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Proc. Natl. Acad. Sci. USA
Vol. 93, pp. 13595–13599, November 1996
Biochemistry
Evidence for a transcriptional activation function of BRCA1
C-terminal region
(breast cancer
y
tumor suppressor gene)
ALVARO N. A. MONTEIRO*, AVERY AUGUST*, AND HIDESABURO HANAFUSA†
Laboratory of Molecular Oncology, The Rockefeller University, New York, NY 10021
Contributed by Hidesaburo Hanafusa, September 11, 1996
ABSTRACT Mutations in BRCA1 account for 45% of
families with high incidence of breast cancer and for 80–90%
of families with both breast and ovarian cancer. BRCA1
protein includes an amino-terminal zinc finger motif as well
as an excess of negatively charged amino acids near the C
terminus. In addition, BRCA1 contains two nuclear localiza-
tion signals and localizes to the nucleus of normal cells. While
these features suggest a role in transcriptional regulation, no
function has been assigned to BRCA1. Here, we show that the
C-terminal region, comprising exons 16–24 (aa 1560–1863) of
BRCA1 fused to GAL4 DNA binding domain can activate
transcription both in yeast and mammalian cells. Further-
more, we define the region comprising exons 21–24 (aa
1760–1863) as the minimal transactivation domain. Any one
of four germ-line mutations in the C-terminal region found in
patients with breast or ovarian cancer (Ala-1708 3Glu,
Gln-1756 C1, Met-1775 3Arg, Tyr-1853 3Stop), had
markedly impaired transcription activity. Together these data
underscore the notion that one of the functions of BRCA1 may
be the regulation of transcription.
Breast cancer is one of the most common diseases affecting
women in the United States. Genetic factors contribute to an
estimated 5% of all breast cancer cases, and up to 36% of the
cases diagnosed before age 30 (1). In 1990, human BRCA1 was
mapped by genetic linkage to the long arm of chromosome 17
(2), firmly establishing the existence of a breast cancer sus-
ceptibility gene. Mutations in BRCA1 alone account for '45%
of the families with high incidence of breast cancer and 80%
of families with high incidence of both breast and ovarian
cancer (3). Identification of human BRCA1 by positional
cloning techniques revealed an ORF coding for 1863 aa (4). No
homology with any known protein was identified with the
exception of a zinc finger domain located in its N-terminal
region. This zinc binding RING finger domain (C3HC4) is
found in several proteins that have their functions mediated
through DNA binding (5). Other features in the sequence were
also recognized, including two putative nuclear localization
signals (aa 500–508 and 609– 615), a leucine zipper (aa 1209–
1231), and an excess of negative charged residues in the
C-terminal region of BRCA1. In many eukaryotic transcrip-
tion activators, the presence of an acidic region in the C
terminus correlates with the transactivation domain (6). With
the exception of the leucine zipper, which is imperfectly
conserved, the other features are highly conserved in human
and mouse Brca1, suggesting that these regions might be
significant for its functions (7–9). Although reports have been
published showing that BRCA1 localizes to the cytoplasm and
secretory granules (10, 11), other investigators have shown that
BRCA1 localizes to the nucleus of normal cells (12, 13). Taken
together these features suggest a function of BRCA1 in
transcription activation.
We have investigated whether the C-terminal region of
BRCA1 is able to activate transcription. We found that
BRCA1 C terminal can act as a transactivation domain when
fused to a heterologous DNA binding domain, and that
germ-line mutations found in patients impair this activity,
suggesting that loss of transcription activation function by
BRCA1 may predispose the carriers to cancer.
MATERIALS AND METHODS
Yeast Strains. Two Saccharomyces cerevisiae strains, HF7c
[MATa, ura3-52, his3-200, lys2-801, ade2-101, trp1-901, leu2-3,
112, gal4-542, gal80-538, LYS::GAL1-HIS3, URA3::(GAL4 17-
mers)
3
-CYC1-lacZ] (14) and SFY526 (MATa, ura3-52, his3-
200, lys2-801, ade2-101, trp1-901, leu2-3, 112, can
r
, gal4-542,
gal80-538, URA3::GAL1-lacZ) (15), were used (CLON-
TECH). HF7c has a HIS3 reporter gene under the control of
the GAL1 upstream activating sequence (UAS), responsive to
GAL4 transcriptional activation. The HF7c transformants
were inoculated in liquid medium lacking either tryptophan or
tryptophan and histidine. The vectors used for expression
confer growth in the absence of tryptophan (see below). If the
fusion proteins activate transcription, yeast transformants are
able to grow in medium lacking histidine. HF7c also has a lacZ
reporter gene under the control of GAL4 17-mers UAS,
responsive to GAL4 transcriptional activation. The SFY526
strain has lacZ under the control of GAL1 UAS. If the fusion
proteins activate transcription, yeast transformants will pro-
duce
b
-galactosidase.
Yeast Expression Constructs. All BRCA1 fragments were
amplified by PCR using the plasmid F3 (a kind gift from
Robert Bogden, Myriad Genetics, Salt Lake City) as a tem-
plate, which contains the C-terminal domain of human BRCA1
(4). The following oligonucleotide primers were used: exons
16–24 (S9503101 and Z9503098, both containing an EcoRI
site 59and a BamHI site 39), exons 16–23 (S9503101 and L23),
exons 16–22 (S9503101 and LX22), exons 16–21 (S9503101
and Z9403097) and exons 22–24 (Z9503100 and Z9503098),
exons 21–24 (S9600330 and Z9503098), exons 21–22 (S9600330
and LX22) (S9503101, 59-CGGAATTCGAGGGAACCCCT-
TACCTG-39; Z9503098, 59-GCGGATCCGTAGTGGCTGT-
GGGGGAT-39; L23, 59-GCGGATCCTGCATGGAAGC-
CATTGTCC-39; LX22, 59-CGGGATCCACCTGTGC-
CAAGG-39; Z9403097, 59-GCGGATCCATCTGTGGGC-
ATGTTGGT-39; Z9503100, 59-CGGAATTCCAACTGGAAT-
GGATGGTA-39; S9600330, 59-CGGAATTCCAGGACAGA-
AAGATC-39). PCR products were gel purified, digested with
BamHI and EcoRI, and ligated into the yeast expression vector
pGBT9 (CLONTECH) (16) similarly digested. Fragments were
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked ‘‘advertisement’’ in
accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviations: X-Gal, 5-bromo-4-chloro-3-indolyl
b
-D-galctopyrano-
side; SD, synthetic medium plus dextrose.
*A.N.A.M. and A.A. contributed equally to this work.
†To whom reprint requests should be addressed at: Laboratory of
Molecular Oncology, Box 169, The Rockefeller University, 1230
York Avenue, New York, NY 10021.
13595
ligated in frame to the GAL4 DNA binding domain. The junc-
tions were sequenced to verify the reading frame. pGBT9 has
TRP1 as a selectable marker, allowing growth in the absence of
tryptophan. For the TA 16y24, the construct 16y24 pGBT9 was
digested with BamHI and EcoRI, and the insert subcloned
in-frame to the GAL4 transactivation domain in pGAD424
(CLONTECH) (16) similarly digested. pGAD424 has LEU2 as a
selectable marker, allowing growth in the absence of leucine.
Mutations were made using splicing by overlap extension
PCR (17) with the following oligonucleotide primers: for the
Ala-1708 3Glu mutation S9503101 and LAE; UAE and
Z9503098 in two separate reactions. Both products were
combined and used as a template for a final round of PCR
using S9503101 and Z9503098. All subsequent mutations used
the same oligonucleotides (S9503101 and Z9503098) for the
final round of PCR. For the first round of PCR the following
oligonucleotides were used: for the Gln-1756 C1mutation
Z9600205 and S9503101; Z9600204 and Z9503098. For the
Met-1775 3Arg mutation S9503101 and S9600207; S9600206
and Z9503098. For the Met-1775 3Lys, Met-1775 3Thr,
Met-1775 3Glu, and Met-1775 3Val the S9600207 was
replaced by primers LMK, LMT, LME, and LMV, respec-
tively; and S9600206 was replaced by primers UMK, UMT,
UME, and UMV, respectively. The Tyr-1853 3Stop mutation
was obtained by direct PCR using S9503101 and L1853 (con-
taining a BamHI site) (LAE, 59-CCCATTTTCCTCCCT-
CAATTCCTAGAAAA-39; UAE, 59-TTTTCTAGGAATT-
GAGGGAGGAAAATGGG-39; Z9600205, 59-GATCTT-
TCTGTCGCTGGGATTCTCTTGCTC-39; Z9600204, 59-
GAGCAAGAGAATCCCAGCGACAGAAAGATC-39;
S9600207, 59-CAGTTGATCTGTGGGCCTGTTGGT-
GAAGG-39; S9600206, 59-CCTTCACCAACAGGCCCACA-
GATCAACTG-39; LMK, 59-CAGTTGATCTGTGGGCTT-
GTTGGTGAAGG-39; UMK, 59-CCTTCACCAACAAGC-
CCACAGATCAACTG-39; LMT, 59-CAGTTGATCTGTG-
GGCGTGTTGGTGAAGG-39; UMT, 59-CCTTCACCAAC-
ACGCCCACAGATCAACTG-39; LME, 59-CAGTTGATCT-
GTGGGCTCGTTGGTGAAGG-39; UME, 59-CCTTCAC-
CAACGAGCCCACAGATCAACTG-39; LMV, 59-CAGTT-
GATCTGTGGGCACGTTGGTGAAGG-39; UMV, 59-
CCTTCACCAACGTGCCCACAGATCAACTG-39; L1853,
59-GCGGATCCAGGTTAGGTGTCC-39). The constructs
were sequenced to confirm the mutations.
Yeast Transformation. Competent yeast cells were obtained
using the yeast transformation system (CLONTECH) based on
lithium acetate, and cells were transformed according to
manufacturer’s instructions.
Interference Assay. Levels of expression were assessed by
transforming the HF7c strain with pCL1 (wild-type full-length
GAL4) (18) and measuring reduction in transcription activa-
tion by pCL1 upon cotransformation with the pGBT9-based
constructs. Three independent clones from each construct
were assayed for
b
-galactosidase production in liquid culture
as described (19).
Growth Curves. HF7c transformants containing different
pGBT9 constructs were grown overnight in synthetic medium
plus dextrose (SD medium) lacking tryptophan. The saturated
cultures were used to inoculate fresh medium lacking trypto-
phan or tryptophan and histidine to an initial OD
600
of 0.0002.
Cultures were grown at 308C in a shaker and the optical density
was measured at different time intervals starting at 12 hr, then
every 4 hr up to 36 hr after inoculation.
Growth on Solid Medium. HF7c transformants were
streaked on solid SDyagar medium lacking tryptophan or
tryptophan and histidine. Growth was scored after 2 days.
Filter
b
-Galactosidase Assay. SFY526 and HF7c transfor-
mants were streaked on a filter overlaid on top of medium
lacking tryptophan and allowed to grow overnight. Cells
growing on the filter were lysed by freeze thawing in liquid
nitrogen, and each filter was incubated in 2.5 ml of Z buffer
(16.1 g/liter Na
2
HPO
4
z7H
2
Oy5.5 g/liter NaH
2
PO
4
zH
2
Oy0.75
g/liter KCly0.246 g/liter MgSO
4
z7H
2
O, pH 7.0) containing 40
m
l of 5-bromo-4-chloro-3-indolyl
b
-D-galctopyranoside (X-
Gal) solution (20 mgyml of X-Gal in N,N-dimethylformamide)
at 308C for up to 24 hr.
Mammalian Expression Constructs: pGBT9-based con-
structs containing the wild-type 16y24 sequences as well as the
mutants were digested with EcoRI and XbaI, and the frag-
ments including vector sequences downstream of the insert
were ligated into similarly digested pSG424 (20) (a gift of Mark
Ptashne, Harvard University). The fragments were subcloned
in frame to GAL4 binding domain similar to the yeast expres-
sion vectors.
Transient Transfections. Cos-7 cells were grown until con-
fluent in DMEM (GIBCOyBRL) supplemented with 10%
fetal bovine serum. Cells were transfected using Lipo-
fectamine (GIBCOyBRL). Briefly, confluent cells were
washed with OptiMEM (GIBCOyBRL) and overlaid with a
mixture containing the following plasmids and Lipofectamine:
0.4
m
g of pSG424 or pSG424 containing 16y24 wild-type,
16y24 (Gln-1756 C1) and 16y24 (Met-1775 3Arg), all
together with 1
m
g of reporter pG5E1bLuc (a gift of Roger
Davis, University of Massachusetts) (21). Cells were exposed
to the transfection mixture for 4 hr, then washed and subjected
to a 2-min 15% glycerol shock. After the shock, fresh medium
was added and cells were harvested 24 and 48 hr after
transfection. Cells were lysed and extracts were assayed for
luciferase activity as described (21).
RESULTS
Transcription Activation by BRCA1 C-Terminal Region.
The vast majority of reported mutations in BRCA1 lead
invariably to truncation of the C-terminal region. The few
missense mutations reported localize either to the zinc finger
motif at the N terminus or to the C-terminal acidic region (22).
Moreover, the C-terminal region includes a highly conserved
stretch between mouse and human BRCA1 (aa 1636 –1795) (8).
We therefore investigated whether the region of a reported
cluster of mutations adjacent to the C-terminal end (22),
comprising exons 16–24 (aa 1560–1863), is involved in tran-
scriptional activation.
Because BRCA1 DNA binding consensus has not been
defined and increasing evidence suggests that many cellular
processes, including transcription, are conserved among eu-
karyotic species, a yeast GAL4 DNA binding domain fusion
system was used (16). We fused the GAL4 DNA binding
domain with human BRCA1 fragments containing either
exons 16y24, 16y23, 16y22, 16y21, 22y24, 21y24, or 21y22. The
16y24 fragment fused to transactivation domain of GAL4,
which is unable to bind DNA (TA-16y24) was used as a
negative control (Fig. 1). These constructs were transformed
into yeast and ability to activate the reporter genes was tested.
As shown in Fig. 2, only the fusions 16y24 and 21y24 were able
to activate transcription of the HIS3 reporter gene. The 21y24
construct however, activated transcription somewhat less ef-
ficiently than the larger 16y24. Scoring HF7c transformants for
growth on solid media gave identical results (Table 1). In
agreement with the results obtained w ith HF7c, only 16y24 and
21y24 were able to activate transcription of lacZ in SFY526
(Table 1). No construct was able to activate
b
-galactosidase
production in HF7c cells (data not shown). The 16y24 con-
struct is thus able to function as a transactivation domain and
sequences contained within exons 21 to 24 of BRCA1 have
sufficient information for activation of transcription, although
sequences contained in adjacent exons may enhance this
activity.
Germ-Line Mutations Impair Transcription Activation. It
has been shown that retroviral transfer of wild-type BRCA1
gene inhibits growth in vitro of breast and ovarian cancer cell
13596 Biochemistry: Monteiro et al. Proc. Natl. Acad. Sci. USA 93 (1996)
lines, thus establishing that BRCA1 acts as a tumor suppressor
(23). Loss or reduction of function of BRCA1 would therefore
confer susceptibility to tumor progression. The fact that a
portion of BRCA1 protein was able to drive expression of the
reporter genes raised the possibility that impairment of this
function could be related to the development of breast and
ovarian cancer.
To test this hypothesis directly we introduced four mutations
reported in afflicted patients, generating the constructs 16y24
(Ala-1708 3Glu, Gln-1756 C1, Met-1775 3Arg, and
Tyr-1853 3Stop) shown in Fig. 1. Ala-1708 3Glu is
associated with very early onset of breast cancer (24). Gln-1756
C1is an insertion of a cytosine in codon 1756 resulting in a
frameshift and is the most frequent mutation found in the
C-terminal (4, 25–29). Met-1775 3Arg is the only recurring
missense mutation in this region (4, 22, 24, 26). Moreover, this
residue lies in a highly conserved stretch between mouse and
human BRCA1 (8). Tyr-1853 3Stop is an insertion of an
adenine in codon 1853 leading to a truncated protein only 11
aa from the C-terminal end that is associated with very
early-onset breast cancer (30). None of the constructs carrying
mutations found in patients was able to activate transcription
of the HIS3 promoter in HF7c (Fig. 3), nor lacZ expression in
SFY526 (Table 1). The difference in activation between the
wild type and the mutants cannot be accounted for different
levels in expression and stability as all constructs were ex-
pressed at roughly the same levels as measured by interference
assays (data not shown).
Transcription Activation in Mammalian Cells. To rule out
the possibility that BRCA1 activates transcription only in the
yeast system, we examined the transcriptional activity of the
wild-type 16y24 and two mutants (Gln-1756 C1and Met-1775
3Arg) using a mammalian cell line, Cos-7. In this system we
used GAL4 DNA binding domain fusions (20) and the lucif-
erase gene as a reporter under the control of GAL4 responsive
elements (21). In Fig. 4 the wild-type fusion activates tran-
scription 10-fold, while the mutants were not able to activate
transcription to a detectable level. Therefore, BRCA1 tran-
scriptional activation function is not restricted to the yeast
system. Similarly to the experiments performed in yeast, the
difference cannot be accounted for different levels of expres-
sion, since all constructs were expressed, with the Met-1775 3
Arg expressed at higher levels than the others, as determined
by Western blotting (data not shown).
FIG. 1. Structure of fusion proteins. Fragments 16y24, 16y23,
16y22, 16y21, 21y24, 22y24, and 21y22 contain exons 16 –24 (aa
1560–1863, top bar), 16 –23, 16–22, 16–21, 21–24, 22–24, and 21–22,
respectively, of the BRCA1 gene fused to the DNA binding domain of
GAL4 (black box). Fragments 16y24 (Gln-1756 C1) and 16y24
(Tyr-1853 3Stop) carry insertions reported to truncate BRCA1.
Fragment 16y24 (Ala-1708 3Glu) is a substitution reported in
patients. Fragments 16y24 (Met-1775 3Lys, Met-1775 3Thr,
Met-1775 3Arg, Met-1775 3Glu, and Met-1775 3Val) are each
individual mutations at codon 1775, with the Met-1775 3Arg mutant
reported in patients with breast cancer. TA 16y24 represents exons
16–24 fused to the activation domain of GAL4. Arrowheads indicate
the positions of the mutations, and shaded areas represent truncated
parts of the mutant protein.
FIG. 2. Transcriptional activation by BRCA1 C terminus. Growth
curves of S. cerevisiae (HF7c) carrying the following fragments of
BRCA1 in pGBT9 grown in medium lacking tryptophan and histidine:
16y24 (
M
), 16y23 (
m
), 16y22 (
E
), 16y21 (
●
), 21y24 (
Ç
), 22y24 (
å
), and
21y24 (
✕
). (Inset) Growth in medium lacking tryptophan only. OD
600
of the cultures (yaxis) is plotted against time in culture (xaxis).
Table 1. Transcriptional activation by BRCA1 C terminus
HF7c SFY526
b
-gal‡COS-7
luciferaseLiquid* Solid†
Wild type
16y24 1(1.0) 111(1.0)
16y23 2(0.0) 22 ND
16y22 2(0.0) 22 ND
16y21 2(0.0) 22 ND
21y24 1(0.6) 11 ND
22y24 2(0.0) 22 ND
21y22 2(0.0) 22 ND
16y24-TA ND 22 ND
16y24 mutants
Ala-1708 3Glu 2(0.0) 22 ND
Gln-1756 C12(0.0) 222(0.0)
Met-1775 3Arg 2(0.0) 222(0.0)
Tyr-1853 3Stop 2(0.0) 22 ND
Met-1775 3Lys 2(0.0) 22 ND
Met-1775 3Val 1(1.0) 11 ND
Met-1775 3Thr 2(0.0) 21 ND
Met-1775 3Glu 2(0.0) 22 ND
ND, not determined.
*Cultures were grown as described in Materials and Methods. Relative
activity shown in parenthesis.
†Several independent colonies were streaked on SD agar lacking
tryptophan and histidine and scored for growth after 3 days at 308C.
‡Several independent clones were assayed for
b
-galactosidase (
b
-gal)
production on filters. To ensure that the negatives were not producing
b
-galactosidase, the assay was also scored after 18 h with the same
results.
Biochemistry: Monteiro et al. Proc. Natl. Acad. Sci. USA 93 (1996) 13597
Hydrophobic Residues Are Major Determinants. Acidic
regions of proteins that are not necessarily transcription acti-
vators can sometimes activate transcription when fused to
heterologous DNA binding domains (31). To exclude this
possibility in our assay using BRCA1 C terminus, we intro-
duced a negative charge at position 1775 (Met-1775 3Glu).
As shown in Table 1 this construct was not able to activate
transcription, eliminating the possibility that a nonspecific
cluster of negative charges in BRCA1 was spuriously activating
transcription.
As has been suggested in other acidic domain-containing
transcription factors, addition of positively charged residues
irrespective of the position results in impairment of the
transcriptional activation function (32). The mutant Met-1775
3Arg described above is a change from a hydrophobic to that
of a positively charged residue, which resulted in drastically
impaired activity of BRCA1 C terminus. We examined the
effects of three other possible mutations at codon 1775: from
the hydrophobic methionine to (i) a positively charged residue
change similar to that seen in the reported patients (Met-1775
3Lys), (ii) a polar residue (Met-1775 3Thr), and (iii)a
hydrophobic residue (Met-1775 3Val), for both growth in
liquid and solid media, as well as in
b
-galactosidase assays.
Changing the methionine at position 1775 to a conservative
residue valine, resulted in wild-type transcriptional activity,
while a change to a positively charged residue (Met-1775 3
Lys) or to a polar residue (Met-1775 3Thr) drastically
diminished transcriptional activation by BRCA1 C terminus
(Table 1). Interestingly, the construct carrying the Met-1775 3
Thr mutation does not change the net charge of the molecule
and is still inactive in HF7c. These data suggest that BRCA1
may function analogously to herpes virus protein VP16 where
negative charges are not the only major determinants but that
hydrophobic residues are also crucial for activity (33).
DISCUSSION
The presence of an acidic region, nuclear localization signals
and a zinc finger domain suggest that BRCA1 may regulate
transcription (4). By using fusion proteins with a heterologous
DNA binding domain (GAL4), we show that the C-terminal
portion of BRCA1 (aa 1560–1863) is able to activate tran-
scription of the HIS3 gene in HF7c (but not lacZ), the lacZ
gene in SFY526, as well as the luciferase gene in mammalian
cells, all driven by GAL4 responsive elements. Although HIS3
and lacZ in HF7c share GAL4-responsive elements, the rest of
the promoter sequences are different (14, 15). The lack of
transcriptional activation of lacZ in HF7c may therefore
reflect the fact that the ability of transcriptional activators to
interact with the basal transcription apparatus varies with
different promoter contexts (34, 35).
Our results also indicate that the smaller C-terminal frag-
ment 21y24 (aa 1760–1863) is also able to activate transcrip-
tion, although less efficiently than the larger fragment 16y24
(aa 1560–1863). Therefore, exons 21y24 have sufficient infor-
mation to activate transcription and represent the minimal
transactivation domain of BRCA1. Moreover, adjacent exons,
16 to 20, contribute to full activity. Interestingly, most muta-
tions found in patients generate proteins lacking all or part of
the minimal transactivation domain defined above. According
to our data, truncated proteins lacking even small regions of
the minimal transactivation domain (e.g., Tyr-1853 3Stop)
would be predicted to have no function in transcriptional
activation, a situation borne out by our data.
While the wild-type BRCA1 C terminus fused to a heter-
ologous DNA binding protein activates transcription, the
proteins carrying missense or nonsense mutations found in
patients with tumors fail to do so. The fact that fusion proteins
carrying mutations known to confer breast cancer susceptibil-
ity abolish activity strongly supports the idea that BRCA1
protein is involved in transcription. We tested four reported
mutations (Ala-1708 3Glu, Gln-1756 C1, Met-1775 3Arg,
and Tyr-1853 3Stop) with association with disease (4, 22,
24–29). Thus we suggest that mutations in BRCA1 which
impair the ability to activate transcription may predispose the
carrier to tumors. These results indicate that BRCA1 may
function to activate transcription of genes involved in sup-
pressing transformation.
Interestingly, a mutation that falls outside the minimal
transactivation domain also abolished function (Ala-1708 3
Glu). The observation that a construct lacking this region is
active suggests that it may be involved in proper folding of the
C-terminal region, which may either stabilize or destabilize the
structure. When the region is deleted, the conformation may
be conserved as in the wild type. An alternative explanation is
that this amino acid is involved in nonessential sites of protein–
protein interactions and that absence of these sites or nondis-
ruptive mutations allow interaction. Other mutations such as
FIG. 3. Mutations occurring in patients impair activity. Shown are
growth curves of S. cerevisiae (HF7c) carrying the following fragments
and mutants in pGBT9 grown in medium lacking tryptophan and
histidine: 16y24 (
M
), 16y24 (Ala-1708 3Glu) (
å
), 16y24 (Gln-1756
C1)(
E
), 16y24 (Met-1775 3Arg) (
●
), and 16y24 (Tyr-1853 3Stop)
(
✕
). (Inset) Growth in medium lacking tryptophan only. The OD
600
of
the cultures (yaxis) is plotted against time in culture (xaxis).
FIG. 4. Transcription activation by BRCA1 C terminus in mam-
malian cells. Luciferase activity of the following fragments and mu-
tants in pSG424, transfected along with luciferase reporter plasmid
into Cos-7 cells plotted as fold activation over control: vector, pSG424
alone; 16y24, wild-type exons 16y24; 16y24 (Met-1775 3Arg),
Met-1775 3Arg mutation; 16y24 (Gln-1756 C1), cytosine insertion
at position 1756. Cells were harvested at 24 hr (black bars) and 48 hr
(shaded bars).
13598 Biochemistry: Monteiro et al. Proc. Natl. Acad. Sci. USA 93 (1996)
Ala-1708 3Glu on the other hand, may destabilize this
interaction.
During the preparation of this manuscript, Chapman and
Verma (36) reported similar results. Using a mammalian
transfection system the authors defined the fragment 16y24 as
a transactivation domain and suggested that the fragment
21y24 represents the minimal transactivation domain, in
agreement with our data. Taken together, these two studies
analyze five different mutations of BRCA1, and all the mu-
tations which have shown disease association have impaired
ability to activate transcription. We believe that a detailed
analysis of BRCA1 C-terminal region may be instrumental to
understanding the etiology of familial breast cancer.
The simple assay used here, testing the ability of the BRCA1
fusion proteins to activate transcription in yeast is a direct way
to assess the functional significance of documented mutations
that localize to the transactivation domain. Furthermore, our
system could be instrumental in discriminating between cancer
predisposing mutations and neutral polymorphisms. The assay
could also be used to screen for therapeutic compounds
capable of restoring the normal function to the mutant pro-
teins.
Definite confirmation that BRCA1 acts as a transcription
activator, however, must await the demonstration of its DNA
binding activity or identification of its target genes.
We thank Alison Egan for help with the constructs. We also thank
Robert Roeder, Alexander Hoffmann, and Curt Horvath for helpful
discussions and Shinya Tanaka and Raymond Birge for critical com-
ments on the manuscript. A.N.A.M. is a Pew Fellow in the Biomedical
Sciences and on leave from The Institute of Chemistry, Federal
University of Rio de Janeiro. A.A. is a recipient of a post-doctoral
fellowship from the National Science Foundation. This work was
supported by Grant CA44356 from the National Cancer Institute.
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