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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 4 (February 15), 1998:
pp. 1418-1425
The Fanconi Anemia Group C Gene Product Is Located in Both the
Nucleus and Cytoplasm of Human Cells
By
Maureen E. Hoatlin,
Tracy A. Christianson,
Winnie W. Keeble,
Adam
T. Hammond,
Yu Zhi,
Michael C. Heinrich,
Paula A. Tower, and
Grover
C. Bagby Jr
From the Division of Hematology and Medical Oncology,
Oregon Health Sciences University, Portland; and The Portland Veterans
Administration Medical Center, Portland, OR.
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ABSTRACT |
The Fanconi anemia (FA) complementation group C (FAC) protein gene
encodes a cytoplasmic protein with a predicted Mr
of 63,000. The protein's function is unknown, but it has been
hypothesized that it either mediates resistance to DNA cross-linking
agents or facilitates repair after exposure to such factors. The
protein also plays a permissive role in the growth of colony-forming
unit-granulocyte/macrophage (CFU-GM), burst-forming unit-erythroid
(BFU-E), and CFU-erythroid (CFU-E). Attributing a specific function to
this protein requires an understanding of its intracellular location.
Recognizing that prior study has established the functional importance
of its cytoplasmic location, we tested the hypothesis that FAC protein
can also be found in the nucleus. Purified recombinant Escherichia
coli-derived FAC antigens were used to create antisera able to
specifically identify an Mr = 58,000 protein in lysates from human Epstein-Barr virus (EBV)-transformed cell
lines by immunoblot analysis. Subcellular fractionation of the cell
lysates followed by immunoblot analysis revealed that the majority of
the FAC protein was cytoplasmic, as reported previously; however,
approximately 10% of FAC protein was reproducibly detected
in nuclear fractions. These results were reproducible by two different
fractionation methods, and included markers to control for
contamination of nuclear fractions by cytoplasmic proteins. Moreover,
confocal image analysis of human 293 cells engineered to express FAC
clearly demonstrated that FAC protein is located in both cytoplasmic
and nuclear compartments, consistent with data obtained from
fractionation of the FA cell lines. Finally, complementation of the FAC
defect using retroviral-mediated gene transfer resulted in a
substantial increase in nuclear FAC protein. Therefore, while
cytoplasmic localization of this protein appears to be functionally
important, it may also exert some essential nuclear function.
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INTRODUCTION |
FANCONI ANEMIA (FA) IS AN autosomal
recessive disorder characterized by cell-cycle defects, cellular
hypersensitivity to agents that damage DNA, bone marrow failure,
diverse congenital anomalies, and a marked increase in the incidence of
acute myelogenous leukemia.1,2 Diagnostically,
the hallmark of this disorder is hypersensitivity of FA cells to the
clastogenic effects of DNA cross-linking agents such as diepoxybutane
(DEB) and mitomycin C (MMC).3,4 Based on these features, FA
has been proposed to result from a disorder in DNA repair. FA is
genetically heterogeneous, with at least five different complementation
groups identified by somatic cell hybrid analysis.5,6
Although the cDNA for FA complementation group C (FAC) has been cloned
by functional complementation, sequenced, and mapped to chromosome
9q22.3,5,7 there is no substantial sequence homology with
any known gene family, and the function of the gene product is
unknown.7 Recent cloning of the FAA gene has likewise been
unable to reveal any clear functional motifs or to shed light on the
roles these genes play in the clinical disease.8,9 It is
clear that the FAC gene product is necessary for optimal growth and
differentiation of hematopoietic progenitor cells,9,10 at
least in part by virtue of the capacity of the protein to modulate
mitotic inhibitory signals induced in progenitor cells by gamma
interferon.10,11
FAC cDNA contains an open reading frame of 558 amino acids and is
predicted to encode an Mr = 63,000 protein that is
predominantly hydrophobic with no obvious transmembrane domain, signal
sequence, or other functional motifs. The first mutation identified in
the gene was a point mutation that results in a leucine to proline substitution at position 554 (L554P).7 Other mutations have been discovered, including a splice mutation (termed IVS4+4A-T) that
occurs in members of the Ashkenazi-Jewish population.12 Both the L554P and IVS4+4A-T mutations are associated with a severe disease phenotype.
Although the cellular phenotype suggests that FA is a DNA repair
disorder, recent reports indicate that FAC is principally, if not
exclusively, cytoplasmic.13-15 Enforced expression of FAC protein in the nucleus by attachment of a nuclear localization signal
renders the wild-type protein incapable of correcting the FAC phenotype
in the MMC assay.15,16 Because the technical approach used
in the latter studies effectively prohibited cytoplasmic FAC
localization, it does not rule out a functional collaboration in the
cytoplasm between other proteins and the FAC protein resulting in
translocation to the nucleus. Consonant with the idea that other
proteins collaborate with the FAC protein is the recent observation
that MMC hypersensitivity occurs when L554P mutant FAC protein is
overexpressed in normal cells17 and that other proteins
coimmunoprecipitate or associate with FAC.13,15
In this report, we describe two new polyclonal antisera raised to
highly purified FAC antigens, both of which are capable of detecting
endogenous FAC protein in human peripheral blood cells and in cell
lines by simple immunoblot and indirect immunofluorescence analyses.
The antisera were used to determine the subcellular location of FAC
protein in human cells. Based on evidence from two different
subcellular fractionation procedures and confocal imaging experiments,
we show that the FAC protein is located both in the cytoplasm and in
the nucleus of human cells.
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MATERIALS AND METHODS |
Cell lines, viruses, plasmids, and chemicals.
Epstein-Barr virus (EBV)-transformed lymphoblast cell lines HSC536N
(originally obtained from Dr Manuel Buchwald, Hospital for Sick
Children, Toronto, Ontario, Canada) and PD4L were supplied by Dr Markus
Grompe (Fanconi Anemia Cell Repository, Oregon Health Sciences
University [OHSU]). The HSC536N cell line has mutations resulting in
a leucine to proline substitution at amino acid position 554 on one
allele and a deletion of the second FAC allele. The PD4L cell line has
a deletion of a G residue in one allele resulting in a severe
truncation, and a mutation that results in a stop codon at amino acid
position 185 on the other allele.12 The HSC536N/FAC cell
line is corrected for MMC hypersensitivity and was produced by
infecting parental cell lines (HSC536N) with FAC-encoding retroviruses,
LFACSN. The HSC536N/neo derivative used as a negative control in some
experiments described was produced by infecting the HSC536N cell line
with an amphotropic- or gibbon ape leukemia virus (GALV)-pseudotyped
virus that encodes neomycin phosphotransferase (LXSN). The LXSN viruses
were produced by PA31718 or PG1319 retroviral
packaging cell lines transfected with the parental vector
pLXSN.20 The EBV-transformed cell line JY, derived from a
normal individual, was a gift from Dr Richard Maziarz (OHSU). All
lymphoblast cell lines were grown in RPMI 1640 (GIBCO-BRL, Grand
Island, NY) supplemented with 15% fetal bovine serum (FBS) (defined,
heat-inactivated, low endotoxin; HyClone, Logan, UT), 1%
L-glutamine (GIBCO-BRL), and 50 µg/mL gentamicin
(GIBCO-BRL) at 37°C and 5% CO2 in a humidified
atmosphere.
Constructs for expression of FAC protein in bacteria were made by
subcloning an EcoRI-XbaI FAC fragment from plasmid pFAC3 (a gift from
M. Buchwald, Toronto) into the vector pUC18 (Pharmacia, Piscataway,
NJ). The resulting vector (pUCFAC3) was digested with EcoRI and SalI
and then inserted into an EcoRI/Sal I-cut pGEX-4T-3 expression vector
(Pharmacia) to produce pGEX-FAC1. To make pGEX-FAN2, the pUCFAC3
subclone was digested with EcoRI and ligated into a pGEX-4T-3 vector
that was linearized with EcoRI. For COS-7 and 293 cell expression,
full-length FAC was subcloned into vector pLXSN and a HpaI/XbaI
fragment containing FAC from this subclone was inserted into an
EcoRV/XbaI-cut pcDNA3 vector (Invitrogen, San Diego, CA). Cells were
transfected with pcFAC or pcDNA3 using DEAE-dextran by standard
methods. Chemicals were purchased from Sigma (St Louis, MO) unless
indicated otherwise.
Purification of FAC antigens.
Glutathione S-transferase (GST) fusion proteins were produced in E
coli strain HB101, which had been transformed with either pGEX-FAN2
or pGEX-FAC1 and induced with isopropyl  -D-galactoside (IPTG). The GST fusion proteins were purified by affinity
chromatography on glutathione Sepharose 4B (Pharmacia) as described by
the manufacturer and modified by Frangioni and Neel.21
Briefly, E coli cell pellets were resuspended in 10 mmol/L Tris
Cl (pH 7.4), 150 mmol/L NaCl, 1 mmol/L EDTA, 5 mmol/L dithiothreitol
(DTT), and 1.5% (wt/vol) sarkosyl and disrupted by passage through a
cold French pressure cell. The resulting crude cell extracts were
centrifuged to remove debris, and Triton X-100 was added to the
supernate to a final concentration of 2% (vol/vol). The extracts were
incubated with glutathione Sepharose 4B and allowed to bind overnight
at 4°C. The affinity matrices were first washed with
phosphate-buffered saline (PBS), then with PBS plus 1% (vol/vol)
Triton X-100, followed by PBS, and finally equilibrated in 50 mmol/L
Tris Cl (pH 8.0). Cleavage of an estimated 9-mg GST fusion protein
bound to the affinity matrix (10-mL bed volume) was performed in 50 mmol/L Tris Cl (pH 8.0), 2.5 mmol/L CaCl2, and 150 mmol/L
NaCl containing approximately 24 to 30 NIH U thrombin for 4 hours at
37°C. The reaction was stopped by adding EDTA to a final
concentration of 5 mmol/L. Preparative SDS-PAGE (model 491 Preparative
Cell; Bio-Rad, Hercules, CA) was used to purify the cleaved proteins to
homogeneity.
Polyclonal antibody production to FAC amino- and carboxy-terminal
regions.
Antibodies were prepared by immunizing New Zealand white rabbits with
purified FAN2 or FAC1 antigens as described previously.22 Briefly, initial injections were given in complete Freund adjuvant (GIBCO-BRL), and injections of approximately 100 µg each in
incomplete Freund adjuvant were given at monthly intervals thereafter.
Antibody production was monitored from test bleeds by immunoblotting
cell lysates from COS-7 cells that were transiently transfected with plasmid pcFAC compared with lysates from cells that had been
transfected with the parental plasmid pcDNA3. Antisera with apparent
specificity for FAC protein were further characterized against
lymphoblast cell lines containing normal or mutant FAC protein. For
confocal studies and for immunoblot analysis of the 293 and 293/FAC
cell lysates, anti-FAC1 was affinity-purified against FAC1 antigen using the strip purification method.23
Immunoblotting.
Cells were washed twice with PBS, and the cell pellets were solubilized
in RIPA (10 mmol/L Tris Cl [pH 7.6], 150 mmol/L NaCl, 1% [wt/vol]
sodium deoxycholate, 1% [vol/vol] Triton X-100, 0.1% [wt/vol]
SDS, 1% [vol/vol] aprotinin, 2 mmol/L
Na3VO4, and freshly added leupeptin [1
µg/mL], pepstatin [1 µg/mL], and 1 mmol/L
phenylmethylsulfonylfluoride [PMSF]). Lysates were centrifuged at
16,000g for 15 minutes at 4°C. Protein concentrations of the
supernates were determined with a commercially available protein
microassay (Bio-Rad) using BSA as a standard. Cell lysates were mixed
with Laemmli sample buffer, heated at 94°C for 5 minutes, and
separated by SDS-PAGE in 7.5% polyacrylamide gels. Proteins were
electroblotted onto Bio-Blot nitrocellulose (Costar, Cambridge, MA) as
previously described.24 Incubations and washes were
performed at room temperature with gentle rocking. Nonspecific binding
was blocked by incubating the blots in 5% (wt/vol) Carnation nonfat
milk (Nestle Foods, Glendale, CA) for 1 hour. Each blot was incubated
with one of the rabbit antisera at a 1:1,000 dilution in 5% nonfat
milk for 1 hour. Primary antibody was followed by six (5-minute) washes of Tris-buffered saline (TBS) containing 0.005% Tween 20, and then
incubation for 30 minutes with goat anti-rabbit IgG-HRP (Bio-Rad) at a
1:10,000 dilution in 5% nonfat milk. After incubation with secondary
antibody, the blot was washed as described with TBS-Tween 20. Antibody-reactive proteins were detected using Amersham ECL reagents
(Arlington Heights, IL) and visualized with x-ray film. Immunoblots
reprobed with different primary antibodies were stripped according to
the ECL manufacturer's recommendations (Amersham) as described by
Kauffman et al.25
Subcellular fractionation.
Cellular fractionation was performed at 4°C as described previously
by Lewis et al.26 Briefly, 5 × 108 JY cells
were lysed in a hypotonic buffer (Tris Cl, pH 7.4, containing freshly
added protease inhibitors: 10 mmol/L Tris Cl [pH 7.4], 1%
[vol/vol] aprotinin, 2 mmol/L sodium vanadate, 1 µg/mL leupeptin, 1 µg/mL pepstatin, and 1 mmol/L PMSF) by 25 to 50 strokes of a Potter-Elvehjem homogenizer (Teflon pestle; clearance, 0.10 to 0.15 mm). Cells were observed microscopically to ensure that they were fully
disrupted and that the nuclei were intact. The homogenate was made
isotonic (final concentration: 10 mmol/L Tris Cl, pH 7.4, 0.1 mmol/L
EDTA, 0.85% NaCl, and 0.1 mmol/L MgSO4) and then centrifuged at 1,000g to separate insoluble material from the cytoplasmic fraction. The pellet was resuspended in buffer C (10 mmol/L
Tris Cl, pH 7.4, 0.1 mmol/L EDTA, 0.25 mmol/L sucrose, and protease
inhibitors as described for RIPA) to a total vol of 5 mL. To separate
plasma membranes, mitochondria, and nuclei, the solution was
centrifuged at 1,000g for 30 minutes. The supernate was
discarded, and the pellet was resuspended in buffer C, loaded onto a
32% to 52% (wt/vol) sucrose gradient cushion (containing 0.1 mmol/L
EDTA, 10 mmol/L Tris Cl, pH 7.4, and protease inhibitors as described
for RIPA), and centrifuged at 100,000g for 45 minutes. Distinct
fractions representing plasma membranes, mitochondria, and nuclei were
removed and prepared for SDS-PAGE analysis. An alternative method for
subcellular fractionation was also performed as described by Tsuda and
Alexander.27 After electrophoresis, the proteins were
electroblotted onto nitrocellulose for immunoblotting as already
described. Blots were reprobed with anti- tubulin to enable
estimation of the signal in various subcellular fractions by
densitometry. Correction for cytoplasmic protein in nuclear fractions
was calculated for each fractionated lysate. Densitometric results were
obtained by analyzing immunoblots with a model 620 Video Densitometer
(Bio-Rad).
Indirect immunofluorescence.
293 cell derivatives were grown on chamber slides, fixed with 3.7%
paraformaldehyde, and permeabilized with 0.2% Triton X-100. The cells
were blocked with a solution of 3% normal goat serum in PBS. Primary
antibody was added (affinity-purified rabbit polyclonal anti-FAC1 and
anti- tubulin monoclonal [Boehringer Mannheim, Indianapolis, IN]
or anti-p300 monoclonal [Upstate Biotechnology, Lake Placid, NY]) and
allowed to bind overnight at room temperature with gentle rocking.
After five 5-minute washes with PBS, secondary antibody was added
(Texas Red-conjugated goat anti-rabbit and Oregon Green-conjugated
goat anti-mouse; Molecular Probes). Primary and secondary antibodies
were diluted in PBS containing 3% normal goat serum. Secondary
antibody incubation was performed in the dark. The stained cells were
mounted in SlowFade (Molecular Probes).
Stained cells were viewed with a Leica 900 confocal
laser-scanning microscope (Leica Inc, Deerfield, IL) equipped with a
krypton-argon laser, a 40× 1.3 NA oil objective lens, a simultaneous
dual-channel detector, and a 24-bit imaging system including Leica's
scanware software function. Appropriate filter sets were used to
distinguish between Texas Red and Oregon Green emissions. For the
optimal z series, slices were taken approximately every 1 µm over an
8-µm thickness with pinhole settings maintained at optimal levels for minimal slice thickness resolution. Settings were optimized using positively stained cells and maintained during scanning of control cells to maintain relative brightness. Collected images were imported into Adobe Photoshop 4.0 (San Jose, CA), pseudo-colored, and overlapped to produce merged images.
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RESULTS AND DISCUSSION |
Expression and purification of FAC proteins from bacteria.
The GST system enables inducible high-level expression of fusion
proteins and subsequent purification by glutathione-affinity chromatography, followed by thrombin cleavage to release the protein of
interest. Two regions of FAC were expressed as GST-fusion proteins in
E coli (Fig 1). GST-FAC1 is a
fusion protein between GST and 453 amino acids of FAC, from amino acid
position 104 to the last amino acid of the FAC protein at position 558. GST-FAN2 is the reciprocal GST-FAC fusion protein that contains
residues 7 through 105 of amino-terminal FAC. Together, these two
proteins encode all but the first six amino acids of the FAC coding
region. GST-FAC1 and GST-FAN2 fusion proteins were expressed in E
coli and purified on glutathione Sepharose 4B.
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| Fig 1.
Expression and partial purification of GST-FAC fusion
proteins. Diagram of the regions of the FAC protein used as antigen to
stimulate antibody production. Numbers correspond to the amino acid
position in the FAC peptide. Arrow indicates the thrombin cleavage site
in the GST fusion proteins.
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We observed that the purification strategy recommended by the
manufacturer resulted in inadequate purification of FAC, which is a
predominantly hydrophobic protein.7 Moreover, minor (but often very antigenic) contaminating proteins in E coli are
difficult to purify away from the desired antigen using purification
strategies that work well for predominantly hydrophilic
proteins.22 In addition, although the antigenic response is
difficult to predict with certainty, the hydrophobicity of FAC
suggested that it might be less antigenic than the GST portion of the
fusion protein. Based on all of these considerations, we designed a
stringent purification strategy that included thrombin cleavage of the
GST portion of the GST-FAC fusion proteins (the cleaved proteins are termed FAC1 and FAN2). After thrombin cleavage, FAC1 and FAN2 were
completely solubilized and denatured before purification by preparative
gel electrophoresis. Purity of the protein collected was estimated by
subjecting a sample of the purified protein to SDS-PAGE,
electroblotting to nitrocellulose, and detecting the proteins by
sensitive colloidal gold staining. Purified antigens were injected into
rabbits according to the procedure already described.
Detection of FAC in EBV-transformed lymphoblast cell lines by
immunoblot analysis.
To determine if the rabbit polyclonal antibodies were specific for FAC,
we analyzed lysates from several different cell lines. Using both
carboxy- and amino-terminal antibodies, a protein of approximate
Mr = 58,000 was detected in lysates from the normal JY cell line, as well as those derived from the HSC536N cell line, which contains full-length FAC with the L554P mutation (Fig 2A and B).
No obvious differences between mutant and wild-type FAC protein
migration patterns were observed. The FAC EBV-transformed cell line PD4
has a C to T transition at nucleic acid position 808 that predicts a
severely truncated protein of Mr = 20,000. As
expected, protein comigrating with FAC protein in the PD4L was absent,
as reported by Yamashita et
al.13
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| Fig 2.
Detection of FAC protein with anti-FAC antisera. One
hundred micrograms of total protein from lymphoblast cell lines was
immunoblotted with anti-FAC polyclonal antibodies as described. (A)
Lane 1, JY; lane 2, PD4L; lane 3, HSC536N; lanes 4 and 6, HSC536N/neo; lanes 5 and 7, HSC536N/FAC transduced with amphotropic- and
GALV-pseudotyped vectors, respectively; lane 8, COS-7/neo; lane 9, COS-7/FAC. FAC protein in A was detected with FAC1 antiserum. B shows
the same blot as A, after stripping and reprobing with FAN2 antiserum. (C) Immunoblot of COS-7/FAC cell lysates (10 µg total cell protein per lane) demonstrating that the strong signal observed in immunoblots with FAC1 antisera and FAN2 antisera is completely blocked by addition
of purified antigen. The first lane of each set represents blocking of
FAC-reactive epitopes with 20 µg purified cognate antigen in a 1-hour
preincubation step with 1 µL antiserum. The second lane of each set
is the unblocked positive control. Complete blocking was also possible
with 10 µg antigen (data not shown).
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For further verification of antibody specificity, we compared lysates
from COS-7 cells that were transiently transfected with pcFAC (a vector
that encodes full-length FAC) and lysates prepared from COS-7 cells
transfected with the backbone vector pcDNA3. Both carboxy-terminal and
amino-terminal antibodies recognized a strong band at
Mr = 58,000 in the COS-7/FAC lysates, but not in
the pcDNA3-transfected COS-7 cell lysates or in experiments where
purified cognate antigen was added to the antibody incubation step (Fig
2C). Immunoblots of COS-7 cells transfected with the plasmid
encoding FAC contained a signal that comigrated with the strong signal
observed in the lysates derived from human hematopoietic cells.
FAC protein is located in the cytoplasm and nucleus.
To examine FAC localization with anti-FAN2 and anti-FAC1 antibodies,
lysates from JY (normal) EBV-transformed cell lines were fractionated
and analyzed by immunoblotting. We found that the majority of FAC
protein was located in the cytoplasm, as reported previously.13-15 However, FAC protein was reproducibly
found in nuclear fractions in multiple separate experiments (Fig 3A;
lanes 1 to 3 represent a fourfold increase in sample loading compared with the cytoplasmic fraction in lane 4). The quality of the
fractionation was assessed by reprobing the blot with anti- tubulin
(Fig 3B). The amount of tubulin present in the nuclear fractions
and cytoplasmic fractions was used to estimate the percentage of
cytoplasmic contamination of the nuclear fractions. We estimate that
the amount of FAC present in the nuclear fraction after correction for
cell number and cytoplasmic contamination was 10%. This observation
was confirmed with anti-FAN2 and anti-FAC1 antisera, which recognize
nonoverlapping epitopes in the FAC protein (data not shown). FAC
protein was not detected in the membrane or mitochondrial fractions
(Fig 3A, lanes 1 and 2).
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| Fig 3.
FAC protein is located in the cytoplasm and nucleus of
normal human lymphoblasts. JY cells were lysed by dounce
homogenization and separated into subcellular fractions according to
the method of Lewis et al.26 Individual fractions were
separated by SDS-PAGE and immunoblotted with FAC1 (A) or tubulin
(B) antiserum. Lane 1, plasma membrane fraction; lane 2, mitochondrial
fraction; lane 3, nuclear fraction; lane 4, cytoplasmic fraction. The
cytoplasmic fraction contains one fourth the cell number contained in
lanes 1 to 3. Similar results were obtained with FAN2 antiserum (data not shown). Values for quality of fractionation were calculated by
desitometric analysis. The total amount of signal observed for tubulin (a cytoplasmic marker) in all fractions was measured and then
used to calculate the amount occurring in the nuclear fraction as an
artifact.
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To confirm and extend these observations, an alternate nuclear
fractionation protocol was performed on normal and FA EBV-transformed lymphoblasts.27 We observed that FAC protein was in both
the cytoplasmic (C) and nuclear (N) fractions of JY and HSC536N cells at an approximate C to N ratio of 3:2 and 9:1 for the two cell types,
respectively, as estimated by densitometry and corrected for cell
number and the amount of tubulin, as already described (Fig
4).
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| Fig 4.
FAC protein is located in the cytoplasm and nucleus of
normal and FA EBV-transformed human lymphoblasts. Cells were
fractionated according to the method of Tsuda et al,27 and
the separated nuclear and cytoplasmic lysates were analyzed by
SDS-PAGE. FAC protein was detected by probing transferred proteins with
FAN2 antisera. The blot was stripped and reprobed with anti- tubulin as a control for artifactual presence of cytoplasmic protein in nuclear
fractions as described in Fig 3. Reprobing with anti-FAC1 gave similar
results (data not shown). Lane 1, JY cytoplasmic fraction; lane 2, HSC536N cytoplasmic fraction; lane 3, JY nuclear fraction; lane 4, HSC536N nuclear fraction. The cytoplasmic fractions (70 µg total
protein loaded) contain approximately one fourth the cell number of the
nuclear fractions (50 µg total protein loaded).
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The definitive method for determining the exact location of a substance
in a three-dimensional volume is to use cross-sectional imaging studies
such as confocal image analysis. This strategy allows virtual
examination of the internal structure of the object, free from
overlapping and potentially confounding images occurring in different
planes (eg, cytoplasm overlapping the nucleus in the cell).
Accordingly, JY and HSC536N cells were examined by confocal
laser-scanning microscopy after immunostaining with anti-FAC1 or
anti- tubulin. Although it is our impression that the pattern of
dual cytoplasmic and nuclear FAC protein localization observed in
fractionation procedures was also observed by this technique, the
endogenous expression of FAC in these cells is at the detection limit
for this assay (data not shown). Therefore, a second approach was taken
to more definitively determine the subcellular distribution of the FAC
protein by confocal analysis. Human 293 cells were transfected with
pcFAC (termed 293/FAC) or the parental plasmid pcDNA3 (293/pcDNA3) as a
control and selected for stable expression in G418. To ensure FAC
expression, cell lysates were prepared from the cell lines and analyzed
by SDS-PAGE followed by immunoblot with affinity-purified anti-FAC1. A
strong unique band was observed at Mr = 58,000 in
cells transfected with pcFAC (Fig 5C),indicating that the 293/FAC cells express FAC abundantly and that no
cross-reacting signals were present in the control cell line by
immunoblot analysis. For immunofluorescence studies, these cell lines
were double-stained with affinity-purified FAC1 and nuclear (anti-p300)
or cytoplasmic (anti- tubulin) markers. The Texas Red and Oregon
Green fluorophores conjugated to the secondary antibodies were selected
to ensure the largest spread between emission wavelengths to maximize
specificity of the signals (615 nm and 522 nm, respectively). As shown
in the representative single optical sections (made parallel to the growing surface) in Fig 5B, the staining of FAC protein with
affinity-purified anti-FAC1 is evident in 293 cells transfected with
pcFAC as compared with an optical section through 293 cells transfected
with pcDNA3 (parental vector). The 293/FAC cells examined were a
population of stably transfected cells rather than a clonal population,
enabling examination of cells expressing different levels of the FAC
protein. The different levels of FAC protein observed in the transduced cells may also be due to involvement of FAC in a cell-cycle pathway or
regulated FAC expression during the cell cycle, as suggested by recent
reports by Kruyt et al28 and Kupfer et al.29
This effect is demonstrated in Fig 5A, where cells were double-labeled with anti-p300 (green) and anti-FAC (red). In the first group (cells
stained with anti-FAC), there is one cell (lower right) that stains
brightly with the antibody, as well as a cell just above with weaker
staining. Other cells in this field have weaker FAC staining, although
all pcFAC-transfected cells that became G418-resistant were positive
compared with the negative control. The nuclei of the stained cells are
visible in the second group of images viewed for p300 staining. p300
staining is not observed in the cytoplasm, as expected. The confocal
images resulting from anti-FAC and anti-p300 staining were merged to
determine if the signals colocalized. The composite panel in Fig 5A
demonstrates colocalization (yellow) of FAC protein and the nuclear
marker p300. Colocalization of p300 and FAC protein can be observed
even in the cell expressing a relatively low level of FAC protein.
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| Fig 5.
FAC protein colocalizes with a nuclear protein marker in
double-immunostained cells. Human 293 cells transfected with a
FAC-encoding plasmid were analyzed by confocal laser-scanning
microscopy after double labeling with affinity-purified anti-FAC1 and
anti-p300 (a nuclear marker). The images shown contain a transfected
cell population that express varying amounts of FAC protein. Nine
optical sections taken from the apical (top left) to basal (bottom
right) cell surface are shown. Composite images were created by merging corresponding images. The FAC protein is red, the p300 protein is
green, and colocalization is yellow. (B) Optical section though a
FAC-expressing cell (293/FAC) and 293 cells transfected with the
parental pcDNA3 plasmid without the FAC gene insert (293/pcDNA3). (C)
293/pcDNA3 and 293/FAC cell lysates were analyzed by SDS-PAGE, transferred to nitrocellulose, and probed with the affinity-purified antibody used in the confocal analysis. Arrow indicates FAC protein. Molecular size markers are indicated at left.
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As permeabilization and fixation procedures can cause artifactual
relocalization of protein,30 the cells were also stained with anti- tubulin to determine if these procedures resulted in the
appearance of tubulin in the nucleus. The images in Fig 6 show that
the nuclear margin is visible in the stained cells and tubulin
staining is not present in the nuclear compartment, although the cells
stained with anti-FAC1 contain FAC protein in the nucleus (Fig
6). The fractionation studies made in
parallel using the EBV-transformed cell lines (JY and HSC536N)
demonstrated that the nuclear localization of FAC protein is not merely
a consequence of overexpression. Moreover, nuclear localization was
decreased or absent in images of some 293 cells overexpressing FAC
(data not shown), further suggesting that nuclear localization is not an artifact of overexpression and that translocation may be dependent on some unknown mechanism.
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| Fig 6.
Confocal laser-scanning of double-stained cells reveals
that FAC protein colocalizes with a cytoplasmic protein marker ( tubulin). The arrangement of images is as described in Fig 5. In the
optical sections shown, FAC protein is stained red, tubulin is
green, and colocalization is yellow. Although the cytoplasmic marker
does not appear in the nuclei of the cells, FAC protein staining is
evident.
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To determine if the amount of FAC protein in nuclear fractions
increases after correction by FAC-encoding retroviral vectors, nuclear
fractions from normal cells, FA group C cells, and FA cells that had
been corrected by transduction with retroviral vectors encoding
wild-type FAC were compared. The specific mutant cells we used for
these experiments endogenously express a full-length mutant FAC protein
in deceased amounts compared with wild-type cells. Figure 7 (column 1)
shows FAC in nuclear fractions of JY cells, a normal EBV-transformed
cell line. The FAC protein was detected by either anti-FAC1 (A and C)
or anti-FAN2 (B and D). Figure 7 (column 2)shows lysates prepared from HSC536N probed with each antibody, as
described for JY cells. The HSC536N cells have lower amounts of FAC
protein detected in nuclear fractions compared with normal JY cells, as
predicted by the loss of one of the FAC alleles in this cell line.
Similarly, HSC536N cells transduced with retroviral vectors expressing
neomycin phosphotransferase, but not wild-type FAC, have reduced
amounts of FAC compared with the normal JY cells (Fig 7, column 3). In
HSC536N cells transduced by retroviral vectors encoding wild-type FAC
and corrected for MMC hypersensitivity, the relative amount of FAC in
nuclear fractions is increased compared with the uncorrected HSC536N
cells and is comparable to normal cells (Fig 7, column 4). In several
separate fractionation experiments using these cell lines, we estimated cytoplasmic and nuclear fractions of FAC and observed that FAC consistently appeared in the nuclear fraction in increased amounts after the cells were corrected by FAC-encoding retroviral vectors (Fig
4). Thus, in both of the corrected cell lines, using two nonoverlapping
FAC antisera, we observed a reproducible increase in the amount of FAC
in the nuclear fractions.
View larger version (44K):
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| Fig 7.
FAC protein is decreased in nuclear fractions of FA cells
compared with normal and corrected FA cells. Cells were fractionated according to the method of Tsuda et al,27 and the nuclear
lysates (50 µg each) were analyzed by SDS-PAGE. FAC protein (arrow)
was detected by probing with FAC1 antisera (A and C) or stripping and
reprobing with FAN2 antisera (B and D). A and B and C and D represent
independent experiments where cells in columns 3 and 4 were transduced
by pseudo-typed viruses encoding neo or FAC, respectively. Column 1, JY; column 2, HSC536N; column 3, HSC536N/neo; column 4, HSC536N/FAC.
The blots were stripped and reprobed with tubulin as shown in the
bottom 2 panels, and the signals for nuclear and cytoplasmic tubulin were estimated by densitometry. These fractionation quality
controls are corrected for cell number, and the numbers shown are the
percent of cytoplasmic protein contained in the nuclear fraction.
Thereby, we estimate that the artifactual component of the signal
detected for FAC in the nuclear fraction of JY cells (for A and B) is
only 6%.
|
|
We attribute our capacity to detect FAC protein in the nuclei of human
cells to technical differences between our methods and those used by
other investigators. First, the subcellular fractionation procedures we
used were unique to our study and may have allowed detection of the
nuclear portion of the FAC protein. Second, one of our detection
methods was based on a simple immunoblot, not a more lengthy
immunoprecipitation procedure that might be associated with a loss of
signal from the nuclear fraction. This is of particular importance
because the FAC protein is labile and requires careful handling to
maintain a signal. Third, individual antibodies often differ in the
capacity to detect protein in different assays, such as
immunoprecipitation, immunoblot, or immunofluorescence (IFA). Finally,
it is possible that the FAC protein located in the nucleus cannot be
immunoprecipitated, or it may be below the detection limit of that
assay.
We have described two independent approaches that indicated that FAC
protein is located not only in the cytoplasm of the cell but in the
nucleus, as well. Interestingly, in studies using an immunofluorescence-based assay, a preliminary report using a FLAG epitope-tagged FAC described both nuclear and cytoplasmic
localization.31 In addition, Youssoufian14
observed an occasional cell with nuclear staining in cells
overexpressing FAC using an IFA.
What is the implication for dual cytoplasmic and nuclear localization
of FAC protein in terms of function? There are several possible
explanations. First, precedent exists for proteins whose predominant
location is distinct from their site of function. For example, the bulk
of the env glycoprotein of the spleen focus-forming virus is
located in the endoplasmic reticulum, yet only a very small portion
(~5%) transported to the cell surface has productive interaction
with the erythropoietin receptor.32-34 Resolving the location of the interaction depended on a combination of genetic and
biochemical evidence34,35; as is likely to be the case for
the FAC protein. Second, since forced nuclear localization abrogates
FAC protein function,17 it is possible that FAC protein
collaborates with proteins in the cytoplasm that modify it for its
function in the nucleus (eg, DNA repair) or that FAC-associated
proteins15,17,36 have nuclear functions themselves that are
activated by FAC protein in the cytoplasm. There are a variety of
examples of this in mammalian cells. For example, the NF-kB protein is
maintained in an inactive form in the cytoplasm by its inhibitor, IkB.
In response to a variety of stimuli, IkB becomes phosphorylated and is
quickly degraded, releasing NF-kB, which then translocates into the
nucleus.37-39 Other examples of translocation in response
to an extracellular signal include MAP kinases
(p42mapk and p44mapk), Stat
molecules,40 the Drosophila homeoprotein
exd,41 and rsk-encoded protein
kinases.42,43 A third possible explanation for the dual
location of the FAC protein is that it may have different functions in
the nucleus and the cytoplasm, similar to the hepatitis B virus HBx
protein. The majority of HBx is in the cytoplasm, but a small amount is
in the nucleus. The cytoplasmic HBx stimulates signal transduction
pathways, while the nuclear form of HBx transactivates transcription
factors in the nucleus.44
Our finding of nuclear localization is compatible with the recent
speculation of Liebetrau et al,45 who examined the murine and human FAC protein for functional motifs. They reported that the FAC
protein has two regions that may be leucine zippers, one of which is
preceded by a basic region and a helix-turn-helix motif. They propose
that FAC has a structure typical of a nuclear protein. Whether these
proposed domains are essential for the observed nuclear localization of
FAC is unknown. How and if the nuclear FAC differs from the cytoplasmic
FAC protein and what factors dictate differential localization will be
an important question for future study.
| |
FOOTNOTES |
Submitted March 6, 1997;
accepted October 1, 1997.
Supported by grants from the National Institutes of Health (HL48546,
G.C.B.), the Department of Veterans Affairs (G.C.B. and M.C.H.), the
Medical Research Foundation of Oregon (M.E.H. and M.C.H.), and the
Fanconi Anemia Research Fund (M.E.H.).
Address reprint requests to Grover C. Bagby Jr, MD,
Division of Hematology and Medical Oncology, Oregon Health Sciences
University, 3181 SW Sam Jackson Park Rd, Portland, OR 97201.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Manuel Buchwald (Toronto, Ontario, Canada) for plasmid pFAC3
containing the full-length FAC cDNA and for cell line HSC536N. Other FA
EBV-transformed cell lines were kindly provided by Marcus Grompe
(Fanconi Anemia Registry, Portland, OR). We are grateful to Lorene
Langeberg (Portland, OR) for advice and help in preparation of the
confocal data for publication. We thank the members of the Fanconi
anemia research community at large for stimulating discussions,
particularly the members of the OHSU Fanconi Anemia Program Project
team.
| |
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Expression of the Fanconi Anemia Group A Gene (Fanca) During Mouse Embryogenesis
Blood,
July 15, 1999;
94(2):
818 - 824.
[Abstract]
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I. Garcia-Higuera, Y. Kuang, D. Naf, J. Wasik, and A. D. D'Andrea
Fanconi Anemia Proteins FANCA, FANCC, and FANCG/XRCC9 Interact in a Functional Nuclear Complex
Mol. Cell. Biol.,
July 1, 1999;
19(7):
4866 - 4873.
[Abstract]
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A. Savoia, I. Garcia-Higuera, and A. D. D'Andrea
Nuclear Localization of the Fanconi Anemia Protein FANCC Is Required for Functional Activity
Blood,
June 1, 1999;
93(11):
4025 - 4026.
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I. Garcia-Higuera, A. D. D'Andrea;, F. A.E. Kruyt, and H. Youssoufian
Regulated Binding of the Fanconi Anemia Proteins, FANCA and FANCC
Blood,
February 15, 1999;
93(4):
1430 - 1432.
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H. Youssoufian, F. A.E. Kruyt, and X. Li
Protein Replacement by Receptor-Mediated Endocytosis Corrects the Sensitivity of Fanconi Anemia Group C Cells to Mitomycin C
Blood,
January 1, 1999;
93(1):
363 - 369.
[Abstract]
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F. A.E. Kruyt, T. Hoshino, J. M. Liu, P. Joseph, A. K. Jaiswal, and H. Youssoufian
Abnormal Microsomal Detoxification Implicated in Fanconi Anemia Group C by Interaction of the FAC Protein With NADPH Cytochrome P450 Reductase
Blood,
November 1, 1998;
92(9):
3050 - 3056.
[Abstract]
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T. Yamashita, G. M. Kupfer, D. Naf, A. Suliman, H. Joenje, S. Asano, and A. D. D'Andrea
The Fanconi anemia pathway requires FAA phosphorylation and FAA/FAC nuclear accumulation
PNAS,
October 27, 1998;
95(22):
13085 - 13090.
[Abstract]
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D. Näf, G. M. Kupfer, A. Suliman, K. Lambert, and A. D. D'Andrea
Functional Activity of the Fanconi Anemia Protein FAA Requires FAC Binding and Nuclear Localization
Mol. Cell. Biol.,
October 1, 1998;
18(10):
5952 - 5960.
[Abstract]
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F. A.E. Kruyt and H. Youssoufian
The Fanconi Anemia Proteins FAA and FAC Function in Different Cellular Compartments to Protect Against Cross-Linking Agent Cytotoxicity
Blood,
October 1, 1998;
92(7):
2229 - 2236.
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Abstract
Introduction
Abstract