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INTRODUCTION |
Breast cancer is the second leading cause of death in American
women, accounting for more than 50,000 deaths each year. Current estimates place the average American woman's lifetime risk of developing breast cancer at approximately 11%. However, women with two
or more first degree relatives with breast cancer have an estimated
13-fold increased risk over the general population (1). Breast cancer
in such families has an inheritance pattern consistent with a highly
penetrant autosomal dominant allele (2, 3). BRCA1, the first
breast cancer susceptibility gene to be identified (4), was isolated in
1994 (5).
An interesting fact regarding BRCA1 is that although the
mutations in the gene in familial breast cancers are of high
penetrance, very few mutations in the BRCA1 gene have been
found in sporadic forms of the cancer. These findings are prompting
researchers to study the possibility of disruption of BRCA1
function through epigenetic mechanisms. Consistent with this notion, it
has been suggested that transcriptional dysregulation of
BRCA1 may play a role in suppressing BRCA1
expression in breast cells, perhaps contributing to the development of
a neoplastic phenotype. Two studies have demonstrated a decrease of
BRCA1 expression in sporadic breast cancer (6, 7). Another
set of studies describe CpG methylation of BRCA1
transcriptional promoter in a number of sporadic breast cancers, in
contrast to the lack of methylation in normal breast tissues
samples (8, 9).
In light of these observations, we initiated a study to characterize
the BRCA1 transcriptional promoter. Previously, the
structural features of the BRCA1 promoter were described
(10, 11), and preliminary descriptions of modest and indirect effects
of estrogen on the BRCA1 promoter activity were reported
(12, 13). However, no information was available regarding regulatory
sites and specific regulatory factors. In this report we provide
evidence for a positive regulatory region
(PRR)1 in the
BRCA1 promoter and show data that suggest that multiple proteins bind specifically to the site.
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EXPERIMENTAL PROCEDURES |
Isolation of the BRCA1 Promoter--
The BRCA1
promoter was subcloned from a bacterial artificial chromosome clone,
BAC 694 (kindly provided by Dr. Sean Tavtigian, Myriad Genetics) (14).
Briefly, PstI linker (5'-GCTGCAGC-3') was ligated into the
blunted HindIII site in the pGL2 vector (Promega vector with
the firefly luciferase reporter gene). BAC 694 was digested with
PstI, and the resulting fragments were shotgun cloned into
the pGL2 vector and transformed into competent DH5-
Escherichia coli cells. The transformed bacterial colonies
were screened by colony hybridization with a radiolabeled
BRCA1 cDNA probe (BRCA1 cDNA was kindly
provided by Frank Calzone) (15) labeled by the random hexamer method
(16), and clones with a 3.8-kilobase insert containing the
BRCA1 5' genomic fragment were selected. The cloned fragment
was sequenced completely and is identical to the previously described
genomic fragment encompassing the BRCA1 promoter
(GenBankTM accession number U37574) (11). The nucleotide
position of mutants are numbers from the P1 promoter initiation site at
nucleotide 1582.
Mutants of BRCA1 Promoter--
Systematic promoter deletions
were constructed by unidirectional exonuclease III digestion. 10 µg
of BRCA1 promoter-luciferase construct was digested with
MluI restriction enzyme and blunted with
-phosphorothioate nucleotides using Klenow enzyme. This treatment
rendered the ends of the linearized plasmid resistant to exonuclease
III digestion. The linearized DNA was purified by
phenol/chloroform/isoamyl alcohol extraction and digested with XhoI restriction enzyme, generating a 5' end susceptible to
the exonuclease digestion. The fragment was purified and then subjected to exonuclease III digestion. Aliquots of the reaction were removed at
regular intervals, and the reactions were terminated. Finally, the
fragments were blunted using S1 nuclease, religated, and transformed into DH5-
competent cells. DNA preparations (using Qiagen columns) made from selected colonies were screened by analytical restriction enzyme digestions. Mutants
202 and +20 were constructed by exploiting the restriction enzyme sites present on the BRCA1 promoter
(EcoRI,
202; SacI, +20) and also the sites
present in the polylinker of the luciferase vector. The
BRCA1-luciferase construct was digested with
EcoRI/XhoI (XhoI site is present in
the polylinker) and SacI (SacI site is also
present in the polylinker), respectively, and blunted with Klenow
enzyme, and the larger fragment gel was purified and religated.
Additional mutants to generate progressive deletions were constructed
by a polymerase chain reaction based strategy. 5' primers were designed
at regular intervals along the sequence of the BRCA1 promoter:
245, 5'-CTC ACG CGT TAG AGG CTA GAG GGC AGG-3';
198, 5'-CTC ACG CGT TCC TCT TCC GTC TCT TTC-3';
177,
5'-CTC ACG CGT TTA CGT CAT CCG GGG GCA-3';
162, 5'-CTC
ACG CGT GCA GAC TGG GTG GCC AAT-3';
152, 5'-CTC ACG
CGT TGG CCA ATC CAG AGC CCC-3';
118, 5'-CTC ACG CGT
CTT TCT GTC CCT CCC ATC-3';
86, 5'-CTC ACG CGT GAT TTC
GTA TTC TGA GAG-3';
43, 5'-CTC ACG CGT GGT TTC CGT GGC AAC GGA-3';
34, 5'-CTC ACG CGT GGC AAC GGA AAA GCG
CGG-3';
26, 5'-CTC ACG CGT AAA GCG CGG GAA TTA CAG-3';
17, 5'-CTC ACG CGT GAA TTA CAG ATA AAT TAA-3';
7,
5'-CTC ACG CGT TAA ATT AAA ACT GCG ACT-3'. The 5' primers
included three bases, CTC, followed by the MluI enzyme site,
which is underlined. The single 3' primer used was: +36, 5'-TAG
CTC GAG GGA AGT CTC AGC GAG CTC-3'. This primer included
TAG followed by a XhoI site, which is underlined, at the 5'
end. The amplification conditions used were as follows (1 cycle for 2 min at 94 °C and 35 cycles at 94 °C for 15 s, 60 °C for
15 s, 72 °C for 30 s), and 1 ng of the cloned
BRCA1 promoter plasmid was used as template DNA. The
amplified products were digested with MluI and
XhoI restriction enzymes and ligated into the restricted
MluI-XhoI site of the pGL3 basic vectors
(Promega). In addition, two synthetic primers (+5, 5'-CGC GTT GCG ACT
GCG CGG CGT GAG CTC GCT GAG ACT TCC TC-3' and +36, 5'-TCG AGA GGA AGT
CTC AGC GAG CTC ACG CCG CGC AGT CGC AA-3') designed to include the
BRCA1 promoter region from +5 to +36 were annealed. The
annealed primers generated a MluI compatible site at the 5'
end and a XhoI compatible site at the 3' end, which were
utilized in ligation of the annealed primers directly into the
MluI and XhoI site of the pGL3 vector. All the
constructs were verified by sequencing.
Cell Culture, Transfections, and Reporter Gene
Assays--
TK-TS13 cells (hamster kidney cells kindly provided by Dr.
Bruno Calabretta) were transfected by calcium phosphate precipitation. A total of 3 µg of DNA was used in each transfection (1 µg of BRCA
promoter, 1 µg of pUC19 plasmid, and 1 µg of pRL-CMV Renilla luciferase vector purchased from Promega). The cells were maintained in
Iscove's modified Dulbecco's medium supplemented with 10% fetal bovine serum and 100 µg/liter gentamycin sulfate. MCF-7 cells (maintained in minimum essential medium, 10% fetal bovine serum, 100 µg/ml gentamycin sulfate, 2 mM sodium pyruvate and
glutamine) were transfected using Fugene-6 reagent manufactured by
Boehringer Mannheim. A total of 2 µg of DNA (0.5 µg of promoter
construct and 1.5 µg of pRL-CMV) with 10 µl of Fugene reagent was
used in each transfection. All the transfections were optimized for
six-well plates. The transfected cells were lysed after 48 h by
addition of 250 µl of passive lysis buffer (Promega). 20 µl of the
lysed cell extract was added to 100 µl of luciferase substrate, and the light emissions were measured in a scintillation counter. Renilla
luciferase readings (which were utilized to normalize the transfection
efficiencies) were measured in the same tube using conditions
recommended by the manufacturer.
DNA Binding Assays--
The experiments were performed
essentially as described previously (17). Electrophoretic mobility
shift assays were performed with a double-stranded probe encompassing
the PRR (see Fig. 3B). Two single-stranded oligonucleotides
(
197 to
161, 5'-CCT CTT CCG TCT CTT TCC TTT TAC GTC ATC CGG GGG CAG
ACT-3', and
161 to
171, 5'-AGT CTG CCC CC-3') were annealed, and
the gap was filled by Klenow polymerase in the presence of
deoxyribonucleotides dATP, dGTP, dTTP,
-32P-radiolabeled
dCTP and 5-bromo-2'-deoxyuridine (BrdUrd) as described (17). Nuclear
extract (5 µg), made from MCF-7 breast cancer line as described
previously, was mixed with 100,000 cpm of probe in Yanos buffer (20 mM Tris, pH 7.8, 50 mM NaCl, 10 mM
MgCl2, 15% glycerol, 0.1 mM EDTA, 0.01%
Nonidet P-40, 100 µg/ml bovine serum albumin, 2 mM
dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, and 0 or 1.5 or 3 µg of poly(dI·dC)) and incubated for 20 min.
Competitions were performed with 50-fold excess of nonradiolabeled annealed double-stranded oligonucleotides. These oligonucleotides contained palindromic C/EBP mutant (5'-TGC AGA GAC TAG TCT CTG CA-3'),
p53 (5'-CCC AAA CAA GCT CCC CTG AAA CAA GCC CGT T-3' and 5'-AAC GGG CTT
GTT TCA GGG GAG CTT GTT TGG G-3'), and palindromic C/EBP canonical
sites (5'-TGC AGA TTG CGC AAT CTG CA-3'). In addition, sheared salmon
sperm DNA (Sigma) and PRR cold probes were also used in competitions.
After the incubations, loading dye was added (final concentrations:
0.06% bromphenol blue, 0.06% Xylene cyanole, and 7.2% glycerol), and
the reactions were electrophoresed in a 5% nondenaturing gel, resolved
for 3 h at 200 V in 0.25× TBE buffer, and after drying the gel
exposed for 5 h to a film.
In experiments involving UV radiation-induced cross-linking of DNA and
protein, each electrophoretic mobility shift assay reaction was
transferred into a 96-well plate at 4 °C and exposed to UV radiation
in a UV-Stratalinker 1800 (Stratagene) for 20 min. After the addition
of sample buffer (1 M Tris, pH 6.8, 10% SDS, 50%
glycerol, 10%
-mercaptoethanol, and 0.2% bromphenol blue) and
boiling for 5 min, the reaction was resolved in a 9% SDS-polyacrylamide gel. The dried gel was exposed to film. Negative control experiments with a random probe were also performed. The random
probe was synthesized (in a manner similar to the PRR probe) by the
extension of a DNA oligonucleotide (5'-CCC GGG AGT AGA-3') annealed to
a longer complementary oligonucleotide (5'-CTA GTC AGA CAC GTA GAC TCT
ACT CCC GGG-3') in the presence of BrdUrd and dCTP containing
-32P label.
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RESULTS |
BRCA1 Minimal Promoter--
In order to identify the minimal
promoter containing the essential regulatory regions, systematically
deleted mutants of the BRCA1 promoter region were
constructed (Fig. 1). The transcriptional activities of these mutants were tested in TK-TS13 and MCF-7 cells by
luciferase reporter gene assays (Fig. 2).
The TK-TS13 cells were used in initial studies (Fig. 2A),
due to the ease with which they are transfected and because of their
ability to support high levels of BRCA1 promoter
activities. Subsequent detailed studies were performed in MCF-7 breast
epithelial cell line (Figs. 2B and
3A).

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Fig. 1.
BRCA1 promoter region and mutants. The
upper panel depicts the BRCA1 promoter region,
with two previously described initiation sites P1 (nucleotide 1582) and
P2 (nucleotide 1858) (11). The lower panel depicts the
generation of unidirectional deletions within the promoter region,
which leave the upstream KpnI site in the polylinker intact
(the adjacent PstI is within the BRCA1 promoter).
Cleavage of the digested fragment with KpnI and
PstI releases the promoter fragment. The numbers of the
mutants correspond to the 5' deletions catalogued and characterized in
Fig. 2A.
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Fig. 2.
Minimal BRCA1
promoter. Luciferase activities of the deletion mutants in
TK-TS13 (A) and MCF-7 cells (B). Deletions that
result in loss of promoter activities are indicated and are found to be
consistent for the two cell lines. The data are representative of three
independent transfections for each line.
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Fig. 3.
PRR in the BRCA1
promoter. A, transfections of polymerase chain
reaction-generated BRCA1 promoter mutants with short
deletions in the minimal promoter region were performed. All the
mutants had the 3' end at nucleotide +36 of the transcription start
site. The 5' ends of each mutant are indicated. The data are
representative of four independent transfections performed in
triplicate for each sample. B, the 5' ends of the deletion
mutants in close proximity to the PRR (which is boxed). The
pyrimidine-rich region and the putative CREB site are indicated.
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Results of transfections of the mutants in TK-TS13 cell indicated that
on the deletion of 1380 bases from mutant
1582 (and generating
202), there is a 2.5-fold drop of luciferase activity, which is not
significant considering the high sensitivity of the luciferase assays
and the number of bases deleted (Fig. 2A). Within the same
tract of promoter DNA (
1582 to
202), the two most wide ranging
luciferase values were 6127 (for
1244) and 308 (for
329) normalized
light units, a difference of 20-fold. However, this difference was
accompanied by a loss of 915 bases from
1244.
Overall, the data suggest that short deletions within the segment
1582 to
202 do not cause a significant change in promoter activity.
However, gross deletions (
1582 to
202) do alter the configuration
of the promoter sufficiently to affect the promoter activities
significantly, because several weak regulatory sites (both enhancers
and repressors) with additive effects may be deleted.
Transfections of selected BRCA1 promoter mutants in MCF-7
cells also indicated that essential transcriptional regulatory sites (which could have a strong effect on the transcription of
BRCA1) were not present in the tract from
1582 to
202
(Fig. 2B). Interestingly, in MCF-7 cells mutant
202 was
observed to possess a transcriptional activity that was comparable (and
slightly higher) to that of construct
1582.
Finally, results from both cell lines indicated the presence of a
sensitive region of 222 bases (
202 to +20), which on deletion resulted in a 100% loss of BRCA1 promoter activities (Fig.
2). These experiments strongly suggested that the essential regulatory elements of the BRCA1 promoter reside within the deleted
segment. Furthermore, the mapped segment encompassed the P1 promoter
region, suggesting that the activity of the P1 promoter was predominant in both the transfected cell lines. Curiously, P2 did not demonstrate any functional activity in either of the cell lines tested (Fig. 2). It
is possible that sequences within the P1 promoter region may regulate
the transcriptional initiation from P2.
Identification of the BRCA1 PRR--
Following the identification
of the
202 to +20 segment as essential for BRCA1
transcription, detailed characterization of the segment was undertaken.
Additional unidirectional, polymerase chain reaction-based deletion
mutants were constructed, and their activities were tested (Fig.
3A). With the aid of these promoter mutants, it was
determined that deletion of a short 22-base pair region between
198
and
177 (Fig. 3B) resulted in a significant loss
(14.5-fold) of luciferase activity. Further removal of 15 more
nucleotides (
162) led to an additional 4-fold loss in activity. Overall, the removal of 36 bases (from
198 to
162) results in a
56-fold loss in luciferase activity, indicating a PRR.
Interestingly, the PRR contains a short polypyrimidine-polypurine
(Py-Pu) tract (the majority of nucleotides on the sense strand are
pyrimidines and by extension the complementary strand is mostly purine)
(Fig. 3B). The first 22 bases in the site are almost
exclusively Py-Pu (21 of 22 or 95%). This Py-Pu-rich tract is followed
by a putative CREB site. Overall the pyrimidines in the sense strand
make up 75% (27 of 36) of PRR.
DNA Binding Assays--
In order to characterize the proteins
binding to the PRR site, electrophoretic mobility shift assays were
performed using MCF-7 nuclear extracts. Retarded protein-DNA complexes
were detected (Fig. 4A,
lanes 2-8). Lane 2 lacked poly(dI·dC), and
therefore the factors binding the probe largely represented nonspecific proteins. Addition of poly(dI·dC) (lanes 3-9), cleared
nonspecific bands, and three protein-DNA complexes were detected
(except in lane 9); a single intense, higher mobility band
and two weaker bands with lower mobility, were observed. Addition of
50-fold excess of double-stranded nonlabeled oligonucleotides or
sheared salmon sperm DNA (lanes 5-8) did not compete away
the protein-DNA complexes. In sharp contrast, the proteins binding the
labeled probe were efficiently competed out by a 50-fold excess of
nonlabeled PRR probe (lane 9). This experiment indicates
that the PRR-binding proteins bind in a sequence-specific manner.

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Fig. 4.
Proteins specifically bind the PRR
sequence. A, electrophoretic mobility shift assay was
performed with a radiolabeled PRR probe. The amounts of poly(dI·dC)
added to each reaction are indicated. Nonspecific competitions were
performed with 50-fold excess of double-stranded, unlabeled
oligonucleotides with indicated binding affinities (lanes
5-7). In addition, 50-fold excess of sheared salmon sperm DNA was
also used as a nonspecific competitor (lane 8). Specific
competition was performed by the addition of 50-fold excess of
unlabeled PRR-binding oligonucleotide (lane 9).
B, DNA-protein UV cross-linking experiments with PRR probe
and MCF-7 nuclear extracts. The protein-DNA complexes are indicated
(arrows). The free PRR probe is at the bottom of
the lanes. In lane 3, 0.5 µg of double-stranded
oligonucleotide (DS Oligo) containing the binding site for
the p53 protein was added. C, comparison of protein
affinities of PRR and random probes in UV cross-linking assays. All the
reactions contain 3 µg of poly(dI·dC) and 0.5 µg of annealed
double-stranded oligonucleotide containing the binding site for the
C/EBP proteins.
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In order to further characterize the components of the protein complex
that assembled on the PRR, UV cross-linking experiments (involving
DNA-protein linkage) were performed (Fig. 4B). This involved
incubation of radiolabeled, BrdUrd-containing PRR probe, with MCF-7
nuclear extracts in the presence of UV radiation. The radiation induced
covalent linking of proteins to the BrdUrd residues present in the PRR
probe. All the reactions contained 3 µg of poly(dI·dC), and one of
the reactions was supplemented with 0.5 µg of double-stranded
oligonucleotides to increase the stringency of binding to the probe
(lane 3). Lane 2 exhibits three diffuse and
indistinct bands. The intensity of these bands increased on the
addition of 0.5 µg of double-stranded oligonucleotides (lane 3), reinforcing the possibility that they represent specific
protein-DNA interactions. The approximate molecular masses of the
protein-DNA complexes observed were 55, 150, and 230 kDa.
Additional experiments were performed to exclude the possibility that
the covalent protein-DNA complexes were formed due to nonspecific
interactions. A random oligonucleotide labeled with
-32P
and BrdUrd residues was used in UV cross-linking experiments (Fig.
4C). Lane 2 of the figure demonstrates the
characteristic three-band pattern representing proteins bound to the
PRR. In contrast, no significant protein-DNA complexes were observed
when the random probe was incubated with MCF-7 nuclear extract
(lane 4). This experiment suggests that the proteins
detected by cross-linking experiments recognized and bound the PRR specifically.
Finally, it is probable that each of the DNA-protein complexes observed
do not represent multiple proteins linked to a single DNA probe but a
single protein molecule bound to one molecule of the PRR probe. This
conclusion is based on the fact that the UV-induced protein-DNA
cross-linking is inefficient; therefore it is unlikely that more than
one protein molecule will link to a single molecule of DNA probe.
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DISCUSSION |
Evidence for important roles of BRCA1 in normal
functioning of cells is accumulating. Specifically, a strong role for
BRCA1 in DNA repair mechanisms (18-20) as well as in
transcriptional regulation (21-23) has been suggested. This theme is
reinforced in a recent report that cites the involvement of
BRCA1 in transcription-coupled repair of DNA (24).
Therefore, it has been suggested that suppression of BRCA1
expression may cause defects in the DNA repair machinery, leading to
chromosomal defects and tumorigenesis.
BRCA1 expression may be suppressed through transcriptional
silencing in a subset of sporadic breast cancers, underscoring the
importance of studies to elucidate the transcriptional mechanisms involved in the regulation of BRCA1 expression. The present
studies strongly suggest that intact and functional PRR may be crucial for normal transcription of BRCA1. Hindrance of the PRR
function may occur either by methylation of proximate sequences or by
alterations in the properties of the regulatory factors, leading to
suppression of BRCA1 expression. Furthermore, elucidation of
the factors that regulate BRCA1 transcription could provide
additional clues regarding its function.
It is interesting that the PRR encompasses a CpG dinucleotides reported
by Mancini et al. (9) to be methylated in one case of
sporadic breast cancer. The methylated cytosine was present in the
putative CREB site present in the PRR (Fig. 3B). CREB
proteins are known to mediate hormone stimulation of a variety of genes (25, 26), and BRCA1 is known to be indirectly responsive to estrogen stimulation (27), prompting speculation of a regulatory role
of CREB in BRCA1 transcription. However, treatment of cells with forskolin (a reagent that stimulates post-translational activation of CREB by phosphorylation; Ref. 28) did not show any effect on the
activities of transiently transfected BRCA1 promoter (data not shown). In addition, forskolin treatment and cotransfection of a
CREB-binding protein (a CREB coactivator; Refs. 29 and 30) expression
plasmid elicited no response from the BRCA1 promoter (data
not shown). Attempts to identify CREB proteins in the DNA-protein complex, either by supershift assays or immunoprecipitation (of cross-linked DNA-protein complexes) with CREB antibodies, were not
successful (data not shown).
The composition of the PRR (21 of the first 22 bases are pyrimidines on
the sense strand) provides possible hints regarding mechanisms involved
in transcriptional regulation of BRCA1. Previous studies
have described the tendency for such Py-Pu domains to form triplex DNA,
which influence transcription (31, 32). These tracts have been reported
to be sensitive to S-1 nuclease digestion and are believed to influence
the conformation of the chromatin assembly in the promoter region. In
addition, a nuclear factor has been reported to bind a Py-Pu tract in
the c-Ki-ras promoter (31). Therefore, it is possible that
1) the Py-Pu domain may alter the chromatin structure of the
BRCA1 promoter region and 2) it may also be involved in
specific recognition and binding by transcription factors.
There is no additional information available at present regarding the
factors binding the PRR. Studies are being initiated to definitively
identify and characterize the binding proteins in order to investigate
their effects on BRCA1 transcription and their potential
role in breast cancer.