From the Department of Molecular Genetics and The
Comprehensive Cancer Center, Ohio State University,
Columbus, Ohio 43210
Received for publication, September 16, 2002, and in revised form, February 3, 2003
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ABSTRACT |
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Ets-2 is a transcriptional activator that can be
modulated by ras-dependent phosphorylation.
Evidence is presented indicating that ets-2 can also act as a
transcriptional repressor. In the breast cancer cell line MCF-7,
exogenous ets-2 repressed the activity of a BRCA1
promoter-luciferase reporter dependent on a conserved ets-2-binding site in this promoter. Conditional overproduction of
ets-2 in MCF-7 cells resulted in repression of endogenous
BRCA1 mRNA expression. To address the mechanism by
which ets-2 could act as a repressor, a biochemical approach was used
to identify proteins that interacted with the ets-2 pointed domain.
From this analysis, components of the mammalian SWI/SNF chromatin
remodeling complex were found to interact with ets-2. Brg-1, the
ATP-hydrolyzing component of the SWI/SNF complex, along with the
BAF57/p50 and Ini1 subunits could be co-immunoprecipitated from cells
with ets-2. The pointed domain of ets-2 directly interacted in
vitro with the C-terminal region of Brg-1 in a
phosphorylation-dependent manner. The combination of
Brg-1 and ets-2 could repress the
BRCA1 promoter reporter in transfection assays. These
results support a role for ets-2 as a repressor and indicate that
components of the mammalian SNF/SWI complex are required as
co-repressors.
The ETS family, encompassing approximately 30 vertebrate members,
encodes for sequence-specific DNA-binding proteins that are
transcriptional activators and repressors (1). The family is defined by
a highly conserved DNA binding domain referred to as the ETS domain.
However, the DNA-binding properties of these factors are similar and
cannot entirely account for the specificity required for the precise
activation of target genes that occurs during the diverse biological
processes mediated by individual family members. Modification of
discrete family members by signal transduction pathways provides an
additional mechanism to determine specificity (1).
For example, members of the ETS family of transcription factors are
important for mediating both transient and persistent changes in gene
expression patterns in response to ras-signaling pathways (2-4). The
ETS family member elk-1 and related factors are directly phosphorylated
by mitogen-activated protein kinases (MAPK),1 a modification
required for the activation of immediate early target genes like
c-fos (2). Similarly, phosphorylation of the ETS family
members ets-1 and ets-2 by ras-dependent
pathways leads to persistent expression of target genes including
extracellular proteases such as urokinase plasminogen activator
(uPA) and stromelysin/MMP-3 (3, 4). Ets-1 and ets-2 are phosphorylated
at a conserved residue (threonine 38 and threonine 72, respectively) by
the well characterized ras-effector pathway, the Raf/MAPK
pathway (4-7). Additionally, the same residue in ets-2 can also be
phosphorylated by another major ras-effector pathway, the
phosphatidylinositol 3-kinase/Akt pathway (8).
Understanding at the molecular level how phosphorylation modifies the
activity of ets-1 and ets-2 will be critical for defining how these
factors selectively regulate target genes. The key phosphorylation event occurs within a region of ets-1 and ets-2 that is conserved through evolution with the Drosophila pointed P2 protein,
and has been termed the pointed domain (9, 10). Phosphorylation of the
conserved threonine residue within this region leads to an increased
ability of ets-1 and ets-2 to activate target promoters (3-7).
Previous work indicated that phosphorylation of ets-1 or ets-2 did not
affect protein turnover, nuclear localization, or intrinsic DNA binding
activity of the factors (3, 4, 7). In addition, the N-terminal region
of ets-2 fused to the heterologous gal4 DNA binding domain is still
regulated by the ras/Erk pathway (11). The pointed domain appears
similar to domains in other transcription factors, for example in the
cAMP responsive enhancer-binding protein, that are regulated by
phosphorylation-dependent protein-protein interactions with
transcription co-activators (12). Ets factors can also act as
repressors of gene expression, and the pointed homology domain has been
implicated in this activity in some family members (1). Thus, whereas
ets-1 and ets-2 have been considered to be activators of gene
expression, it is possible that they also repress target gene expression.
Genetic and biochemical evidence demonstrate that the products of the
SWI/SNF genes, first defined in Saccharomyces cerevisiae as
co-activators of gene expression, form a complex with the ability to
remodel chromatin (13). The SWI/SNF complex is conserved in mammals and
can act as a co-activator for steroid hormone receptors (13-16). The
complex has ATP-dependent chromatin remodeling activity and
can alter the conformation of the nucleosome core in a reversible fashion (13, 14). The mammalian ATPase hydrolyzing subunit Brg-1
interacts with the retinoblastoma tumor suppressor protein (Rb) (17).
The Brg-1 remodeling complex is required for E2F/Rb-mediated repression
of gene expression (18, 19). Recently, experiments performed in
S. cerevisiae have definitively established the ability of
the ATPase-hydrolyzing subunit of the complex (SNF2) to act as a
co-repressor of gene expression (20). Therefore, SWI/SNF appears to act
as both co-activator and co-repressor (20).
In the present report evidence is presented indicating that ets-2 can
repress the BRCA1 promoter, a promoter previously reported to contain functional ets-binding sites (21-24), in the breast cancer
cell line MCF-7. Ets-2 can bind to the BRCA1 proximal
promoter, and ets-2 co-expression results in repression of a
BRCA1 promoter-luciferase in transient transfection assays.
Conditional overexpression of ets-2 in MCF-7 cells resulted in
repression of the endogenous BRCA1 promoter, whereas
expression of other targets, uPA and MMP3, were stimulated by ets-2
overexpression. Biochemical evidence is presented indicating that ets-2
physically interacted with discrete sets of cellular proteins dependent
on the phosphorylation status of the factor. We show that components of
the mammalian SWI/SNF (mSWI/SNF) chromatin remodeling complex comprised
one set of proteins that interacted with unphosphorylated ets-2. The pointed homology region of ets-2 directly interacted with the C-terminal region of Brg-1, the ATP-hydrolyzing component of the mammalian complex, in a phosphorylation-dependent manner
in vitro. Co-expression of Brg-1 with ets-2 resulted in
repression of a stably integrated BRCA1 reporter gene in
SW13 cells, a Brg-1 null cell line. These results
demonstrate that ets-2 can act as a repressor and that a chromatin
remodeling complex is necessary for this activity.
Cell Lines--
To generate the tetracycline-controlled
inducible ets-2 MCF-7 cells, an influenza virus hemagglutinin
(HA)-epitope tagged version of wild-type ets-2 (3) was cloned into the
tetracycline operator-expression vector, pUHD10-3 (25), to generate
pUHD10-3/HA-ets-2. The MCF-7/Tet-off cell line
(Clontech) was transfected with
pUHD10-3/HA-ets-2 to establish stable expression cell lines
that can be induced to express HA-ets-2. Transfections were done using
LipofectAMINE Plus (Invitrogen, Carlsbad, CA) as described previously
(26). The tetracycline-controlled inducible ets-2 MCF-7 cells were
grown in Dulbecco's modified Eagle's medium (DMEM) without phenol red with 10% charcoal-treated fetal bovine serum and 1 µg/ml
tetracycline (Sigma). To induce ets-2 expression, adherent cells were
washed 3 times with phosphate-buffered saline and then re-fed with 10% charcoal-treated fetal bovine serum/DMEM without tetracycline.
For generating stable cell lines expressing ets-2 Ala-72
protein, NIH 3T3 cells were transfected with the HA-tagged version of
ets-2 Ala-72 by the calcium phosphate method as described (3), and
hygromycin-resistant clones were selected in 200 units/ml hygromycin
(Calbiochem, Inc.) and analyzed by Western blotting. Cells were grown
in DMEM with 10% calf serum containing 100 units/ml hygromycin.
For metabolic labeling of cellular proteins with
[35S]methionine, cells were placed in DMEM that lacked
both serum and methionine for 45 min, then incubated with DMEM
containing 90 µCi/ml [35S]methionine (1200 Ci/mmol;
ICN, Irvine, CA) for 2 h.
Human carcinoma SW13 cells were obtained from American Type Culture
Collection (Bethesda, MD) and cultured in DMEM plus 5% bovine calf
serum. SW13 cells that contained stably integrated BRCA1
promoter-luciferase reporter were generated by the co-transfection of 3 µg of BRCA1-luciferase plasmid and 1 µg of a
neomycin-resistance vector using LipofectAMINE Plus (Invitrogen).
48 h after transfection, cells were selected with G418 (400 µg/ml, Invitrogen) and media was changed every 3 days. 14 days after
selection, there were about 100 colonies on the dish. The cells were
pooled and cultured with 200 µg/ml G418 and used for subsequent experiments.
Plasmids, DNA Transfections, and Northern Blots--
The
vector used for expression of the HA-tagged versions of ets-2 and ets-2
Ala-72 proteins were previously described (3, 5). The expression
vectors for Brg-1 and Brg-1 K798R were previously described (16). The
luciferase reporters for the human BRCA1 (21), and murine
MMP14/MT1-MMP (27) promoters were kindly provided by Ellen Soloman
(Guy's Hospital, London) and Joseph Madri (Yale University),
respectively. The BRCA1 promoter with the ets-site
double-point mutation CGTAAGAGT was constructed
by site-directed PCR mutagenesis as previously described (3). The
mutation was verified by sequencing.
Transient transfections in MCF7 or SW13 cells were performed by the
calcium phosphate method as described (3, 16). For the transient assays
either a Rous sarcoma virus-
The SW13 cells containing the stably integrated BRCA1
promoter reporter were transfected using LipofectAMINE Plus
(Invitrogen). Brg-1 and ets-2 expression plasmids
(see Fig. 7B) were cotransfected with 0.5 µg of a
puromycin-resistant expression vector, pBABE-puromycin (6).
36 h after transfection, cells were selected with 4 µg/ml puromycin and subsequently harvested 36 h after the initial
selection. Luciferase activity was adjusted by cell lysate protein concentration.
RNA was isolated and analyzed by Northern blotting as previously
described (5). Levels of RNA expression were quantified using a
Amersham Biosciences PhosphorImager.
Immune Reagents and Analysis--
The antibodies specific for
ets-2 and phosphorylated ets-2 (pT72-ets-2) have been previously
described (6). The anti-HA antibody was purchased from Babco, Inc.
(Richmond, CA). Polyclonal anti-rabbit antibodies specific for Brg-1
and Brm-1 were gifts from Said Sif (Ohio State University), BAF57/p50
from Gerry Crabtree (Stanford University), and Ini1 from Anthony
Imbalzano (University of Massachusetts).
For standard immunoprecipitations, 2.5-3 × 106 cells
were placed in 500 µl of lysis buffer consisting of 50 mM
Tris-Cl, pH 7.4, 150 mM NaCl, 3 mM
MgCl2, and 1% Nonidet P-40. For more stringent analysis,
the same buffer was used except that it contained 500 mM
NaCl and 0.5% deoxycholate in addition to 1% Nonidet P-40. The buffer
contained a mixture of protease and phosphatase inhibitors that was
previously described (5, 6). The pre-cleared cell lysates were
incubated with specific antibody (2-4 µg) and 20 µl of a 50%
slurry of protein-G beads (Amersham Biosciences) overnight at
4 °C. The immunoprecipitates were washed 4 times in lysis buffer and
analyzed by SDS-PAGE.
Affinity Chromatography and Protein Interaction
Assays--
Recombinant ets-2 protein corresponding to the pointed
homology region (amino acids 60-167) was prepared as described (5, 6)
and covalently linked to Affi-Gel 10 beads (10 mg of ets-2/ml of beads;
Bio-Rad). The ets-2 column was phosphorylated in a reaction that
contained 10 µl of ets-2 affinity column, 30 µl of kinase buffer
(30 mM Hepes, pH 7.2, 20 mM MgCl2,
2 mM dithiothreitol), 100 µM ATP, 10 µCi of
[
For direct protein interaction assays, a portion of Brg-1 corresponding
to amino acids 1108-1686 at the C-terminal of the protein (17) were
expressed as a GST fusion protein in Escherichia coli, and 1 µg of fusion protein was immobilized using glutathione beads
(Amersham Biosciences). The beads were incubated with 1 µg of the
recombinant ets-2 pointed domain that was unphosphorylated, or
phosphorylated by recombinant MAPK p42 in a reaction that contained 10 µCi of [
Western analysis was performed as previously described (5, 6). A
Lumi-Imager (Roche Diagnostics) was used for quantification of
chemiluminescent signals. For estimation of ets-2 concentration, standard curves for both ets-2 antibodies were prepared using recombinant protein corresponding to the ets-2 pointed domain that was
phosphorylated with MAPK p42 as described above.
Electrophoretic Mobility Shift Assays--
Recombinant ets-2
corresponding to the DNA binding domain (amino acids 334-466) was
produced using the pGEX expression vector system and purified by
glutathione-Sepharose chromatography (Amersham Biosciences) as
previously described (28). Purified protein was used in the
electrophoretic mobility shift assays using standard conditions as
previously described (28). Double stranded oligonucleotides were
end-labeled with polynucleotide kinase. The sequences of the
oligonucleotides used were (sense strand) (BRCA1 oligos correspond to
Expression of ets-2 Correlates with Expression of ets-2 Target
Genes in Breast Cancer Cells--
Previous work demonstrated that
expression of phosphorylated ets-2 correlates with a more invasive,
mesenchymal phenotype in both ovarian and prostate cancer tumor cell
lines (29, 30). To determine whether a similar correlation was detected
in breast cancer cell lines, we studied expression of ets-2 and est-2
target genes in MCF-7 cells, an estrogen receptor-positive non-invasive cell line, and in MDA231 cells, representative of invasive breast tumor
cell lines (Fig. 1, A and
B).
Western analysis of these two cell lines indicated that ets-2 was
expressed at ~10-fold higher levels in MDA231 cells than in MCF-7
(Fig. 1A). Furthermore, the pT72 phosphorylated, activated form of ets-2 could be detected in MDA231 cells but not MCF-7 cells, as
assayed using a pT72 ets-2 phospho-specific antibody (5). Similar
results were observed if ets-2 were first immunoprecipitated from these
two cell lines using the non-phosphodiscriminating antibody, then
analyzed by Western analysis using the same antibody or the pT72 ets-2
antibody (Fig. 1B, lanes 3 and 4,
respectively). From these Western blots, we estimated that ~40-50%
of ets-2 was phosphorylated at position Thr-72 in MDA231 (see
"Experimental Procedures").
Correlating with phospho-ets-2 expression, the mRNA expression
levels for three known ets-2 target genes, uPA (3, 28), MMP3/stromelysin (31), and bcl-X (8, 32), were all higher levels in
MDA231 cells versus MCF-7 cells (Fig. 1C).
Additionally, the expression of another metalloprotease implicated in
mammary tumorigenesis MMP14/MMP-MT1 (27, 33, 34) was higher in MDA231 cells than in MCF-7. MMP14 expression is reported to be increased by
ras signaling pathways (35), and the proximal promoter for the mouse
and human genes contain a consensus ets-like site that is related to
sites found in other ets-regulated genes (Fig. 1D). Interestingly, the expression of ets-2 mRNA is identical
in MCF-7 and MDA231 cells, indicating that differences in ets protein
expression between the two cell lines (see Fig. 1, A and
B) are because of a post-transcriptional regulatory mechanism.
In contrast to the known ets-2 target genes, the expression of tumor
suppressor BRCA1 was ~5-fold lower in MDA231 in comparison to MCF-7 (Fig. 1C). The BRCA1 Ets-2 Binds to the BRCA1 Promoter and Represses BRCA1 Promoter
Activity in MCF-7 Cells--
To test the hypothesis that ets-2 might
repress BRCA1 expression, we first performed electrophoretic
mobility shift assay analysis to determine whether ets-2 could bind to
the ets-consensus site in the BRCA1 promoter (Fig.
2A). Recombinant ets-2 bound to the BRCA1 oligonucleotides containing the wild-type
ets-binding site (ACGGAAGAGG, wild-type). However, 10-fold lower levels
of ets-2·DNA complex was formed when the consensus site was mutated by one base outside of the GGA core (ACGGAAGAGT, M2).
Mutation of the G residue in the ets core motif (ACGTAGAGG,
M1), or a mutation of both G residues
(ACGTAAGAGT, M1 + M2) ablated ets-2 recognition of the BRCA1 sequence (Fig. 2A). These mutated
oligonucleotides also failed to compete effectively for binding to
wild-type sequence in competition assays (data not shown).
To determine whether ets-2 binding to the BRCA1 promoter had
functional significance, transient transfections were performed in
MCF-7 using BRCA1 and MMP14 promoter-luciferase reporters. The BRCA1 promoter was highly active in MCF-7 cells, but
co-transfection of an ets-2 expression vector resulted in a
concentration-dependent repression of BRCA1
promoter activity, with a maximum repression of ~10-fold observed
(Fig. 2B). In contrast, MMP14 promoter activity was low in
MCF-7 cells, and ets-2 co-expression stimulated MMP14 promoter activity
by 12-fold in MCF-7 cells (Fig. 2B). When a uPA promoter
reporter was tested, results were similar to those observed for the
MMP14 promoter (data not shown).
To demonstrate whether ets-2 repression of BRCA1 promoter
activity required a functional ets-binding site, a BRCA1
promoter luciferase reporter with either a wild-type ets-binding site
(ACGGAAGAGG) or a mutated ets-binding site
(ACGTAAGAGT, M1 + M2) were studied by transient transfection in MCF-7 cells. Titration of ets-2 expression vector resulted in a concentration-dependent repression of
the wild-type BRCA1 promoter with a maximum repression of
10-fold observed, as above. However, ets-2 was not able to repress
activity of the mutated BRCA1 promoter, indicating that the
ets-binding site is necessary for ets-2-mediated repression (Fig.
2C).
Previous work has demonstrated that the ets-2 transactivation potential
is enhanced by phosphorylation at position Thr-72 (3, 4, 7). To
determine the role of residue Thr-72 in ets-2 activity in the transient
assays in MCF-7 cells, we studied the effect of co-transfection of a
vector encoding the ets-2 T72A mutation with both MMP14 and
BRCA1 reporters (Fig. 2D). In this analysis,
MMP14 promoter activity was activated ~2-fold in the assays, and this
activation was not dose dependent, results that are consistent with
what was observed with ets-2 T72A in NIH 3T3 fibroblasts
(3). In contrast, ets-2 T72A repressed the activity of the
BRCA1 promoter in the same way as ets-2 Thr-72 (Fig.
2D). These results indicate that an intact Thr-72 site is
required for transactivation of MMP14 and similar targets, but not for repression of BRCA1.
Ets-2 Overexpression in MCF-7 Cells Inhibits BRCA1 Expression and
Increases uPA and MMP3 Expression--
To test whether expression of
exogenous ets-2 would affect the expression of the endogenous
BRCA1 gene in MCF-7 cells, we constructed MCF-7 cell lines
that conditionally express an HA epitope-tagged ets-2. Conditional
expression of ets-2 was accomplished by putting the gene
under the control of the tetracycline operator and tetracycline-VP16
activator, such that ets-2 was expressed when tetracycline was removed
from the cell culture media (26). The results with one cloned cell
line, MCF-7 clone 21B, are presented in Fig.
3.
After growing cells in the absence of tetracycline for 0, 1, 8, and
24 h, cell lysates were subjected to Western analysis using the
anti-HA epitope tag antibody. HA-ets-2 could be detected 1 h after
stimulation, and expression levels were maximal following 24 h of
induction (Fig. 3A, first lane). The
phosphorylated pT72 form of HA-ets-2 was present (Fig. 3A,
second lane) but the levels of the pT72 form were estimated
to be 5-10% of the total ets-2 pool, significantly lower than in
MDA231 cells (see Fig. 1). The level of expression of endogenous ets-2
remained low in the cells following induction of the HA-ets-2 protein
(Fig. 3A, third lane).
The expression of BRCA1, uPA, and MMP3 mRNA was studied
by Northern analysis in the tetracycline-regulated cell line (Fig. 3B). Induction of HA-ets-2 repressed BRCA1
mRNA levels 5-fold (Fig. 3B, lane 1), to a
level similar to the level of expression observed in MDA231 cells
(compare lanes 5 and 6). In contrast, the
expression of uPA mRNA and MMP3 mRNA levels were increased ~4-5-fold following induction of HA-ets-2 expression (Fig.
3B, lanes 2 and 3, respectively).
However, the levels of uPA and MMP3 RNA induced demonstrated that
levels of expression were approximately still 5-fold lower than in
MDA231 cells (compare lanes 5 and 6), consistent
with the lower levels of phosphorylated ets-2 expressed in the
tetracycline-regulated MCF-7 cell line. Similar results were obtained
for all 4 genes studied with an independent cell clone that expressed
3-fold lower levels of ets-2 than clone 21B (data not shown).
The ets-2 Pointed Domain Interacts with Distinct Sets of Nuclear
Proteins in a Phosphorylation-dependent Manner--
The
results presented above indicated that ets-2 could act as both a
repressor and activator of gene expression. We postulated that ets-2
might interact with both co-repressors and co-activators via the
conserved pointed domain.
To begin testing this hypothesis, NIH 3T3 cells were constructed that
expressed the HA-tagged ets-2 T72A mutation. As shown in Fig.
2D, this mutated from of ets-2 could act as a repressor but
was only a weak activator, and thus we might expect ets-2 T72A to
interact preferentially with co-repressors. Cells that expressed ets-2
T72A (Fig. 4A, lanes
3 and 4) were compared with serum-starved NIH 3T3 cells
that also have a low level of the phosphorylated form of ets-2 (5, 6)
(Fig. 4A, lanes 1 and 2). Extracts
from cells labeled with [35S]methionine were lysed and
immunoprecipitated under native conditions with antibody specific for
ets-2 (Fig. 4A, lanes 1 and 3) or for the HA tag (Fig. 4A, lanes 2 and 4).
In preliminary experiments the positions of ets-2 and HA-ets-2
proteins, indicated by arrowheads in the figure, were
determined in the denaturing gels by Western blotting; the positions of
authentic proteins were subsequently confirmed by comparing their
mobility with the mobility of ets-2 and HA-ets-2 proteins overexpressed
in COS cells and run on the same gels (data not shown).
These immunoprecipitation experiments demonstrated that the HA-tagged
T72A version of ets-2 was expressed at the same level as endogenous
protein (Fig. 4A, arrowheads). Additionally,
several other proteins were reproducibly present in both
immunoprecipitates, in particular species of apparent molecular mass
200 and 50 kDa (p200 and p50, indicated by
arrows in Fig. 4A, lanes 3 and
4). These proteins were also detected in ets-2
immunoprecipitates obtained serum-starved NIH 3T3 cells (Fig.
4A, lane 1).
In contrast, these bands were not detected in HA immunoprecipitates
prepared from NIH 3T3 cells (Fig. 4A, lane 1).
Furthermore, when the immunoprecipitation was performed with higher
salt concentration and inclusion of an ionic detergent, these bands are
no longer contained in the ets-2 or HA-ets-2 immunoprecipitates (Fig.
4B). These control experiments indicate that the p200 and
p50 bands are present in a complex with either endogenous or HA forms
of ets-2, and are not likely to be nonspecific or cross-reacting proteins found in the immunoprecipitates.
In a complementary biochemical approach a recombinant ets-2 protein,
corresponding to the pointed domain (amino acids 67-170) that includes
residue threonine 72 (5, 6), was overexpressed in E. coli
and covalently linked to an Affi-Gel 10 matrix. Whole cell lysates
prepared from [35S]methionine-labeled NIH 3T3 cells were
incubated with the ets-2 affinity column. After washing, the proteins
that bound to the column were eluted and analyzed by SDS-PAGE along
with the immunoprecipitated proteins (Fig. 4A, lane
5). The experiment demonstrated that proteins with the same
mobility as those detected in the HA immunoprecipitate, in particular
p200 and p50, also bound to the ets-2 pointed domain affinity column.
The results support the conclusion that these proteins form a specific
complex with ets-2.
In a parallel experiment, the ets-2 affinity column was first incubated
with recombinant, active MAPK p42 to phosphorylate threonine 72. Previous studies demonstrated that threonine 72 is the only site in the
recombinant protein phosphorylated by MAPKs (5, 6). By using a trace
amount of [
To determine whether the phosphorylation of ets-2 in vivo
could affect interaction with the p200 and p50 proteins,
immunoprecipitations were performed in ER-Raf/3T3 cells (6). These
cells contain an estrogen responsive form of the activated Raf
oncogene, and estrogen treatment persistently stimulates MAPK kinases
activity and ets-2 phosphorylation at Thr-72, as well as ets-target
gene activation (6). In these experiments, the p200 and p50 proteins could again be co-immunoprecipitated with endogenous ets-2, but stimulation of Raf signaling had no significant effect on the relative
amount of these proteins found in the immunoprecipitates (Fig.
4C, lane 1 compared with lane 2).
Western analysis of a portion of these immunoprecipitates demonstrated
that phosphorylated ets-2 could be detected following activation of Raf
(Fig. 4C, lower panels). We estimated that about
50-60% of ets-2 was phosphorylated at position Thr-72 in the
experiment shown (see "Experimental Procedures"). Thus, a
significant pool of unphosphorylated ets-2 was present in the cells and
immunoprecipitates analyzed in this experiment.
Ets-2 Co-immunoprecipitates with Brg-1 and Components of the
Mammalian SWI/SNF Complex--
As a first attempt at identifying the
proteins that co-immunoprecipitated with ets-2, we took a candidate
protein approach and obtained antibodies for known co-activators and
co-repressors in the 200-kDa size range. Fortuitously, one of the first
antibodies tested was directed against Brg-1, the ATPase hydrolyzing
subunit of the mSWI/SNF complex (13, 14), and recognized the p200 protein (Fig. 5A). In
addition, antibody against the BAF-57/p50 subunit of the mSWI/SNF
complex (13) recognized the p50 protein present in anti-ets-2
immunoprecipitates (Fig. 5A). The Ini1 subunit of mSWI/SNF
(12-14) could also be detected in a complex with ets-2 in NIH 3T3
cells (Fig. 5A). In contrast, the Brm-1 protein, a protein
highly related to Brg-1 that can be found in a distinct mSWI/SNF
complex (13, 14), was not immunoprecipitated with ets-2 (Fig.
5A).
The MCF-7 cells engineered to conditionally express ets-2
were used to determine whether the ets-2/mSWI/SNF interaction could be
detected in human cells (Fig. 5B). For these experiments,
cells were grown for 8 h after removing tetracycline from the
culture media followed by isolation of ets-2 complexes by
immunoprecipitation. These experiments showed that Brg-1 and Ini1 could
be detected in complex with ets-2 after tetracycline removal, but not
in cells maintained in tetracycline (Fig. 5B). The
BAF157/p50 antibody did not cross-react with proteins present in MCF-7
cells in our hands (data not shown).
The ets-2 Pointed Domain Forms a
Phosphorylation-dependent Complex with Brg-1 in
Vitro--
To determine whether the interaction of Brg-1 with ets-2
was direct and dependent on ets-2 phosphorylation, GST pull-down experiments were performed. A recombinant GST-Brg-1 fusion protein containing the entire C-terminal portion of the protein from amino acids 1108 to 1686 was used in "pull-down" assays with the
recombinant ets-2 pointed region (Fig.
6A). In these experiments,
GST-Brg-1 could form a complex with the ets-2 pointed protein, whereas
GST alone did not (Fig. 6A, top panel, lane
2 versus lane 1). Approximately 30% of the ets-2 input
was present in the complex (lane 3 represents 50% of ets-2
input).
In a parallel experiment, the ets-2 pointed region was phosphorylated
in vitro using recombinant MAPK p42 and
[ Brg-1 and ets-2 Repress the BRCA1 Promoter in SW13 Cells--
To
test the functional significance of the Brg-1/ets-2 interaction, the
effects of these nuclear factors on the activity of the
BRCA1-luciferase reporter were studied (Fig.
7). For these experiments, the tumor cell
line SW13, which lacks detectable Brg-1 and Brm-1 proteins (15, 16,
37), was used. First, the BRCA1-luciferase reporter was
introduced into cells with the combination of expression vectors for
ets-2 and Brg-1 in transient transfection assays
(Fig. 7A, left panel). The results of the experiments, expressed as fold-repression, indicated that neither expression vectors for ets-2 nor Brg-1 alone
repressed the BRCA1 reporter. However, the combination of
the two resulted in an approximate 3-fold repression of reporter
activity (Fig. 7A, left panel). If an expression
vector for a Brg-1 gene encoding a protein with a mutation
in the ATP-binding domain, Brg-1 (K798R), was used in the assay,
repression of the BRCA1 reporter was not observed in either
the presence or absence of ets-2 (Fig. 7A,
left panel). As a control, we also studied the ability of
Brg-1 to act as a co-activator for the glucocorticoid
receptor (GR) using an artificial glucocorticoid responsive reporter
that contained 8 GR-binding sites. As previously reported, the
combination of Brg-1 and GR expression vectors could
stimulate a target reporter more efficiently than either gene alone
(16) (Fig. 7A, right panel).
Because transient DNA templates may not always be organized as
chromatin (16, 38), we also performed assays with SW13 cells that
contained stably integrated BRCA1-luciferase reporter genes
(Fig. 7B). In these experiments, ets-2 expression
by itself had little effect on the reporter activity. Brg-1
expression alone could repress the BRCA1 reporter ~7-fold,
whereas the combination of ets-2 and Brg-1
resulted in a further dose-dependent repression of
BRCA1 promoter activity (Fig. 7B). A 14-fold
reduction was observed with the higher concentration of
ets-2 expression vector. As in the transient assays,
Brg-1 (K798R) did not repress BRCA1 reporter activity alone or in combination with ets-2.
Ets-2 has previously been characterized as an activator of gene
expression, and the results presented here indicate that this factor
can also act as a repressor. Results from transient transfection assays
and following conditional expression of ets-2 demonstrate that ets-2
could both repress and activate target genes in MCF-7 cells.
Furthermore, ets-2 directly interacted with Brg-1, the ATPase component
of the mSWI/SNF complex, and Brg-1 behaved as a transcriptional
co-repressor along with ets-2. Specificity for target gene regulation
within the ets family of transcription factors can be achieved in part
through the ability of signaling pathways to selectively activate ets
family members (1). For example, the ras pathway selectively activates
only a subset of ets family members, including ets-2. The results
presented here may begin to provide insight into how signaling pathways
modulate the activity of ets-2.
At least in vitro, the pointed domain of ets-2 interacted
with distinct sets of cellular proteins, including Brg-1, dependent on
phosphorylation of residue threonine 72, a target of ras/MAPK signaling. These results suggest the hypothesis that
ras-dependent phosphorylation of ets-2 switches
the activity of this factor from repressor to activator by modulating
interactions with co-repressors like Brg-1. However, the fact that
pools of both phosphorylated and non-phosphorylated ets-2 are present
even in cells that have high activation of the ras/raf/MAPK signaling
pathways as can be achieved with the ER-Raf system (6), makes it
difficult to determine in vivo whether phosphorylation of
ets-2 strictly modulates its activity as repressor, or even its
interaction with Brg-1. Whereas the model that signaling pathways can
modulate the pools of ets-2 that act as either repressor or activator
within cells is attractive, additional work is required to validate
this model. It is possible that signaling pathways never lower the level of non-phosphorylated ets-2 sufficiently to affect repression of target genes. A significant portion of ets-2 may always be present
in a repressor complex within cells, and phosphorylation may only
increase the pool of ets-2 that is able to activate target genes.
The results presented here provide further evidence that the mammalian
SWI/SNF complex can also repress gene expression, in addition to the
well characterized role as a co-activator in mammalian cells (13, 15,
16). At least two models can be proposed to account for the activity of
the ets-2·Brg-1 complex as a repressor instead of as an activator of
gene expression. One possibility is that direct interaction with ets-2
may cause a conformational change in the mSNF/SWI complex resulting in
an increased ability to form closed nucleosome structures over the
open, altered nucleosome structure (14).
A second possibility that may account for our results is
that distinct subcomplexes may mediate activation and repression functions of mSWI/SNF (20, 39). In mammalian cells, discrete Brg-1 and
Brm-1 subcomplexes containing mSin3 co-repressors have been
characterized (39), whereas recent work in yeast suggests that the
co-repressor function may be mediated by the ATPase-hydrolyzing SNF2
subunit alone, and not require additional complex subunits (20). Our
analysis indicates that at least two known components of mSWI/SNF (Ini1
and BAF57/p50) in addition to Brg-1 are present in the putative ets-2
co-repressor complex. The closely related gene product Brm-1 could not
be detected in a complex with ets-2, consistent with evidence
suggesting that Brg-1 and Brm-1 complexes are biochemically and
functionally distinct (39, 40). However, additional antibodies that are
available for mSWI/SNF complex subunits, for example, p155 and p170,
were not sufficiently specific, at least in our hands, to allow us to
determine with certainty whether other components were associated with
ets-2. Defining the exact composition of the complex, and demonstrating
that the ets-2 containing complex has chromatin remodeling activity,
are important questions that need to be addressed by future work.
New mechanisms by which the tumor suppressor BRCA1
might be down-regulated in mammary tumor cells are biologically
significant. Germline mutations in BRCA1 account for
approximately one-half of inherited breast cancers, but mutations of
BRCA1 are infrequent in sporadic breast cancer (41). Several
studies indicate that BRCA1 expression is down-regulated in
primary breast tumors versus normal breast tissue (42-45).
Aberrant methylation of CpG islands in the BRCA1 promoter
may be one mechanism that leads to decreased gene expression in
sporadic breast cancer (42-44). However, hypermethylation of the
BRCA1 promoter region is only found in ~13% of sporadic breast cancer cases (46), suggesting that additional mechanisms may be
involved in BRCA1 silencing. Ets-2 as both a repressor of
BRCA1 and an activator of extracellular proteases like uPA, MMP3, and MMP14 in a subset of breast cancer cases provides an attractive model.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase (MCF-7 cells) or an
expression vector for Renilla luciferase (pRL-CMV, Promega,
SW13 cells) was included as an internal control for transfection efficiency (0.1 µg/DNA precipitate). Relative luciferase activity is
equal to (raw luciferase activity)/(raw activity of the internal control × the protein concentration of the extract) (3).
Fold-repression is the ratio of relative luciferase activity for the
BRCA1-luciferase reporter alone (with empty expression
vectors) to the activity in the presence of ets-2,
Brg-1, or the combination of both ets-2 and
Brg-1, as indicated in the figure legends.
-32P]ATP (500 Ci/mmol, PerkinElmer Life
Sciences), and 5 µl of activated recombinant MAPK p42 (Upstate
Biotechnology, Lake Placid, NY). Either the unphosphorylated or the
phosphorylated ets-2 affinity columns were incubated with cell extracts
prepared in lysis buffer as above for 16 h at 4 °C. Washing
procedures and analysis were performed as for the immunoprecipitations above.
-32P]ATP but no cold ATP (see above for
details of the kinase reaction). Beads and ets-2 protein were incubated
in lysis buffer for 16 h at 4 °C. Beads were washed and the
material bound analyzed by Western blotting using the ets-2 specific
antibody, or for 32P-labeled protein by autoradiography and phosphorimaging.
208 to
182 relative to the ATG): BRCA1 wild-type,
5'-GGTAGAATTCTTCCTCTTCCGTCTCTTGG; BRCA1 M1,
5'-GGTAGAATTCTTACTCTTCCGTCTCTTGG; BRCA1 M2,
5'-GGTAGAATTCTTCCTCTTACGTCTCTTGG; and BRCA1 M1 + M2, 5'-GGTAGAATTCTTACTCTTACGTCTCTTGG. A
Amersham Biosciences PhosphorImager was used to quantify the amount of protein-DNA complex formed.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (35K):
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Fig. 1.
Expression of phosphorylated ets-2 correlates
with expression of target genes in the breast cancer cell lines MCF-7
and MDA231. A, nuclear extracts from MCF-7 and MDA231
breast cancer cells were analyzed by SDS-PAGE, followed by Western
blotting with the anti-pT72 ets-2 (top panel).
The same membrane as in the top panel was reprobed with a
pan-ets-2 antibody (middle panel). Blot was reprobed with
antibody specific for the ets factor elf-1 as a loading control
(bottom panel). The arrows indicate the position
of ets-2. B, ets-2 was immunoprecipitated from MCF-7
(lane 3) and MDA231 (lane 4) cells using the
non-phosphodiscriminating anti-ets-2 antibody. The immunoprecipitate
was analyzed by Western blotting using the same two antibodies used in
panel A, as indicated in the figure. Lanes 1 and
2, preimmune ets-2 antiserum was used for control
immunoprecipitations from MCF-7 and MDA231, respectively.
Arrows indicate the position of ets-2. The unlabeled band
present in each lane is immunoglobulin heavy chain. C,
mRNA expression of ets-2 target genes in MCF-7 (lane 1)
and MDA231 (lane 2). Total RNA was prepared and analyzed by
Northern blotting (10 µg per lane) with uPA (first panel),
MMP3/stromelysin (second panel), bcl-x (third
panel), MMP14 (fourth panel), BRCA1
(fifth panel), and ets-2 (sixth panel)
probes utilized as indicated. Note that there are two ets-2 mRNA
isoforms of 2.2 and 4.5 kb that differ in the 3' non-coding region. The
blot was reprobed with a -actin probe (seventh panel) as
a control for sample loading. D, comparison of the target
gene promoter sequences encompassing the ets-2-binding site. uPA
(GenBankTM number X02419), MMP3 (GenBankTM
number U43511), bcl-x (GenBankTM number D30746), MMP14
(GenBankTM number AF158733), and BRCA1
(GenBankTM number U37574) sequences are displayed. Also
shown is the consensus sequence for an ets-1/ets-2-binding site
(1).
promoter, the
predominant promoter active in breast cells (36), also contains a
consensus ets-binding site conserved in both mouse and human promoters
(21-24) (Fig. 1D). Thus, the expression of ets-2 protein
and BRCA1 mRNA were inversely correlated in MCF-7 and
MDA231 cells.
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Fig. 2.
Ets-2 binds to the BRCA1
promoters and represses BRCA1 promoter activity in
MCF-7 cells. A, electrophoretic mobility shift analysis
with recombinant ets-2. The wild-type BRCA1 sequence ( 208
to
182 relative to the ATG) along with nucleotides mutated (shown by
asterisk) are represented above the gel.
Wild-type BRCA1 (lane 1), mutated
BRCA1 oligonucleotides M2 (lane 3), M1
(lane 5), and M1 + M2 (lane 7) were end-labeled
with 32P and incubated with 1 µg of recombinant ets-2
protein, as indicated. Probes incubated without recombinant protein
were included in even numbered lanes (WT, M2, M1, and M1 + M2 in lanes 2-8, respectively). B, MCF-7 cells
were transfected with 5 µg of human BRCA1 or mouse MMP14
luciferase reporters and 100 ng of Rous sarcoma virus-
-galactosidase
reporter (an internal control). Either empty expression vector or
vector for ets-2 (either 100 or 500 ng) as indicated, were
co-transfected. Relative luciferase activity, adjusted for the internal
control and for protein concentration of the cell-free extracts, is
presented. C, MCF-7 cells were transfected with either 5 µg of BRCA1 promoter luciferase reporter containing the
wild-type ets-binding site (BRCA1) or the mutated
ets-binding site M1 + M2 (MT-BRCA1, see panel A),
with internal control as above, and with or without the addition of
ets-2 expression vector (either 100 or 500 ng) as indicated.
The activity is expressed as fold-repression, which is, the ratio of
relative luciferase activity for the BRCA1 or
MT-BRCA1 reporter alone (with empty expression vector) to
the relative luciferase activity in the presence of ets-2.
D, MCF-7 cells were transfected with 5 µg of human
BRCA1 or mouse MMP14 luciferase reporters, internal control,
and with or without mutated ets-2 T72A expression vector
(either 100 or 500 ng) as indicated. Relative luciferase activity, as
above, is presented. For panels B-D, the average of three
independent experiments performed in duplicate are presented.
Error bars indicate the standard deviation obtained from the
three independent experiments.
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Fig. 3.
Conditional expression of Ets-2 alters target
gene expression in MCF-7 cells. A, after growing
MCF-7/tetracycline activator cells in the absence of tetracycline
( Tet) for 0, 1, 8, and 24 h, as indicated, equal
amounts of cell lysates were analyzed by SDS-PAGE, followed by Western
blot with the anti-HA antibody (top panel), the anti-pT72
ets-2 (middle panel), or with an ets-2 non-discriminating
antibody (lower panel). Arrows indicate the
position of ets-2. B, total RNA was prepared from cells
treated as in panel A, and analyzed by Northern blotting (10 µg/lane) with probes specific for BRCA1, uPA, MMP3, and
-actin, as indicated.
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Fig. 4.
Ets-2 interacts with nuclear proteins in a
phosphorylation-dependent manner. A,
serum-starved NIH 3T3 cells (lanes 1 and 2), or
NIH 3T3 cells that expressed HA-tagged Ala-72 ets-2 (lanes 3 and 4) were labeled with [35S]methionine and
protein extracts were prepared. The labeled extracts were incubated
with either an ets-2 specific antibody (E2, lanes
1 and 3) or with the anti-HA antibody (HA,
lanes 2 and 4). In parallel experiments, labeled
NIH 3T3 extracts were incubated with an ets-2 pointed domain affinity
column that was either unphosphorylated (lane 5) or
phosphorylated by MAPK p42 (lane 6). Proteins present in the
immunoprecipitates or bound to the affinity column were separated by
SDS-PAGE and visualized by autoradiography. The position of ets-2
proteins (endogenous and HA-tagged, determined by Western blotting) are
indicated by arrowheads, and co-immunoprecipitated proteins
of relative mass 200 and 50 kDa are indicated by arrows.
Note that the band with similar mobility to HA-ets-2 in lanes
5 and 6 does not cross-react with the HA or ets-2
antibodies. The position of molecular weight markers are also indicated
in the figure. B, immunoprecipitation as in panel
A, using ets-2 and HA antibodies as indicated. NIH 3T3 extracts
were in lanes 1 and 3, whereas extracts from
cells expressing HA-ets T72A were in lanes 2 and
4. Lanes 1 and 2 were performed under
standard conditions (150 mM NaCl and 1% Nonidet P-40),
whereas lanes 3 and 4 were performed with more
stringent conditions (500 mM NaCl and 0.5% deoxycholate).
C, immunoprecipitation as in panel A, using ets-2
antibody and labeled extracts prepared from ER-Raf/NIH 3T3 cells (6)
grown in the absence (lane 1) or presence of
10 6 M
-estradiol (lane 2).
Position of endogenous ets-2 is indicated by the arrowhead,
whereas the p200 and p50 bands are shown by arrows. The
lower panels show the results of Western blotting with these
cells, using pT72 or non-discriminating ets-2 antibody, as
indicated.
-32P]ATP in the kinase reaction, we were
able to calculate that >90% of the covalently linked ets-2 protein
was phosphorylated (data not shown). Analysis of proteins from NIH 3T3
cells that bound to the phospho-ets-2 affinity column indicated that a
discrete set of proteins distinct from those bound to the
unphosphorylated column were detected (Fig. 4A, lane
6). Notably, p200 and p50 did not bind to the phospho-ets-2 column
(Fig. 4A, lane 5 versus lane 6).
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Fig. 5.
Ets-2 forms a complex with components of
mammalian SWI/SNF. Panel A, Western analysis of
proteins that co-immunoprecipitate with HA-ets-2 Ala-72 in NIH 3T3
cells that express this exogenous form of ets-2 (lane 2).
Components of the mSWI/SNF complex, Brg-1, BAF57/p50, and Ini1, all
were found in complex with the HA-ets-2 T72A (panels 1-3), but Brm-1
is not found in the complex (lane 2, panel 4). Lane
1 is a Western blot performed on total nuclear extracts prepared
from these cells. As a control, immunoprecipitations with HA antibody
were performed on normal NIH 3T3 cells that do not express the
HA-tagged protein (lane 3). Panel B, Western
analysis of proteins that co-immunoprecipitated with HA-ets-2 in the
tetracycline-off MCF-7 cells (see Fig. 3). Tetracycline was removed
from cells for 8 h, HA-ets-2 was immunoprecipitated with HA
antibody, and the immunoprecipitates were analyzed by Western blots
with antibodies as indicated (lane 3). Lane 1 represents Western analysis of crude nuclear extracts with the same
antibody, whereas lane 2 is a Western blot performed on
material in the HA immunoprecipitate prepared from cells grown in the
presence of tetracycline (non-inducing conditions).
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Fig. 6.
Phosphorylation-dependent direct
interaction between Brg-1 and Ets-2 in vitro.
A, recombinant GST-Brg-1 (1 µg) was immobilized on GST
beads and then incubated with the recombinant, unphosphorylated pointed
domain of ets-2 (1 µg). Protein bound to beads was analyzed by
SDS-PAGE and Western blotting, using ets-2 or Brg-1 specific antibodies
as indicated. Lane 1, GST only + ets-2 pointed; lane
2, GST-Brg-1 + unphosphorylated ets-2; lane 3, 50% of
input of ets-2 and Brg-1. B, pull-down assay with GST-Brg-1
and ets-2 pointed phosphorylated by Erk using
[ -32P]ATP. Upper panel is an autoradiograph
to detect 32P-labeled ets-2 pointed. Radioactivity was
quantified using a Amersham Biosciences PhosphorImager. Two lower
panels are Western blots probed with Brg-1 and ets-2 antibodies,
respectively. Lane 1, GST only + ets-2 pointed; lane
2, GST-Brg-1 + 32P-labeled ets-2; lane 3,
50% of input of ets-2 and Brg-1.
-32P]ATP. Cold ATP was not added to the kinase
reaction, so that only a trace amount of the ets-2 pointed protein
would be phosphorylated. The 32P-labeled ets-2 pointed
protein was used in the pull-down assay with GST-Brg-1 as above (Fig.
6B). In this case, less than 5% of the
32P-labeled ets-2 protein was found in a complex with the
GST-Brg-1 protein (Fig. 6B, top panel, lane
2 versus lane 3). In contrast, Western analysis
demonstrated that ~30% of the unlabeled ets-2 pointed domain could
still be detected in the pull-down fraction.
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Fig. 7.
Brg-1 and ets-2
repress BRCA1 promoter reporter in SW13 cells.
A, transient transfections performed in SW13 cells. 5 µg
of human BRCA1-luciferase reporter was co-transfected with
100 ng of expression vector for ets-2 and 250 ng of expression vector
for Brg-1, or Brg-1 K798R, or with a combination
of 100 ng of ets-2 vector and 250 ng of Brg-1, or
Brg-1 K798R vectors, as indicated (left bar
graph). In parallel experiments shown in the bar graph
to the right, 5 µg of an 8 × GRE reporter was
co-transfected with 100 ng of rat GR expression vector (47), 250 ng of
expression vector for Brg-1, or with a combination of 100 ng
of GR vector and 250 ng of Brg-1 vector. All experiments
were performed in the presence of 10 7 M
dexamethasone in this analysis. Fold-induction indicates the
ratio of 8 × GRE relative activity alone compared with activity
seen with GR or Brg-1 alone, or a combination of the two.
B, transfection with stably integrated
BRCA1-luciferase reporter. For these experiments, 1 µg of
Brg-1 or Brg-1 K798R alone or in combination with
ets-2 (0.2 or 1 µg, as indicated) were transfected into SW13 cells
that contained stably integrated copies of the
BRCA1-luciferase reporter. For both panels A and
B, fold-repression is the ratio of relative luciferase
activity (see "Experimental Procedures") for the
BRCA1-luciferase reporter alone (with empty expression
vectors) to the activity in the presence of ets-2,
Brg-1 (Brg-1 K798R), or a combination of both
ets-2 and Brg-1 (Brg-1 K798R). Results
of three independent experiments performed in duplicate are presented.
Error bars indicate standard deviation of the
measurements.
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Matt Hartman and Lori Nelsen for excellent technical assistance. We acknowledge Ellen Solomon for the human BRCA1 promoter plasmid, Joseph Madri for the murine MMP14 promoter reporter, Trevor Archer and Steve Goff for Brg-1 cDNAs and expression vectors, and Said Sif, Anthony Imbalzano, and Gerry Crabtree for mSWI/SNF complex antibodies.
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FOOTNOTES |
---|
* This work was supported by National Cancer Institute Grant R01-CA-53271 (to M. C. O.).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.
§ Both authors contributed equally to this work.
¶ Present address: Dept. of Biology, Carleton College, Northfield, MN 55057.
Present address: Dept. of Science and Mathematics, Cedarville
University, Cedarville, OH 45314.
** To whom correspondence should be addressed: Dept. of Molecular Genetics, Ohio State University, 484 W. 12th Ave., Columbus, OH 43210. Tel.: 614-688-3824; Fax: 614-292-4466; E-mail: ostrowski.4@osu.edu.
Published, JBC Papers in Press, March 10, 2003, DOI 10.1074/jbc.M209480200
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ABBREVIATIONS |
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The abbreviations used are: MAPK, mitogen-activated protein kinase; uPA, urokinase plasminogen activator; HA, hemagglutinin; DMEM, Dulbecco's modified Eagle's medium; GST, glutathione S-transferase; GR, glucocorticoid receptor; MMP, matrix metalloproteinase.
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