From the Department of Biochemistry and Molecular
Biology, University of Texas Health Sciences Center, Houston, Texas
77030, the § Department of Experimental Therapeutics,
University of Texas M. D. Anderson Cancer Center, Houston Texas
77030, and the ¶ Verna and Mars McLean Department of Biochemistry
and Molecular Biology,
Department of
Molecular and Human Genetics, and §§ Howard
Hughes Medical Institute, Baylor College of Medicine, Houston,
Texas 77030
Received for publication, December 9, 2002, and in revised form, January 24, 2003
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ABSTRACT |
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p53-binding protein-1 (53BP1) is phosphorylated
in response to DNA damage and rapidly relocalizes to presumptive sites
of DNA damage along with Mre11 and the phosphorylated histone 2A variant, DNA damage-response mechanisms ensure the fidelity of chromosomal
transmission, and their failure may lead to the development of diseases
such as cancer (1). In response to
One protein that contains numerous (S/T)Q motifs and two C-terminal
BRCT repeats is p53-binding protein 1 (53BP1). 53BP1 was discovered as
a p53-interacting factor in a two-hybrid screen (15) and was
subsequently proposed to function as a transcriptional co-activator of
p53 (16). Although the relationship between 53BP1 and p53 has not been
fully established, 53BP1 and p53 from both Xenopus and
humans have been shown to interact either directly or indirectly in
experimental settings that express high levels of 53BP1 protein from
plasmids or that naturally occur in eggs (15, 17). We, as well as
others, have demonstrated previously that 53BP1 is involved in the DNA
damage-response network (17-20). 53BP1 proteins are phosphorylated in
response to Antibodies and Indirect Immunofluorescence--
Three antibodies
that recognize both the human and murine 53BP1 proteins were generated
for this study. We found that our 53BP1 antibodies recognize both the
murine and human proteins. Polyclonal antibodies raised against
glutathione S-transferase fusion proteins encoding
the first 524 amino acids of human 53BP1 ( Mouse Genetics and Genotyping of
m53BP1tr/tr Animals--
Murine animals
defective in m53BP1 (m53BP1tr/tr)
were generated with a random retrovirus as described previously (29).
Genomic DNA was isolated from mouse tail snips by standard methods.
Insertion of VICTR54 introduces new XbaI sites into the
intron preceding exon 14 of the wild-type allele. Therefore, a
disrupted m53BP1 allele will be broken into multiple
XbaI fragments including a 5'-proximal, 1.5-kb fragment. To
detect this fragment, we PCR-amplified and labeled with 32P
a probe downstream of the 5' naturally occurring XbaI site
but upstream of the one introduced by VICTR54, as shown in Fig.
2A. The primers used to amplify the 700-base pair probe for
Southern analysis were 5'-CTCAGCATCCATGCTGGGC-3' and
5'-TACTTAATGAGGCTAGAGCACAGC-3'. The sequences of the primers used for
RT-PCR analysis were as follows: A, 5'-CCTCAGGCAGAGTGAACA-3'; B,
5'-CTCTGTGTCGTCCACGGGAGCACT-3'; C, 5'-GTGGCGATGCAAGACATGGCCA-3'; D,
5'-GCCAAGAACAGATGGAACAGCTGA-3'. Either poly(A)+ or total
RNA was isolated by standard methods and used to prepare cDNA with
the Superscript one-step PCR system (Invitrogen).
Immune System Analysis--
Bone marrow, thymus, and spleen
tissue were analyzed in 8-week-old male and female mice. Bone marrow
was flushed with Hank's balanced salt solution (Invitrogen)
from two femurs per animal. Cells were counted in 3.0% acetic acid
with a hemocytomter. Bone marrow cells were stained in Hank's balanced
salt solution/2.0% fetal bovine serum, with Fc block, CD11b (Mac-1),
Gr-1, ter119, CD19, anti-IgM, CD45R/B220, and CD43 (all from
Pharmingen). Flow cytometric analysis was performed on a BD Biosciences
FACScan, with CellQuest software, and appropriate negative isotype
control antibodies (Pharmingen) were used in all analyses. Spleens and thymuses were excised and gently crushed through 100-µm cell
strainers (Falcon) in Hank's balanced salt solution/fetal bovine
serum. Red blood cells were lysed with ACK buffer (0.15 M NH4Cl, 1.0 mM KHCO3,
0.1 mM EDTA, pH 7.2) (5 ml/spleen or thymus, 5 min at room
temperature). After centrifugation and washing with phosphate-buffered saline/2.0% fetal bovine serum, the cells were counted as above and
stained for flow cytometric analysis. Thymic cells were stained with
CD4, CD8, CD25, CD44, and CD3 (Pharmingen). Flow cytometry was
performed as above.
Chromosome Aberration Studies--
Exponentially growing passage
2 MEFs were irradiated at room temperature with 0, 0.5, or 1.5 Gy of
Dynamic Nuclear Localization of 53BP1 in the Absence and Presence
of DNA Damage--
It has been recently shown that 53BP1 localizes to
the kinetochore during mitosis (28). However, the behavior of 53BP1
during interphase in the absence of extrinsic DNA damage has not been fully investigated. To examine the interphase behavior of 53BP1 during
the course of normal, unperturbed MCF-7 cell cycles, we used a laser
scanning cytometer to determine the nuclear localization of 53BP1 and
the cellular DNA content for any given cell. In G1, 53BP1
exists in a diffuse nuclear pattern as well as in large nuclear
"dots" (Fig. 1A). In
S-phase, 53BP1 can be found in a discrete, punctate pattern (Fig.
1A). The nuclear distribution pattern of 53BP1 in
G2 cells appeared in two types, one similar to S-phase but
with fewer foci (Fig. 1A) and one that exhibited few, if
any, large dots (not shown). It is well established that 53BP1
relocalizes to nuclear foci in response to DNA damage (17-20). We
found that 53BP1 and ATR co-localized to nuclear foci in response to
hydroxyurea (Fig. 1B). We also found that 53BP1 physically associates with ATR in nuclear extracts derived from K562 cells (Fig.
1C). 53BP1 can be detected in ATR immunoprecipitates and ATR
is present in 53BP1 immunoprecipitates, and the association occurs
independently of DNA damage (Fig. 1C). Moreover, ATR
phosphorylates 53BP1 in vitro (not shown). Thus, 53BP1
interacts with various factors implicated in genomic stability
including ATR, p53, H2AX, BRCA1, and Chk2. To address which structural
elements of 53BP1 are required for the formation of irradiation-induced
foci, we created a series of mutant constructs in the 53BP1-expression vector pCMH6K53BP1 (16). We generated mutant forms of 53BP1 that
deleted the C-terminal BRCT motifs ( Generation of Mice Defective in m53BP1
(m53BP1tr/tr)--
To begin to decipher the
functional role for 53BP1 in the DNA damage response, we identified
embryonic stem cells (OST94324) from Omnibank (29) containing a single,
~5.0-kb retroviral insertion (VICTR54; Fig.
2A) in murine 53BP1 (m53BP1;
1,957 amino acids; 80% identity to human 53BP1; see Ref. 28). VICTR54
was found inserted within a 4.9-kb intron located between exons 13 and
14 (Fig. 2A). VICTR54, and its related vectors, are usually
found within introns and contain splice acceptor (SA) and
donor (SD) sequences such that a neomycin (NEO)
resistance gene and flanking sequences are spliced into the mature
transcript as an exon (Fig. 2A) (29). These
transcriptional fusions disrupt the coding sequence through the
introduction of premature stop codons. Such gene trapping methodologies
have been applied previously to understanding gene function (29).
OST94324 cells were used to generate transgenic animals heterozygous in
m53BP1 (m53BP1+/tr) as described previously (29). Southern
blotting with DNA isolated from tail biopsies confirmed the disruption
in m53BP1 and was used to genotype the animals (Fig. 2B; see
"Experimental Procedures"). Crosses between heterozygous animals
produced m53BP1tr/tr progeny born at the
expected frequencies. The m53BP1tr/tr animals
were found to be fertile, but we did observe that crosses between
mutant animals produced smaller litters as some embryos spontaneously
aborted and were reabsorbed by the mother (data not shown). RT-PCR
analysis with various primers 5' and 3' to the insertion demonstrated
that exon 13 failed to properly splice next to exon 14 in the
m53BP1tr/tr mice (Fig. 2C). Rather,
the "artificial" exon containing neomycin from VICTR54 was
spliced adjacent to exon 13 as verified with primers specific for exon
13 and the neomycin gene (primer set D/A; Fig. 2C).
Sequencing of a cloned RT-PCR product spanning the insertion event
revealed that the natural coding sequence of m53BP1 had stopped after
residue 1,205, where it then fused to 21 residues derived from VICTR54
before terminating (Fig. 2D). Therefore the disrupted allele
of m53BP1 encodes a truncated 1,226 residue protein
(m53BP1tr), and notably, m53BP1tr is missing
over 700 residues including its functional nuclear localization signal,
kinetochore binding domain (KINET), and two BRCT motifs (Fig.
2D). To determine whether m53BP1tr was
expressed, we performed immunoprecipitation/Western blotting (IP/WB)
analysis from brain extracts derived from
m53BP1+/+, m53BP1+/tr,
and m53BPtr/tr animals. By using antibodies
specific for the N and C termini of 53BP1 ( Immune Deficiencies in m53BP1tr/tr
Mice--
We observed that m53BP1tr/tr animals
were growth-retarded as the males and females were found on average to
weigh 25 and 15% less, respectively, than their wild-type littermates
(Fig. 3A). We found that
thymuses derived from m53BP1tr/tr animals were
significantly smaller and possessed fewer cells than those from
m53BP1+/+ animals (Fig. 3B). This
suggests that defects in m53BP1 may contribute to immune
deficiencies, a result that has been observed for various DNA
damage-response factors, including H2AX (24). We found that the
lymphoid organ architecture of thymuses, as assayed by hematoxylin and
eosin staining of tissue sections, appeared normal in
m53BP1tr/tr mice (data not shown). In addition,
flow cytometric analysis with a variety of markers (e.g.
B220, CD43, Gr-1, CD11a, and Ter119) revealed that bone marrow pro-B,
pre-B, myeloid, and erythroid progenitor populations were normal in
m53BP1tr/tr mice (not shown). Although CD4 and
CD8 T cell populations were proportionately similar in
m53BP1tr/tr and m53BP1+/+
thymuses, we observed that progression out of the DNIII stage of early
thymocyte development was impaired in
m53BP1tr/tr animals (Fig. 3C), the
stage at which Genomic Instability in m53BP1tr/tr
Mice--
Mice with defects in double-stranded break repair are highly
sensitive to
53BP1 interacts with a variety of factors known to be involved in the
maintenance of genomic stability including ATR, p53, H2AX, BRCA1, Chk2,
and ATM (15, 20, 25, 27). The generation of murine animals defective in
m53BP1 provides a valuable tool to further understand the
role of the protein in the DNA damage response. The
m53BP1tr/tr allele expresses a truncated version
of m53BP1, and this likely represents a significant impairment in some
aspects of its function. m53BP1tr/tr is missing
over 700 amino acids including the nuclear localization signal, the
C-terminal BRCT motifs, and a kinetochore-binding domain. We have
observed that this domain is also necessary for forming
irradiation-induced nuclear foci. Indeed, the lack of detectable,
irradiation-induced foci in mutant MEFs suggests that the protein
cannot fully perform its functions as a DNA damage-response element.
Moreover, the lethality observed for m53BP1tr/tr
mice at higher doses of radiation (7 Gy) suggests that there are no
other factors acting redundantly with m53BP1 with respect to this
aspect of radiation resistance and indicates that m53BP1 is a critical
element for double-stranded break repair. Therefore, the C-terminal 700 amino acids of m53BP1 encode important, functional determinants of the protein.
We observed that m53BP1tr/tr animals are
growth-retarded as the males weigh, on average, 25% less than their
wild-type littermates. The decreased thymus size, reduced T cell count,
immature B cell population, and lack of progression out of DNIII for
thymus T cells reveal that m53BP1tr/tr animals
are immune-deficient. How m53BP1 contributes to this process remains to
be established, but one possibility is that the protein participates in
the maturation of T-cell receptors and immunoglobulins during V(D)J
recombination, a process known to utilize DNA repair proteins (30).
This is particularly interesting given the involvement of m53BP1 in
double-stranded break repair as revealed by several factors including,
most notably, the sensitivity of m53BP1tr/tr
animals after exposure to 7 Gy of ionizing radiation. Indeed, sensitivity to ionizing radiation often correlates with impaired V(D)J
joining (30). Moreover, H2AX defective-animals are also immune-deficient (24). As H2AX is required for the formation of 53BP1
foci and because it physically associates with 53BP1 (20), it is
possible that an ordered pathway of assembly of DNA damage-response
proteins at these programmed breaks may facilitate V(D)J recombination
and maximize antibody diversity.
Our results show that genetic defects in m53BP1 result in a
pleiotropic phenotype consistent with defects in DNA repair and checkpoint control. The phenotype of 53BP1-defective animals is quite
similar to H2AX-deficient ones, consistent with the notion that H2AX
operates upstream of 53BP1 in a DNA damage-response pathway. When such
pathways are defective, cells cannot properly repair damaged DNA, a
situation that may lead to increased genomic instability and the
development of diseases such as cancer. For example, given the immune
deficiencies in m53BP1tr/tr mice, one may
anticipate the generation of lymphomas. In light of this, we have not
observed the development of any cancerous phenotypes in our
m53BP1-defective mice. Although there are a variety of
possible reasons for this (i.e. genetic background, allele,
etc.), it is interesting to note that mice nullizygous for H2AX also
apparently fail to generate
cancers.3 As H2AX and 53BP1
are not required for viability, it is possible that mutations in 53BP1,
when combined with other mutations in critical DNA damage-response
elements (i.e. H2AX, ATM, and p53) will lead to more severe
defects in genomic stability, a process that may then lead to the
development of cancer. The analysis of cells derived from these crosses
is likely to provide more insight into how 53BP1 functions in the DNA
damage response in concert with its various interacting partners.
-H2AX. 53BP1 associates with the BRCA1 tumor suppressor, and knock-down experiments with small interfering RNA have
revealed a role for the protein in the checkpoint response to DNA
damage. By generating mice defective in m53BP1
(m53BP1tr/tr), we have created an animal model
to further explore its biochemical and genetic roles in
vivo. We find that m53BP1tr/tr animals
are growth-retarded and show various immune deficiencies including a
specific reduction in thymus size and T cell count. Consistent with a
role in responding to DNA damage, we find that m53BP1tr/tr mice are sensitive to ionizing
radiation (
-IR), and cells from these animals exhibit chromosomal
abnormalities consistent with defects in DNA repair. Thus, 53BP1 is a
critical element in the DNA damage response and plays an integral role
in maintaining genomic stability.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-IR,1
phosphoinositide-like kinases (PIKs) such as ATM (mutated in ataxia-telangiectasia) transduce damage signals to kinases,
transcription factors, and DNA repair proteins by targeting
(S/T)Q motifs (2). A second PIK, ATR (ATM- and Rad3-related), also
responds to
-IR, but it appears to respond primarily to agents that
create replicational stress (i.e. hydroxyurea and
aphidicolin) (2). ATM and ATR have distinct but overlapping substrate
specificities including the ability of both enzymes to target p53
serine residue 15 (Ser-15) as well as the product of breast cancer
susceptibility gene 1, BRCA1, at Ser-1423 (3, 4). BRCA1 is a
major target of the DNA damage response, and mutations in
BRCA1 contribute to nearly 50% of familial forms of breast
and ovarian cancer (5). BRCA1 had been found associated with RNA
polymerase II (6), chromatin-remodeling factors (7), and a variety of
DNA repair and replication factors (8-10). Indeed, BRCA1 has been
shown to function in genomic stability by controlling homologous
recombination, transcription-coupled repair of oxidative DNA damage,
and cell cycle checkpoints (11-14).
-IR, and this is likely governed by the action of PIKs
like ATM (17, 19, 20).
-IR also induces 53BP1 to rapidly relocalize
to DNA repair foci, and this response is delayed or inhibited by
treatment with the PIK inhibitors caffeine and wortmannin. 53BP1 foci
also overlap with those formed by the Mre11 complex, BRCA1, and the
phosphorylated form of the histone variant H2AX (
-H2AX; see Refs.
18-20). As both the Mre11 complex and
-H2AX are believed to
localize to physical sites of DNA damage (21-23) and to recruit
various DNA repair factors to these sites, 53BP1 has been inferred to
localize to these sites as well. This notion is further supported by
the fact that
-H2AX recruits 53BP1 to nuclear foci and physically interacts with 53BP1 (20, 24). Recent studies have revealed a role for
53BP1 in cell cycle checkpoints (25-27) as well as in maintaining p53 levels in response to
-IR (27). Here we show that a
380-amino-acid region of 53BP1 that includes a recently described
kinetochore-binding domain (28) is necessary for the formation of
irradiation-induced foci. We further deciphered the role of 53BP1 in
the DNA damage response by generating mice defective in
m53BP1. We report that murine animals expressing a truncated form of m53BP1 (m53BP1tr/tr) exhibit
a pleiotropic phenotype that includes growth retardation, immune
deficiencies including defects in T cell maturation, sensitivity to
-IR, as well as increased chromosomal aberrations. Taken together, these results reveal that 53BP1 is an integral component of the DNA
damage-response network and indicate that the protein plays an
important role in maintaining genomic stability.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
53BP1) or the last 200 residues of the protein (
53BP1-C) were affinity-purified by
established procedures and used as described in the text.
53BP1-N is
a polyclonal, anti-peptide antibody that was raised against an
N-terminal sequence GVLELSQSQDVEE that is conserved between human and
murine 53BP1 proteins. Polyclonal antibodies were affinity-purified by
standard methods. Anti-HA antibodies were purchased from Covance, and
anti-ATR antibodies were obtained from Oncogene Research Products.
-IR using a Nasatron irradiator, returned to 37 °C for 30 min to
allow cells irradiated in mitosis to exit, and then incubated with 1 µg/ml colcemid for 2 h prior to cell harvest, hypotonic (0.075 M KCl) fixation (3:1 methanol:glacial acetic acid), and
metaphase spread preparation. Dried slide preparations were stained
with Giemsa and examined for the presence of chromatid gaps, breaks,
and exchanges by light microscopy. Between 50 and 100 metaphases were
scored for each treatment.
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
BRCT), the kinetochore-binding region (
KINET), the N-terminal 1,234 residues except for the initiation codon (
NH3), and a protein mutated at 15 potential phosphorylation sites (15AQ), some of which are known to be targeted during the DNA damage
response.2 All constructs
maintained the nuclear localization signal. Transient tranfections with
these various constructs into MCF-7 cells revealed that the mutant
proteins were being expressed (not shown). We confirmed that the
wild-type, HA-tagged version of 53BP1 encoded by pCMH6K53BP1 generated
nuclear foci in response to DNA damage when immunostained with an
antibody specific for the HA tag (Fig. 1D). Untransfected
cells were found to stain negative with anti-HA antibodies (not shown).
Our results indicate that the majority of 53BP1 appears dispensable for
DNA damage-inducible focus formation, including the N-terminal 1,234 residues (which includes numerous (S/T)Q motifs) as well as the
C-terminal BRCT motifs (Fig. 1D). Surprisingly,
KINET, a
380-amino-acid deletion (residues 1,236-1,615) that removes the
kinetochore binding region (28) of 53BP1, failed to form
irradiation-induced foci as the protein persisted in a diffuse nuclear
pattern after irradiation (Fig. 1D).
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Fig. 1.
Dynamic nuclear
localization of human 53BP1 during interphase, association with ATR,
and structural requirements for DNA damage inducible focus
formation. As shown in A,
immunofluorescence analysis with an antibody specific for 53BP1
( 53BP1; see "Experimental Procedures") reveals the nuclear
staining pattern for cells unambiguously assigned to the
G1-phase (left), S-phase (middle),
and G2-phase (right) of the cell cycle as
determined by DNA content with a lasing scanning cytometer. Black and
white images were captured with a ×100 objective on a Zeiss Axiophot
and pseudocolored in Adobe PhotoShop. As shown in B, ATR and
53BP1 co-localize to nuclear foci in response to hydroxyurea (2.0 mM). Left panel, immunostaining for 53BP1.
Middle, immunostaining with an antibody specific for ATR
(see "Experimental Procedures"). Right, merged images to
show co-localization between 53BP1 and ATR. As shown in C,
ATR and 53BP1 physically interact before and after DNA damage. K562
cells were grown and either left untreated (lanes 1 and
2) or treated with 2.0 mM hydroxyurea
(HU; lane 3) for 18 h prior to the
preparation of nuclear extracts for immunoprecipitation with an
antibody against 53BP1 (left panel) or ATR (right
panel). Immunoblotting was then performed with the reciprocal
antibodies as shown. D, schematic representation of primary
structure of 53BP1 (not drawn to scale). Hatched lines
represent locations of (S/T)Q sites mutated in 15AQ.
KINET, kinetochore-binding region (28); NLS,
nuclear localization signal (28). E, identification of a
region of 53BP1 required for irradiation-induced focus formation.
Wild-type, HA-tagged 53BP1 and various mutant derivatives were
transfected into MCF7 cells and treated with 10 Gy of ionizing
radiation prior to fixation and immunostaining with an antibody
specific for the HA tag (Covance). The following constructs expressing
in-frame 53BP1 deletions or mutations were made:
BRCT (deletes amino
acid residues 1,786-1,964),
KINET (deletes amino acid residues
1,236-1,615),
NH3 (deletes the first 1,234 amino acids residues
except for the initiation codon), and 15AQ, a construct with mutations
in 15 (S/T)Q sites. The following serine or threonine residues were
mutated to alanines in 15AQ: Ser-6, Ser-13, Ser-25, Ser-29, Ser-105,
Ser-166, Ser-176, Ser-178, Thr-302, Ser-452, Ser-523, Ser-543, Ser-625,
Ser-784, Ser-892. All constructs were verified by DNA sequencing and
expressed in either 293T or MCF7 cells (not shown).
53BP1-N and
53BP1-C, respectively; see "Experimental Procedures"), we
determined that a truncated m53BP1 protein corresponding to
m53BP1tr appeared in heterozygous and mutant extracts but
not in wild-type ones (Fig. 2E). The levels of
m53BP1tr appeared much lower than the full-length protein
and, in some cases, we observed an apparent isoform of m53BP1 in
wild-type and heterozygous animals (Fig. 2E). The
disappearance of full-length m53BP1 in the mutant samples was
accompanied by the appearance of a smaller protein corresponding to
m53BP1tr (Fig. 2E). We observed that
53BP1-N
cross-reacted with m53BP1tr but not with
53BP1-C,
demonstrating that m53BP1 is indeed truncated in
m53BP1tr/tr animals (Fig. 2, E and
F).
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Fig. 2.
Generation and characterization
of mice defective in m53BP1
(m53BP1tr/tr). A,
schematic diagram of insertion event in m53BP1 (not drawn to
scale). The thick horizontal lines represent positions of
probes for Southern blotting as described in B. Arrows represent the position and orientation of PCR primers
used in C. The insertion of VICTR54 was determined by DNA
sequencing to reside within the intron preceding exon 14 at nucleotide
position 1,730 (marked by *). Splicing of the neomycin gene and
flanking DNA produces a transcript that potentially disrupts the proper
splicing of exons 13 and 14. LTR, long-terminal repeat;
NEO, neomycin resistance gene; PGK,
phosphoglycerate kinase-1; BTK, Bruton's tyrosine kinase;
SA and SD, splice acceptor and donor,
respectively. B, top, Southern blotting to
determine the genotype of m53BP1-defective animals. 10 µg
of genomic DNA was digested with XbaI and was probed with a
radiolabeled fragment (see "Experimental Procedures") capable of
discerning wild-type (WT) and mutant alleles as
discussed under "Experimental Procedures." Bottom, a
700-bp probe derived from the neomycin gene was used to help genotype
the animals. +/+, wild type; +/tr, heterozygous;
tr/tr, homozygous. As shown in C,
RT-PCR analysis indicates that improper splicing occurs between exons
13 and 14 in m53BP1tr/tr mice. Positions and
orientation of primers for PCR are indicated in A. Control
reactions without reverse transcriptase showed essentially no amplified
products (not shown). As shown in D,
m53BP1tr/tr encodes a truncated protein of 1,226 amino acids. RT-PCR products derived from primer set A/D (as shown in
C) using RNA isolated from
m53BP1tr/tr animals as template were cloned into
the TA vector (Invitrogen). DNA sequencing and conceptual translation
indicated that m53BP1tr/tr animals potentially
encode a truncated m53BP1 protein (m53BP1tr) of 1,205 natural residues along with an additional 21 residues derived from the
VICTR54 vector. m53BP1tr is missing over 700 C-terminal
residues, including those that specify the kinetochore binding domain
(KINET; amino acids 1,220-1,601), the nuclear localization
signal (NLS; mapped to amino acids 1,601-1,703; ref), and
the C-terminal BRCT repeats (amino acids, 1,665-1,957). The small,
vertical rectangle in m53BP1tr represents the additional
vector-derived 21 residues. E and F, detection of
m53BP1tr protein by IP/WB analysis. 1.0 mg of total brain
protein extracts derived from +/+, +/tr, and tr/tr animals was
immunoprecipitated with 5 µg of affinity-purified antibody
( 53BP1-N) raised against an N-terminal peptide sequence (see
"Experimental Procedures") and split into two equal parts. One part
(E) was blotted with
53BP1-N, and the other
(F) was immunoblotted with an affinity-purified antibody
specific for the C terminus (
53BP1-C). E, lane
1, IP from 100 µg of MCF-7 nuclear extract. Lane 2,
IP with nonspecific IgG control. Lanes 3-5, IP with +/+,
+/tr, and tr/tr animals as determined in B. IP/WB analysis
shows the presence of m53BP1tr in
m53BP1+/tr and
m53BP1tr/tr animals but not of m53BP
+/+ ones. In some cases, we observed an apparent isoform of
m53BP1 in brain tissue, as designated by the asterisk.
F, WB with
53BP1-C, an affinity-purified antibody against
the C-terminal 200 residues of human 53BP1. As shown,
53BP1-C
recognizes full-length m53BP1 but fails to immunoreact with
m53BP1tr, indicating that the protein is indeed missing
C-terminal residues of m53BP1.
-gene rearrangement occurs. This indicates that
m53BP1 participates in proper T cell development, a process known to
require various DNA repair factors (30). We also found that spleens
derived from m53BP1tr/tr animals were similar in
size and organ architecture to those from
m53BP1tr/tr animals and that the lack of
functional m53BP1 did not affect the proportions of B and T lymphocytes
(data not shown). We did observe, however, that
m53BP1tr/tr spleens were deficient in
mature B cells (IgMloIgDhi; Fig.
4D), suggesting that
deficiencies in m53BP1 may also result in defective B lymphocyte
development.
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Fig. 3.
Growth retardation and immune deficiencies in
mice defective in m53BP1. A, mean body
weight, in grams, of m53BP1+/+ and
m53BP1tr/tr mice. (n = 10). As
shown in B, reduced thymus size in
m53BP1tr/tr animals results in lower
tissue cell count. Mean cell numbers are × 106 (per
mouse) of bone marrow (two femurs per mouse), spleens, and thymuses
from m53BP1+/+ and
m53BP1tr/tr mice (n = 4).
C, defective T cell development in
m53BP1tr/tr mice as revealed by double-negative
thymocyte populations. CD4+ and CD8+ cells were
removed by gating, leaving DNI (CD44+CD25 ),
DNII (CD44+CD25+), DNIII
(CD44
CD25+), and DNIV
(CD44
CD25
) thymocytes. Numbers
in the histogram quadrants are average percentages for four mutant or
control animals. D, defective B cell development in
m53BP1tr/tr animals. Immature
(IgMhiIgDlo), transitional
(IgMhiIgDhi), and mature
(IgDhiIgMlo) B cells in
m53BP1+/+ and m53BP1tr/tr
mouse spleens. Numbers in the histogram quadrants are
average percentages for four mutant or control mice.
View larger version (22K):
[in a new window]
Fig. 4.
Characterization of animals and cells
defective in m53BP1tr/tr.
A, survival of 4-6-week-old
m53BP1tr/tr and m53BP1+/+
mice after exposure to 7 Gy of -IR. Six animals from each genotype
were used in the experiment. B, survival of 4-6-week-old
m53BP1tr/tr animals after exposure to 1.5 Gy of
ionizing radiation.
-IR. To evaluate whether m53BP1 contributes
to increased sensitivity to DNA damage, we treated
m53BP1tr/tr or wild-type animals with 7 Gy of
-IR. After this whole body irradiation treatment, we found that
100% of the mutant animals died between 9 and 15 days post-irradiation
in contrast to only 16% of the control littermates (Fig.
4A). This shows that animals defective in m53BP1
are highly sensitive to
-IR, a result that parallels previous
observations with H2AX-deficient mice (24). Despite this, we found that
m53BP1tr/tr animals treated with lower doses of
-IR (1.5 Gy) remained viable (Fig. 4B). To further
explore m53BP1 function, we generated embryonic fibroblasts (MEFs) from
wild-type and m53BP1tr/tr animals.
m53BP1tr/tr MEFs proliferated more slowly than
their wild-type counterparts (Fig.
5A). Immunofluorescence
analysis indicated that the truncated m53BP1 protein expressed in
m53BP1tr/tr animals failed to form foci in
response to DNA damage as it was essentially absent from the nucleus
(data not shown). This result is consistent with our transfection
studies, which have shown that C-terminal determinants (
KINET) are
necessary for focus formation. The relative growth of the mutant and
the wild-type MEFs was reminiscent of what has been recently described
for H2AX (24). To further characterize cells defective in
m53BP1, we examined the cytological consequences of impaired
m53BP1 function in early passage MEFs derived from
m53BP1tr/tr and m53BP1+/+
animals. For this, exponentially growing MEFs (passage 2) were treated
with 0, 0.5, or 1.5 Gy of
-IR, and metaphase preparations were
examined 2.5 h post-irradiation. Untreated MEFs derived from m53BP1tr/tr animals showed increased levels of
chromatid gaps, breaks, and, to a lesser extent, exchanges when
compared with those derived from m53BP1+/+ mice,
suggesting an intrinsic genomic stability defect in the mutant cells
(Fig. 5, B and C). More strikingly, irradiated
MEFs derived from m53BP1tr/tr animals showed an
~2-fold increase in levels of chromatid breaks and gaps when compared
with MEFs derived from wild-type mice (Fig. 5, B and
C). Although MEFS from m53BP1tr/tr
animals showed relatively high chromatid exchange rates at 0.5 Gy when
compared with those from m53BP1+/+ animals, this
difference was less apparent at 1.5 Gy, perhaps due to the limited
progression to mitosis of the most damaged cells from both populations
during this time frame. One possible explanation for the increased
frequencies of chromosomal aberrations observed in the
m53BP1tr/tr MEFs following irradiation might be
a deficiency in a G2 checkpoint response whereby more
damaged cells would still be permitted to enter mitosis and would be
available for chromosome analysis. In fact, recent reports have
implicated 53BP1 in the G2/M checkpoint (25-27). To
examine this in our MEFs, either m53BP1tr/tr or
wild-type MEFs were treated with 0, 1.5, or 10 Gy of
-IR, and
cultures were analyzed for the fraction of cells showing
phospho-histone H3 immunostaining (mitotic cells) either after 1 or
16 h post-irradiation (in the presence of colcemid). Although all
cell types showed evidence of a partial G2/M block
following irradiation, MEFs derived from
m53BP1tr/tr mice showed only a slight decrease,
if any, in the G2 block when compared with MEFs derived
from wild-type mice (data not shown). The minimal effects on the
G2/M checkpoint observed in our
m53BP1tr/tr MEFs may be due to the nature of the
truncated protein produced from the m53BP1tr/tr
allele that is expressed in our mutant animals described here.
View larger version (25K):
[in a new window]
Fig. 5.
Chromosomal abnormalities in
m53BP1tr/tr cells. A,
growth curve of MEFs derived from m53BP1tr/tr
(open diamonds) and m53BP1+/+
(closed circles). B, metaphase preparation of
mutant MEF following 1.5 Gy of -IR. Note the presence of a chromatid
gap, two chromatid breaks, and one chromatid exchange in the metaphase
sample. C, relative frequencies of chromatid gaps, breaks,
and exchanges in metaphases of wild-type and mutant MEFs following 0, 0.5, and 1.5 Gy of ionizing radiation.
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ACKNOWLEDGEMENTS |
---|
We thank Jian Kuang and Randy Legerski for help in setting up our makeshift laboratory at M. D. Anderson in the aftermath of Hurricane Allison. We thank Heladio Ibarguen for help with the metaphase spreads. We are grateful to David Cortez for providing useful suggestions during the course of the project. We are also indebted to Mike Blackburn, Rob Kirken, Jeff Frost, Hays Young, and Jose Molina for technical advice. We thank Jungie Chen for communicating results prior to publication.
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FOOTNOTES |
---|
* This work was supported by grants from The Robert A. Welch Foundation (to P. B. C.) and The Ellison Medical Foundation (to P. B. C.) as well as National Institute of Health Grants GM65812-01 (to P. B. C.), DK46207 (to R. E. K.), and 5P30 CA16672 (to W. N. H.).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.
A fellow of the U. S. Army Breast Cancer Postdoctoral Trainee Award.
** A Sophie Caroline Steves Distinguished Professor in Cancer Research.
¶¶ An investigator with the Howard Hughes Medical Institute, an Ellison Medical Foundation Senior Scholar, and the Welch Professor of Biochemistry.
An Ellison Medical Foundation Junior Scholar who
is grateful for their support. To whom correspondence should be
addressed. Fax: 713-500-0652; E-mail:
Phillip.B.Carpenter@uth.tmc.edu.
Published, JBC Papers in Press, February 10, 2003, DOI 10.1074/jbc.M212484200
2 Z. Xia, J. C. Morales, and P. B. Carpenter, unpublished data.
3 Dr. A. Nussenzweig, personal communication.
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ABBREVIATIONS |
---|
The abbreviations used are:
-IR, ionizing radiation;
PIK, phosphatidyl inositol-like kinase;
BRCT, BRCA1
C-terminal repeats;
MEF, murine embryonic fibroblast;
ATM, mutated in
ataxia-telangiectasia;
ATR, ATM- and Rad3-related;
RT, reverse
transcription;
HA, hemagglutinin;
IP, immunoprecipitation;
WB, Western
blotting;
DN, double negative.
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