Activation of Transcription in Vitro by the BRCA1 Carboxyl-terminal Domain*

Dawit T. HaileDagger and Jeffrey D. Parvin§

From the Dagger  Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138 and § Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115

    ABSTRACT
Top
Abstract
Introduction
References

The breast and ovarian specific tumor suppressor protein, BRCA1, has been shown to be a transcription factor because its carboxyl terminus, when fused to the GAL4 DNA binding domain, activates gene expression in cells. In this study, purified GAL4-BRCA1 protein functions in transcriptional activation assays using a minimal in vitro system. When compared with a standard activator, GAL4-VP16, the levels of activation produced by the BRCA1 fusion protein were stronger when in the presence of certain coactivators. The transcriptional activation by BRCA1 is maximal when in the presence of the PC4 (positive component 4) coactivator but not HMG2 (high mobility group protein 2) and when the template is negatively supercoiled. By contrast, transcriptional activation by VP16 was highest in the presence of HMG2 as well as PC4 and when DNA templates had linear topology. Activation by VP16 was largely unaffected by the concentration of TFIIH, whereas activation by BRCA1 was strongly affected by TFIIH concentrations. The differing cofactor and template requirements suggest that GAL4-BRCA1 and GAL4-VP16 regulate different steps in the pathways that lead to transcriptional activation.

    INTRODUCTION
Top
Abstract
Introduction
References

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, pDelta 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 Delta 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, Delta 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.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Comparison of BRCA1- and VP16-mediated transcriptional activation

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 Delta 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.

    ACKNOWLEDGEMENTS

We thank M. Meisterernst for the gift of expression vectors for PC4 and NC2, J. DeJong and R. G. Roeder for expression vectors for TFIIA, and C. Chiang for the FLAG-tagged TFIID cell line. We thank B. P. Schlegel and S. F. Anderson for advice during the course of this research and A. Dutta, E. Silverman, and B. Schlegel for critical reading of the manuscript.

    FOOTNOTES

* This work was supported in part under a National Science Foundation graduate fellowship (to D. T. H.) and by National Institutes of Health Grant GM53504 and an American Cancer Society Junior Faculty Research Award (to J. D. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Pathology, Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115. Fax: 617-732-7449; E-mail: jparvin{at}rics.bwh.harvard.edu.

The abbreviations used are: Pol II, RNA polymerase II; PC4, positive component 4; NC2, negative component 2; HMG2, high mobility group protein 2.
    REFERENCES
Top
Abstract
Introduction
References

  1. Miki, Y., Swensen, J., Shattuck-Eidens, D., Futreal, P. A., Harshman, K., Tavtigian, S., Liu, Q., Cochran, C., Bennett, L. M., Ding, W., et al.. (1994) Science 266, 66-71[Medline] [Order article via Infotrieve]
  2. Futreal, P. A., Liu, Q., Shattuck-Eidens, D., Cochran, C., Harshman, K., Tavtigian, S., Bennett, L. M., Haugen-Strano, A., Swensen, J., Miki, Y., et al.. (1994) Science 266, 120-122[Medline] [Order article via Infotrieve]
  3. Somasundaram, K., Zhang, H., Zeng, Y. X., Houvras, Y., Peng, Y., Zhang, H., Wu, G. S., Licht, J. D., Weber, B. L., and El-Deiry, W. S. (1997) Nature 389, 187-190[CrossRef][Medline] [Order article via Infotrieve]
  4. Ouchi, T., Monteiro, A. N. A., August, A., Aaronson, S. A., and Hanafusa, H. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 2302-2306[Abstract/Free Full Text]
  5. Zhang, H., Somasundaram, K., Peng, Y., Tian, H., Zhang, H., Bi, D., Weber, B. L., and El-Deiry, W. S. (1998) Oncogene 16, 1713-1721[CrossRef][Medline] [Order article via Infotrieve]
  6. Scully, R., Chen, J., Plug, A., Xiao, Y., Weaver, D., Feunteun, J., Ashley, T., and Livingston, D. M. (1997) Cell 88, 265-275[Medline] [Order article via Infotrieve]
  7. Wu, L. C., Wang, Z. W., Tsan, J. T., Spillman, M. A., Phung, A., Xu, X. L., Yang, M. C., Hwang, L. Y., Bowcock, A. M., and Baer, R. (1996) Nat. Genet. 14, 430-440[Medline] [Order article via Infotrieve]
  8. Chapman, M. S., and Verma, I. M. (1996) Nature 382, 678-679[CrossRef][Medline] [Order article via Infotrieve]
  9. Monteiro, A. N., August, A., and Hanafusa, H. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 13595-13599[Abstract/Free Full Text]
  10. Anderson, S. F., Schlegel, B. P., Nakajima, T., Wolpin, E. S., and Parvin, J. D. (1998) Nat. Genet. 19, 254-256[CrossRef][Medline] [Order article via Infotrieve]
  11. Scully, R., Anderson, S. F., Chao, D. M., Wei, W., Ye, L., Young, R. A., Livingston, D. M., and Parvin, J. D. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 5605-5610[Abstract/Free Full Text]
  12. Neish, A. S., Anderson, S. F., Schlegel, B. P., Wei, W., and Parvin, J. D. (1998) Nucleic Acids Res. 26, 847-853[Abstract/Free Full Text]
  13. Koleske, A. J., and Young, R. A. (1994) Nature 368, 466-469[CrossRef][Medline] [Order article via Infotrieve]
  14. Parvin, J. D., and Young, R. A. (1998) Curr. Opin. Genet. Dev. 8, 565-570[CrossRef][Medline] [Order article via Infotrieve]
  15. Kim, Y. J., Bjorklund, S., Li, Y., Sayre, M. H., and Kornberg, R. D. (1994) Cell 77, 599-608[Medline] [Order article via Infotrieve]
  16. Chao, D. M., Gadbois, E. L., Murray, P. J., Anderson, S. F., Sonu, M. S., Parvin, J. D., and Young, R. A. (1996) Nature 380, 82-85[CrossRef][Medline] [Order article via Infotrieve]
  17. Maldonado, E., Shiekhattar, R., Sheldon, M., Cho, H., Drapkin, R., Rickert, P., Lees, E., Anderson, C. W., Linn, S., and Reinberg, D. (1996) Nature 381, 86-89[CrossRef][Medline] [Order article via Infotrieve]
  18. Pan, G., Aso, T., and Greenblatt, J. (1997) J. Biol. Chem. 272, 24563-24571[Abstract/Free Full Text]
  19. Nakajima, T., Uchida, C., Anderson, S. F., Parvin, J. D., and Montminy, M. (1997) Genes Dev. 11, 738-747[Abstract]
  20. Cho, H., Orphanides, G., Sun, X., Yang, X. J., Ogryzko, V., Lees, E., Nakatani, Y., and Reinberg, D. (1998) Mol. Cell. Biol. 18, 5355-5363[Abstract/Free Full Text]
  21. Meisterernst, M., and Roeder, R. G. (1991) Cell 67, 557-567[Medline] [Order article via Infotrieve]
  22. Meisterernst, M., Roy, A. L., Lieu, H. M., and Roeder, R. G. (1991) Cell 66, 981-993[Medline] [Order article via Infotrieve]
  23. Goppelt, A., Stelzer, G., Lottspeich, F., and Meisterernst, M. (1996) EMBO J 15, 3105-3116[Abstract]
  24. Shykind, B. M., Kim, J., and Sharp, P. A. (1995) Genes Dev. 9, 1354-1365[Abstract]
  25. Parvin, J. D., and Sharp, P. A. (1993) Cell 73, 533-540[Medline] [Order article via Infotrieve]
  26. Pil, P. M., Chow, C. S., and Lippard, S. J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 9465-9469[Abstract]
  27. Werten, S., Langen, F. W., van Schaik, R., Timmers, H. T., Meisterernst, M., and van der Vliet, P. C. (1998) J. Mol. Biol. 276, 367-377[CrossRef][Medline] [Order article via Infotrieve]
  28. Werten, S., Stelzer, G., Goppelt, A., Langen, F. M., Gros, P., Timmers, H. T. M., Van der Vliet, P. C., and Meisterernst, M. (1998) EMBO J. 17, 5103-5111[Abstract/Free Full Text]
  29. DeJong, J., and Roeder, R. G. (1993) Genes Dev. 7, 2220-2234[Abstract]
  30. DeJong, J., Bernstein, R., and Roeder, R. G. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 3313-3317[Abstract]
  31. Kretzschmar, M., Kaiser, K., Lottspeich, F., and Meisterernst, M. (1994) Cell 78, 525-534[Medline] [Order article via Infotrieve]
  32. Ge, H., and Roeder, R. G. (1994) Cell 78, 513-523[Medline] [Order article via Infotrieve]
  33. Parvin, J. D., Timmers, H. T., and Sharp, P. A. (1992) Cell 68, 1135-1144[Medline] [Order article via Infotrieve]
  34. Kim, J., Parvin, J. D., Shykind, B. M., and Sharp, P. A. (1996) J. Biol. Chem. 271, 18405-18412[Abstract/Free Full Text]
  35. Chiang, C. M., and Roeder, R. G. (1993) Pept. Res. 6, 62-64[Medline] [Order article via Infotrieve]
  36. Manley, J. L., Fire, A., Samuels, M., and Sharp, P. A. (1983) Methods Enzymol. 101, 568-582[Medline] [Order article via Infotrieve]
  37. Thompson, N. E., Aronson, D. B., and Burgess, R. R. (1990) J. Biol. Chem. 265, 7069-7077[Abstract/Free Full Text]
  38. Chasman, D. I., Leatherwood, J., Carey, M., Ptashne, M., and Kornberg, R. D. (1989) Mol. Cell. Biol. 9, 4746-4749[Medline] [Order article via Infotrieve]
  39. Buratowski, S., Hahn, S., Sharp, P. A., and Guarente, L. (1988) Nature 334, 37-42[CrossRef][Medline] [Order article via Infotrieve]
  40. Tantin, D., and Carey, M. (1994) J. Biol. Chem. 269, 17397-17400[Abstract/Free Full Text]
  41. Pan, G., and Greenblatt, J. (1994) J. Biol. Chem. 269, 30101-30104[Abstract/Free Full Text]
  42. Holstege, F. C., Tantin, D., Carey, M., van der Vliet, P. C., and Timmers, H. T. (1995) EMBO J. 14, 810-819[Abstract]
  43. Schaeffer, L., Roy, R., Humbert, S., Moncollin, V., Vermuelen, W., Hoeijmakers, J., Chambon, P., and Egly, J. M. (1993) Science 260, 58-63[Medline] [Order article via Infotrieve]
  44. Xiao, H., Pearson, A., Coulombe, B., Truant, R., Zhang, S., Regier, J. L., Triezenberg, S. J., Reinberg, D., Flores, O., Ingles, C. J., and Greenblatt, J. (1994) Mol. Cell. Biol. 14, 7013-7024[Abstract]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.