From the
Department of Medicine, Mount Sinai
School of Medicine, New York, New York 10029 and the
Laboratory of Molecular Oncology and Cell Cycle
Regulation, Howard Hughes Medical Institute, University of Pennsylvania School
of Medicine, Philadelphia, Pennsylvania 19104
Received for publication, March 25, 2003
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ABSTRACT |
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INTRODUCTION |
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A considerable body of data supports a role for BRCA1 in gene transcription. First, it was shown in both yeast and mammalian cells that a GAL4-BRCA1 fusion protein activates transcription of target genes, whereas tumor-derived missense mutants in the C terminus of BRCA1 failed to do so (9). Second, the C-terminal domain of BRCA1 binds to the RNA helicase, a component of the RNA polymerase II holoenzyme, as well as the p300/CBP1 co-activator (1012). In addition, chromatin-modifying proteins, such as the SWI/SNF-related complex, bind to the N terminus of BRCA1 (13). Third, BRCA1 is able to bind transcription factors, such as p53, ATF1, STAT-1, and the Jun family of proteins, to co-activate transcription (1417). BRCA1 also binds to Myc to inhibit transcription (18). Finally, BRCA1 was shown to regulate cell cycle progression by activating the p21waf1/Cip1 promoter in cells that express wild type and mutant p53 (17, 19). BRCA1 also was found to transactivate the expression of p27kip1, a member of the universal cyclin-dependent kinase inhibitor family (20). Additionally, BRCA1 appears to regulate transcriptionally the expression of GADD45, a protein that is involved in G2 arrest. Cells that are null for BRCA1 or are deficient in GADD45 have a G2/M cell cycle checkpoint defect as well as genomic instability (2123). Recently, it was shown that BRCA1 is essential for activating the Chk1 kinase, which represses the expression of cyclin B1 and induces G2/M arrest (24).
In this study, we show that BRCA1 interacts with another transcription
factor, p65/RelA. p65/RelA is a member of the NF-B family of
transcription factors, which consists of five members: p65/RelA, c-Rel, RelB,
p50/p105, and p52/p100. The p105 and p100 proteins are processed into smaller,
active ones
(2527).
All of the members have a conserved N-terminal region of
300 amino acids
termed the Rel homology domain (RHD). The DNA-binding site, dimerization
domain, nuclear localization signal, and I
B-binding site reside within
this domain. Three of the family members (p65/RelA, c-Rel, and RelB) have a
transactivation domain in the C terminus
(25,
26). The NF-
B
transcription factor is found as an inactive homodimer or heterodimer within
the cytoplasm. The most abundant form is the p65/p50 heterodimer. The dimer
associates through the Rel homology domain with an inhibitory molecule,
I
B
, a member of the I
B protein family
(26,
28). The inactive
NF-
B-I
B complex also associates with the catalytic subunit of
cAMP-dependent protein kinase, whose activity is inhibited by its association
with the NF-
B-I
B complex
(29). NF-
B is activated
by exogenous stimuli, such as oxidative (H2O2) or
radiation stress (UV and
-radiation), inflammatory cytokines
(TNF
or IL-1
), T cells activation, and growth factor stimulation
(28,
30,
31). Upon activation,
I
B
is phosphorylated by the I
B kinase complex, leading to
the ubiquitination of I
B
and its subsequent degradation by the
26 S proteosome (32). Once
phosphorylated by cAMP-dependent protein kinase on serine 276, the NF-
B
dimer translocates to the nucleus
(33,
34), binds to
sequence-specific promoter elements, and activates the transcription of target
genes that are involved in immune and inflammatory reactions, anti- and
pro-apoptotic processes, and cell cycle regulation
(35).
In the present study, we show by both in vitro and in
vivo assays that the p65/RelA interacts through the Rel homology domain
with BRCA1. The association between these two molecules enhances the
transcriptional activation of NF-B target genes, such as Fas
and interferon
(IFN
). This process is dependent upon the
phosphorylation of p65/RelA at serine 276 and the presence of
NF-
B-binding sites in the reporter gene. The transcriptional effect can
be diminished by a super-stable inhibitor I
B
N, a chemical
inhibitor of NF-
B, BARD1, and a BRCA1 RING domain mutant.
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MATERIALS AND METHODS |
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Luciferase AssayHuman 293T cells and MCF-7 cells were grown
at 37 °C and 5% CO2 in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin
(Invitrogen). The cells were seeded at a density of 3 x 105
cells/well in a 12-well dish 1 day prior to transfection and transfected by
LipofectAMINE Plus (Qiagen, Valencia, CA) according to manufacturer's
recommendation. Transfections were performed with a total amount of 1
µg/well DNA, consisting of 30100 ng of reporter, 5 ng of
tk-Renilla (Promega, Madison, WI), 50100 ng of pcr-3
BRCA1 or on a molar basis 2550 ng of empty vector and various
amounts of pBluescript (Stratagene, La Jolla, CA). 10 ng/ml TNF
(Calbiochem) or 2.5 ng/ml IL-1
(Invitrogen) were added for 12 h prior to
lysis. Cells were harvested 48 h post-transfection by detergent-mediated lysis
(Promega Dual luciferase lysis buffer), and 20 µl of cell lysate were
assayed for luciferase activity (Dual Luciferase, Promega, Madison, WI).
GST Protein Affinity AssayGlutathione
S-transferase (GST) fusion proteins were expressed in Escherichia
coli strain BL-21. Expression was induced with 0.4 mM
isopropyl-1-thio--D-galactopyranoside for 4 h, 37 °C. The
bacteria were lysed in phosphate-buffered saline, containing 3% Triton X-100
and protease inhibitors (Roche Applied Science), and sonicated. GST fusion
proteins were isolated from the supernatant by incubation (15 h, 4 °C)
with glutathione-agarose beads (Amersham Biosciences). The beads were
collected by centrifugation and extensively washed in lysis buffer. To
quantify the amount of protein bound to the agarose, an aliquot of beads was
boiled in 1x SDS loading buffer (62.5 mM Tris, pH 6.9, 10%
glycerol, 2% SDS, 5%
-mercaptoethanol), separated by electrophoresis
through a 10% polyacrylamide gel, Coomassie-stained, and compared with known
quantities of bovine serum albumin.
Equal amounts of GST fusion proteins (1015 µg) immobilized on glutathione-agarose beads were used for each in vitro protein affinity assay. To reduce nonspecific binding, GST fusion proteins were preincubated (30 min, 25 °C) in 3% bovine serum albumin/NETN buffer (20 mM Tris, pH 8.0, 10 mM NaCl, 0.2% Nonidet P-40) supplemented with protease inhibitors. Binding assays were performed with 20 µl of in vitro transcribed/translated (Promega, Madison, WI) [35S]methionine-labeled protein (1 h, 25 °C) in 1 ml of NETN. Beads were collected by centrifugation, washed three times in NETN buffer, and boiled in 1x SDS loading buffer. Bound proteins were resolved by 12% SDS-PAGE. The gel was stained with Coomassie Blue, soaked in AMPLIFY (Amersham Biosciences), dried, and exposed to film (1272 h, 80 °C).
Immunoprecipitation293T cells (3 x 106)
were seeded 1 day prior to transfection in 10-cm2 dishes.
Subconfluent plates were transfected with 520 µg of the indicated
combination of expression vectors by LipofectAMINE Plus. Forty eight hours
post-transfection cells were treated (1 h, 37 °C) with TNF (10
ng/ml) and lysed in 1% Nonidet P-40 lysis buffer (50 mM Tris, pH
8.0, 150 mM NaCl, 1% Nonidet P-40, and protease inhibitors). Cell
lysates were incubated with anti-FLAG antibody (M2; Sigma) or anti-BARD1 (gift
from Dr. R. Baer, Columbia University) for 12 h at 4 °C. Immune complexes
were collected by incubation (1 h, 4 °C) with protein G-agarose (Roche
Applied Science). After extensive washing, immunoprecipitated proteins were
resolved by 8% SDS-PAGE and analyzed by Western blotting with anti-FLAG,
anti-HA (Covance, Richmond, CA), anti-CBP (Santa Cruz Biotechnology, Santa
Cruz, CA), anti-BARD1, or anti-HA horseradish peroxidase antibodies (Santa
Cruz Biotechnology). Membranes were developed with enhanced chemiluminescence
(Amersham Biosciences).
For endogenous immunoprecipitations, cellular extracts were lysed in 1%
Nonidet P-40 lysis buffer and incubated with polyclonal anti-BRCA1 antibodies
(Santa Cruz Biotechnology) or IgG as a control. Western blotting for detection
of endogenous BRCA1 and endogenous p65/RelA was performed with mouse
monoclonal anti-BRCA1 (AB1; Oncogene, Boston) and anti-p65 antibody (F-6;
Santa Cruz Biotechnology), respectively. Detection of IB
in the
cytoplasm after TNF
treatment was performed with an anti-
I
B
antibody (Santa Cruz Biotechnology).
Subcellular FractionationSubcellular fractionation was
described previously (36).
Briefly, cells were rinsed twice in ice-cold phosphate-buffered saline, pH
7.4, and gently resuspended in lysis buffer B (10 mM Tris, pH 8.4,
140 mM NaCl, 1.5 mM MgCl2, 0.5% Nonidet P-40,
1 mM dithiothreitol, and complete protease inhibitors (Roche
Applied Science). The lysate was centrifuged at 1000 x g for 3
min at 4 °C, and the supernatant was saved as the cytoplasmic fraction.
The pellets were resuspended in lysis buffer B, and a one-tenth volume of the
detergent (3.3% (w/v) sodium deoxycholate and 6.6% (v/v) Tween 40) was added
under slow vortexing, and the suspension was incubated on ice for 5 min.
Nuclei were pelleted by centrifugation at 1000 x g for 3 min at
4 °C, and the supernatant (post-nuclear fraction) was saved and added to
the cytoplasmic fraction. Nuclei were rinsed once in lysis buffer B. For
Western blot analysis the nuclear and the cytoplasmic fractions were lysed in
RIPA buffer supplemented with complete protease inhibitor (Roche Applied
Science) on ice. A 50-µg aliquot of each fraction was analyzed. This
protocol yielded intact nuclei as determined by the presence of RNApolII
(Santa Cruz Biotechnology) without significant cytoplasmic contamination as
determined by the absence of -actin (Sigma).
Reverse Transcription and Real Time PCRMCF-7 cells were
infected with an adenovirus containing BRCA1 or GFP (multiplicity of
infection = 30). Forty eight hours after infection cells were treated (6 h, 37
°C) with TNF (10 ng/ml). RNA from treated and untreated cells was
isolated using the Qiagen RNeasy kit (Qiagen, Valencia, CA) and DNase-treated,
and then reverse transcription was performed according to instructions for
Superscript II, RNase H-reverse transcriptase (Invitrogen). Quantitative real
time PCRs were performed in triplicate using 2 µl of cDNA product from the
reverse transcription reaction. The PCR contained 0.5 units of AmpliTaq Gold,
1x Syber buffer, 2 mM MgCl2, 1 mM dNTP
with dUTP (PE Biosystems, Warrington, UK), and 0.4 µM of each
5' and 3' primer. Reactions were performed with an initial hot
start incubation at 95 °C for 10 min, followed by denaturation at 95
°C for 30 s, annealing at various temperatures depending upon the primer
pairs for 30 s, and extension at 72 °C for 30 s. Forty amplification
cycles were performed. The data were collected on an ABI Prism 7700
(PerkinElmer Life Sciences) and analyzed using the Sequence Detector version
1.7 program. The PCR end products were checked on 1.8% agarose gel. The
primers pairs used for amplification of specific NF-
B target genes are
as follows: IFN
5'-CACGACAGCTCTTTCCATGA-3' and
3'-AGCCAGTGCTCGATGAATCT-5'; Fas
5'-GCACAGTCAATGGGGATGAACC-3' and
3'-GCACTTGGTATTCTGGGTCCG-5'; actin
5'-GCCCAGAGCAAGAGAGGTAT-3' and
3'-GGCCATCTCTTGCTCGAAGT-5'.
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RESULTS |
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Because treatment of transfected cells with TNF induces other
transcription factors in addition to NF-
B
(38), subsequent experiments
were performed to determine whether the increase in reporter activity was due
to the specific activation of NF-
B by TNF
. For this purpose, we
used a truncated version (Fas 0.4 kb) of the above-mentioned
Fas promoter that only contains two NF-
B-binding sites and a
mutant form of this short promoter (Fas-mut 1,2), in which both
NF-
B sites were mutated
(36). Cells transfected with
the wild-type promoter showed a 4-fold increase in transcriptional activity in
the presence of TNF
and an almost 8-fold increase in the presence of
BRCA1. The mutant Fas promoter was not stimulated by
addition of TNF
, and co-transfection of BRCA1 did not enhance
the transcriptional activity (Fig.
1B). Next, a deletion mutant of I
B
(I
B
N) was used as a super-stable inhibitor of
NF-
B. This inhibitor lacks two phosphorylation sites that are important
for targeting I
B for degradation, as a result, I
B
N
fails to dissociate from NF-
B and blocks the translocation of
NF-
B to the nucleus after treatment of the cells with TNF
(Fig. 1C, compare
lanes 1 and 2 to lanes 3 and 4). In the
presence of I
B
N, the ability of BRCA1 to augment
transcription in the presence of TNF was reduced by 40%
(Fig. 1D).
I
B
N had no affect on the localization of BRCA1
(Fig. 1C). Similar
results were observed when transfected cells were treated with SN-50
(Calbiochem), a peptide that competes with the binding of NF-
B to the
nuclear membrane and thus blocks NF-
B translocation into the nucleus
(Fig. 1E).
Collectively, these results indicate that the effect of BRCA1 on these
reporter genes is mediated by NF-
B.
Association of BRCA1 with the Rel Homology Domain of
p65/RelATo determine whether BRCA1 binds to the p65/RelA
subunit of NF-B in response to treatment with TNF
, we performed
endogenous co-immunoprecipitation experiments. 293T cells were seeded at low
concentration on 10-cm2 plates and 24 h later treated with
TNF
, whereas control cells were left untreated. Lysates from these
cells were subjected to immunoprecipitation with either rabbit polyclonal
anti-BRCA1 antibody or with IgG as a control. Western blot analysis performed
with a mouse monoclonal anti-BRCA1 antibody revealed that endogenous BRCA1 was
immunoprecipitated from treated and untreated cells by the BRCA1 antibody but
not with the control IgG (Fig.
2). In contrast, the p65/RelA subunit was only
co-immunoprecipitated with the BRCA1 antibody from cells treated with
TNF
(Fig. 2). The
endogenous expression of BRCA1 and p65/RelA is shown by Western blot analysis
(Fig. 2). The
co-immunoprecipitation of BRCA1 and p65/RelA was also observed in MCF-7 cells
(data not shown). As a marker for the translocation of p65/RelA into the
nucleus, the reduction of I
B
levels was demonstrated after
treatment with TNF
(Fig.
2).
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The p65/RelA subunit of NF-B consists of two main regions, the RHD
located at the N terminus (aa 1332) and the transactivation domain
located at the C terminus of the molecule (aa 299550)
(Fig. 3A). To identify
which region of p65/RelA interacts with BRCA1, full-length p65/RelA and each
individual domain of the molecule were HA-tagged
(Fig. 3A) and
expressed in 293T cells in the absence or presence of FLAG-BRCA1. The cells
were treated with TNF
, and lysates were immunoprecipitated with a FLAG
antibody, and precipitated proteins were immunoblotted with FLAG antibody to
detect BRCA1 and an HA-antibody to detect p65. As shown, full-length p65/RelA
and RHD were co-immunoprecipitated with BRCA1 in TNF
-treated cells
(Fig. 3B, lanes
4 and 6). In contrast, the fragment containing only amino acids
299550 of p65/RelA failed to associate with BRCA1
(Fig. 3B, lane
8), even though this protein was expressed in the cells
(Fig. 3C, lanes
7 and 8). Taken together, these results indicate that
NF-
B interacts through the Rel homology domain with BRCA1.
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Identification of the p65-binding Region within the BRCA1 MoleculeTo determine which region of the BRCA1 molecule binds to p65/RelA, six fragments of BRCA1 (1304, 260553, 502802, 7581064, 10051313, and 13141863) were expressed as glutathione S-transferase (GST) fusion proteins and used for in vitro protein affinity assays with three fragments of p65/RelA metabolically labeled with [35S]methionine (Fig. 4A). Equivalent levels of GST fusion proteins were used in each protein affinity assay (data not shown). The GST-BRCA1 fragments 1304, 260553, and 502802 bound to the full-length p65/RelA protein as well as to the 1332 p65/RelA fragment. p65/RelA bound most strongly to GST-BRCA1 fragment 260553 (Fig. 4A). A very weak binding was observed with the GST-BRCA1-(13141863) fragment. In accordance with our earlier results, the 299550 p65/RelA did not bind to any of the BRCA1 fragments (Fig. 4A, bottom panel). The inverse experiment was also performed. Specifically, the 1332 p65/RelA fragment was expressed as a GST fusion protein and used in an in vitro protein affinity assay with four fragments of BRCA1 (1802, 1260, 261553, and 554802) metabolically labeled with [35S]methionine. All the BRCA1 fragments bound to GST-(1332)-p65/RelA but not to GST alone (Fig. 4B). These data demonstrate that a broad, N-terminal region of BRCA1 is important for binding to the Rel homology domain of p65/RelA.
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The RING finger domain, located in the N terminus of BRCA1, is important
for protein-protein interactions. It is characterized by the presence of a
conserved C3HC4 sequence, which coordinates the binding
of two zinc atoms. Missense mutations in BRCA1 occur, with varying
frequencies, at four out of seven cysteine residues of the BRCA1 RING domain
(cysteine 29, 34, 61, and 64). Mutation of cysteine 61 to glycine (C61G) was
shown to disrupt the zinc binding and formation of BRCA1 N-terminal homodimers
in vitro (39). In
order to determine whether mutation in the RING finger domain influences the
transcriptional effect of BRCA1 on NF-B target genes, 293T cells were
transfected with two concentrations (100 or 150 ng/ml) of either wild type
BRCA1 or BRCA1 mutated at cysteine 64 or cysteine 61. The
cysteine mutants were 30 and 50% less active than the wild type BRCA1
transfected at the same concentration (Fig.
5A). The wild-type BRCA1 and mutant forms were expressed
at comparable levels at both concentrations
(Fig. 5B).
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BARD1 Inhibits Transcriptional Activation by BRCA1 BARD1 is
a RING finger protein that binds to the N-terminal domain of BRCA1, including
the RING finger and stabilizes the molecule
(40). Because BARD1 and
NF-B both bind the N-terminal portion of BRCA1, we determined whether
BARD1 could affect the functional interaction between BRCA1 and NF-
B.
BARD1 was co-transfected into 293T cells with or without BRCA1. BRCA1
induced a 4-fold increase in reporter activity when cells were treated with
TNF
. However, BARD1 blocked the ability of BRCA1 to augment
transcription by 50% (Fig.
5C), even though the level of BRCA1 was elevated in cells
co-transfected with BARD1 (Fig.
5D). BARD1 had no significant effect on the ability of
TNF
to activate the reporter gene in the absence of BRCA1
(Fig. 5C). A truncated
form of BARD1, which lacks the RING finger domain, did not inhibit the
transcriptional activity of BRCA1 (data not shown). To determine whether BARD1
blocked co-activation by BRCA1 by inhibiting the formation of a BRCA1-p65
complex, HA-p65/RelA and FLAG-BRCA1 were expressed in 293T cells in the
absence or presence of BARD1. In this triple transfection experiment, BARD1
did not decrease the level of expression of BRCA1 in transfected cells
(Fig. 5E, lanes
2 and 6) nor did it alter the subcellular localization of BRCA1
or p65 (data not shown). We consistently noted that p65/RelA expression
decreased expression of BRCA1 in transfected cells, an effect possibly due to
promoter or competition or squelching due to the overexpression of p65/RelA.
Despite this, several important pieces of information could be determined from
the experiment. When p65/RelA alone was transfected into cells, it could not
be detected in BARD1 precipitates (Fig.
5E, lane 3). When cells were transfected with
HA-p65/RelA and BARD1, HA-p65/RelA was detected in the BARD1
immunoprecipitates (Fig.
5E, lane 7). Curiously, a similar level of
HA-p65 was also immunoprecipitated in the presence of FLAG-BRCA1 using BARD1
antibodies. This presumably occurred through endogenous BARD1 present in the
cell (Fig. 5E,
lane 4) and may be due to the mutual stabilization of BRCA1 and
BARD1. Importantly, when BARD1 and BRCA1 were expressed together
along with p65, the amount of p65 found in BARD1 immunoprecipitates was
strongly enhanced. Similar results were obtained when BRCA1 was used to
immunoprecipitate the proteins (data not shown). Therefore, the ability of
BARD1 to inhibit trans-activation of TNF
-mediated
transcription by BRCA1 could not be explained by blocking the formation of the
BRCA1-p65-RelA complex. On the contrary, BARD1 and BRCA1 together more readily
interacted with p65 than alone.
Mutation of Serine 276 on the p65/RelA Subunit Abolishes the
Effect of BRCA1The p65/RelA subunit is phosphorylated by
cAMP-dependent protein kinase at serine 276 in the Rel homology domain. This
phosphorylation event is important for the binding of CBP. In order to
determine whether the phosphorylation of the Rel homology domain is also
important for the enhancement of the transcriptional activity by BRCA1, 293T
cells were co-transfected with p65/RelA, p65/RelA (S276A) mutant, or empty
vector in the presence or absence of BRCA1. In the presence of
TNF and wild-type p65/RelA, there is a 3-fold enhancement in the
transcriptional activity induced by BRCA1. Transfection of the mutated form of
p65/RelA was found to abolish this effect
(Fig. 6A). To verify
if this mutated form also influences the physical interaction between BRCA1
and p65/RelA, we performed a co-immunoprecipitation experiment. The mutant
p65/RelA (S276A) was HA-tagged and expressed in 293T cells in the absence or
presence of FLAG-BRCA1. Lysates from cells treated with TNF
were
immunoprecipitated with anti-FLAG antibody. Precipitated proteins were
analyzed by Western blot with anti-FLAG antibody to detect BRCA1 and an
anti-HA antibody to detect mutated p65/RelA. As shown, mutant p65/RelA was
immunoprecipitated only in the presence of BRCA1
(Fig. 6B, top
panel). This suggests that phosphorylation at serine 276 is not required
for the physical interaction between BRCA1 and p65/RelA but is important for
the enhancement of the transcriptional activity induced by BRCA1.
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It was shown previously
(11) that CBP also binds to
BRCA1. Therefore, we next determined whether co-expression of CBP and BRCA1
would have a greater effect on transcription of NF-B target genes than
either one alone. Whereas BRCA1 stimulated transcription from the reporter
gene by nearly a factor of 3 and CBP stimulated transcription by 2.5-fold, the
combination of BRCA1 plus CBP led to a 10-fold increase in transcriptional
activity (Fig. 6C).
Co-transfection of BRCA1 with a deletion mutant of CBP devoid of the
histone acetyl-transferase domain (CBP
HAT) resulted in significantly
less stimulation of transcription. BRCA1 appeared to stabilize CBP, and in the
presence of BRCA1, CBP and CBP
HAT were expressed at equivalent levels
(Fig. 6D, lanes
4 and 6). In contrast, expression of CBP had no effect on the
level of expression of BRCA1 (Fig.
6D, lanes 2, 4, and 6). We next
wondered whether BRCA1, CBP, and p65/RelA could be found as a complex. For
this purpose, 293T cells were co-transfected with HA-p65 and CBP in the
absence or presence of FLAG-BRCA1. The cells were treated with
TNF
; lysates were immunoprecipitated with FLAG antibody, and
precipitated proteins were immunoblotted with FLAG antibody to detect BRCA1,
HA antibody to detect p65/RelA, and CBP antibody to detect CBP. CBP or
p65/RelA alone could be co-precipitated with BRCA1
(Fig. 6E, lanes
4 and 7, respectively) and also when all three proteins were
expressed, both HA-p65 and CBP could be co-immunoprecipitated with FLAG-BRCA1
(Fig. 6E, lane
8). Immunoprecipitation with a CBP antibody also precipitated both BRCA1
and p65 (data not shown). Together, these results suggest that CBP, by
complexing with the activated and phosphorylated form of NF-
B as well
as with BRCA1, may mediate the transcriptional effects of BRCA1.
BRCA1 Stimulates Expression of Endogenous NF-B Target
GenesBecause BRCA1 enhanced the TNF
-induced activity of
IFN
and Fas reporter genes, we determined whether BRCA1 could
affect expression of endogenous NF-
B target genes. RNA was isolated
from MCF-7 cells infected with an adenovirus expressing BRCA1 or GFP and
treated with TNF
. Quantitative real time PCR analysis revealed that
BRCA1 significantly augmented the expression of IFN
and Fas
mRNA in infected cells above the increase mediated by TNF
alone
(Fig. 7). Importantly, these
results were in agreement with those obtained in reporter experiments and
suggest that the ability of BRCA1 to interact with the NF-
B system has
relevance in vivo.
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DISCUSSION |
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The importance of the N-terminal region of BRCA1 for transcriptional
activity was also highlighted by the ability of BARD1 to inhibit
trans-activation by BRCA1. BARD1 and BRCA1 interact in their N
terminus in a region immediately adjacent to each RING finger
(47). The RING fingers of the
proteins then together form a structure with considerable ubiquitin ligase
activity (40). BARD1
expression, although generally increasing BRCA1 protein accumulation within
the cell, actually inhibited the ability of BRCA1 to activate transcription
through NF-B. The N terminus of BARD1 including the RING finger and
sequences required for heterodimerization with BRCA1 was required for this
effect. BARD1 did not change the subcellular localization of BRCA1 or p65/RelA
and did not inhibit the formation of the BRCA1-p65 complex. On the contrary
BRCA1, BARD1, and p65 readily interacted when co-expressed. This suggests that
the transcriptional function of BRCA1 is altered when complexed by BARD1. This
may reflect the ability of BARD1 to bind with BRCA1 in nuclear dots which
might represent sites of DNA repair rather than transcription
(48). The reduced
transcriptional function of BRCA1 when co-expressed with BARD1 might also be
related to the fact that BARD1 enhances the ubiquitin ligase activity of BRCA1
in vitro (43,
49), potentially directing the
activity of the complex to histones, particularly H2AX, found at sites of DNA
damage. Alternatively, BARD1 might inhibit transcriptional activity of BRCA1
by association with the polymerase II holoenzyme, inhibiting polyadenylation
though interaction with the CstF factor
(50).
NF-B is primarily found in the cytoplasm in a complex with
I
B
and the catalytic subunit of protein kinase A. Upon
activation by the cytokines TNF
or IL-1
, p65/RelA is
phosphorylated on serine 276 by protein kinase A and is translocated to the
nucleus (55,
56). This phosphorylation
facilitates the docking of the transcriptional co-activator CBP, which
enhances the ability of NF-
B to activate transcription
(33,
34). It has been shown that
the KIX domain of p300/CBP interacts with both the N- and the C-terminal
domains of BRCA1 (11). We
demonstrated by co-immunoprecipitation that BRCA1 can complex with both
p65/RelA and CBP and cooperates with these proteins to activate NF-
B
target genes. Mutation of serine 276 to alanine in the p65/RelA subunit
abrogates the ability of BRCA1 to enhance transcription, even though the
interaction between p65 (S273A) and BRCA1 is not disrupted. This reduction in
the transcriptional activity may be due to the inability of CBP to bind to the
unphosphorylated form of p65/RelA. An analogous type of complex interaction
occurs with another BRCA1-binding protein, ATF1. Like p65, the DNA-binding
C-terminal domain of ATF1 is required for interaction with BRCA1. The
N-terminal KID domain of ATF1, which is also phosphorylated by protein kinase
A, mediates an interaction between ATF1 and CBP
(14). The phosphorylated form
of ATF1 is important for its transcriptional activity but is not important for
the physical interaction between BRCA1 and ATF1
(14). This theme is found in
yet another system. Stimulation of cells by another cytokine, IFN
,
leads to serine phosphorylation of STAT-1
and ultimately activation of
IFN
-inducible genes. Phosphorylation at Ser-727 in the C-terminal
region of STAT-1
enhances transcription by recruiting p300/CBP and also
BRCA1. BRCA1 acts in concert with STAT-1 to differentially regulate a subset
of IFN
target genes
(16,
51) involved in growth
control. Similarly, BRCA1 may only activate a subset of NF-
B genes. How
specificity in BRCA1-dependent gene regulation is achieved remains
unclear.
Apart from binding to co-activators, such as CBP/p300 or to transcription
factors, such as ATF1 and p65/RelA, BRCA1 also binds to RNA polymerase II
through its C-terminal domain
(10,
12). Based on these multiple
interactions, we propose a model whereby BRCA1 functions as a scaffold protein
by tethering together the various transcriptional elements
(Fig. 8). In the absence of
BRCA1 (Fig. 8A), CBP
binds to the p65/RelA subunit and to the TATA box-binding protein and TFIIB,
which binds to RNA polymerase II
(5254)
and co-activates the transcription of NF-B target genes. Addition of
BRCA1, which interacts physically with p65/RelA, CBP, and RNA polymerase II as
demonstrated in Fig.
8A, enhances the transcriptional activation of
NF-
B target genes (Fig.
8B).
|
Both BRCA1 and NF-B have been reported to be involved in
pro-apoptotic functions in various cell types. We show BRCA1 significantly
increases the transcriptional activity of NF-
B target promoters, such
as those of IgK, IFN
, and Fas, above the activity observed by
stimulation by TNF
alone. This activity is inhibited by a super-stable
inhibitor of NF-
B, as well as by a chemical inhibitor of NF-
B.
The mutated form of the Fas gene could not be activated by BRCA1,
demonstrating that BRCA1 is exerting its effect through NF-
B. The
potential physiological relevance of this finding was heightened by our result
showing that overexpression of BRCA1 increased expression of the endogenous
FAS and IFN
genes above the levels seen with TNF
treatment alone. The FAS receptor along with FASL activate a pathway leading
to apoptosis. IFN
is a cytokine that can promote apoptosis in response
to viral infection (51).
Hence, through the induction of NF-
B targets genes BRCA1 might induce
an antiviral and anti-proliferative state in the cell.
In multiple systems, NF-B has been implicated in the apoptotic
response. In T cells, topoisomerase poisons and UV irradiation activate the
stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) pathway,
which leads to the induction of the NF-
B transcription factor.
NF-
B activates FASL, which in turn binds to its receptor, FAS/CD95,
thus resulting in apoptosis
(55,
56). In addition, in neuronal
cells glutamate induces cell death by activating the NF-
B transcription
factor (57). Overexpression of
a dominant-negative p65/RelA protein inhibits apoptosis induced by serum
starvation in HEK 293 cells
(58). Moreover, incubation of
osteoblastic mc3T3-E1 cells with TNF
induces apoptosis and stimulates
nuclear translocation of NF-
B. Treatment with an anti-TNF
antibody prevents apoptosis and decreases NF-
B activation, indicating
that activation of NF-
B through TNF
may be an important
apoptotic signaling pathway in osteoblasts
(59,
60).
BRCA1 has also been implicated in the SAPK/JNK pathway leading to apoptosis
(21). Thangaraju et
al. (37) reported that
overexpression of BRCA1 in breast and ovarian cancer cell lines induces
expression of FAS and FASL, ultimately leading to apoptosis after serum
withdrawal through the activation of the SAPK/JNK pathway. Because both
NF-B and BRCA1 were shown to activate the expression of FAS through the
JNK pathway, it is possible that BRCA1 is functioning as a transcriptional
co-activator with NF-
B in this system.
NF-B has also been linked to the prevention of apoptosis.
NF-
B is often constitutively active in breast cancer cells lines and
diverse solid tumor-derived cell lines
(6163).
NF-
B can induce expression of anti-apoptotic genes, such as inhibitor
of apoptosis protein (IAP) family and superoxide dismutase, and genes that
contribute to proliferation, such as c-MYC and
BCL-XL
(64). Therefore, persistent
activation of NF-
B could provide a mechanism that protects cancer cells
from apoptosis. In breast cancers where the tumor suppressor BRCA1 is mutated,
the balance between pro-apoptotic and anti-apoptotic pathways may be shifted
toward the latter. Future studies will be aimed at determining whether BRCA1
acts as a co-activator of only pro-apoptotic genes or as a general activator
of all NF-
B target genes.
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FOOTNOTES |
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¶ To whom correspondence should be addressed: Box 1130, Mount Sinai School of Medicine, New York, NY 10029. Tel.: 212-659-5487; Fax: 212-849-2523; E-mail: jonathan.licht{at}mssm.edu.
1 The abbreviations used are: CBP, CREB-binding protein (CREB, cAMP-response
element-binding protein); RHD, Rel homology domain; TNF, tumor necrosis
factor-
; GST, glutathione S-transferase; STAT, signal
transducers and activators of transcription; aa, amino acids; HA,
hemagglutinin; IL, interleukin; GFP, green fluorescent protein; JNK, c-Jun
N-terminal kinase; SAPK, stress-activated protein kinase; IFN, interferon.
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ACKNOWLEDGMENTS |
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REFERENCES |
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