used them for
the bulk of the work presented. In order to show that these data are
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BLOOD, 15 JANUARY 2005 䡠 VOLUME 105, NUMBER 2
FA AND CHROMATIN DURING S PHASE
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Figure 3. The FA proteins form foci at G1-S and during S phase. HeLa cells transfected with either EGFP-FANCA/ECFP-FANCG or ECFP-FANCG/EYFP-FANCC were
grown on slides and synchronized by double thymidine block to the G1-S border. Whole cells and chromatin were visualized at the indicated times after release: (A) G1-S, 0
hours; (B) S phase, 4 hours; (C) early G2 phase, 6 hours; (D) late G2 phase, 8 hours; (E) mitosis, after 16 hours in 1 ␮M nocodazole; and (F) early G1, after collection of floating
mitotic cells, followed by 4 hours in regular media to allow for anchorage and cytokinesis. (G) Representative FACScan analysis of transfected, synchronized cells. (H)
Asynchronous HeLa cells cotransfected with GFP-FANCA and CFP-FANCG were sorted by FACScan into G1, S, and G2-M groups. Microscopy revealed that FA proteins were
cytoplasmic during G1 and G2-M and nuclear during S phase.
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BLOOD, 15 JANUARY 2005 䡠 VOLUME 105, NUMBER 2
MI and KUPFER
Figure 4. FA core complex localizes to chromatin fibers. HeLa cells transfected
with EGFP-FANCA/CFP-FANCG were synchronized and collected as in Figure 3.
The mitotic cells were cytospun onto a new slide. Chromatin fibers were then
prepared on slides by vertical urea extraction. Micrographs show foci, then diffusion
of FA proteins on fibers through S phase, clumping in G2, and absence by mitosis.
applicable to primary cells as well, we transfected primary FA-A
mutant cells with EGFP-FANCA and ECFP-FANCG. In the
untreated cells, analysis of both whole cells and chromatin revealed
that FANCA and FANCG colocalized to foci in the nucleus of
whole cells (Figure 6A) and chromatin (Figure 6B). Cells treated
with MMC displayed increased chromatin staining, consistent with
the data presented in the HeLa cells. Even in the whole cell, the
nucleus can be seen homogeneously stained after MMC treatment.
Thus, for FA core complex function, behavior of the complex in
immortalized and primary cells appears similar.
Figure 6. FA proteins exist as foci and diffuse on chromatin after MMC
treatment in primary cells. Primary FA-A fibroblasts were transfected with EGFPFANCA/CFP-FANCG and treated with 0.1 ␮M MMC for 24 hours. (A) Whole cells and
(B) chromatin were prepared and visualized.
FA proteins localize to chromatin predominately after MMC
treatment and during S phase
Chromatin fiber experiments and synchronization experiments suggest
similarities in appearance of FA proteins during S phase and after MMC
treatment. This suggests that the DNA damage inducibility of the FA
protein localization on chromatin may be a function of engagement of
an S-phase checkpoint. This has been suggested by work done by
Akkari et al,29 who showed that cytotoxicity induced by MMC was
dependent on traverse through S phase.
In order to compare the magnitude of MMC effect upon
chromatin localization of the FA proteins, we treated HeLa cells
synchronized to the G1-S border and asynchronous HeLa cells with
MMC. S-phase cells were prepared by release into regular media
for 4 hours; cells were treated with MMC during this time. After
chromatin extract preparation, we performed FANCA immunoblotting. Unexpectedly, peak levels of FANCA in chromatin were seen
during S phase, which were greater than the increased levels
induced by MMC in asynchronous cells (Figure 7A). Concomitant
treatment with MMC during S phase did not stimulate higher FA
protein levels in chromatin. A negative control lane with 100 ␮g of
whole cell extract of EUFA143 FA-G mutant cells was included, as
these cells do not express FANCG and express little FANCA.
FACS data showed that MMC did not alter the DNA histogram in
synchronized cells but, as has been well documented, did induce a
marked degree of S (12 hours) and G2-M (24 hours) accumulation
(Figure 7B). These data suggest that the MMC inducibility of FA
proteins to chromatin is achieved through traverse in S phase and is
consistent with the data from Akkari et al.29
Discussion
Figure 5. FA protein foci diffuse after MMC treatment. HeLa cells transfected with
EFGP-FANCA/CFP-FANCG were treated for 24 hours with 0.1 ␮M MMC. (A) Whole
cells, (B) chromatin, and (C) chromatin fibers were prepared and examined. FA
proteins diffused on chromatin after MMC treatment.
We previously have implicated the chromatin-nuclear matrix as a
functional unit upon which the FA core complex performs its
normal function.39 In this paper, we confirm and extend these
findings by showing that this is an S-phase–specific process by (1)
demonstrating that the FA core complex forms chromatin foci at the
G1-S border and at the beginning of S phase, (2) showing that these
foci diffuse in response to DNA damage and during S phase, and
(3) showing that the proteins exit the nucleus by the onset of
mitosis as a multiprotein complex.
These data suggest a dynamic movement during the cell cycle
whereby FA protein foci form on chromatin by G1-S, the foci
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BLOOD, 15 JANUARY 2005 䡠 VOLUME 105, NUMBER 2
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Figure 7. FA proteins localize to chromatin predominately after MMC treatment and during S phase. HeLa cells were synchronized by double thymidine block and
collected. G1-S cells were not released but were treated with 0.1 ␮M MMC for 4 hours. S-phase cells were prepared by simultaneous release for 4 hours in regular media and
treated with 0.1 ␮M MMC for 4 hours. Chromatin was prepared and run on SDS-PAGE. After transfer, the membrane was blotted with anti-FANCA, anti-FANCG, and
anti–topoisomerase II antisera. Neg indicates negative mutant EUFA143 FA-G cells that express no FANCG and very little FANCA. (B) 100 000 cells from the indicated
treatment group were counted on FACScan at each time point.
diffuse, and proteins increase in amount during S phase. This is
recapitulated by the chromatin fiber experiments. The MMC
treatment appears to induce a similar change in both chromatin foci
as well as in fibers, suggesting that the response to DNA damage
and during S phase may be similar. This is supported by literature
from the Grompe and Rosselli groups suggesting the importance of
the FA pathway in an S-phase process as well as from the D’Andrea
group showing that FANCD2 is mono-ubiquitinated during S phase
and in response to DNA damage.29,46-49
These data have implications for potential function of the FA core
complex. First, a specific function for the FA proteins remains obscure in
spite of the cloning of 8 genes. Second, the emergence of chromatin
regulation as important to genomic stability has become part of the
forefront of current research, exemplified by SWI/SNF and by chromatin remodeling activities of cancer related proteins such as core binding
protein (CBP) and BRCA1.50 Examples of regulation and involvement
of histone modification have been shown to affect transcription,
replication, and DNA repair. Indeed, although no associated chromatin
remodeling activity could be detected, FANCA has been shown to bind
to human SWI/SNF.51 Also, recent work has shown that BRCA1 binds
to FANCD2 and that BRCA2 is indeed FANCD1.48,52 Since FANCD2
ubiquitination is dependent upon wild-type FA core complex, and
this ubiquitination occurs in response to DNA damage, it is indeed
plausible that the genomic surveillance function of the FA pathway
occurs at the level of chromatin. Recently, FANCD2 and its
importance in chromatin has been supported by work detailing that
mono-ubiqutinated FANCD2 localizes to chromatin and is responsible for BRCA2 loading onto chromatin.40,41
Our future studies now will focus on a biochemical function for
the FA pathway. These studies suggest a link between replication
and repair of DNA damage. Our prior work detailing MMC
induction of FA protein to chromatin did not compare directly
MMC effect versus S phase. Our direct comparison implies that the
2 are likely inseparable.
Localization to chromatin implies that these biochemical activities may play a role directly in DNA repair, DNA replication, and
other genomic surveillance activities. While direct DNA binding of
the FA core complex has not been shown, it is plausible that the FA
core complex can interact with the functional unit of DNA, namely
chromatin. The recent report of Bloom helicase (BLM) binding
with the FA core complex suggests work to show how the
chromatin interaction occurs.53 We will attempt to show colocalization with BLM and other S-phase proteins in situ, including those
involved in recombinatorial repair, replication machinery, and
other components of chromatin such as histones.
Acknowledgments
We thank Han Joenje for the GANO cell line and Maureen Hoatlin
and members of the Kupfer laboratory for useful discussions.
References
1. Alter BP, Young NS. The Bone Marrow Failure
Syndromes. In: Nathan DG, Oski FA, eds. Hematology of infancy and childhood, vol 1. Philadelphia, PA: WB Saunders; 1993:216-316.
7. Levitus M, Rooimans MA, Steltenpool J, et al.
Heterogeneity in Fanconi anemia: evidence for
two new genetic subtypes. Blood. 2004;103:
2498-2503.
2. Auerbach A, Buchwald M, Joenje H. Fanconi
Anemia. In: Vogelstein B, Kinzler K, eds. Genetics of cancer. New York, NY: McGraw-Hill; 1997:
317-332.
8. Strathdee CA, Gavish H, Shannon WR, Buchwald M. Cloning of cDNAs for Fanconi’s anaemia
by functional complementation. Nature. 1992;
356:763-767.
3. Fanconi G. Familiar infantile perniziosaartige
Anamie (pernizioses Blutbild und Konstitution). Z
Kinderheil. 1927;117:257-280.
9. Lo Ten Foe JR, Rooimans MA, Bosnoyan-Collins
L, et al. Expression cloning of a cDNA for the major Fanconi anemia gene, FAA. Nat Genet. 1996;
14:320-323.
4. Fanconi G. Familial constitutional panmyelocytopathy, Fanconi’s anemia (FA), I: clinical aspects.
Semin Hematol. 1967;4:233-240.
5. Joenje H, Oostra A, Wijker M, et al. Evidence for
at least eight Fanconi anemia genes. Am J Hum
Genet. 1997;61:940-944.
6. Joenje H, Levitus M, Waisfisz Q, et al. Complementation analysis in Fanconi anemia: assignment of the reference FA-H patient to group A.
Am J Hum Genet. 2000;67:759-762.
10. Apostolou S, Whitmore SA, Crawford J, et al. Positional cloning of the Fanconi Anaemia Group A
gene. Nat Genet. 1996;14:324-328.
11. de Winter JP, Rooimans MA, van Der Weel L, et
al. The Fanconi anaemia gene FANCF encodes a
novel protein with homology to ROM. Nat Genet.
2000;24:15-16.
12. de Winter JP, Leveille F, van Berkel CG, et al. Isolation of a cDNA representing the Fanconi ane-
mia complementation group E gene. Am J Hum
Genet. 2000;67:1306-1308.
13. de Winter JP, Waisfisz Q, Rooimans MA, et al. The
Fanconi anaemia group G gene FANCG is identical
with XRCC9. Nat Genet. 1998;20:281-283.
14. Hejna JA, Timmers CD, Reifsteck C, et al. Localization of the Fanconi anemia complementation
group D gene to a 200-kb region on chromosome
3p25.3. Am J Hum Genet. 2000;66:1540-1551.
15. Whitney M, Thayer M, Reifsteck C, et al. Microcell mediated chromosome transfer maps the
Fanconi anemia group D gene to chromosome
3p. Nat Genet. 1995;11:341-343.
16. Timmers C, Taniguchi T, Hejna J, et al. Positional
cloning of a novel Fanconi anemia gene,
FANCD2. Mol Cell. 2001;7:241-248.
17. Meetei AR, de Winter JP, Medhurst AL, et al. A
novel ubiquitin ligase is deficient in Fanconi anemia. Nat Genet. 2003;35:165-170.
18. Rathbun RK, Faulkner GR, Ostroski MH, et al.
Inactivation of the Fanconi anemia group C gene
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
766
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
BLOOD, 15 JANUARY 2005 䡠 VOLUME 105, NUMBER 2
MI and KUPFER
augments interferon-gamma-induced apoptotic
responses in hematopoietic cells. Blood. 1997;
90:974-985.
Whitney M, Royle G, Low M, et al. Germ cell defects and hematopoietic hypersensitivity to
gamma-interferon in mice with a targeted disruption of the Fanconi anemia C gene. Blood. 1996;
88:49-58.
Cumming RC, Liu JM, Youssoufian H, Buchwald
M. Suppression of apoptosis in hematopoietic
factor-dependent progenitor cell lines by expression of the FAC gene. Blood. 1996;88:4558-4567.
Marathi UK, Howell SR, Ashmun RA, Brent TP.
The Fanconi anemia complementation group C
protein corrects DNA interstrand cross-link-specific apoptosis in HSC536N cells. Blood. 1996;88:
2298-2305.
Ridet A, Guillouf C, Duchaud E, et al. Deregulated apoptosis is a hallmark of the Fanconi anemia syndrome. Cancer Res. 1997;57:1722-1730.
Kupfer GM, D’Andrea AD. The effect of the Fanconi Anemia polypeptide, FAC, upon p53 induction and G2 checkpoint regulation. Blood. 1996;
88:1019-1025.
Dutrillaux B, Aurias A, Dutrillaux AM, Buriot D,
Prieur M. The cell cycle of lymphocytes in Fanconi anemia. Hum Genet. 1982;62:327-332.
Kaiser TN, Lojewski A, Dougherty C, Juergens L,
Sahar E, Latt SA. Flow cytometric characterization of the response of Fanconi’s Anemia cells to
mitomycin C treatment. Cytometry. 1982;2:291297.
Haneline L, Broxmeyer H, Cooper S, et al. Multiple inhibitory cytokines induce deregulated progenitor growth and apoptosis in hematopoietic
cells from fac⫺/⫺ mice. Blood. 1998;91:40924098.
Pang Q, Fagerlie S, Christianson TA, et al. The
Fanconi anemia protein FANCC binds to and facilitates the activation of STAT1 by gamma interferon and hematopoietic growth factors. Mol Cell
Biol. 2000;20:4724-4735.
Cumming RC, Lightfoot J, Beard K, Youssoufian
H, O’Brien PJ, Buchwald M. Fanconi anemia
group C protein prevents apoptosis in hematopoietic cells through redox regulation of GSTP1. Nat
Med. 2001;7:814-820.
Akkari YM, Bateman RL, Reifsteck CA, Olson SB,
Grompe M. DNA replication is required to elicit
cellular responses to psoralen-induced DNA interstrand cross-links. Mol Cell Biol. 2000;20:82838289.
30. Kupfer GM, Naf D, Suliman A, Pulsipher M,
D’Andrea AD. The Fanconi anaemia proteins,
FAA and FAC, interact to form a nuclear complex.
Nat Genet. 1997;17:487-490.
nents of the human SNF/SWI complex, are phosphorylated and excluded from the condensed
chromosomes during mitosis. EMBO J. 1996;15:
3394-3402.
31. Naf D, Kupfer GM, Suliman A, Lambert K,
D’Andrea AD. Functional activity of the Fanconi
anemia protein FAA requires FAC binding and
nuclear localization. Mol Cell Biol. 1998;18:59525960.
43. Blower MD, Sullivan BA, Karpen GH. Conserved
organization of centromeric chromatin in flies and
humans. Dev Cell. 2002;2:319-330.
32. Yamashita T, Kupfer G, Naf D, et al. The Fanconi
anemia pathway requires FAA phosphorylation
and FAA/FAC nuclear accumulation. Proc Natl
Acad Sci U S A. 1998;95:13085-13090.
44. Nakanishi K, Moran A, Hays T, et al. Functional
analysis of patient-derived mutations in the Fanconi anemia gene, FANCG/XRCC9. Exp Hematol. 2001;29:842-849.
33. Christianson TA, Bagby GC. FANCA protein binds
FANCG proteins in an intracellular complex.
Blood. 2000;95:725-726.
45. Kupfer G, Naf D, Garcia-Higuera I, et al. A patient-derived mutant form of the Fanconi anemia
protein, FANCA, is defective in nuclear accumulation. Exp Hematol. 1999;27:587-593.
34. de Winter JP, van Der Weel L, de Groot J, et al.
The Fanconi anemia protein FANCF forms a
nuclear complex with FANCA, FANCC and
FANCG. Hum Mol Genet. 2000;9:2665-2674.
46. Pichierri P, Rosselli F. The DNA crosslink-induced
S-phase checkpoint depends on ATR-CHK1 and
ATR-NBS1-FANCD2 pathways. EMBO J. 2004;
23:1178-1187.
35. Garcia-Higuera I, Kuang Y, Naf D, Wasik J,
D’Andrea AD. Fanconi anemia proteins FANCA,
FANCC, and FANCG/XRCC9 interact in a functional nuclear complex. Mol Cell Biol. 1999;19:
4866-4873.
47. Taniguchi T, Garcia-Higuera I, Andreassen PR,
Gregory RC, Grompe M, D’Andrea AD. S-phase–
specific interaction of the Fanconi anemia protein,
FANCD2, with BRCA1 and RAD51. Blood. 2002;
100:2414-2420.
36. Waisfisz Q, de Winter JP, Kruyt FA, et al. A physical complex of the Fanconi anemia proteins
FANCG/XRCC9 and FANCA. Proc Natl Acad Sci
U S A. 1999;96:10320-10325.
37. Hussain S, Witt E, Huber PA, Medhurst AL, Ashworth A, Mathew CG. Direct interaction of the
Fanconi anaemia protein FANCG with BRCA2/
FANCD1. Hum Mol Genet. 2003;12:2503-2510.
38. Thomashevski A, High AA, Drozd M, et al. The
Fanconi anemia core complex forms 4 different
sized complexes in different subcellular compartments. J Biol Chem. 2004;279:26201-26209.
39. Qiao F, Moss A, Kupfer GM. Fanconi anemia proteins localize to chromatin and the nuclear matrix
in a DNA damage- and cell cycle-regulated manner. J Biol Chem. 2001;276:23391-23396.
40. Meetei AR, Yan Z, Wang W. FANCL replaces
BRCA1 as the likely ubiquitin ligase responsible
for FANCD2 monoubiquitination. Cell Cycle.
2004;3:179-181.
41. Wang X, Andreassen PR, D’Andrea AD. Functional interaction of monoubiquitinated FANCD2
and BRCA2/FANCD1 in chromatin. Mol Cell Biol.
2004;24:5850-5862.
42. Muchardt C, Reyes JC, Bourachot B, Leguoy E,
Yaniv M. The hbrm and BRG-1 proteins, compo-
48. Garcia-Higuera I, Taniguchi T, Ganesan S, et al.
Interaction of the Fanconi anemia proteins and
BRCA1 in a common pathway. Mol Cell. 2001;7:
249-262.
49. Rothfuss A, Grompe M. Repair kinetics of
genomic interstrand DNA cross-links: evidence
for DNA double-strand break-dependent activation of the Fanconi anemia/BRCA pathway. Mol
Cell Biol. 2004;24:123-134.
50. McKenna NJ, Xu J, Nawaz Z, Tsai SY, Tsai MJ,
O’Malley BW. Nuclear receptor coactivators: multiple enzymes, multiple complexes, multiple functions. J Steroid Biochem Mol Biol. 1999;69:3-12.
51. Otsuki T, Furukawa Y, Ikeda K, et al. Fanconi
anemia protein, FANCA, associates with BRG1, a
component of the human SWI/SNF complex.
Hum Mol Genet. 2001;10:2651-2660.
52. Howlett NG, Taniguchi T, Olson S, et al. Biallelic
inactivation of BRCA2 in Fanconi anemia. Science. 2002;297:606-609.
53. Meetei AR, Sechi S, Wallisch M, et al. A multiprotein nuclear complex connects Fanconi anemia
and Bloom syndrome. Mol Cell Biol. 2003;23:
3417-3426.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2005 105: 759-766
doi:10.1182/blood-2004-01-0001 originally published online
July 15, 2004
The Fanconi anemia core complex associates with chromatin during S
phase
Jun Mi and Gary M. Kupfer
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