 |
INTRODUCTION |
The BRCA1 gene encodes a breast and ovarian-specific tumor
suppressor protein (1, 2), but how the protein functions remains
unclear. Transfection of DNA, which overexpresses full-length BRCA1
protein, results in cell cycle arrest via activation of the p21
promoter (3), and data suggest that the BRCA1 protein is a regulatory
component in the pathway by which the p53 protein regulates the cell
cycle (4, 5). BRCA1 is a large 220-kDa protein that may be divided into
several functional and structural domains. A domain in the middle of
the protein has been shown to associate with the recombination repair
factor RAD51 (6). The amino-terminal 100-amino acid residues bind
BARD1, a protein of unknown function (7). The carboxyl-terminal
300-amino acid domain has been associated with the activation of
transcription. For example, when the BRCA1 carboxyl terminus is fused
to the GAL4-DNA binding domain and transfected into cells, it activates transcription of GAL4 site-dependent promoters (8-10).
BRCA1 has been identified as a component of the RNA polymerase II
holoenzyme (11, 12). Pol II1
exists in at least two forms in the cell: core polymerase and the Pol
II holoenzyme (Ref. 13, reviewed in Refs. 14 and 15). The core
polymerase contains 10 to 12 subunits and a total molecular mass of 500 kDa. The holoenzyme form of Pol II, whose mass is estimated in the
multi-megadalton range, contains in addition to Pol II, basal
transcription factors, factors bound to the carboxyl-terminal domain of
the largest Pol II subunit known as SRB (suppressors of
RNA polymerase B mutations) proteins, and
regulatory proteins such as BRCA1 and CBP (11-14, 16-20). In HeLa
cells in culture, the interaction of BRCA1 carboxyl terminus with the
holoenzyme is essential for its function as a transcriptional activator
(10).
This study establishes optimal conditions under which the BRCA1
carboxyl terminus functions as a transcriptional activator. The
regulation of transcription in vitro with purified basal
transcription factors and core Pol II is dependent upon a set of
factors collectively known as coactivators (21, 22). Under certain
conditions, some of these coactivators boost basal transcription, for
example the positive components PC1 through PC5, whereas other factors may repress basal transcription, for example the negative components NC1 and NC2 (23). High mobility group protein 2 (HMG2) is another coactivator (24). The topology of the DNA templates is yet another parameter in transcription with profound effects on the basal transcription process, making some basal factors nonessential (25).
Some of these coactivators have specific effects on DNA topology. For
example, HMG2 binds to bent DNA (26), and PC4 binds to single-stranded
DNA (27, 28).
To better understand how BRCA1 protein regulates gene expression, we
established an in vitro transcriptional activation assay using the GAL4 DNA binding domain fused to the BRCA1 carboxyl terminus.
This is the first in vitro activity that can be ascribed to
a domain from BRCA1. Moreover we find that GAL4-BRCA1 activates transcription with core Pol II, suggesting a second, non-holoenzyme pathway by which BRCA1 regulates gene expression. It is this second pathway that is the subject of these experiments. The function of
GAL4-BRCA1 as an activator is now shown to be highly dependent upon
specific coactivator components and upon the negative superhelical topology of the template DNA. In this way, GAL4-BRCA1 functions markedly differently from the GAL4-VP16, the standard model activator for in vitro studies.
 |
MATERIALS AND METHODS |
Transcription Factors--
The transcription factors used in
this study were purified using established techniques. Recombinant
TFIIA was expressed in Escherichia coli using vectors that
expressed a fusion of the two larger subunits and the smallest subunit
of the human factor (gift of J. DeJong and R. G. Roeder; Refs. 29
and 30). The TFIIA subunits were separately purified under denaturing
conditions using nickel nitrilotriacetate matrix, and the subunits were
mixed at equimolar concentrations and renatured by dialysis. High
activity TFIIA complex, as determined by complex formation assay using TATA-binding protein and promoter DNA probe, was purified using a
BioScale Q2 column and a BioLogic high pressure chromatography system
(Bio-Rad). The expression and purification of TFIIB and TFIIE have been
described (25). TFIIF was expressed in E. coli using a
vector for each subunit (gift of Z.F. Burton) followed by column
chromatography of each subunit separately using a BioScale Q2 column.
Recombinant coactivator PC4 was expressed in E. coli (vector, gift of M. Meisterernst; Refs. 31 and 32) and purified by
heparin-agarose chromatography followed by gel filtration. The amount
of PC4 included in transcription reactions, 100 ng, was optimized by
titrating the factor and scoring for the ratio of transcription from
the stimulated template to that from the basal template when in the
presence of the GAL4 fusion factor. The two subunits of NC2 were
separately expressed in E. coli (vectors, gift of M. Meisterernst; Ref. 23) and purified by nickel nitrilotriacetate matrix
followed by chromatography on a BioScale S2 ion exchange column. The
activity for recombinant NC2 was determined using a complex formation
assay with TATA-binding protein and promoter DNA (33, 34). TFIID was
purified from a HeLa cell line carrying a FLAG-tagged TATA-binding
protein molecule using an established protocol (cell line, gift of
C. M. Chiang; Ref. 35). TFIIH was purified from BJA-B cells. Whole
cell extracts (36) of BJA-B cells were applied to Biorex70 matrix in
0.15 M potassium acetate, as described before (11, 12) and
washed in 0.6 M potassium acetate, and bound protein was
eluted from the matrix with 1.5 M potassium acetate. TFIIH
was then chromatographed using a BioScale Q2 column (Bio-Rad) in which
it was in the unbound fraction at 0.1 M KCl followed by a
BioScale S2 column and eluted at about 0.5 M KCl in a
linear gradient. The TFIIH prepared in this way was free of other
contaminating basal factor activities as determined by transcription
assay (data not shown). The core Pol II was immunopurified using an
established protocol (37).
The GAL4-VP16 construct used in this study was the same as developed by
Chasman et al. (38), and it contains the GAL4 DNA binding
domain sequences from amino acid residues 1-147 and the 90 carboxyl-terminal amino acids of the VP16 activator. This factor was
purified by chromatography on DEAE-Sepharose followed by a BioScale Q2
matrix, resulting in a pure 29-kDa factor at a concentration of 0.03 mg/ml. The GAL4-BRCA1 construct contains the GAL4 DNA binding domain
from amino acid residues 1-100; thus, it lacks the weak activation
domain present between residues 100-147. The BRCA1 sequences from
1560-1863 were fused directly to the carboxyl-terminal end of the GAL4
sequences. A hexahistidine tag was ligated to the amino-terminal end of
the GAL4 DNA binding domain. After expression of the fusion protein in
E. coli strain BL21(DE3), the protein was purified by metal
ion chromatography, and by analysis using SDS-polyacrylamide
electrophoresis gels stained with Coomassie Blue, the protein was pure
and at a concentration of 0.2 mg/ml.
Plasmid Templates--
G-less cassette construct G5-E4 contained
the adenoviral E4 promoter upstream of a DNA sequence in which there
were no guanines in the coding strand from the transcription start site
to 390 base pairs downstream. Upstream of the E4 promoter were five DNA binding elements that can be bound by the GAL4 protein (38). By
comparison to the pµ(-47)-(G-)-I template (25), using agarose gels
containing chloroquine it was found that the G5-E4 template preparation
contained on the average 20 superhelical turns in the negative
orientation (data not shown). All transcription reactions include an
internal basal control template, p
ML-200, consisting of a core
adenoviral major late promoter upstream of a shortened 210-base pair
G-less cassette (39).
Transcription Reactions--
Transcription reactions were
performed in 25-µl volumes containing 20 mM HEPES-NaOH,
pH 7.9, 20% glycerol, 1 mM EDTA, 5 mM MgCl2, 90 mM potassium acetate, 3 mM dithiotreitol, 4 µM ZnSO4, 0.2 mg/ml bovine serum albumin, 100 µM each ATP and UTP, 2.5 µM CTP, 50 µM 3'-OMe-GTP, and 30 ng of each
template. Proteins included were 100 ng of TFIIA, 60 ng of TFIIB, 4 ng
of TFIIE, 100 ng of TFIIF, 100 ng of Pol II, 0.5 µl of
immunoaffinity-purified fTFIID, and 1 µl of TFIIH fraction. As
indicated, the following coactivators were added: 100 ng of PC4, 50 ng
of HMG2, 100 ng of NC2. The reactions were incubated at 30 °C for 90 min, terminated by the addition of 0.2 ml of 7 M urea, 1%
sodium dodecyl sulfate, 10 mM EDTA, 0.35 M
ammonium acetate, 0.1 mg/ml tRNA, and a radioactive 550-nucleotide RNA
recovery control (not shown in figures). Reactions were extracted in
phenol, precipitated in ethanol as per standard procedures, and
subjected to electrophoresis on 6% polyacrylamide gels containing 8.3 M urea. Dried gels were exposed to film, generally for
16-24 h, and were quantified using a PhosphorImager. Activation was measured as the ratio of RNA product of the G5-E4 template to RNA
product of the
ML template (in the presence of activator protein),
all divided by the same ratio in a control reaction without activator.
 |
RESULTS |
GAL4-BRCA1 Is a Potent Activator of Transcription in Vitro--
To
establish an in vitro reaction that will allow the
dissection of BRCA1 protein function in the regulation of
transcription, a fusion protein encoding the DNA binding domain of the
GAL4 transcription factor and the BRCA1 carboxyl terminus (amino acids
1560-1863) was analyzed for the activation of transcription in
vitro using purified transcription factors and core Pol II. The
transcription reaction included a template containing five GAL4 DNA
binding sites upstream of the adenoviral E4 promoter, which produced a 390-nucleotide G-less RNA. As an internal control in all experiments, an additional template was added lacking GAL4 sites but containing a
minimal adenoviral major late promoter upstream of a 210-base pair
G-less cassette. The addition of the GAL4-BRCA1 protein to transcription reactions containing TFIIA, TFIIB, TFIID, TFIIE, TFIIF,
TFIIH, core Pol II, and coactivator PC4 resulted in a robust transcriptional activation of the G5-E4 template (Fig.
1). The level of activation even when
using very low amounts of GAL4-BRCA1 (2 ng/25-µl reaction) was as
high as we normally observe with other potent transcriptional
activators, such as GAL4-VP16 (Fig. 2),
and at high concentration, the activator squelched all transcription. As can be seen in the figure, even at low GAL4-BRCA1 concentration, the
protein inhibited transcription from the basal promoter, presumably by
squelching.

View larger version (44K):
[in this window]
[in a new window]
|
Fig. 1.
Transcriptional activation by BRCA1
carboxyl-terminal domain. In vitro transcription
reactions contained highly purified basal factors, coactivator PC4, and
the indicated volumes of GAL4-BRCA1. Transcription from the GAL4
site-dependent promoter, G5-E4, resulted in accumulation of
390-nucleotide RNA, and transcription from the basal control template
lacking GAL4 response elements, ML, resulted in accumulation of a
210-nucleotide RNA. Activation of transcription by GAL4-BRCA1 is
observed as the stimulated accumulation of RNA from the G5-E4 promoter
at low levels of BRCA1 addition. The amount of GAL4-BRCA1 protein
preparation added is indicated (µl/reaction).
|
|

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 2.
BRCA1 transcriptional activation is
condition-specific. A, comparison of DNA binding
activities of GAL4-BRCA1 and GAL4-VP16. A DNA fragment containing three
GAL4 DNA binding elements was used as a probe for complex formation
assays. The indicated volumes (µl) of recombinant factor used in each
assay are indicated at the top of the gel. The different DNA-protein
complexes are indicated. B, comparison of transcriptional
activation function of BRCA1 and VP16. All reactions contained TFIIA
and PC4 as well as the other basal factors. HMG2 was included in
reactions in lanes 5-8. GAL4-BRCA1 was included in
lanes 2 and 6. GAL4-VP16 was included in
lanes 4 and 8. The level of relative
transcriptional activation produced by a GAL4 fusion protein,
quantified using PhosphorImager analysis, is indicated at the
bottom.
|
|
To quantitatively compare the level of activation due to the BRCA1
carboxyl-terminal domain with that observed with the VP16 carboxyl-terminal domain, we normalized the protein preparations of
GAL4 fusions by their DNA binding activity using the electrophoretic mobility shift assay (Fig. 2A). The DNA probe encoded three
GAL4 binding sites, and occupation of one site resulted in a single band, whereas occupation of two sites results in two shifted bands, depending on which site was occupied. Occupation of three sites resulted in a single slowly migrating band. The GAL4-VP16 fusion protein containing only the carboxyl-terminal tail of the VP16 protein
is smaller than the GAL4-BRCA1 (29 kDa versus 59 kDa); thus,
the shifts occur at different positions. This analysis revealed that
the DNA binding activity for GAL4-BRCA1 at 0.1 µl (20 ng), as used in
Fig. 1, was slightly less than that with 0.5 µl (15 ng) from the
GAL4-VP16 preparation. Transcriptional activation by GAL4-VP16 is
optimal when using 1 µl (30 ng) of this pure preparation/standard transcription reaction, and in the following analysis, 1 µl of the
VP16 preparation was compared with 0.1 µl (20 ng) of the BRCA1 preparation. Thus, in all of the following comparisons, more GAL4-VP16 DNA binding activity was present than the GAL4-BRCA1 DNA binding activity.
PC4 Coactivator Facilitates Activation by
GAL4-BRCA1--
Transcriptional activation by GAL4-BRCA1 and by
GAL4-VP16 is dependent upon the addition of coactivators (Table
I), consistent with prior observations
(22). Direct comparison of the transcriptional activation by BRCA1 and
VP16 when in the presence of the PC4 coactivator and TFIIA revealed
that the activation due to BRCA1 was about 29-fold, and the activation
due to VP16 was about 6-fold (lanes 1-4, Fig.
2B). Thus, even when more DNA binding activity of the GAL4-VP16 protein was used, the BRCA1 fusion protein was more potent in
the activation of transcription. Activation by BRCA1 was
condition-specific. For example, the inclusion of HMG2 in the
transcription reactions resulted in a dramatic decrease in the
activation by BRCA1 and, conversely, an increase in the activation by
the model activator, VP16.
Different constellations of coactivators and TFIIA were assayed for
activation by GAL4-BRCA1 versus GAL4-VP16. As seen in Fig.
3, lanes 1-4, in the absence
of PC4 but in the presence of TFIIA and HMG2, the activation by BRCA1
is about the same as for VP16. The BRCA1 fusion protein reproducibly
caused a greater decrease in the basal control template than the VP16
fusion protein regardless of the coactivator present in the reaction;
thus, in Fig. 3 the activation ratios were equal even though the total
level of transcription of the G5-E4 template was higher when VP16 was
the activator. The NC2 coactivator was fairly neutral with regard to
coactivating either BRCA1 and VP16 and yielded lower levels of
coactivation than either PC4 and HMG2 (Fig. 3, lanes 5-8;
Table I).

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 3.
Transcriptional activation using HMG2 and NC2
as coactivators in the presence of TFIIA. All transcription
reactions included TFIIA and the other basal factors. Addition of HMG2
(lanes 1-4) or a combination of PC4 and NC2 (lanes
5-8) were tested in transcription activation assays. GAL4-BRCA1
was included in reactions analyzed in lanes 2 and
6, and GAL4-VP16 was included in reactions analyzed in
lanes 4 and 8. Transcriptional activation ratios
are indicated at the bottom.
|
|
Omission of TFIIA from the transcription reactions did not affect the
specificity of the BRCA1 and VP16 fusion proteins with regard to
activation when in the presence of either PC4 or HMG2 (Fig.
4). The levels of activation were
reproducibly lower, possibly because the basal reaction was repressed
to a lesser extent by the coactivators. Under some conditions, the
addition of TFIIA was required. For example, inclusion in reactions of
both PC4 and HMG2 resulted in a total repression of all transcription, which was reversed by the addition of TFIIA (Table I). The data in
Table I summarize the activation ratios by GAL4-BRCA1 and GAL4-VP16
observed with various combinations of coactivators and TFIIA. The
parameters such as basal factors, template concentrations, and salt
concentrations were as in Fig. 2B. What is most striking about the activation ratios is that neither activator is either consistently stronger or weaker. For example, when PC4 is the only
coactivator present, BRCA1 is more potent than VP16; however, with the
inclusion of HMG2, the relative strength of the activation is reversed,
with VP16 being more potent than BRCA1.

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 4.
TFIIA is not required for transcriptional
activation but does potentiate the activation response. All
reactions included the basal factors with the exception of TFIIA.
Included in reactions was PC4 (lanes 1-4) or HMG2
(lanes 5-8). GAL4-BRCA1 was included in lanes 2 and 6, and GAL4-VP16 was included in lanes 4 and
8.
|
|
Activation by BRCA1 Is Dependent upon Template DNA
Supercoiling--
The different coactivator specificities for BRCA1
and VP16 in otherwise identical reactions suggested the possibility
that part of their regulatory function was dependent upon different mechanisms of activation. Among the properties of the transcription reaction tested, the topology of the DNA template was found to have a
significant effect. The templates in Figs. 1-4 were all supercoiled plasmids. When the G5-E4 plasmid was linearized using a restriction endonuclease and used as a transcriptional template, the activation because of BRCA1 was sharply diminished (Fig.
5, lanes 4-6). The transcription from the
ML (supercoiled) internal control plasmid was
still repressed, yielding a net transcriptional activation for BRCA1
reduced from 26-fold to 11-fold. This loss in activation by BRCA1 was,
thus, because of a 3-fold reduction in the accumulation of RNA from the
G5-E4 template. Most of the residual activation was because of
repression of the basal control template by GAL4-BRCA1. For comparison,
the VP16 activation increased from 7-fold on the supercoiled G5-E4
template to 23-fold when the same DNA template had linear topology.
Negatively supercoiled plasmids in solution are in a dynamic
equilibrium between two states: 1) totally double-stranded with
superhelical turns in the negative direction and 2) partially unwound
with no superhelical turns. For example, the G5-E4 template averages
about 20 negatively supercoiled turns per plasmid. This template could
then have as many as 200 base pairs unwound at any given time, and some
of the time the promoter will be unwound. Negative supercoiling and
promoter unwinding are associated with increasing the level of
transcription (25, 40-42). In contrast, linear DNA templates have no
supercoils and, thus, have entirely double-stranded properties. The
levels of activation due to VP-16 are higher when using linear DNA
template (Fig. 5), suggesting that VP16 interacts more effectively than
does BRCA1 with the factors that generate single-stranded DNA at the
promoter site.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 5.
DNA topology differentially affects
transcriptional activation by BRCA1 and VP16. Transcription
reactions included all the basal factors, TFIIA, and PC4. Negatively
supercoiled G5-E4 template was included in reactions (lanes
1-3), and G5-E4 DNA that had been linearized using a restriction
endonuclease was included in reactions analyzed in lanes
4-6. GAL4-BRCA1 was included in lanes 2 and
5, and GAL4-VP16 was included in lanes 3 and
6. Activation ratios are indicated at the bottom and were
determined by comparison with the appropriate control reaction.
S.C., supercoiled.
|
|
TFIIH Concentration Is Critical for Optimal Activation by
BRCA1--
It had been shown that basal transcription on supercoiled
templates could be independent of TFIIH because of the presence of
single-stranded DNA over the promoter (25, 40-42). Furthermore, TFIIH
has helicase activity that can generate single-stranded DNA at the
promoter (43), and PC4 is a single-stranded DNA-binding protein (27,
28). Thus, the effect of varying the concentration of TFIIH was
investigated. As shown in Fig. 6, the
concentration of TFIIH was critical to the level of transcriptional
activation observed with the BRCA1 fusion protein. In contrast, the
TFIIH concentration had little effect on the RNA synthesized from the GAL4-dependent template in the presence of the VP16 fusion
protein. At low TFIIH concentration, basal transcription was low, and
the VP16 fusion protein was quite effective at stimulating
transcription from the G5-E4 promoter under these conditions,
suggesting that VP16 may recruit the TFIIH to the appropriate template
(Fig. 6, lane 3). The activation ratio was only 4-fold in
this reaction, because the RNA from the basal control template also was
increased. In contrast, the BRCA1 fusion did not activate transcription
with these conditions of low TFIIH concentration (Fig. 6, lane
2). When using the standard amount of TFIIH, just as was observed in Fig. 2B, the BRCA1 fusion protein was a more powerful
activator than VP16. When using excess TFIIH, the repression by PC4 was completely reversed, and thus, there was no activation from either BRCA1 or VP16 (Fig. 6, lanes 7-9). This effect of TFIIH on
coactivation by PC4 suggests that most of the activation in the
presence of PC4 is because of the activator reversing the repression by
PC4 (anti-repression).

View larger version (41K):
[in this window]
[in a new window]
|
Fig. 6.
Concentration of TFIIH in transcription
reactions strongly affects the level of transcriptional activation by
GAL4-BRCA1. All reactions included TFIIA, PC4, and all the basal
factors with the exception that TFIIH was included at one-fourth the
standard concentration (lanes 1-3), standard concentration
(lanes 4-6), or at 4-fold higher concentration (lanes
7-9). Templates were all supercoiled. GAL4-BRCA1 was included in
reactions in lanes 2, 5, and 8.
GAL4-VP16 was included in reactions in lanes 3,
6, and 9. The indicated transcriptional
activation ratios were determined by comparison with the appropriate
basal control reaction. S.C., supercoiled.
|
|
 |
DISCUSSION |
These experiments are the first to demonstrate an activity for a
BRCA1 domain using in vitro reactions. Transcriptional
activation by the BRCA1 carboxyl terminus fused to the minimal GAL4 DNA
binding domain (amino acids 1-100) was found to be highly
condition-specific. Compared with the standard VP16 carboxyl terminus
fused to GAL4, the BRCA1 fusion was more potent than the VP16 fusion
under certain conditions, and under other conditions, VP16 was a more
powerful transcriptional activator. Various constellations of
coactivators were compared for activation by the BRCA1 and VP16
carboxyl termini. The most striking differences were noted between PC4
alone versus PC4 plus HMG2. Under the former conditions,
BRCA1 yielded about five times more activation than VP16, whereas under
the latter conditions, VP16 was twice as effective than was BRCA1.
We hypothesized that template topology may be an important factor
accounting for the differences between BRCA1 and VP16. PC4 has been
shown to bind single-stranded DNA (27). Supercoiled DNA has much higher
single-stranded content than does linear DNA (reviewed in Ref. 25) and,
thus, may impact the transcription. Activation by BRCA1 was highly
dependent upon the supercoiled DNA, whereas the VP16 was more effective
on a linear template. These data suggest that activation by BRCA1 may
be dependent upon other factors generating single-stranded DNA. These
data are consistent with the observation that the concentration of
TFIIH was critical for the activation levels observed with these two
proteins. VP16 appeared to be more effective at recruiting TFIIH when
at limiting concentrations, consistent with earlier data (44). Although BRCA1 appeared to be less effective at recruiting TFIIH when the factor
was in low concentration, it was dependent upon TFIIH, presumably to
generate single-stranded DNA at the promoter site. These data
demonstrate that the BRCA1 and VP16 activators function via different
mechanisms leading to the regulation of transcription. Given that PC4
binds to single-stranded DNA, TFIIH has an essential helicase activity,
and the activation by BRCA1 is most effective on a supercoiled DNA
template, we suggest that the generation of single-stranded DNA at the
promoter site is essential for optimal transcriptional activation by BRCA1.
Data are accumulating to suggest that BRCA1 plays an important role in
transcription. GAL4-BRCA1 fusions expressed in cells have been shown to
activate transcription (8-10). The BRCA1 protein in a cell has been
shown to be a component of the Pol II holoenzyme (11), and interaction
with holoenzyme components were key to the transcriptional activation
by GAL4-BRCA1 (10). In this study we demonstrate that BRCA1 activates
transcription by a second, holoenzyme-independent pathway via the basal
factors and coactivators, and ongoing studies are aimed at identifying
which factors are being directly contacted by BRCA1 and which amino
acid residues of BRCA1 are involved in the protein-protein
interactions. This study describes optimal conditions required by the
carboxyl-terminal domain of BRCA1 to activate transcription using an
in vitro system consisting of purified proteins.
Optimization of this system will help understand the role of
full-length BRCA1 protein as a transcriptional regulator interacting
with authentic promoters.