(Received for publication, May 20, 1997)
From the Division of Medical and Molecular Genetics, UMDS, 8th Floor, Guy's Tower, Guy's Hospital,London SE1 9RT, United Kingdom
We have analyzed the promoter region of the human
BRCA1 gene in detail and demonstrate that the expression of
the BRCA1 gene is under complex regulation. First, its
transcription is under the control of two promoters generating two
distinct transcripts and
, and second, promoter
is shared
with the adjacent NBR2 gene and is bi-directional. Both
promoter
and promoter
are responsive to estrogen stimulation.
We also discerned that there are striking differences in both the
genomic organization and immediate cis-control elements of
the BRCA1 gene between humans and mice.
Since its isolation in 1994 (1), much effort has been devoted toward unraveling the biological function of the breast and ovarian cancer susceptibility gene, BRCA1. The fact that many germline mutations have been found has firmly established the involvement of the BRCA1 gene in familial breast and/or ovarian cancer (2). However, unlike the precedent of other tumor suppressor genes where mutant forms of the gene products are responsible for both the inherited and sporadic forms of the same type of cancer, no sporadic breast cancers and only a handful of sporadic ovarian cancers have been found to harbor BRCA1 mutations (3-5). It is thus postulated that either BRCA1 is involved only in the etiology of inherited breast cancer or that BRCA1 disruption in sporadic cancer occurs by mechanisms other than mutations in the coding region. A few lines of evidence are consistent with the latter hypothesis: in sporadic breast cancer the BRCA1 mRNA levels are decreased (6), while in familial breast cancer inferred regulatory mutations are present (2). Therefore, studies aimed at determining the regulation of the BRCA1 gene will be critical in helping to resolve this enigma as well as to understand the normal function of the gene product.
We have previously established a comprehensive map of approximately
50-kilobase pair genomic DNA encompassing not only the 5 end of the
BRCA1 gene but also the nearby NBR1 gene
(previously named 1A1-3B) (7), which was isolated as a
candidate for the ovarian cancer marker CA125 (8). The 5
ends of both
genes are duplicated with a partial copy of the BRCA1 gene
lying head to head with the NBR1 gene and a partial copy of
the NBR1 gene lying head to head with the BRCA1
gene (Fig. 1) (7). Further analysis of
the expression of the partial copies of both genes showed that the
partial copy of the NBR1 5
exons is in fact part of a new
gene, NBR2 (9). The fact that the transcription start sites
of the BRCA1 and NBR2 genes are only 218 bp1 apart suggests that the
intergenic region may function as a bi-directional promoter. In
addition, we have previously identified two distinct BRCA1
transcripts differing by the alternative use of the first exons,
predicting that the BRCA1 gene is regulated by two promoters (10). This study is therefore aimed at unravelling the regulation of
the BRCA1 gene by functional analysis of the
BRCA1 promoters.
Plasmid p3ba containing the 5
region of both BRCA1 and NBR2 genes was described
previously (10). Constructs pGL1-4, -6, -8, -9, and -10 were generated
by inserting various restriction enzyme digested promoter fragments
derived from p3ba into pGL3-basic vector (Promega), upstream of the
firefly luciferase gene in either the BRCA1 or
NBR2 orientation. Constructs pGL5, -7, and -11-13 were
generated by polymerase chain reaction amplification, using Pfu polymerase (Stratagene), of the relevant promoter
fragments (Fig. 2) and subsequently subcloned into the pGL3-basic
vector. Plasmid pGL12M and pGL12RM, in which the CCAAT site
(nucleotide 1433) was mutated to CACCT, were generated by a
standard polymerase chain reaction-based site-directed mutagenesis
method (11). Plasmid DNA was prepared with Qiagen columns and the
sequences of all the constructs were verified by automated sequencing
analysis using a 373A sequencer (ABI).
Cell Culture and Transfections
All cell lines were from stocks maintained by the Imperial Cancer Research Fund. MCF7 between T47D and JAR were grown in RPMI 1640 (Sigma) with 10% fetal calf serum (FCS, Life Technologies, Inc.). SKOV3 was grown in Eagle's medium (Sigma) with 10% FCS and BT20 in minimal essential medium (Sigma) supplemented with bicarbonate and 15% FCS. Cells were cultured until approximately 70% confluent. Qiagen prepared plasmid DNA (15 µg of test plasmid and 10 µg of pJ7 control plasmid) was transfected by electroporation (250-270 volts, 960 µF) into approximately 5 ×106 cells.
Reporter Gene AssaysAll assays were carried out using
assay kits from Promega. Cells were harvested 40-48 h after
transfection by the addition of reporter lysis buffer (Promega),
followed by cell scraping. The cell lysates were analyzed for both the
luciferase and -galactosidase activities using assay kits from
Promega. After normalization to the
-galactosidase control, the
transactivation activity of each test construct was calculated relative
to the pGL3-basic vector, the activity of which was arbitrarily defined
as 1. Each experiment was done at least twice, and the relative
promoter activities shown represent the mean value.
The mouse sequence used in this analysis was from cosmid clone, MCHCA1, as described previously (12) with additional mouse genomic sequence generated from further sequencing of this clone (GenBankTM number U73040). Human and mouse sequences were compared using the "BestFit" program (GCG). Potential transcription factor binding sites within the human and mouse sequences were identified by searching against the "tfsites.dat" data base using the "FindPatterns" program (GCG).
Estrogen Stimulation of Promoter ActivityTo examine the
promoter activities in response to estrogen stimulation, MCF7 cells
were cultured in phenol red-free RPMI 1640 supplemented with 10%
charcoal/dextran-stripped fetal calf serum for at least 3 days. Ten
micrograms of the luciferase constructs and 10 µg of pJ7 plasmid were
co-transfected by electroporation into MCF7 cells. After 16-18 h,
fresh medium containing 10 nM 17-estrodiol was added to
the transfected cells. Twenty-four hours later, the cells were
harvested and analyzed for both the luciferase and
-galactosidase
activities as above. Relative luciferase activities upon estrogen
stimulation were calculated with respect to that obtained in the
absence of the hormone, which was defined as 1. Each experiment was
performed in duplicate and repeated at least twice.
We have
previously isolated a genomic clone p3ba containing the 5 region of
the BRCA1 and NBR2 genes (Fig.
2) (9, 10). The expression of the two
distinct BRCA1 transcripts
and
(where exon 1A and
exon 1B are the first exons respectively) is predicted to be controlled
by their respective promoters
and
(Fig. 2). In addition, due to
the close proximity of the transcription start sites for
BRCA1 transcript
and the NBR2 transcript,
BRCA1 promoter
is therefore predicted to act as a
bi-directional promoter. To address these issues, a series of
luciferase reporter constructs were generated by inserting various
fragments of p3ba into the pGL3-basic vector (Fig. 2). The promoter
activities were assessed by transient transfection of these reporter
constructs initially into the breast carcinoma cell line, MCF7. All of
the 5
-deletion constructs of the BRCA1 and NBR2
promoters tested were expressed in MCF7 cells, but with different
efficiencies (Fig. 2). For the BRCA1 promoter
, the
strongest activity was detected from the 267-bp fragment (pGL12)
encompassing 43 bp of the BRCA1 exon 1A, the 218-bp
intergenic region, and 6 bp of the NBR2 exon 1 (Fig. 2).
When tested in the NBR2 orientation, the same 267-bp
fragment also displayed the strongest promoter activity (pGL12R),
which, interestingly, is 2.5-fold more potent than that orientated in the BRCA1 direction (Fig. 2). These results confirm our
hypothesis that the promoter
of the BRCA1 gene is
bi-directional, and the cis-control elements harbored in the
intergenic region are of crucial importance in co-ordinated expression
of the BRCA1 and NBR2 genes.
The promoter activities of pGL11 and pGL11R are less active than that
of pGL12 and pGL12R, respectively, indicating that the sequence from
positions 1191 to 1357 (GenBankTM number U37574, Fig. 2) contains a
silencer negatively regulating the expression of both genes. The
inhibitory effect of this silencer is much more pronounced on the
expression of the NBR2 gene (pGL12R/pGL11R = 837/16, a
52-fold decrease) than that on the BRCA1 gene
(pGL12/pGL11 = 335/97, a 3.5-fold decrease) (Fig. 2). Strong
enhancers and silencers for the NBR2 gene were also
predicted to reside in the sequence between position 1 and 1191. This
is reflected by the sharp increase in promoter activity of pGL10R
compared with that of pGL11R (259 versus 16, a 16-fold
increase) and significant decreases in the activity of pGL9R compared
with that of pGL10R (37 versus 259, a 7-fold decrease) and
the activity of pGL8R compared with that of pGL9R (2.2 versus 37, a 17-fold decrease) (Fig. 2). In contrast, this
fragment (nucleotide positions 1-1191) only reduced the activity of
the BRCA1 promoter by 2.5-fold (pGL11/pGL8 = 97/40,
Fig. 2). These observations indicate that the basic promoter activities
of both the BRCA1 and NBR2 genes are modulated by
enhancers and silencers located between positions 1 and 1357. The
ultimate expression of both genes could therefore be influenced by the cross-talk among an assembly of different transcription factors, determined by the cis-elements located in this region and
the availability of these factors during development and perhaps
tumorigenesis.
For the BRCA1 promoter (pGL7 and pGL6), the activity was
much weaker (approximately 1/150) than that of promoter
(pGL12) (Fig. 2). The activity of promoter
can, however, be modulated by
upstream cis-elements located in promoter
, as reflected
by a 12-fold increase in promoter activity of pGL5 compared with that
of pGL7 (24 versus 2, Fig. 2). This suggests that the
expression of transcripts
and
of the BRCA1 gene may
be co-regulated.
To confirm these results, the promoter activities of a subset of the constructs presented in Fig. 2 were determined in a number of other human cell lines: breast carcinoma cell lines T47D and BT20, an ovarian cancer cell line SKOV3, and a placenta choriocarcinoma cell line JAR. Similar data to that observed in the MCF7 cell lines were obtained (Table I).
|
In light of the results from the above deletion
analysis of the BRCA1 and NBR2 promoters, we
directed our attention to the 862-bp region encompassing both the
BRCA1/NBR2 bi-directional promoter and the
BRCA1 promoter , i.e. from positions
1191-2052 (GenBankTM number U37574). DNA sequence homology search of
this region with a transcription factor data base indicates the
presence of several CCAAT boxes, GC boxes and PEA3 binding sites, and
one putative binding site for each of the transcription factors CREB, GH, and AP1 (Fig. 3). These sites were
obviously candidates for co-ordinated regulation of the
BRCA1 and NBR2 genes. Since the BRCA1
and NBR2 promoters are TATA-less promoters, and the
significance of the CCAAT element in modulating the function of such
promoters has been documented (13), we chose to examine the regulatory effect of the CCAAT box located in the intergenic region (nucleotide 1433-1437, Fig. 3). This CCAAT box present in reporter constructs pGL12 and pGL12R was targeted by site-directed mutagenesis changing CCAAT to CACCT. Compared with the respective wild type constructs, the activities of the mutated pGL12M and pGL12RM decreased by 65 and 56%, respectively, demonstrating a role for the
CCAAT element in the co-ordinated activation of both the
BRCA1 and the NBR2 genes.
Hormonal Regulation of BRCA1 Expression
Estrogen is known to
modulate the growth and differentiation of human breast epithelium (14)
and several studies have shown that BRCA1 levels are
elevated upon estrogen stimulation (15, 16). We therefore examined the
responses of the BRCA1 promoters to estrogen induction.
Sequence homology searches reveal that neither promoter nor
promoter
contains a classic estrogen response element (ERE)
(5
-GGTCANNNTGACC-3
) (17). However, an alternative ERE
(5
-GGTCA(N)3TGGTC(N)9TGACC-3
) (18), was identified in the BRCA1 promoter
(Figs. 2 and 3). Upon
estrogen stimulation, the promoter activity of the ERE-containing
construct pGL7 was induced by 1.5-fold, while no change in activity was detected in the non-ERE-containing construct pGL6 (Fig.
4). This result implies that
BRCA1 promoter
is regulated by estrogen. For
BRCA1 promoter
, sequence homology search recognized a
putative AP1 site (Fig. 3), which is known to bind the cellular
proto-oncogene products c-Fos and c-Jun and has recently been shown to
mediate estrogen effects (19). As presented in Fig. 4, a 2-fold
increase in promoter activity of the pGL12 construct was observed upon estrogen induction, while no significant effect was seen for the pGL12R
construct. The magnitudes of increase in activities upon estrogen
stimulation in both BRCA1 promoters are in line with that
observed in other promoters mediated by the alternative ERE and the AP1
site, which ranged from 1.4- to 8-fold (18, 19).
Comparison of the Regulatory Region of the BRCA1 Gene in Humans and Mice
To gain insight into the biological function of the
BRCA1 gene in the human, several studies have been performed
using the mouse as an animal model (20-23). However the relevance of
expression and function of the BRCA1 gene in mice to that in
humans is, at present, unknown. To address this issue, we analyzed the
5 end of the mouse Brca1 gene. Dramatic differences in
genomic organization at the 5
-flanking region of the BRCA1
gene between the two species are observed (12) (Fig. 1). A fragment of
approximately 30 kilobase pairs of genomic DNA housing both the
NBR2 gene and a pseudocopy of the 5
end of the
BRCA1 gene found in humans is absent in mice. Since
eukaryotic transcriptional machinery functions in a chromatin environment, the difference in the chromosomal organization of the 5
end of the BRCA1 gene between humans and mice raises the possibility that the expression of the BRCA1 gene is
regulated differently. Furthermore, alignment of the 5
sequence of the BRCA1 gene from the human and the mouse revealed that the
human exon 1B is not conserved in the mouse (GenBankTM number U73040), which is further supported by the fact that exon 1B of the human BRCA1 transcript
contains the primate-specific
Alu sequence. This result indicates there is no mouse
homologue of the human BRCA1 transcript
, implying that
the mouse Brca1 gene is unlikely to be regulated by two
promoters. Last, sequence homology comparison between the
cis-control elements in the promoter regions of the human
and mouse BRCA1 genes revealed that only a SP1 binding site and a CCAAT box are conserved (Fig. 3). In particular, neither the ERE
nor the AP1 site is found in the 5
-flanking region of the mouse
Brca1 gene, indicating that the regulation of the
BRCA1 levels by estrogen is unlikely to be the same across
species. Therefore for both genomic organization and immediate
cis-control elements in the BRCA1 promoters,
significant differences have been found between humans and mice.
This study analyzed the BRCA1 promoters in detail and demonstrates that the expression of the human BRCA1 gene is under the control of two promoters, one of which is bi-directional. The different transcripts may have distinct biological functions, and the maintenance of a correct ratio between them may be important for normal function. Further studies aimed at determining the proportion of the distinct BRCA1/NBR2 transcripts to each other in the growth and/or differentiation of human breast epithelial cells and in mammary tumorigenesis should shed light on the function of both genes.
We have also studied the regulation of the BRCA1 promoter
activity by estrogen. Evidence for estrogen regulation of BRCA1 first
came from studies in the mouse where increased BRCA1
expression was observed in mammary gland during puberty, pregnancy, and
lactation and following treatment of ovariectomized animals with
estrogen and progesterone (23, 24). Consistent with these observations, Gudas et al. (15) reported that estrogen induces
BRCA1 expression in ER-positive breast cancer cell lines.
Our data show that both BRCA1 promoters and
are
responsive to estrogen stimulation, albeit being a less pronounced
effect than that observed for a classic perfect ERE (17). We detected a
newly described alternative ERE (18) in BRCA1 promoter
(Figs. 2 and 3). It is therefore possible that the estrogen stimulation
effect seen in promoter
is mediated by the classical ER pathway,
where estrogen-bound ER interacts with DNA and subsequently interacts
with the basic transcription machinery to stimulate transcription. In
contrast, no conventional ERE was detected in BRCA1 promoter
, suggesting that more complicated mechanisms may underlie the
estrogen regulation of this promoter. It has been proposed that
estrogen·ER complex can activate transcription directly through the
AP-1 motif (19, 25, 26). In BRCA1 promoter
, a putative
AP-1 site is detected. Estrogen may therefore regulate BRCA1
expression via protein-protein interaction between ER and the
c-Fos·c-Jun complex at the AP1 site. Alternatively, the estrogen
activation of the BRCA1 promoter
may be indirect,
secondary to hormone-induced synthesis of other transcription factors,
which in turn transactivate the BRCA1 promoter
. This
hypothesis is supported by the observation that estrogen regulation of
BRCA1 expression is blocked by cyclohexamide (27), suggesting de novo protein synthesis is required for
estrogen-induced BRCA1 expression. However, these
investigators failed to detect any response to estrogen induction using
genomic fragments near the 5
end of the BRCA1 gene (27).
Neither of the two promoter constructs made by these authors contains
the transcription initiation sites of the BRCA1 gene;
therefore no direct comparison to our results can be made.
Through targeted germline inactivation, mice have provided a valuable animal model to study the role of tumor suppressor genes in normal growth control pathways and in human cancer. For the BRCA1 gene, however, contrasting data regarding the function of the encoded protein in development and in mammary tumorigenesis have been documented between mice and humans. Brca1-null mice die during early embryogenesis (20-22), suggesting that Brca1 is indispensable for normal cell growth and differentiation during murine embryonic development. This is in contrast with the human phenotype observed, in which a woman homozygous for a BRCA1 mutation is normal in growth and development with the only mutation-associated phenotype being a predisposition to breast/ovarian cancer (28). In addition, mice heterozygous for the Brca1 deletion were phenotypically normal and did not develop any type of cancer at least by 1 year of age, whereas humans heterozygous for BRCA1 mutations are susceptible to early onset breast and/ovarian cancer. We demonstrate that both the genomic organization and the immediate cis-control elements of the BRCA1 gene are significantly different between humans and mice. This implies that the temporal and spatial expression pattern of the BRCA1 gene may differ between the two species, and caution needs to be exercised when interpreting experiments involving either endogenous murine BRCA1 expression or transgenic models of BRCA1 function.