Binding of CtIP to the BRCT Repeats of BRCA1 Involved in the
Transcription Regulation of p21 Is Disrupted Upon DNA Damage*
Shang
Li
,
Phang-Lang
Chen
,
Thirugnana
Subramanian§,
G.
Chinnadurai§,
Gail
Tomlinson¶,
C. Kent
Osborne
,
Z.
Dave
Sharp
, and
Wen-Hwa
Lee
**
From the
Departments of Molecular Medicine/Institute
of Biotechnology and
Medicine, University of Texas Health
Science Center, San Antonio, Texas 78245, § Institute for
Molecular Virology, St. Louis University Medical Center, St. Louis,
Missouri 63110, and ¶ Department of Pediatrics, Hamon Center for
Therapeutic Oncology Research, University of Texas Southwestern
Medical Center, Dallas, Texas 75235
 |
ABSTRACT |
Mutations in BRCA1 are responsible
for nearly all of the hereditary ovarian and breast cancers, and about
half of those in breast cancer-only kindreds. The ability of BRCA1 to
transactivate the p21 promoter can be inactivated by mutation of the
conserved BRCA1 C-terminal (BRCT) repeats. To explore the mechanisms of this BRCA1 function, the BRCT repeats were used as bait in a yeast two-hybrid screen. A known protein, CtIP, a co-repressor with CtBP, was
found. CtIP interacts specifically with the BRCT repeats of BRCA1, both
in vitro and in vivo, and tumor-derived
mutations in this region abolished these interactions. The association
of BRCA1 with CtIP was also abrogated in cells treated with
DNA-damaging agents including UV,
-irradiation, and adriamycin, a
response correlated with BRCA1 phosphorylation. The transactivation of the p21 promoter by BRCA1 was diminished by expression of exogenous CtIP and CtBP. These results suggest that the binding of the BRCT repeats of BRCA1 to CtIP/CtBP is critical in mediating transcriptional regulation of p21 in response to DNA damage.
 |
INTRODUCTION |
Mutations in BRCA1 are responsible for nearly all of
the hereditary ovarian and breast cancers, and about half of those in breast cancer-only kindreds (1-3). How BRCA1 inactivation leads to
tumor formation remains unclear. Studies of homozygous mutation of
Brca1 in mice showed a phenotype of early embryonic
lethality (4-6). Interestingly, Brca1
/
mouse embryonic stem cells are hypersensitive to ionizing radiation and
hydrogen peroxide, and defective in transcription-coupled repair of
oxidative DNA damage (7). An extension of development to embryonic day
11-12 was observed in Brca1
/
mice carrying
additional p53 or p21waf/cip1 null mutations (8, 9). A role
for BRCA1 in transcription regulation was provided by observations
showing that expression of p21, a known target for p53 transcriptional
activation, is increased significantly in Brca1 mutant
embryos (8). Consistent with this observation, wild-type, but not
mutant BRCA1, was able to transactivate the expression of p21 and
inhibit cell cycle progression from G1 into S phase in
human cells (10). Taken together, these results suggested that BRCA1
may have a role in the DNA repair process involving p21 and p53
expression. However, the molecular basis for these observations is
largely unknown.
The C terminus of BRCA1 contains a transcription activation region
(10-13) and two conserved
BRCT1 repeats frequently
found in proteins involved in DNA repair and cell cycle regulation
(14-16). Although the general function of the BRCT motif is unclear,
several lines of evidence suggest that it may be involved in
protein-protein interactions (17, 18). Here, we report specific
interactions between CtIP and the BRCT repeats of BRCA1 both in
vitro and in vivo. Apparently, complex formation of
BRCA1, CtIP and CtBP plays an important role in the regulation of p21 expression.
 |
EXPERIMENTAL PROCEDURES |
Plasmid Constructs--
The pAS-BRCT plasmid was generated by
polymerase chain reaction amplification of nucleotides 4898-5592 of
BRCA1 using pBSK-BRCA1a (19) as the template and the following
primers: 5'-CCGGAATTCCGGGGCCGCAGGGAGAAGCCAGAATTGA-3' and
5'-ATAGGATCCTCAGTAGTGGCTGTGGGGGAT-3'. The 0.7-kilobase polymerase chain
reaction product was cloned into the EcoRI/BamHI
sites of pAS2-1 vector (CLONTECH, Palo Alto, CA).
The pGST-BRCT plasmid was constructed by digestion of pAS-BRCT with
EcoRI, Klenow fill-in, and ligation of BamHI
linkers (New England Biolab, Beverly, MA). The construct was then
digested with BamHI to release the BRCT fragment and
subsequently cloned into BamHI site of pGEX-2T vector (Amersham Pharmacia Biotech). The GST-BRCT
mutant was engineered using site mutagenesis kit (Stratagene, La Jolla, CA) with the following primers: 5'-CCAGGAGCTGGACACCTAACTGATA CCCCAGATCC-3' and
5'-GGATCTGGGGTATCAGTTAGGTGTCCAGCTCCTGG-3'. pGAL4-BRCT was constructed
by digestion of pGST-BRCT with BamHI to release the BRCT
fragment and subsequently cloned into the BamHI
site of pM2 vector (20). The pGAL4-BRCT mutants, including A1708E,
P1749R, and Y1853term, were generated by site-directed
mutagenesis with the following primers:
5'-CATTTTCCTCCCTCAATTCCTAG-3' and 5'-CTAGGAATTGAGGGAGGAAAATG-3' for A1708E mutant; 5'-CTCTTGCTCGCTTTCGACCTTGGTGG-3' and
5'-CCACCAAGGTCGAAAG CGAGCAAGAG-3' for P1749R mutant; the primers
used to generate Y1853term mutant was shown above. The
pVP16-CtIP plasmid was constructed by cloning full-length CtIP into the
NotI site of pVP-Flag5 vector (20). The pGST-CtIP plasmid
was engineered by digestion of pVP16-CtIP with
XhoI/StuI and subsequently cloned into pGST4
vector. For the transfection study, full-length BRCA1 was cloned into
NotI/XhoI sites of pcDNA3.1 vector
(Invitrogen, Carlsbad, CA). The pcDNA-CtIP vector that expresses
full-length CtIP, the pRcCMV-CtBP vector that expresses the full-length
CtBP, and GST-CtBP were previously described (21, 22). The pWWW-luc
(10), pSV40-
-gal and pGAL4-VP16 (23) plasmids were provided as described.
Yeast Two-hybrid Screen--
The pAS-BRCT plasmid, which
contains the GAL4 DNA-binding domain fused to BRCA1 (amino acids
1634-1863), was used as the bait for screening a cDNA library
prepared from human B lymphocytes as described (24). Because the bait
has weak transactivation activity, 50 mM of
3-amino-1,2,4-triazole was used in the screening to reduce the background.
In Vitro Binding Assay--
Bacterially expressed and purified
GST or GST fusion proteins were incubated with in vitro
synthesized [35S]methionine-labeled CtIP, BRCA1, or CtIP
and BRCA1 proteins as described (25).
Mammalian Two-hybrid Assay--
Human kidney 293 cells were used
in this assay as described (20). The expression of GAL4-X fusion
proteins was verified by straight Westerns using a specific antibody
that recognizes the GAL4 DNA-binding domain (Santa Cruz Biotechnology,
Inc. Santa Cruz, CA).
Immunoprecipitation and Western Blot Analyses--
Cells were
lysed in lysis 250 buffer and immunoprecipitated as described (24). The
immunoprecipitates were separated by SDS-polyacrylamide gel
electrophoresis and analyzed by immunoblotting.
Cell Transfection and Luciferase Assay--
Human kidney 293 cells were transfected with 0.5 µg of pWWW-luc or pG5E-luc, 0.5 µg
of pSV40-
-gal, and 2 µg of each different plasmid DNA as indicated
(control vector pcDNA3.1 was used to bring the final amount of DNA
to 10 µg) using calcium phosphate/DNA co-precipitation method.
Luciferase activity was measured 48 h after transfection as
described (26). For each 10-cm dish of HCT116 cells, 10 µg of
pRcCMV-CtBP plasmid was transfected, using the Lipofectin transfection method.
Treatments with DNA-damaging Agents--
Human colon cancer
cells, HCT116, were treated with UV (1 mJ/cm2) or
-irradiation (10 Gy), and harvested 1 h after treatment. For
adriamycin treatment, HCT116 cells were incubated with adriamycin (0.2 µg/ml) for 24 h before harvest.
 |
RESULTS |
CtIP Interacts Specifically with Wild-type BRCT Repeats of
BRCA1--
To explore the potential function of the BRCT repeats of
BRCA1, we used them as bait in a yeast two-hybrid screen for
interacting proteins (24). One of the 20 clones isolated encodes amino
acids 17 to 713 of the known protein CtIP (Fig.
1A), which was also identified
by others using a different approach (27, 28).

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Fig. 1.
Identification of BRCA1-interacting protein,
CtIP. A, amino acids 1634-1863 of BRCA1 were fused
in-frame to the GAL4 DNA binding domain in pAS vector. The schematic
shows the clone isolated from the yeast two-hybrid screen containing
amino acids 17-713 of CtIP relative to full-length CtIP. B,
GST fusion proteins that contain BRCT repeats either wild-type or with
the 1853 tyrosine nonsense mutation. C, Coomassie gel
showing the GST and GST-fusion proteins used in the in vitro
binding assay. The mutant form of fusion protein (GST-BRCT ) migrates
slightly faster than the wild-type form. D, in
vitro transcribed and translated
[35S]methionine-labeled protein from full-length CtIP
(lane 1) binds to the wild-type GST-BRCT fusion protein
(lane 3), but not the GST (lane 2) or the mutated
GST-BRCT fusion proteins (lane 4). E,
mammalian two-hybrid analysis of the interactions between CtIP and
wild-type or mutated BRCT repeats of BRCA1. Each culture of human 293 cells was transiently co-transfected with the pG5E-Luc reporter vector,
the pSV40- -gal control vector, and two of the indicated expression
vectors. The GAL4 expression vector expresses either the GAL4
DNA-binding domain (denoted by "+" in the GAL4 column in
lanes 1 and 2), or the GAL4-BRCT fusion protein
(WT; lanes 3-6), and variants of the BRCT
sequence that have the A1708E, P1749R, or Y1853term
mutations (lanes 7-12). The VP16 vector encodes either the
VP16 transactivation domain alone (denoted by "+" in the VP16
column in lanes 1-4) or the VP16-CtIP full-length fusion
construct (CtIP, lanes 5-12). The luciferase
activities were normalized to -galactosidase activities.
F, expression of the GAL4-BRCT fusion proteins in the
mammalian two-hybrid assays. Equivalent aliquots of lysate from
untransfected 293 cells (lane 1); those transfected with
GAL4 parental vector (lane2); GAL4-BRCT wild-type, A1708E,
P1749R, or Y1853term constructs (lanes 3-6,
respectively) were analyzed by straight Westerns using a rabbit
polyclonal antibody specifically against GAL4 DNA-binding domain (Santa
Cruz Biotechnology, Inc.).
|
|
To test whether CtIP directly binds to the BRCT repeats, an in
vitro binding assay using GST-fusion proteins was performed. The
BRCT repeats that served as the bait in the above screen and a mutant
containing a familial 1853 tyrosine nonsense mutation were fused with
GST (GST-BRCT and GST-BRCT
, Fig. 1B). Bacterially expressed and purified GST-BRCT, but not GST-BRCT
or GST (Fig. 1C), can bind to in vitro synthesized
[35S]methionine-labeled CtIP protein (Fig. 1D,
lane 3, compare lanes 2 and 4).
To ascertain whether CtIP and the BRCT repeats of BRCA1 can interact in
cells, a mammalian two-hybrid assay (20) was performed. Full-length
CtIP was fused to the VP16 transactivation domain of herpesvirus in an
expression vector (pVP16-CtIP), and a panel of expression vectors
encoding the DNA binding-domain of GAL4 fused to either wild type
(GAL4-BRCT, Fig. 1E) or mutated (GAL4-BRCTM, Fig. 1E) BRCT repeats of BRCA1 were constructed. The BRCT
mutants contain individual alterations identified in familial breast
cancers including missense (A1708E, first BRCT repeat; and P1749R,
spacer region) and nonsense (Y1853term, second repeat)
mutations. Human kidney 293 cells were co-transfected with a
GAL4-responsive luciferase (pG5E-luc) and
-galactosidase
(pSV40-
-gal) reporters, expression plasmids for either GAL4 or
GAL4-BRCT (wild type or mutant), and VP16 or VP16-CtIP. A significant
increase in luciferase activity was observed upon co-expression of
wild-type GAL4-BRCT and VP16-CtIP (Fig. 1E, lanes
5 and 6). In contrast, no obvious activity was observed
upon co-expression of GAL4 or wild-type GAL4-BRCT with VP16 (Fig.
1E, lanes 1-4), or the mutated BRCT repeats in
GAL4-BRCTM with VP16-CtIP (Fig. 1E, lanes
7-12). Because the cells transfected with GAL4, GAL4-BRCT and
GAL4-BRCTM (Fig. 1F, lanes 2-6)
expressed these fusion proteins at comparable levels, the reduction of
luciferase activity was not attributable to lack of protein expression.
Identification of Cellular CtIP Protein--
To study the in
vivo interactions of endogenous BRCA1 and CtIP, mouse polyclonal
antibodies recognizing CtIP (C21) were generated using an antigen
consisting of GST translationally fused with amino acids 324-537 of
CtIP. [35S]methionine-labeled human colon carcinoma cell
(HCT116) lysates immunoprecipitated with C21 identified a band (Fig.
2iA, lane 2) whose mobility
was consistent with the predicted molecular mass (125 kDa) of CtIP
(21). A band of similar size was not detected in the immunoprecipitates
of pre-immune serum (Fig. 2A, compare lanes 1 and
2). Pre-incubation of the antiserum with GST-CtIP, but not
GST resulted in specific depletion of the 125-kDa band (Fig.
2A, compare lanes 3 and 4).
Reprecipitation of the immunoprecipitates by a second incubation with
anti-CtIP antibodies resulted in a specific 125-kDa band (Fig.
2A, lane 5). Based on these results, we concluded
that the 125-kDa protein is the cellular CtIP protein.

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Fig. 2.
In vivo interactions between CtIP and
BRCA1. A, specificity of the anti-CtIP antibodies.
Lysates of HCT116 cells labeled with [35S]-methionine
were immunoprecipitated with pre-immune serum (lane 1),
anti-CtIP antibody (C21) alone (lane 2), anti-CtIP antibody
pre-incubated with GST-CtIP antigen (lane 3), or anti-CtIP
antibody pre-incubated with GST (lane 4). Lane 5 shows a double immunoprecipitation with anti-CtIP antibody. The
anti-CtIP antibody specifically recognizes a cellular protein
(arrow) whose mobility is consistent with a molecular mass
of 125 kDa. B, co-immunoprecipitation of CtIP with wild-type
BRCA1, but not with mutated BRCA1 in vivo. Lysates from
HCT116 (lanes 1, 2, 4, 6, and 7) and HCC1937
cells (lanes 3, 5, and 8) were immunoprecipitated
with the following antibodies; anti-p84 control monoclonal antibody
(lane 1), 6B4, an anti-BRCA1 monoclonal antibody
(lanes 2 and 3), C20, a rabbit polyclonal
antibody against BRCA1 C-terminal peptide (Santa Cruz Biotechnology,
Inc.) (lanes 4 and 5), pre-immune serum
(lane 6), and C21, anti-CtIP mouse polyclonal serum
(lanes 7 and 8). In the upper panels
(lanes 1-8), the blots of immunoprecipitated proteins were
probed with anti-BRCA1 monoclonal antibody to detect BRCA1; in the
lower panels (lanes 1'-3' and
6'-8'), the blots were probed with anti-CtIP polyclonal
serum to detect CtIP. CtIP can be co-immunoprecipitated with the
wild-type (lanes 2 and 2'), but not mutated
(lanes 3 and 3') BRCA1. Conversely, wild-type
(lanes 7 and 7'), but not mutated (lanes
8 and 8') BRCA1 can be co-immunoprecipitated with
CtIP.
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Co-immunoprecipitation of CtIP and BRCA1 in Vivo--
The in
vivo interaction between CtIP and BRCA1 was further examined in
HCT116 and breast cancer cells, HCC1937 (29), by co-immunoprecipitation. HCC1937 cells contain an insertion of cytosine
at nucleotide 5382 of BRCA1 that generates a frameshift at amino acid
1794, which stops translation at 1829 (29). Anti-BRCA1 monoclonal
antibody 6B4 (30), but not control antibody against p84 (N5), a nuclear
matrix protein (31), specifically immunoprecipitated a 220-kDa protein
in HCT116 cell lysates (Fig. 2B, compare lanes 1 and 2). Consistent with the 5382insC mutation in BRCA1, a
faster migrating product was detected in HCC1937 cell lysates (Fig.
2B, lane 3). This protein is likely to be the
HCC1937 BRCA1 product because it cannot be recognized by
C-20 antibodies (against amino acids 1843-1862 of BRCA1), which
readily immunoprecipitated a 220-kDa protein from HCT116 (Fig.
2B, compare lanes 5 and 4).
Furthermore, using anti-BRCA1 6B4, but not control antibody N5, CtIP
was co-immunoprecipitated with BRCA1 in HCT116 cell lysates (Fig.
2B, compare lanes 1' and 2'). No
detectable CtIP was present in the 6B4 immunoprecipitates from HCC1937
cell lysate (Fig. 2B, lane 3'). Consistent with
this result, BRCA1 was reciprocally co-immunoprecipitated with
anti-CtIP C21 antibody from HCT116 cell lysate, but not from HCC1937
cell lysate (Fig. 2B, compare lanes 7 and
8). Levels of CtIP immunoprecipitates detected from both
cell lines were comparable (Fig. 2B, lanes 7' and
8'). These data indicated the existence of an in
vivo complex of BRCA1 and CtIP, that, in HCC1937 cells, is
disrupted presumably by an altered C terminus lacking intact BRCT repeats.
Dissociation of CtIP from BRCA1 upon Treatment with DNA-damaging
Agents--
Since BRCA1 has a potential role in DNA repair, its
interaction with CtIP may mediate cellular responses to DNA damage.
Therefore, it is possible that their interaction might be altered upon
treatment of cells with DNA-damaging agents. To address this
possibility, HCT116 cells were treated with UV,
-rays, or
adriamycin, and the cell lysates were immunoprecipitated with
anti-BRCA1 antibody (6B4). Consistent with previous data (32, 33), a
slower migrating form of phosphorylated BRCA1 was detected after
treatment of the cells with DNA-damaging agents (Fig.
3A, lanes 3-5
compare with lane 2). Importantly, the association of CtIP
was undetectable in the BRCA1 immunoprecipitates subsequent to DNA
damage (Fig. 3A, compare lanes 3'-5' and
lane 2'). This change in BRCA1/CtIP association appeared to
be correlated with increased BRCA1 phosphorylation. The undetectable
levels of CtIP in BRCA1 immunoprecipitates were not due to lower
expression because the amount of CtIP did not change subsequent to
treatment with genotoxic agents (Fig. 3B). Similar results
were also observed in breast epithelial cell line MCF10A (data not
shown).

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Fig. 3.
Dissociation of BRCA1 from CtIP upon
DNA-damaging agents treatment. A, BRCA1/CtIP
interactions were altered in response to DNA damage. HCT116 cells
treated with UV radiation (1 mJ/cm2), -irradiation (10 Gy) were harvested 1 h subsequently. Cells treated with adriamycin
(0.2 mg/ml) were harvested 24 h later. Lysates from untreated and
treated HCT116 cells were immunoprecipitated with anti-p84 control
antibody (lane 1) or anti-BRCA1 antibody-6B4 (lanes
2-5), separated by SDS-PAGE, and transferred to membranes. In the
upper panel, the membrane was probed with anti-BRCA1
antibody, 6B4. Note the appearance of slower migration forms of BRCA1
after treatment (lanes 3-5, compare with lane
2). In the lower panel, the membrane was probed with
anti-CtIP antibody, C21. CtIP was not detected in the anti-BRCA1
immunoprecipitates in lysates prepared from treated cells (lanes
3'-5') but is readily detected in untreated cells (lane
2'). B, expression levels of CtIP in cells treated with
DNA-damaging agents was not altered. Aliquots of cell lysates used for
the above immunoprecipitations were assayed by straight Westerns using
the indicated antibodies. The upper panel shows a membrane
probed with anti-CtIP antibody. Note that the levels of CtIP expression
were comparable before or after treatment. As a protein loading
control, the lower panel shows a membrane probed with
anti-p84 antibody.
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|
Association of CtBP and BRCA1 Mediated by CtIP--
CtIP was
originally identified in a yeast two-hybrid screen for proteins that
interacted with an adenovirus E1A C-terminal-binding protein, CtBP
(22). The binding of CtBP to E1A represses CR1-dependent transcriptional activation and tumorigenesis (21, 34). Both CtIP and
E1A use a conserved PLDLS motif to interact with CtBP. Several
transcription factors including Knirps, Snail (35), and Hairy (36),
which contain P-DLS-K/V motifs, bind to CtBP to repress transcription
during Drosophila development. Based on this observation, it
is possible that BRCA1 is linked to the CtBP co-repressor through
CtIP.
To address this possibility, we used an in vitro binding
assay to test whether CtIP has different binding motifs for BRCA1 and
CtBP. As shown, mutation of the PLDLS motif in CtIP to LASQC abolished
the interaction between CtIP and GST-CtBP (Fig.
4A, compare lanes 4 and 8). However, the interaction between the mutated CtIP
and GST-BRCT was unaffected (Fig. 4A, compare lanes
3 and 7), suggesting that CtIP binds to CtBP and the
BRCT repeats of BRCA1 using different motifs. Thus, it is possible that
CtIP can bridge BRCA1 and CtBP to form a complex. To test this
possibility, HCT116 cells were transiently transfected with pRcCMV-CtBP
to overexpress the T7-tagged CtBP full-length protein. As shown, anti-T7-tag antibody can co-immunoprecipitate the full-length CtBP
protein, cellular CtIP, and BRCA1 (Fig. 4B, lane
2), but not the control antibody (lane 1). In the
reciprocal experiment, anti-BRCA1 antibody can bring down cellular
BRCA1, CtIP and T7-tagged CtBP (Fig. 4B, lane 4).
These results suggest that CtBP, CtIP, and BRCA1 can form a
complex.

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Fig. 4.
BRCA1 binds to CtBP through CtIP.
A, in vitro synthesized
[35S]methionine-labeled protein from full-length
wild-type (lane 1) or mutated CtIP (PLDLS-LASQC, lane
5), was incubated with GST (lanes 2 and 6),
GST-BRCT (lanes 3 and 7), or GST-CtBP
(lanes 4 and 8). GST-CtBP binds to wild-type, but
not the mutated CtIP (compare lanes 4 and 8).
B, HCT116 cells were transfected with pRcCMV-CtBP plasmid.
The cell lyates were immunoprecipitated with 8G11 (anti-GST monoclonal
antibody, lanes 1 and 3), -T7 (anti-T7-tag
monoclonal antibody, lane 2), or 6B4 (anti-BRCA1 monoclonal
antibody, lane 4). The membranes were probed with 6B4, C21,
or -T7 antibody as indicated. T7-tagged CtBP full-length protein is
indicated by an arrow. The IgG heavy chain is marked by an
asterisk.
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Repression of BRCA1-dependent Transactivation of the
p21 Promoter by CtIP and CtBP--
Previous studies (10, 12) showed
that BRCA1 was able to transactivate p21 expression. Formation of the
CtBP, CtIP, and BRCA1 complex predicts that ectopic expression of CtIP
or CtBP may affect BRCA1-dependent transactivation of the
p21 promoter. To test this hypothesis, transient transfections of human
293 cells were performed with a p21 promoter-luciferase reporter
plasmid pWWW-luc, pSV40-
-gal transfection control plasmid, and
combinations of BRCA1, CtIP, and CtBP expression vectors. The
expression of BRCA1 resulted in a 5-fold activation of the p21 promoter
compared with empty vector alone. Co-expression of CtIP moderately
inhibited BRCA1-dependent transactivation of the p21
promoter. However, co-expression of CtIP and CtBP repressed p21
promoter activity to background levels (Fig. 5A).
Interestingly, co-expression of CtBP and BRCA1 also resulted in
significant repression of p21 promoter, which was likely due to the
abundance of endogenous CtIP in cells. Likewise, the modest inhibition
of BRCA1-dependent transcription by CtIP may be due to the
recruitment of cellular CtBP. Overexpression of CtBP alone does not
have an effect on p21 promoter (Fig.
5A). The repression of
BRCA1-dependent transactivation of p21 promoter by CtBP and
CtIP depends on their association with BRCA1, because overexpression of
CtIP and CtBP cannot repress the transactivation of GAL4 promoter
(pG5E-Luc) by the GAL4-VP16 hybrid (Fig. 5B). These
experiments suggest that a potential biological function of the
BRCA1/CtIP interaction is to repress target promoters through contacts
with the CtBP co-repressor.

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Fig. 5.
CtIP/CtBP specifically represses
BRCA1-dependent transactivation of p21 promoter.
A, human 293 cells were transfected with pWWW-luc (10),
pSV40- -gal, and the expression plasmids containing BRCA1, CtIP, and
CtBP as indicated. B, similar transfection was performed as
shown in panel A, with pG5E-Luc, pSV40- -gal and the
expression plasmids containing GAL4-VP16, CtIP, and CtBP as indicated.
In both experiments luciferase activity was measured 48 h after
transfection and normalized to -galactosidase activity. Expression
of CtIP and CtBP repress the BRCA1-dependent
transactivation of p21 promoter, but not the
GAL4-VP16-dependent transactivation of GAL4 promoter. The
data represent three independent transfections, each assayed for
luciferase activity twice.
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 |
DISCUSSION |
The BRCT repeats were first identified as a highly conserved
structural domain among more than 50 nonorthologous proteins, many of
which are involved in DNA repair and cell cycle check point control
(14-16). Familial mutations have been frequently found in the BRCT
repeats of BRCA1, suggesting that the function of the BRCT repeats of
BRCA1 is important for the BRCA1 tumor suppression function. Using BRCT
repeats as bait, we have identified a BRCA1-associated protein,
CtIP. CtIP interacts specifically with the BRCT repeats of BRCA1
both in vitro and in vivo, and tumor-derived
mutations in BRCT repeats abolish this specific interaction. The three
tumor-derived mutations A1708E, P1749R, and Y1853term, are
either located in the first motif, second motif, or the spacer region
of BRCT repeats. However, all of them abolish the interaction of BRCT
repeats with CtIP. Interestingly, these mutations also abolish the
transactivation activity of BRCA1(13). This tight correlation suggests
that the interaction between BRCA1 and CtIP is relevant to its
transcription regulation activity.
CtIP was originally identified as a CtBP-associated protein in a yeast
two-hybrid screen. Further study has mapped the PLDLS motif in CtIP as
the binding site for CtBP (22). This motif was also found in E1A and
several Drosophila transcriptional factors (22, 35, 36).
BRCA1 does not have a PLDLS motif, and cannot bind to CtBP directly.
Apparently, CtIP has different binding motifs for CtBP and BRCA1
respectively, allowing CtBP to associate with BRCA1 through CtIP, as
shown in Fig. 4. The formation of this complex is important for the
repression of BRCA1-dependent transcription activation of
the p21 promoter. Data from previous studies (10) and in this report
(Fig. 5) suggested that overexpression of exogenous BRCA1 can
transactivate the p21 promoter. A reasonable explanation for these
observations is that overexpression of BRCA1 titrates the CtIP/CtBP
complex and allows the additional copies of BRCA1 to act on the p21 promoter.
The up-regulation of p21 was observed in cells treated with
DNA-damaging agents (37). Similarly, treatment with DNA-damaging agents
disrupts the interaction between BRCA1 and CtIP/CtBP, thus, allowing
BRCA1 to transactivate p21 promoter. Dissociation of CtIP and BRCA1 is
correlated with the hyperphosphorylation of BRCA1 upon treatment with
DNA-damaging agents. These results suggest that CtIP/CtBP may
negatively regulate the transactivation activity of BRCA1 on the p21
promoter. However, whether BRCA1 transactivates p21 promoter directly
or binds to additional transcription factors remains to be explored.
 |
ACKNOWLEDGEMENTS |
We thank K. Somasundaram and W. El-Deiry for
the p21 promoter constructs, R. Baer for pM2, pVP-flag5, pG5E-luc, and
pSV40-
-gal vectors, A. Farmer for the GST-CtIP construct, Paula
Garza for antibody preparations, and S. Post and S. Van Komen for help
with the BRCT constructs.
 |
FOOTNOTES |
*
This work was supported by Grants from the NCI (to W. H. L.) (CA58183 and CA30195) and from the Susan G. Komen
Foundation for Breast Cancer Research (to P. L. C.) (9733).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. E-mail: leew{at}uthscsa.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
BRCT, BRCA1 C
terminus;
CtBP, C-terminal-binding protein;
CtIP, CtBP interacting
protein;
GST, glutathione S-transferase.
 |
REFERENCES |
-
Gayther, S. A.,
Warren, W.,
Mazoyer, S.,
Russell, P. A.,
Harrington, P. A.,
Chiano, M. S.,
Hamoudi, R.,
van Rensburg, E. J.,
Dunning, A. M.,
Love, R.,
Evans, G.,
Easton, D.,
Clayton, D.,
Stratton, M. R.,
and Ponder, B. A. J.
(1995)
Nat. Genet.
11,
428-433[Medline]
[Order article via Infotrieve]
-
Easton, D. F.,
Ford, D.,
and Bishop, D. T.
(1995)
Am. J. Human Genetics
56,
265-271[Medline]
[Order article via Infotrieve]
-
Bertwistle, D.,
and Ashworth, A.
(1998)
Curr. Opin Genet. Dev.
8 (1),
14-20[CrossRef][Medline]
[Order article via Infotrieve]
-
Liu, C. Y.,
Flesken-Nikitin, A.,
Li, S.,
Zeng, Y. Y.,
and Lee, W. H.
(1996)
Genes Dev.
10,
1835-1843[Abstract]
-
Gowen, L. C.,
Johnson, B. L.,
Latour, A. M.,
Sulik, K. K.,
and Koller, B. H.
(1996)
Nat. Genet.
12,
191-194[Medline]
[Order article via Infotrieve]
-
Hakem, R.,
de la Pompa, J. L.,
Sirard, C.,
Mo, R.,
Woo, M.,
Hakem, A.,
Wakeham, A.,
Potter, J.,
Reitmair, A.,
Billia, F.,
Firpo, E.,
Hui, C. C.,
Roberts, J.,
Rossant, J.,
and Mak, T. W.
(1996)
Cell
85,
1009-1023[Medline]
[Order article via Infotrieve]
-
Gowen, L. C.,
Avrutskaya, A. V.,
Latour, A. M.,
Koller, B. H.,
and Leadon, S. A.
(1998)
Science
281,
1009-1012[Abstract/Free Full Text]
-
Hakem, R.,
de la Pompa, J. L.,
Elia, A.,
Potter, J.,
and Mak, T. W.
(1997)
Nat. Genet.
16,
298-302[Medline]
[Order article via Infotrieve]
-
Ludwig, T.,
Chapman, D. L.,
Papaioannou, V. E.,
and Efstratiadis, A.
(1997)
Genes Dev.
11,
1226-1241[Abstract]
-
Somasundaram, K.,
Zhang, H. B.,
Zeng, Y. X.,
Houvras, Y.,
Peng, Y.,
Zhang, H. X.,
Wu, G. S.,
Licht, J. D.,
Weber, B. L.,
and El-Deiry, W. S.
(1997)
Nature
389,
187-190[CrossRef][Medline]
[Order article via Infotrieve]
-
Monteiro, A. N. A.,
August, A.,
and Hanafusa, H.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
13595-13599[Abstract/Free Full Text]
-
Ouchi, T.,
Monteiro, A. N. A.,
August, A.,
Aaronson, S. A.,
and Hanafusa, H.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
2302-2306[Abstract/Free Full Text]
-
Chapman, M. S.,
and Verma, I. M.
(1996)
Nature
382,
678-679[CrossRef][Medline]
[Order article via Infotrieve]
-
Callebaut, I.,
and Mornon, J. P.
(1997)
FEBS Lett.
400,
25-30[CrossRef][Medline]
[Order article via Infotrieve]
-
Bork, P.,
Hofmann, K.,
Bucher, P.,
Neuwald, A. F.,
Altschul, S. F.,
and Koonin, E. V.
(1997)
FASEB J.
11,
68-76[Abstract/Free Full Text]
-
Koonin, E. V.,
Altschul, S. F.,
and Bork, P.
(1996)
Nat. Genet.
13,
266-267[Medline]
[Order article via Infotrieve]
-
Critchlow, S. E.,
Bowater, R. P.,
and Jackson, S. P.
(1997)
Curr. Biol.
7,
588-598[Medline]
[Order article via Infotrieve]
-
Nash, R. A.,
Caldecott, K. W.,
Barnes, D. E.,
and Lindahl, T.
(1997)
Biochemistry
36,
5207-5211[CrossRef][Medline]
[Order article via Infotrieve]
-
Chen, C. F.,
Li, S.,
Chen, Y.,
Chen, P. L.,
Sharp, Z. D.,
and Lee, W. H.
(1996)
J. Biol. Chem.
271,
32863-32868[Abstract/Free Full Text]
-
Wu, L. J. C.,
Wang, Z. W.,
Tsan, J. T.,
Spillman, M. A.,
Phung, A.,
Xu, X. L.,
Yang, M. C. W.,
Hwang, L. Y.,
Bowcock, A. M.,
and Baer, R.
(1996)
Nat. Genet.
14,
430-440[Medline]
[Order article via Infotrieve]
-
Schaeper, U.,
Boyd, J. M.,
Verma, S.,
Uhlmann, E.,
Subramanian, T.,
and Chinnadurai, G.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92 (23),
10467-10471[Abstract]
-
Schaeper, U.,
Subramanian, T.,
Lim, L.,
Boyd, J. M.,
and Chinnadurai, G.
(1998)
J. Biol. Chem.
273,
8549-8552[Abstract/Free Full Text]
-
Sadowski, I.,
Ma, J.,
Triezenberg, S.,
and Ptashne, M.
(1988)
Nature
335,
563-564[CrossRef][Medline]
[Order article via Infotrieve]
-
Durfee, T.,
Becherer, K.,
Chen, P.-L.,
Yeh, S.-H.,
Yang, Y.,
Kilburn, A. E.,
Lee, W.-H.,
and Elledge, S. J.
(1993)
Genes Dev.
7,
555-569[Abstract]
-
Li, S.,
Ku, C. Y.,
Farmer, A. A.,
Cong, Y. S.,
Chen, C. F.,
and Lee, W. H.
(1998)
J. Biol. Chem.
273,
6183-6189[Abstract/Free Full Text]
-
el-Deiry, W. S.,
Tokino, T.,
Velculescu, V. E.,
Levy, D. B.,
Parsons, R.,
Trent, J. M.,
Lin, D.,
Mercer, W. E.,
Kinzler, K. W.,
and Vogelstein, B.
(1993)
Cell
75,
817-825[Medline]
[Order article via Infotrieve]
-
Yu, X.,
Wu, L. C.,
Bowcock, A. M.,
Aronheim, A.,
and Baer, R.
(1998)
J. Biol. Chem.
273,
25388-25392[Abstract/Free Full Text]
-
Wong, A. K.,
Ormonde, P. A.,
Pero, R.,
Chen, Y.,
Lian, L.,
Salada, G.,
Berry, S.,
Lawrence, Q.,
Dayananth, P.,
Ha, P.,
Tavtigian, S. V.,
Teng, D. H.,
and Bartel, P. L.
(1998)
Oncogene
17,
2279-2285[CrossRef][Medline]
[Order article via Infotrieve]
-
Tomlinson, G. E.,
Chen, T.-L.,
Stastny, V. A.,
Virmani, A. K.,
Spillman, M. A.,
Tonk, V.,
Blum, J. L.,
Schneider, N. R.,
Wistuba, I. I.,
Shay, J. W.,
Minna, J. D.,
and Gazdar, A. F.
(1998)
Cancer Res.
58,
3237-3242[Abstract]
-
Chen, Y.,
Chen, P. L.,
Riley, D. J.,
Lee, W. H.,
Allred, D. C.,
and Osborne, C. K.
(1996)
Science
272,
125-126
-
Durfee, T.,
Mancini, M. A.,
Jones, D.,
Elledge, S. J.,
and Lee, W.-H.
(1994)
J. Cell Biol.
127,
609-622[Abstract]
-
Chen, Y.,
Farmer, A. A.,
Chen, C.-F.,
Jones, D. C.,
Chen, P.-L.,
and Lee, W.-H.
(1996)
Cancer Res.
56,
3168-3172[Abstract]
-
Scully, R.,
Chen, J. J.,
Ochs, R. L.,
Keegan, K.,
Hoekstra, M.,
Feunteun, J.,
and Livingston, D. M.
(1997)
Cell
90,
425-435[Medline]
[Order article via Infotrieve]
-
Sollerbrant, K.,
Chinnadurai, G.,
and Svensson, C.
(1996)
Nucleic Acids Res.
24,
2578-2584[Abstract/Free Full Text]
-
Nibu, Y.,
Zhang, H.,
and Levine, M.
(1998)
Science
280,
101-104[Abstract/Free Full Text]
-
Poortinga, G.,
Watanabe, M.,
and Parkhurst, S. M.
(1998)
EMBO J.
17,
2067-2078[Abstract/Free Full Text]
-
Elledge, S. J.
(1996)
Science
274,
1664-1672[Abstract/Free Full Text]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.