From the Department of Pathology, School of Medicine, Yale University, New Haven, Connecticut 06510
Received for publication, November 7, 2002, and in revised form, December 23, 2002
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ABSTRACT |
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NFBD1/KIAA0170 is a nuclear
factor with an N-terminal FHA
(forkhead-associated) domain and a
tandem repeat of BRCT (breast cancer susceptibility gene-1 C terminus)
domains, both of which are present in a number of proteins
involved in DNA repair and/or DNA damage signaling pathways. We have
investigated the association of NFBD1 with DNA damage responses. We
found that the NFBD1 transcript is abundant in the
testis relative to other tissues. NFBD1 is a chromatin-associated
protein and is modified in G2/M phase or after DNA
damage. NFBD1 phosphorylation in response to ionizing radiation (IR)
was ATM-dependent. NFBD1 exhibited diffuse
nuclear staining in the majority of untreated cells analyzed by
indirect immunofluorescence and formed discrete nuclear foci after
exposure to IR, UV radiation, and hydroxyurea treatment. IR induced
NFBD1 foci within 1 min. The foci colocalized with The faithful duplication and segregation of genetic information to
daughter cells are of primary importance for maintaining genome
stability. DNA double-strand breaks
(DSBs)1 pose serious threats
to genome stability. DSBs can be induced by exogenous agents such as
ionizing radiation (IR), by endogenously generated free radicals
produced during normal cellular metabolic reactions, or by replication
stress (1). DSBs are also naturally occurring intermediates in normal
cellular processes such as meiotic recombination and V(D)J
recombination (1).
A sophisticated surveillance network, including checkpoint controls,
exists in all eukaryotes to detect and repair damaged DNA before a cell
moves on to the next phase of the cell cycle (2). One of the earliest
events in the cellular response to DSBs is the phosphorylation of a
histone H2A variant, H2AX, at sites of DNA damage (3-5). H2AX is
rapidly phosphorylated (within 1 min) at an evolutionarily conserved
residue, Ser139, when DSBs are induced in mammalian cells,
resulting in discrete phosphorylated H2AX ( DNA damage activates a cascade of protein kinases that relay the signal
to downstream effectors to halt the cell cycle and that facilitate
repair of the damage (2). In budding yeast, the serine/threonine kinase
Mec1 is a master regulator of cellular responses to DNA damage. In
response to DNA damage, Rad9 is phosphorylated in a
MEC1-dependent manner (10-12); and in turn,
phosphorylated Rad9 apparently recruits another serine/threonine
kinase, Rad53, to the Mec1 complex for activation (13, 14). (Note that
budding yeast Rad9 is unrelated to human RAD9.) Alternatively,
phosphorylated Rad9 oligomers may act as a scaffold to bring Rad53
molecules into close proximity to each other, facilitating
cross-phosphorylation between Rad53 molecules and subsequent release of
activated Rad53 (15). Rad9 also regulates activation of Chk1, another
important effector kinase of Mec1, in the G2/M checkpoint
(16).
Mec1 belongs to the family of phosphatidylinositol
3'-kinase-related kinases (PIKKs). In mammals, these kinases
include ATM (ataxia-telangiectasia
mutated) and ATR (ATM- and
Rad3-related). ATM, which is not
essential for development, mediates the early response to DNA DSBs, and
its inactivation in patients with ataxia-telangiectasia leads to
checkpoint defects and genome instability (17). Rad53 is an FHA
(forkhead-associated)
domain-containing kinase, and its human ortholog is CHK2.
CHK2 mutations have been found in some Li-Fraumeni syndrome
kindreds that do not have p53 mutations (18, 19) and in a variety of
sporadic cancers (20-23). IR-dependent CHK2 activation
requires that its FHA domain be functional (24) and is
ATM-dependent (25). The Rad9 checkpoint protein
in Saccharomyces cerevisiae and its ortholog Crb2/Rhp9 in
Schizosaccharomyces pombe share sequence similarity
primarily within their tandem repeat of C-terminal BRCT
(BRCA1 C terminus) domains. The only human genes encoding proteins with tandem repeats of C-terminal BRCT domains
are BRCA1, 53BP1, and
NFBD1/KIAA0170. This has suggested that one or
more of these proteins may serve budding yeast Rad9-like functions in
coupling ATM and ATR to CHK2. Although BRCA1 interacts with and is a
substrate of CHK2, BRCA1 is not required for CHK2 activation after IR
(26). However, BRCA1 regulates CHK1 activation in the G2/M
checkpoint (27). 53BP1 is phosphorylated in an
ATM-dependent manner in response to IR (28, 29).
53BP1 forms discrete nuclear foci after IR, UV radiation, and
replication block (28-30). 53BP1 foci colocalize with NFBD1/KIAA0170 was first described as an expressed sequence tag clone
in the HUGE (human unidentified
gene-encoded large proteins) Database
(32).2 The NFBD1
gene was mapped to 6pter-6p21.31, within the major histocompatibility
complex class I locus. It is one of the genes located within 1.7 megabases flanked by chromosomal markers D6S265 and D6S273. Loss
of heterozygosity at these markers occurs in >50% of cervical
carcinomas (33). NFBD1 encodes a nuclear
factor with an N-terminal FHA domain and a tandem repeat of
BRCT domains, both of which are present in a
number of proteins involved in the DNA repair and/or DNA damage
signaling pathways (34-36). Therefore, we sought to determine whether
NFBD1 is involved in DNA damage signaling pathways. We found that NFBD1
quickly localized to DSB sites along with other DNA damage signaling
and repair factors.
Plasmids--
A clone in the HUGE Database
(GenBankTM/EBI accession number D79992) containing the
entire coding sequence of NFBD1/KIAA0170 was
obtained from Takahiro Nagase (Kazusa DNA Research Institute, Chiba,
Japan). For expression in mammalian cells, NFBD1-coding sequences were
amplified by PCR and cloned into the pcDNA3xHA-Neo, resulting in
pcDNA-HANFBD1. Internal deletion mutants were generated from
pcDNA-HANFBD1 by subcloning using appropriate restriction enzymes.
Plasmid constructs were verified by sequence
analysis.3
Antibodies--
A SmaI-XbaI fragment
encoding a polypeptide from residues 142 to 568 was released from
pcDNA-HANFBD1 and subcloned into the glutathione
S-transferase (GST) vector pGEX4T-3. The resulting construct
was used to produce GST fusion protein in Escherichia coli.
The GST fusion protein, purified as described (26), was used to
immunize rabbits for antibody production (Yale Animal Resources
Center). Antibodies were purified from crude sera on a protein
G/protein A-agarose column (Calbiochem) and then an affinity column
conjugated to the antigenic GST fusion protein. The final concentration
of the purified antibodies was adjusted to 1 mg/ml. Mouse anti-53BP1
monoclonal antibody was a kind gift of Thanos D. Halazonetis (Wistar
Institute) (30). Also used were rabbit and mouse IgG (Sigma), mouse
anti-hemagglutinin (HA) monoclonal (16B12, Covance), anti-BRCA1 (Ab-1,
Calbiochem), anti-ORC2 (clone 920-4-41, Pharmingen), anti-GRB2 (clone
81, Transduction Laboratories), anti- Northern Blotting--
Human tissue mRNA blots
(Clontech and OriGene) were probed using a
SmaI-XbaI fragment from pcDNA- HANFBD1
labeled with [32P]dCTP using a random-primed DNA
labeling kit (Roche Molecular Biochemicals) according to the
manufacturer's instructions. A probe for mouse Nfbd1
was derived from a mouse expressed sequence tag clone
(GenBankTM/EBI accession number AW106340) encoding putative
mouse NFBD1. The sequence of this expressed sequence tag clone and
similarity to a portion of human NFBD1 were verified by end-to-end
sequencing. A mouse tissue mRNA blot (Clontech)
was probed with an EcoRI-SacI fragment encoding
the first 490 amino acids derived from this cDNA.
Cell Culture and
Transfections--
ATM-deficient SV40-transformed
human GM5849C fibroblasts were obtained from the Coriell Institute for
Medical Research (Camden, NJ). Other cell lines were obtained from the
American Type Culture Collection. Cells were grown in Dulbecco's
modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 50 units/ml penicillin, and 50 mg/ml
streptomycin. Transfection was performed with FuGENE 6 (Roche Molecular
Biochemicals) at a ratio of 1 µg of plasmid to 2 µl of FuGENE 6. The small interfering RNA (siRNA) duplex targeting NFBD1 was
prepared by annealing two 21-ribonucleotide oligonucleotides, 5'-UCC
UGA GAC CUC CUA AGG UTT-3' and 5'-ACC UUA GGA GGU CUC AGG ATT-3'
(Dharmacon Research). The scrambled siRNA duplex sequences were 5'-GUU
CAC UCA UGA UCG AGC CTT-3' and 5'-GGC UCG AUC AUG AGU GAA CTT-3'. HeLa
cells were transfected with the siRNA duplex using OligofectAMINE
(Invitrogen) and analyzed 60 h after transfection. 48-60 h after
transfection, cells were irradiated in a Shepherd Mark I
137Cs irradiator at a dose rate of 1.75 gray
(Gy)/min. Cells were UV-irradiated at a dose of 50 J/m2
with a Stratagene cross-linker 48-60 h after transfection. 48-60 h
after transfection, cells were treated with 1 mM
hydroxyurea (Sigma) for 24 h.
Synchronization of HeLa Cells--
HeLa cells were grown in the
presence of 2 mM thymidine (Sigma) for 18 h, washed
with phosphate-buffered saline (PBS), and grown in fresh medium without
thymidine for 8 h. Thymidine was added again to 2 mM
to block cells at G1/S. After another 18 h, cells were
transferred to fresh medium, and samples were harvested every 2 h
for a period of 16 h. To arrest cells in mitosis, cells were first
treated with 2 mM thymidine, released into fresh medium for
3-4 h, and then blocked with medium containing 100 ng/ml nocodazole for 16 h. Floating cells were collected, washed with PBS, and seeded into fresh medium. Synchronization and cell cycle state were
examined by propidium iodide staining and fluorescence-activated cell
sorter analysis.
Chromatin Fractionation--
Chromatin fractionation was
performed essentially as described (37). Briefly, ~3 × 106 cells were washed with PBS and resuspended in 200 µl
of buffer A (10 mM HEPES (pH 7.9), 10 mM KCl,
1.5 mM MgCl2, 0.34 M sucrose, 10%
glycerol, 1 mM dithiothreitol, and protease inhibitor
mixture (Roche Molecular Biochemicals)). Triton X-100 was added to a
final concentration of 0.1%, and the cells were incubated for 5 min on
ice. Nuclei were collected in the pellet (P1) by low speed centrifugation (1500 × g, 4 min, 4 °C). The
supernatant (S1) was further clarified by high speed centrifugation
(13,000 × g, 10 min, 4 °C) to remove cell debris
and insoluble aggregates. The supernatant was designated S2. Nuclei
were washed once with buffer A and then lysed in 200 µl of buffer B
(3 mM EDTA, 0.2 mM EGTA, 1 mM
dithiothreitol, and protease inhibitor mixture). After a 10-min
incubation on ice, soluble nuclear proteins (S3) were separated from
chromatin by centrifugation (2000 × g, 4 min).
Isolated chromatin (P3) was washed once with buffer B and spun
down at high speed (13,000 × g, 1 min).
Immunoprecipitation and Immunoblotting--
Cell lysate was
harvested after treatment in high salt buffer containing 20 mM Tris-HCl (pH 8.0), 0.4 M NaCl, 1 mM EDTA, 0.5% Nonidet P-40, and protease inhibitor
mixture. 2 µg of antibodies was used for immunoprecipitation from 400 to 500 µg of total lysate plus an equal volume of buffer containing
20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.5%
Nonidet P-40, and protease inhibitor mixture at 4 °C overnight.
Precipitates were washed with 20 mM Tris-HCl (pH 8.0), 0.2 M NaCl, 1 mM EDTA, and 0.5% Nonidet P-40.
Immunoblots on nitrocellulose were blocked with 5% nonfat milk in PBST
(PBS with 0.5% Tween 20) and washed with PBST. Primary antibodies were incubated with 5% bovine serum albumin in PBST. Secondary antibodies were incubated with 5% nonfat milk in PBST.
Phosphatase Treatment--
Immunoprecipitates of endogenous
NFBD1 from HEK293 and HeLa cells before and after exposure to
Indirect Immunofluorescence--
Cells grown on
poly-D-lysine-coated eight-chamber slides were either
mock-treated or exposed to 50 J/m2 UV light or 1 mM hydroxyurea for 24 h or the specified dose of IR
(0.1-5 Gy from a 137Cs source). In some experiments, 10 or
50 µM wortmannin was added to the cells 1 h before
irradiation. At the indicated time points, cells were fixed in 4%
paraformaldehyde in PBS for 15 min, followed by permeabilization for 15 min in 0.5% Triton X-100 in PBS. Slides were blocked with 5% bovine
serum albumin in PBST for 30 min at 37 °C, incubated with primary
antibody for 30 min at 37 °C, washed with PBST, and incubated with
secondary antibody (rhodamine-conjugated donkey anti-mouse IgG (1:1000)
or fluorescein isothiocyanate-conjugated anti-rabbit IgG (1:200)) for
30 min at 37 °C. Dilutions of primary antibodies were 1:1000 for
anti-NFBD1; 1:500 for anti- NFBD1 Is a Chromatin-associated Nuclear Protein--
Human and
mouse tissue mRNA blots were probed with NFBD1 cDNA
sequences to determine the tissue-specific distribution of
NFBD1 transcripts. Both human and mouse blots
revealed an abundant transcript at a size of ~7.5 kb in testis and
low levels in placenta, kidney, brain, heart, liver, thymus, smooth
muscle, lung, colon, and peripheral blood leukocytes (data not shown).
Although the high expression in testis could be connected with meiotic
functions such as recombination, the significance is not clear because
of the strong transcriptional induction of many genes during spermatogenesis.
The human NFBD1 clone includes an open reading frame
of 6270 nucleotides that would encode a polypeptide of 2089 amino acids with a predicted molecular mass of 226 kDa. This polypeptide has a
C-terminal tandem repeat of BRCT domains and an N-terminal FHA domain
(Fig. 1A). In the middle of
the polypeptide is a tandem array of nine serine- and threonine-rich
24-amino acid repeats interspersed with other sequences (Fig.
1B). Data mining did not reveal other proteins with
significant homology to these repeats.
We produced and affinity-purified rabbit polyclonal antibodies against
the N terminus of the putative polypeptide. This antibody preparation
recognized three bands with a slower electrophoretic mobility than that
of the 250-kDa protein marker in immunoblots of a variety of cell
lines, including HEK293, U2OS osteosarcoma cells, normal human WI38
fibroblasts, ATM-deficient human GM05849C fibroblasts, and
HeLa cells (Fig. 1C and data not shown). These three bands
detected in HeLa cells were dramatically diminished after transfection
with a pair of siRNA oligonucleotides specific for the NFBD1-coding
region, but not after transfection with scrambled siRNA
oligonucleotides (Fig. 1D). These three bands were probably derived from translation of alternatively spliced transcripts or
alternative transcriptional initiation sites because expression of
HA-tagged NFBD1 cDNA yielded only a single band
(Fig. 1C, lanes 2, 5, and
9). HA-NFBD1 comigrated with the largest endogenous polypeptide recognized by anti-NFBD1 antibody and was recognized as
well by anti-HA antibody (Fig. 1C). This also indicates that the cDNA clone contains, at a minimum, most of the coding sequence for NFBD1.
Indirect immunofluorescence of cells transfected with a plasmid
encoding HA-NFBD1 with anti-NFBD1 antibody revealed diffuse nuclear
staining (Fig. 1E). The staining intensity was increased in
the subset of cells expressing HA-NFBD1 (Fig. 1E, compare
HA and NFBD1 panels). The staining was blocked if
the antibody was preincubated with the antigenic GST fusion protein,
but not with GST fusion proteins derived from other NFBD1 fragments
(data not shown). The exclusively nuclear staining indicates that
NFBD1 encodes a nuclear protein, which is also
supported by a previous report using green fluorescent protein-tagged
NFBD1 (38).
To further verify the antibody specificity and to identify domains
required for nuclear localization, we generated a series of overlapping
deletion mutants with three copies of the HA epitope tag at their N
termini (Fig. 1A). Transiently transfected HEK293 cells were
co-immunostained with anti-NFBD1 and anti-HA antibodies. In cells
transfected with expression constructs with sequences encoding the
antigenic polypeptide used to produce anti-NFBD1 antibody (Fig.
1F, N2 and N3 panels), but not with
other deletion mutants (panels N1, C1, and
C2), the subset of cells that reacted with anti-HA antibody
also showed increased intensity with anti-NFBD1 antibody. All of the
deletion mutants except the N1 fragment showed an exclusively nuclear
staining pattern with anti-HA antibody (Fig. 1F). The N1
fragment contains the FHA core sequence homology domain, which
apparently is not sufficient for nuclear localization. The N2 fragment
is an extension of the N1 fragment and was sufficient for nuclear
localization. Thus, a nuclear localization signal may be present
between residues 145 and 568, consistent with previous observations
(38). The C1 fragment was also sufficient for nuclear localization
(Fig. 1F). The earlier report (38) did not identify any
nuclear localization signal within this fragment because only progressive deletions from the C terminus were analyzed. Canonical nuclear localization signals are characterized by short, single, or
bipartite stretches of basic amino acids separated by a flexible spacer
with the relative consensus sequence K(K/R)X(K/R) (39). This
fragment contains at least two stretches of basic residues within a
highly basic region
(1860KPGKRKRDQAEEEPNRIPSRSLRRTK1885).
Thus, NFBD1 may have a second nuclear targeting signal near its C terminus.
We performed nuclear fractionation to determine whether NFBD1 is
associated with chromatin. NFBD1, like chromatin-bound ORC2, but not
the cytoplasmic protein GRB2, was present exclusively in the
chromatin-enriched P3 fraction (Fig. 1G). This association did not change dramatically after NFBD1 Is Modified in G2/M Phase--
NFBD1
contains FHA and BRCT domains, both of which are present in a variety
of genes involved in cell cycle controls. NFBD1/KIAA0170 has been flagged as a G2/M-regulated transcript in the
genome-wide microarray analysis of cell cycle-regulated transcription
in HeLa S3 cells (40). Using cells synchronized by double thymidine block, we found that NFBD1 protein levels were low in S phase and
higher in cell populations enriched for G2/M and
G1 (Fig. 2A),
consistent with the transcriptional profile.
A number of DNA damage-response proteins undergo regulated
phosphorylation. Therefore, we sought to determine whether NFBD1 is
phosphorylated during the cell cycle. HeLa cells were synchronized at
the beginning of S phase by using a double thymidine block, and cell
synchrony was monitored by flow cytometry of propidium iodide-stained
cells. Mobility shift of NFBD1, consistent with phosphorylation,
occurred 8 h after release from the thymidine block, coincident
with a large subpopulation of G2/M cells, and was
maintained until a shift to more rapid mobility 12 h after release, at which time G1 cells predominated, and continued
in this form through S phase (Fig. 2A). Thus, NFBD1 is
enriched and modified in G2/M cells.
NFBD1 Is Hyperphosphorylated in Response to the Spindle Checkpoint
Activated by Nocodazole--
Nocodazole is an antimitotic agent that
disrupts mitotic spindle function and arrests the cell cycle at
G2/M (41). HeLa cells were arrested in mitosis by
consecutive incubation with thymidine and nocodazole. Nocodazole
blockade resulted in a supershift of NFBD1 to a form that migrated
significantly more slowly than the G2/M form detected with
double thymidine block/release (Fig. 2A). NFBD1 returned to
normal mobility 8 h after release from the nocodazole block (data
not shown). Nocodazole-induced supershift of NFBD1 was due to
phosphorylation because it was eliminated by treatment of NFBD1 with
protein phosphatase (Fig. 2B, compare lanes 6,
7, and 9-11). Thus, NFBD1 is hyperphosphorylated
in response to nocodazole blockade.
ATM Is Required for IR-induced Hyperphosphorylation of
NFBD1--
DNA damage activates a cascade of protein kinases and
induces phosphorylation of many DNA damage-response and repair proteins (2, 17). Similarly, we found that endogenous NFBD1 in
The PIKK ATM is a central signaling protein in the response to IR and
other sources of DSBs. We sought to determine whether NFBD1
phosphorylation after IR is ATM-dependent. In
ATM-deficient fibroblasts, IR-dependent
retardation of HA-NFBD1 occurred with expression of wild-type, but not
kinase-defective (ATMKD), ATM (Fig. 3B,
lanes 6 and 8). When expressed in HEK293 cells
and exposed to NFBD1 Forms Nuclear Foci in Response to DNA Damage or Replication
Block--
A number of proteins involved in DNA damage signaling and
repair, including 53BP1 (28-30), BRCA1 (42), MRE11/RAD50/NBS1 (43), NFBD1 Colocalizes with H2AX, MRE11, and 53BP1--
Histone H2AX
becomes hyperphosphorylated in response to IR and forms discrete
nuclear foci on sites of DSBs within 1 min (5). Several essential DNA
repair factors, including BRCA1 (5, 8) and MRE11/RAD50 (5, 45),
form foci that colocalize with Dose Dependence and Time Course of NFBD1 Focus Formation--
H2AX
focus formation on the DSB sites is one of the earliest events in
response to forms of DNA damage that induce DSBs (3). The
colocalization between the NFBD1 and H2AX foci suggested that NFBD1 is
an early participant involved in DSB sensing and damage signaling. We
determined the time course of NFBD1 focus formation in HEK293 cells in
response to 5 Gy of irradiation (Fig. 6,
A and B). Within 1 min, NFBD1 foci
were visible and colocalized with the H2AX foci. NFBD1 foci peaked at
30 min and lasted at least 2 h after IR. NFBD1 foci
significantly declined 4 h after IR. Although the majority of
cells did not have a significant number of nuclear foci 12 h after
irradiation, a small population of the cells (<20%) retained
~10-20 nuclear foci/cell. Throughout this time course, the number of
H2AX foci was similar to that of NFBD1 foci, and the two foci were
always colocalized. Similar kinetics of NFBD1 focus formation in
response to
Rapid focus formation and colocalization with H2AX foci suggest that
NFBD1 may relocalize to the same DSB sites as H2AX after DNA damage. If
this is the case, the number of NFBD1 foci should correlate with the
dose of ATM Is Involved in NFBD1 Focus Formation--
Having found that
NFBD1 underwent ATM-dependent phosphorylation
and formed discrete nuclear foci after IR, we determined whether NFBD1
focus formation requires ATM. The fungal
phosphatidylinositol 3'-kinase inhibitor wortmannin inhibits the kinase
activities of ATM and DNA-dependent protein kinase in
intact cells, with half-maximal inhibition at concentrations of ~5
µM. The kinase activity of ATR is significantly more
resistant to this drug, with half-maximal inhibition at concentrations
>100 µM (46). HEK293 cells were pretreated with various
concentrations of wortmannin 1 h prior to exposure to 5 Gy of
We provide here the first identification of endogenous NFBD1
protein. We found that NFBD1 is heterogeneously expressed and modified
during the cell cycle and that NFBD1 is phosphorylated in response to
IR. Like many DNA damage-response proteins, NFBD1 relocalizes to sites
of DNA damage upon irradiation. The phosphorylation and relocalization
of NFBD1 in response to IR require ATM.
NFBD1 is modified, probably by phosphorylation, in the G2/M
phase in a normal cell cycle and is hyperphosphorylated after nocodazole treatment, which activates the spindle checkpoint (41). We
speculate that NFBD1 may be involved in G2/M checkpoint
control, and it will be of interest to determine whether protein
kinases activated by the spindle checkpoint are involved in NFBD1 regulation.
Central to all DNA damage-induced checkpoint responses is a pair of
PIKKs: the ATM and ATR kinases (17, 47). The deployment of ATM
versus ATR depends upon the initiating DNA lesion. Although ATR is more important for the response to UV light, alkylating agents,
and replication inhibitors, ATM is foremost in the response to IR.
Proteins regulated directly by ATM are important in control of the
G1 (p53, CHK2, and MDM2) (25, 48-51), S (NBS1 and CHK2) (52-55), and G2 (BRCA1 and human RAD17) (56-58)
checkpoint pathways. Identification of additional substrates of ATM
will be critical for understanding checkpoint controls.
Taken together, our results strongly suggest that NFBD1 is a substrate
of ATM. Phosphorylation and relocalization of NFBD1 in response to IR
depend upon ATM. NFBD1 was phosphorylated in response to IR
(Figs. 2B and 3A). This phosphorylation was
ATM-dependent (Fig. 3B). The PIKKs
prefer to phosphorylate substrates at the (S/T)Q motifs (59, 60). There
are 32 (S/T)Q motifs present in NFBD1. However, we cannot at present
rule out the possibility that
ATM-dependent phosphorylation of NFBD1 is
mediated indirectly by ATM-regulated kinases, including CHK1 and CHK2.
Recent studies have shed light on how damaged DNA is detected by the
sensor complexes (47). In response to DNA damage, both the ATR-ATRIP
(ATR-interacting protein) kinase
complex and the proliferating cell nuclear antigen-related
RAD9-RAD1-HUS1 complex are independently loaded onto the chromatin at
or near the sites of DNA damage (61-64). Loading of the RAD9-RAD1-HUS1
complex is RAD17-dependent (61). The interaction between
RAD17 and ATM/ATR is DNA damage-inducible (57). Phosphorylation and
relocalization of H2AX are two of the earliest events after DNA damage
(3-5) and are ATM/ATR-dependent (8,
65). Thus, it is plausible that H2AX is recruited by the ATR-ATRIP
and/or RAD9-RAD1-HUS1 complex to ATM/ATR kinases for phosphorylation.
In turn, phosphorylated H2AX may mark the region of chromatin targeted
for further assembly of signaling machinery and DNA repair. H2AX is
critical in facilitating recruitment of signaling and repair factors
such as 53BP1, MRE11/RAD50, and BRCA1 to the sites of DNA damage
(9).
We have provided evidence that NFBD1, together with H2AX, relocates to
DSBs after IR. First, NFBD1 is a nuclear protein that constitutively
bound to chromatin (Fig. 1). Second, NFBD1 formed discrete nuclear foci
after IR (Figs. 4-6), and the number of foci correlated with the dose
of Once NFBD1 is recruited to the sites of damaged DNA and is
phosphorylated by upstream kinases such as ATM, NFBD1 may, in turn, recruit downstream effectors for activation. Such effectors may include
CHK1 and CHK2. H2AX and 53BP1 foci colocalize with
phospho-Thr68 CHK2 foci after DNA damage (44). This
suggests that phospho-Thr68 CHK2 relocates to and binds to
DSBs. Recently, 53BP1 has been shown to interact with CHK2 and to be
required for CHK2 activation (31). A bacterially produced GST
fusion of the first 337 residues of NFBD1 containing the FHA domain was
capable of binding to bacterially produced wild-type CHK2 (which
undergoes autophosphorylation (26)), but not to kinase-defective CHK2
(data not shown). These results suggest a link between NFBD1 and CHK2.
Our preliminary results indicate that depletion of endogenous NFBD1 in
HeLa cells by siRNA may reduce early phospho-Thr68 CHK2
after IR (data not shown). And, ectopic overexpression of the NFBD1
BRCT domains compromises production of phospho-Thr68 CHK2
foci after IR (66). Thus, timely IR-dependent CHK2
activation may be dependent on NFBD1.
In budding yeast, in response to DNA damage, CHK1 and CHK2
homologs are coupled to PIKK through the intermediary Rad9. Orthologs of Rad9 should ideally fulfill criteria of sequence homology, regulation through phosphorylation by PIKKs, and physical interaction with CHK proteins. These criteria are fulfilled by fission yeast Crb2/Rhp9, where the only extended sequence homology is in the C-terminal tandem BRCT domains. There are only three genes in the human
genome encoding C-terminal tandem BRCT domains, which are
BRCA1, 53BP1, and NFBD1. Both
53BP1 (28, 29) and NFBD1 (this study) are probable substrates of ATM.
BRCA1 is a substrate of both ATM and ATR (58, 67). 53BP1 is
required for CHK2 activation and is involved in S and G2/M
checkpoint controls (31). BRCA1 regulates CHK1 activation in the
G2/M checkpoint. BRCA1 is also required for the S phase
checkpoint (42). Therefore, all three of these BRCT domain-containing
proteins may serve overlapping functions analogous to those of budding
yeast Rad9. However, information is still scant on the means through
which these proteins affect CHK activity.
In summary, our data demonstrate that NFBD1 is a component of the early
ATM/ATR response to DNA damage and localizes, along with several DNA
damage-response proteins, to DNA damage foci. With the importance of
DNA damage signaling and repair factors in genome stability and the
intriguing chromosomal location of this gene, it will be interesting to
determine whether NFBD1 is mutated in cancer, particularly cervical carcinoma.
-H2AX foci, which have been previously shown to localize at sites of DNA double-strand breaks. IR-induced NFBD1 foci also colocalized with 53BP1 and MRE11/RAD50 foci. Taken together, these results suggest that NFBD1 is a
mediator of DNA damage-dependent signaling.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-H2AX) foci at or near
the DNA damage sites. Immunofluorescence studies have revealed that
-H2AX forms nuclear foci at the sites of DSBs (4, 5). The number of
foci increases in the first 10-30 min after irradiation before they
gradually decline, correlating with the number of slowly rejoining DSBs (5).
-H2AX foci are also found at sites of V(D)J
recombination-induced DSBs in developing thymocytes (6), at sites of
recombinational DSBs during meiosis (7), and at sites of stalled
replication forks (8). Thus,
-H2AX foci are indicators of the
presence of DSBs in vivo. Lack of H2AX causes genome
instability in mice (9). H2ax
/
cells
fail to recruit DNA damage signaling and repair factors such as NBS1
(Nijmegen breakage
syndrome-1), 53BP1
(p53-binding protein-1), and BRCA1 (breast
cancer susceptibility gene-1), but not RAD51,
to irradiation-induced foci (9). Thus, H2AX is critical for
facilitating the assembly of specific DNA repair complexes on damaged DNA.
-H2AX foci
(29, 30). Thus, 53BP1 may bind to DNA DSBs (29, 30). Recently, 53BP1
was shown to interact with CHK2 and to be required for CHK2 activation
(31). Thus, Rad9-like checkpoint functions in mammals may be carried
out by multiple BRCT domain-containing proteins.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin (clone AC15, Sigma),
anti-MRE11 (clone 12D7, GeneTex), anti-phospho-Ser139 H2AX
(clone JBW301, Upstate Biotechnology, Inc.), and rabbit anti-phospho-Ser345 CHK1 (Cell Signaling Technology)
antibodies. For immunoprecipitations, antigen-antibody complexes were
recovered with protein G/protein A-agarose. Proteins were detected
using horseradish peroxidase-conjugated rat anti-HA monoclonal antibody
(3F10, Roche Molecular Biochemicals), horseradish peroxidase-conjugated
secondary antibodies and chemiluminescence reagents (Pierce), and
fluorescein isothiocyanate- and rhodamine-conjugated secondary
antibodies (Jackson ImmunoResearch Laboratories, Inc.).
-irradiation or from HeLa cells treated with 250 µg/ml nocodazole
for 18 h were incubated with 1000 units of
-phosphatase (New
England Biolabs Inc.) in the presence of 2 mM
MnCl2 for 1-2 h at 30 °C in a 50-µl reaction volume.
-H2AX, anti-MRE11, and anti-HA (16B12);
1:15 for anti-53BP1; and 1:50 for anti-BRCA1. After washing, the slides
were mounted with coverslips with mounting medium containing
4,6-diamidino-2-phenylindole (Vector Labs, Inc.). Images were acquired
using a Nikon Microphot-FX microscope with a ×40 or ×60 objective and
a SPOT digital camera.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
NFBD1 is a chromatin-associated nuclear
protein. A, schematic diagram of NFBD1
structure and deletion mutants. The known structural domains, FHA and
BRCT domains, are indicated by the striped and
filled boxes, respectively. Solid bars mark
repeats shown in Fig. 1B. Deletion mutants N1-N3, C1, and
C2 were produced by restriction enzyme digestions. Restriction enzymes
used were EcoRI and ApaI for N1, EcoRI
and XbaI for N2, EcoRI and XhoI for
N3, EcoRI and XhoI for C1, and BamHI
and XbaI for C2. B, tandem repeats in NFBD1. Each
repeat is shown as an individual solid bar in A. C, antibody specificity evaluated by immunoprecipitation
(IP) and/or immunoblotting (IB). Cell lysates
were extracted from HEK293 cells (lanes 1, 3,
4, 6, 8, 10, 11,
and 13) or from HEK293 cells transfected with HA-NFBD1
(lanes 2, 5, 7, 9,
12, and 14). Total lysates, not
immunoprecipitates, are analyzed in lanes 6, 7,
13, and 14. The remaining lanes depict
immunoprecipitations with the antibodies indicated. D,
antibody specificity by immunoblotting after siRNA depletion of NFBD1.
HeLa cells were non-transfected (Untreated) or transfected
with siRNA duplex against NFBD1 (siRNA) or with scrambled
siRNA duplex (Scrambled) using OligofectAMINE. Transfectants
were exposed to 5 Gy of irradiation 60 h after transfection.
Non-transfected cells were treated with the same dose of
-irradiation. Lysates were extracted at different time points after
irradiation as indicated and analyzed by immunoblotting for NFBD1 and
-actin. E, antibody specificity in immunofluorescence.
HEK293 cells were transfected with HA-NFBD1. Transfectants were seeded
on poly-O-lysine-coated eight-chamber slides 48 h after
transfection. The following morning, cells were either mock-treated or
exposed to 5 Gy of
-irradiation and, after 1 h, analyzed by
dual immunofluorescence with anti-HA and anti-NFBD1 antibodies. DNA was
stained with 4,6-diamidino-2-phenylindole (DAPI).
F, nuclear localization of NFBD1. The experiment was
performed as described for E, except that HEK293 cells were
transfected with the deletion mutants of NFBD1 depicted in A with N-terminal HA tags, and the cells
were not irradiated. G, NFBD1 binds to chromatin. Extracts
of HEK293 cells were fractionated as described under "Experimental
Procedures." The resultant fractions were resolved by SDS-PAGE.
S2, cytoplasmic fraction; S3, nuclear soluble
proteins; P3, chromatin-enriched pellet.
-irradiation, UV radiation, or
hydroxyurea treatment (data not shown). Thus, NFBD1 may constitutively bind to chromatin.
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Fig. 2.
NFBD1 is modified in G2/M
phase. A, HeLa cells were synchronized as
described under "Experimental Procedures." Cells were stained with
propidium iodide. Cell cycle profiles were determined by flow
cytometry. B, NFBD1 is hyperphosphorylated after IR or
nocodazole block. Cell lysates were extracted from HeLa cells 1 h
after 10 Gy of irradiation or nocodazole treatment for 18 h. Total
lysates, not immunoprecipitates, are analyzed in lanes 5,
6, 7, and 12. Affinity-purified
immunocomplexes using anti-NFBD1 antibody from mock-treated cells,
irradiated cells, and nocodazole-treated cells were left untreated
(lanes 1, 2, 8, and 9) or
were mock-treated (lanes 3 and 10) or incubated
with protein phosphatase (PPase; lanes 4 and
11). IB, immunoblotting; IP,
immunoprecipitation; Noc, nocodazole; Asn,
asynchronous cells; DT, double thymidine block.
-irradiated HeLa and HEK293 cells had reduced electrophoretic mobility (Figs. 2B, lanes 2, 3, and 5; and
3A, lanes 2,
3, and 6). This retardation was due to
phosphorylation because it was eliminated by treatment of
immunoprecipitated NFBD1 with protein phosphatase (Figs. 2B, lane 4; and 3A, lane 4). It is
noteworthy that phosphatase treatment of the hyperphosphorylated forms
of NFBD1 induced by IR or nocodazole resulted in faster migration forms
than in untreated cells (Figs. 2B, compare lanes
4 and 5 and lanes 11 and 12; and
3A, compare lanes 4 and 5). These
results suggest that NFBD1 is basally phosphorylated. Ectopically
expressed HA-NFBD1 in HEK293 cells was also phosphorylated after IR
(data not shown).
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Fig. 3.
ATM-dependent
hyperphosphorylation of NFBD1 after IR. A, NFBD1 is
phosphorylated in response to IR. HEK293 cells were either mock-treated
or exposed to 10 Gy of irradiation. Lysates were extracted 2 h
after irradiation. Total lysates, not immunoprecipitates, are analyzed
in lanes 5 and 6. Affinity-purified
immunocomplexes using anti-NFBD1 antibody from mock-irradiated and
irradiated cells were left untreated (lanes 1 and
2) or were mock-treated (lane 3) or incubated
with protein phosphatase (PPase; lane 4).
B, NFBD1 hyperphosphorylation after IR is
ATM-dependent. ATM-deficient human
GM5849C fibroblasts were transfected with vector (lanes 1 and 2) or HA-NFBD1 only (lanes 3 and
4) or were cotransfected with HA-NFBD1 and wild-type ATM
(lanes 5 and 6) or kinase-defective ATM
(ATMKD; lanes 7 and 8). Transfectants
were exposed to 10 Gy of irradiation 48 h after transfection.
Lysates were extracted 2 h after irradiation and used for
immunoblotting (IB) with anti-HA antibody. IP,
immunoprecipitation.
-irradiation, the HA-tagged N3 fragment, but not the
other deletion mutants, was detected with anti-phospho-(S/T)Q antibody, which recognizes the phosphorylated (S/T)Q motif present in the ATM/ATR
substrates (data not shown). These results indicate that NFBD1 is
likely a substrate of ATM.
-H2AX (3, 5), and phospho-Thr68 CHK2 (44), relocalize
and form subnuclear foci in the cells in response to DNA damage or
replication blocks. In untreated HEK293 cells, HeLa cells, U2OS cells,
and WI38 fibroblasts, anti-NFBD1 antibody staining yielded diffuse
nuclear staining and few nuclear foci (Fig.
4 and data not shown). Numerous
nuclear foci were apparent in all cells 2 h after irradiation at 5 Gy (Fig. 4). NFBD1 foci were also evident in HEK293 and HeLa cells
2 h after exposure to UV light (50 J/m2) or 24 h
after hydroxyurea treatment (Fig. 4 and data not shown).
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Fig. 4.
NFBD1 focus formation in response to DNA
damage and replication block. HEK293 cells were either
mock-treated or exposed to 5 Gy of irradiation, 50 J/m2 UV
light, or 1 mM hydroxyurea (HU). Cells were
fixed and co-immunostained with anti-NFBD1 and
anti-phospho-Ser139 H2AX antibodies 2 h after
irradiation or 24 h after hydroxyurea treatment. DNA was stained
with 4,6-diamidino-2-phenylindole (DAPI).
-H2AX. 53BP1, a mediator of the DNA
damage checkpoint that is required for CHK2 activation in response to
IR (31), also colocalizes with
-H2AX. HEK293 cells induced NFBD1
foci that colocalized with H2AX foci within 2 h after IR (Fig.
5). This colocalization was also observed
in HeLa and U2OS cells (data not shown). In BRCA1-deficient
HCC1937 cells, spontaneous NFBD1 and H2AX foci were evident and
colocalized (data not shown). As shown in Fig. 5, IR-induced NFBD1 foci
colocalized with MRE11 and 53BP1 and partially with BRCA1. NFBD1 foci
also colocalized with
-H2AX after UV radiation or replication block
by hydroxyurea treatment (Fig. 4).
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Fig. 5.
NFBD1 foci colocalize with
-H2AX, 53BP1, and MRE11 foci and
partially with BRCA1 foci after IR. HEK293
cells were either mock-treated or exposed to 5 Gy of irradiation. Cells
were fixed and analyzed by immunofluorescence with anti-NFBD1,
anti-53BP1, anti-MRE11, or anti-BRCA1 antibody. DNA was stained with
4,6-diamidino-2-phenylindole (DAPI).
-irradiation were observed in HeLa and U2OS cells.
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Fig. 6.
Time course (A and
B) and dose dependence (C and
D) of NFBD1 focus formation. A,
immunofluorescence images of HEK293 cells exposed to 5 Gy of
irradiation and co-immunostained with anti-NFBD1 and
anti-phospho-Ser139 H2AX antibodies at the indicated times after irradiation; B, average number of
NFBD1 foci/cell calculated by counting at least 40 cells/time point;
C, immunofluorescence images of HEK293 cells exposed to
different doses of -irradiation as indicated and co-immunostained
with anti-NFBD1 and anti-phospho-Ser139 H2AX antibodies
1 h after irradiation; D, average number of NFBD1
foci/cell calculated by counting at least 40 cells/dose point.
-irradiation. Indeed, as shown in Fig. 6 (C and
D), with increased doses of
-irradiation from 0.1 to 5 Gy, NFBD1 foci increased from several foci to ~40 foci/cell. NFBD1
foci also colocalized with H2AX foci at all doses.
-irradiation. Cells were fixed and co-immunostained with anti-NFBD1
and anti-
-H2AX antibodies 1 h after irradiation. Formation of
both NFBD1 and H2AX foci was greatly impaired in the presence of 10 µM wortmannin, and this impairment was more severe with
50 µM wortmannin (Fig. 7).
This is consistent with previous reports that wortmannin treatment inhibits H2AX focus formation in response to IR (5). We also observed
that NFBD1 focus formation in DNA-dependent protein
kinase-deficient cells was intact after
-irradiation (data not
shown). These results indicate that ATM is involved in NFBD1 focus
formation in response to
-irradiation.
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Fig. 7.
ATM is involved in NFBD1 focus
formation after -irradiation. HEK293
cells were mock-treated with Me2SO or treated with 10 µM (W10) or 50 µM
(W50) wortmannin before exposure to 5 Gy of irradiation.
Cells were fixed and co-immunostained with anti-NFBD1 and
anti-phospho-Ser139 H2AX antibodies 1 h after
irradiation.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-irradiation (Fig. 6C). Third, NFBD1 foci colocalized
with
-H2AX and MRE11/RAD50 (Fig. 5), known DNA repair and
damage signaling factors bound to DSBs after DNA damage. Fourth, NFBD1
focus formation occurred very rapidly (within 1 min) and peaked 30 min
after IR (Fig. 6A). These kinetics are similar to those of
-H2AX focus formation. Whether NFBD1 relocalization and focus
formation, like 53BP1 (9), are dependent on the phosphorylation and
relocalization of H2AX is unclear. Knockdown of NFBD1 by siRNA in HeLa
cells did not prevent or delay H2AX focus formation in response to IR
(data not shown). Thus, H2AX functions either in parallel to or
upstream of NFBD1.
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ACKNOWLEDGEMENTS |
---|
We thank Michael Kastan and Takahiro Nagase for plasmids, Thanos D. Halazonetis for mouse monoclonal antibody against 53BP1, and JoAnn Falato for secretarial assistance. We thank Soo-Jung Lee for critically reading the manuscript and other members of the Stern laboratory for helpful comments.
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FOOTNOTES |
---|
* This work was supported by United States Army Research and Material Command Grants DAMD 17-98-1-8272 (to D. F. S.) and DAMD 17-01-1-0465 (to X. X.), United States Public Health Service Grant R01CA82257 (to D. F. S.), and a Leslie H. Warner fellowship from the Yale Comprehensive Cancer Center (to X. X.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Pathology,
School of Medicine, Yale University, 310 Cedar St., BML 342, New Haven,
CT 06510. Tel.: 203-785-4832; Fax: 203-785-7467; E-mail: Df.stern@yale.edu.
Published, JBC Papers in Press, December 23, 2002, DOI 10.1074/jbc.M211392200
2 Available at www.kazusa.or.jp/huge/gfpage/KIAA0170/.
3 Primer sequences and detailed cloning strategies are available upon request.
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ABBREVIATIONS |
---|
The abbreviations used are: DSBs, double-strand breaks; IR, ionizing radiation; PIKK, phosphatidylinositol 3'-kinase-related kinase; GST, glutathione S-transferase; HA, hemagglutinin; siRNA, small interfering RNA; Gy, gray; PBS, phosphate-buffered saline.
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