|Home | About | Journals | Submit | Contact Us | Français|
There have been few published analyses of the effects of missense mutations of the BRCA1 gene on BRCA1 protein function. In this study, we adapted a previously described homology directed recombination (HDR) assay to the analysis of the effects of BRCA1 point substitutions on its function in recombination. We established a HeLa-derived cell line, which has integrated in its genome a recombination substrate. Following transfection of a plasmid that expresses the endonuclease that creates a double-stranded break in the recombination substrate, HDR is readily scored by the percentage of GFP-positive cells. By combining RNAi specific for the cellular BRCA1 mRNA with expression of BRCA1 mutants resistant to the RNAi, we could effectively replace the endogenous BRCA1 protein with selected point mutants of BRCA1 and test these in the recombination assay. We found that both, the amino- and carboxy-terminal ~300 residues of BRCA1 were essential for directing HDR. Sixteen missense mutants from the amino terminus of BRCA1 were analyzed for function in HDR, and we found that several point mutants fully replaced the wild-type BRCA1 and are neutral in this process. Mutation of any single zinc-coordinating residue was fully defective in this assay. Several protein variants due to missense mutations, including methionine-18 to threonine and threonine-37 to arginine were also found to be defective for recombination. We have thus established a robust assay system for the analysis of the effects of specific missense mutations of BRCA1 in regulating the homologous recombination process.
The Breast Cancer associated gene 1 (BRCA1) is a tumor suppressor that is specifically associated with familial cases of breast cancer and ovarian cancer (1). In familial breast cancer, a woman may inherit a BRCA1 allele with a mutation, and during her lifetime there is a high likelihood that in at least one breast epithelial cell the second, non-mutated, allele of BRCA1 will be lost by deletion. Such a cell is hemizygous for BRCA1 and the one remaining allele has a mutation. If that mutation renders the BRCA1 defective for a key biological function, then that cell has increased likelihood of transforming into a breast tumor.
The BRCA1 gene has been sequenced from many individuals with a family history of breast cancer, and many of the characterized mutations result in a frame shift or a stop codon and truncate the encoded BRCA1 protein. Clearly, such truncating mutations predispose to breast cancer. Missense mutations, however, can present a diagnostic dilemma since specific point mutations have low prevalence in the population and the disease association is often unclear. Several genetic and evolutionary analyses can predict whether a given point mutation is likely to predispose to breast cancer, but the interpretation can be ambiguous (2–4).
Although there have been many studies assessing the functional implication of BRCA1 mutations, there has been no systematic functional studies that are designed for analysis of the full length protein or that assess a biological process such as homologous recombination. Several in vitro biochemical assays have tested the effects of point mutations in protein-protein interactions (5), in BRCA1-dependent ubiquitin ligase activity (6), and in the control of the centrosome (7). A tissue culture cell-based assay fuses 468 amino acids from the BRCA1 carboxy-terminus to a DNA-binding domain and analyzes the effect of the mutant on a transcription-based reporter assay (8). This latter assay has been very successful in assigning whether a missense mutation is associated with loss of transcription function, but the assay cannot evaluate BRCA1 sequences in the amino-terminal three-fourths of the protein, and the assay does not address non-transcription functions of BRCA1, such as regulation of DNA repair.
In this study, we have adapted the homologous recombination assay that BRCA1 regulates (9–11) to a robust method to analyze specific BRCA1 protein variants. The modified assay tests specific BRCA1 missense mutations in the homologous recombination process. We have characterized a set of point mutations in the amino terminus of BRCA1 in this assay as an initial step in determining whether a BRCA1 variant of unknown function may predispose to breast cancer.
The plasmid encoding the recombination substrate, pDR-GFP, and the I-SceI expression plasmid, cBAS, were both kindly provided by Drs. K. Nakanishi and M. Jasin (Memorial Sloan Kettering Cancer Center, NY, NY). BRCA1 expression plasmid, pcDNA-5’HA-BRCA1, has been previously described (12), and site-specific point mutations identified on the Breast Cancer Information Core (BIC) were generated using the Quick Change Kit from Stratagene. Details of the subcloning are available upon request. Plasmids for the expression of BRCA1 deletion mutants have been previously described (12, 13).
Sequences of the 3’-UTR of BRCA1 were targeted with siRNA oligonucleotides based on the sequence GCUCCUCUCACUCUUCAGU specific for nucleotides 80,780 to 80,798 of the BRCA1 gene (accession number AY273801). BRCA2 was targeted using siRNA based on the sequence UAAAUUUGGACAUAAGGAGUCCUCC. The control siRNA, called GL2, targets the luciferase gene (14).
A stable derivative of HeLa cells was established by transfection with pDR-GFP using standard procedures and selection in 1.5 µg/ml puromycin. Puromycin-resistant cells were cloned by limiting dilution in 96 well plates, and an initial screen selected those colonies that had no detectable GFP fluorescence. Remaining colonies were replica plated and transfected in parallel with cBAS to express the I-SceI. The untransfected stocks of those clones with intense fluorescence in a high percentage of cells were selected and re-cloned by limiting dilution. The number of integration sites in the final cell line, HeLa-DR13-9, was determined by digesting genomic DNA with XhoI and analyzing by Southern blotting.
On day 1, HeLa-DR13-9 cells, approximately 4.0 × 104 cells in a 1.5 cm well were transfected with 5 pmoles of BRCA1 siRNA targeting the 3’-UTR of BRCA1 gene and 0.3 µg of the appropriate BRCA1 expression plasmid in the presence of 0.5 µl of lipofectamine-2000 (Invitrogen). On day 2, the transfected cells were transferred to 3.5 cm well dishes. On day 3, the cells were transfected with 25 pmoles of the BRCA1 siRNA, 0.75 µg of the appropriate BRCA1 expression plasmid, plus 0.75 µg of cBAS in the presence of 2.5 µl of lipofectamine-2000. On day 6, cells were trypsinized and 10,000 cells from each well were counted by flow cytometry using a Becton Dickinson FACSCalibur instrument in the OSU Comprehensive Cancer Center Analytical Cytometry shared resource.
All point mutants of BRCA1 analyzed were tested in triplicate and the percentage of cells with recombined locus encoding GFP were normalized to the same percentage from the control siRNA transfected cells. In the figures, the averages are shown with the standard error of the mean depicted by error bars.
HEK293T cells were transfected with the same BRCA1 variant expression constructs as used in the HDR assay. Whole cell lysates were harvested 2 days post transfection and subjected to immunoaffinity purification using the HA.11 anti-HA tag antibody (Covance) by standard procedures (12). Western blots were analyzed with anti-HA antibody and anti-BARD1 antibody H-300 (Santa Cruz) by standard methods.
We adapted an established assay (15) for homologous recombination in which two inactive alleles of green fluorescent protein (GFP) are integrated in a single locus in the genome of the cell. One allele contains the 18 bp recognition element for the I-SceI endonuclease. Transfection into the cells of a plasmid for expressing the I-SceI results in a double strand break in one GFP allele, and this break can be repaired by homology directed recombination (HDR) using the second inactive allele of GFP. (The plasmids encoding the recombination substrate and the I-SceI endonuclease were the gift of M. Jasin, Memorial Sloan-Kettering Cancer Center, NY, NY.) This repair results in gene conversion that creates a GFP allele that encodes an active protein, and the recombination can be detected by identifying green fluorescing cells. This plasmid and strategy (Figure 1A) has been successfully used in a variety of experiments in which a cell line carrying a mutant version of a protein under study has had this recombination substrate inserted in the cell and the effect of the specific protein was then evaluated (15–17). We instead inserted a copy of this recombination substrate into HeLa cells and carefully selected a clone, called HeLa-DR13-9, which has no background GFP fluorescence, but following transfection of the I-SceI expressing plasmid there is a high level of GFP-positive cells. In many repeated experiments, between 10% and 20% of the cells will undergo recombination following I-SceI expression. This cell line is then useful for the analysis of RNAi-depletion of proteins and testing for their effects in HDR.
HeLa-DR13-9 cells were transfected with either a control siRNA or an siRNA specific to BRCA1. Two days later these cells were re-transfected with the appropriate siRNA plus the plasmid for expression of I-SceI, and three days later the percentage of GFP-positive cells was determined by flow-cytometry. The reduction in GFP-positive cells when BRCA1 is depleted is evident from inspection of the resultant monolayers (Figure 1B) and is quantified by flow cytometry (10,000 cells counted per sample). In the experiment for Figure 2, in the absence of transfected I-SceI expression plasmid, there are no GFP-positive cells (Figure 2, lane 1). In the presence of I-SceI and a control siRNA 16% of the cells were GFP-positive (lane 2). Depletion of BRCA1 or BRCA2 reduced the number of cells with recombined GFP-alleles by eight-fold or 40-fold, respectively. Similar reductions in HDR were observed with siRNAs targeting other BRCA1 and BRCA2 sequences, indicating that these results are not due to off-target effects of the siRNA (data not shown). Further, these results are consistent with published observations (9, 18). Importantly, when the siRNA targets the 3’-UTR of the BRCA1, expression of exogenous BRCA1 from a plasmid with a different 3’-UTR results in complete restoration of HDR activity (Figure 2, lane 4). This result suggests that we have a robust assay for determining the effects of specific BRCA1 mutations in the regulation of the homologous recombination process.
We next assayed a series of synthetic BRCA1 deletion mutants for function in the HDR assay. In Figure 3A, the four deletion mutants are diagrammed. Using the same time course as in Figure 2, the endogenous BRCA1 protein was depleted by 3’-UTR-targeting siRNA, and the test BRCA1 protein was expressed from a co-transfected plasmid. Multiple repeat experiments were done, and in each experiment the maximal recombination varies from 10% to 20%, depending on transfection efficiency. Within an experiment the ratio of GFP-positive cells in the BRCA1 depleted sample relative to the control RNAi was consistently 8- to 10-fold reduced. We thus normalized data in each experiment, allowing us to average the results from multiple experiments. Replacing the endogenous BRCA1 with ΔN-BRCA1, which has deleted residues 1–302, results in a 4- to 5-fold reduction in homologous recombination. Expression of the ΔM1-BRCA1, which has deleted residues 305–770, reduced recombination by about 40%. Similarly, expression of the ΔM2-BRCA1, which has deleted residues 775–1292, resulted in a decrease in recombination by about 60%. Expression of the ΔC-BRCA1, which has deleted residues 1527–1863, resulted in a 10-fold loss of recombination. Clearly, the amino-terminus and carboxy-terminus each had a significant role in controlling the recombination reaction. Expression of the two internal domains had intermediate effects. In each case, the concentrations of BRCA1 protein expressed from the transfected plasmid were at or higher levels than the endogenous BRCA1 protein (Figure 3C), indicating that the reduction in HDR activity was not due to failure to express the test protein. Since the expression level of the ΔN, ΔM1, and ΔM2 mutants were significantly higher than the endogenous BRCA1 protein, it is possible that the magnitudes of the deficiencies in HDR activity with these BRCA1 deletion mutants were underestimated. We decided to focus on point mutations in the amino terminus.
We decided to test point mutations in the amino-terminal 71 amino acid residues of BRCA1 that were derived from individuals with a family history of breast cancer and identified from the BIC database. The reasons for using mutations identified in breast or ovarian cancer clinics were two-fold: first, this is the most direct way to identify candidate amino acid residues for critical function without performing saturating mutagenesis and testing a large number of variants; and second, the results of our functional tests might be useful in counseling such individuals who carry these mutations. Sixteen different variants were produced by site-directed mutagenesis of the BRCA1 cDNA-expressing plasmid. One of these variants, C27A, is a synthetic mutation and is not derived from a variant obtained from an individual with a family history with breast cancer. Rather, the C27A variant completes the set of eight different Zn-coordinating residues of BRCA1 in this analysis. The results of multiple experiments are shown in Figure 4. Consistent with our previous experiments, depletion of BRCA1 by transfection of siRNA targeting the 3’-UTR and co-transfection of the empty plasmid vector, resulted in a 10-fold reduction in GFP-positive cells relative to the control siRNA. Transfection of this BRCA1-specific siRNA along with the wild-type BRCA1 expression plasmid fully restored homology-directed recombination (Figure 4A, lane 3). Strikingly, transfection of each mutant either fully restored recombination to 100% or was fully negative, at the same level of homologous recombination as the vector-transfected control. We note that the deletion mutants used in Figure 3 produced recombination levels that had intermediate results: the ΔM1 and ΔM2 deletions caused partial decrements in homologous recombination. Even deleting the amino terminus, containing all of the residues being tested did not have as severe an effect on the HDR assay as did the point mutants. The expression levels of the point mutants of BRCA1 all were similar to the endogenous protein level (Figure 4B, compare lane 3 to all other lanes) whereas the ΔN deletion, which encompassed all of these point mutants, was significantly overexpressed relative to the endogenous protein and perhaps causing a partial masking of the HDR defect. Alternatively, it is a formal possibility that the point mutations in the BRCA1 protein have both a loss of function phenotype and a dominant negative phenotype.
Eight of the residues that were tested coordinate zinc ions in the RING domain: C24R, C27A, C39Y, H41R, C44F, C47G, C61G, and C64G. It is anticipated that substitution of any of these amino acid residues would have major structural consequences to the protein. Consistent with that concept, replacement of the endogenous BRCA1 with any of these BRCA1 molecules with mutated zinc-coordinating residues was non-functional in HDR (Figure 4A, lanes 6, 7, 10, 11, 13, 14, 16, 17).
Eight other substitution mutants were tested for function in the homologous recombination pathway. The M18T and T37R variants did not complement the HDR assay. Variants that could complement the HDR activity were I21V, I31M, I42V, L52, D67Y, and R71G.
We tested whether the mutant BRCA1 proteins used in this study could bind to BARD1 (Figure 5). We transfected the HA-epitope tagged BRCA1 into HEK293T cells and purified proteins in complex with the variant BRCA1 protein by immunoaffinity purification (IP) using antibody recognizing the HA-tag. Purified proteins were analyzed by immunoblots specific for the HA-epitope to evaluate the effectiveness of expression and purification of the mutant BRCA1 protein and specific for the endogenous BARD1 protein. We found that BRCA1 variant proteins I21V, I31M, I42V, L52F, D67Y, and R71G effectively purified cellular BARD1 protein. These are the same proteins that functioned in homologous recombination. Of note, the BRCA1 variant proteins M18T and H41R had detectable low level of BARD1 binding, but these BRCA1 variants did not complement the HDR assay.
We have summarized the available information for each mutant in Table 1, including results of HDR function and BARD1 binding (this study), E3 ubiquitin ligase activity and resistance to ionizing radiation (6), and available clinical information. For many of these variants family history was unavailable, and this limited the analysis we could do. Examples of available family data are supplied in Supplemental Table 1. We applied an algorithm called VUS Predict (19) to the 16 variants. VUS Predict calculates the odds of a variant being detrimental based on a variety of characteristics of tumors including estrogen receptor, progesterone receptor, and Her2 status, tumor grade, histopathology, age of onset, and the position of the amino acid residue in a functional domain or evolutionary conserved sequence. If there were no clinical data, then the VUS Predict output was based on the single criterion of evolutionary conservation (asterisked values in Table 1).
Of the 16 BRCA1 variants in this study, four of the eight Zn-coordinating residues have been classified as deleterious when mutant (Table 1). One other variant, R71G, has been classified as deleterious (2, 20), and one, D67Y, had been classified as neutral (4). One of the substitution mutants, M18T, has a published odds ratio of being deleterious of 31:1 (4). Using VUS Predict, the M18T mutant has 170.8:1 odds of being deleterious. While these analyses clearly indicate the M18T variant has a trend as a deleterious allele, the magnitude of the ratio did not exceed the threshold of 1000:1 for making a clinical classification. We find that this mutation of BRCA1 causes a complete loss of HDR activity. Though this substitution, when expressed in a truncated BRCA1 peptide in the absence of the BARD1 did have ubiquitin ligase enzymatic activity, it was defective in protection of a cell against ionizing radiation (6). In another study using the BRCA1/BARD1 heterodimer, the M18T variant was inactive as a ubiquitin ligase (21). The M18T substitution could affect the BRCA1-BARD1 heterodimerization interface (22), resulting in reduced BARD1 association with the BRCA1 variant (Figure 5). Our results in the HDR assay are consistent with this substitution being deleterious.
Genetic analysis of one of these mutants, D67Y, has suggested that it is neutral with regard to cancer predisposition (4), and consistent with that notion it has the same phenotype as does wild-type BRCA1 in the HDR assay. Though the R71G substitution is considered cancer-promoting, the mutation is thought to affect the splicing of the mRNA (20), such a feature would be missed when expressing a cDNA as in this experiment. Mutant BRCA1(R71G) protein functioned in the HDR at similar levels as did wild-type BRCA1.
Among the remaining five variants of unknown clinical consequence, the T37R variant was defective in HDR (Figure 4A, lane 9). In new research published while this study was being written has indicated that the similar T37K variant is likely to be deleterious based on clinical and evolutionary analysis (3). The T37R substitution was also found to defective in providing ionizing radiation resistance (6), consistent with our HDR results. Taken together, the threonine-37 residue is likely critical to the homologous recombination process, and consequently to radio-resistance. The threonine-37 side chain is solvent exposed, but in a small cavity near the BRCA1-BARD1 heterodimerization interface (22). Perhaps the bulky lysine or arginine substitutions result in disruption of BRCA1-BARD1 binding, or alternatively another protein important to HDR binds to this pocket.
This study establishes a new assay for evaluating the function of BRCA1 protein variants. Critical components include the HeLa-derived cell line containing an integrated recombination substrate that is readily scored using flow cytometry. The very low background fluorescence in this cell, combined with the very high response to I-SceI expression, make this cell line ideal for analyzing the homologous recombination process. Since HeLa cells are readily transfected, we routinely obtain greater than 70% of the cells transfected in a monolayer (data not shown), this cell line is ideal for silencing a given gene by RNAi and re-expressing the gene via an RNAi-resistant plasmid expression vector. Though BRCA1 is associated with breast and ovarian cancers, this function for BRCA1 has been found in all cell types, and is thus valid to study in the HeLa cell line. While not high-throughput, the assay is relatively quick once the mutant plasmids are prepared. Several variants can be analyzed simultaneously in a one week experiment. We plan to expand this analysis to a number of variants in the amino- and carboxy-termini of the BRCA1 protein. This continuing effort will greatly expand the analysis of the functional consequences of missense mutations in BRCA1 function.
When a BRCA1 variant protein is defective in the homologous recombination process, is it cancer associated? So far, the results indicate a strong correlation with this HDR assay and cancer predisposition, but the analysis of more mutants will be important to determine whether there is an association. Conversely, are BRCA1 variants that have full HDR activity neutral mutations? The R71G variant has full HDR activity, but it is certainly cancer-associated but via splicing (20), a mechanism that would not be identified in this assay using fully spliced cDNAs. Thus, we already have one example indicating that the converse is not true. In addition, we are developing a similar assay for centrosome control by BRCA1 variants, and initial results suggest that some of these variants do indeed function differently in the two assays (Z. Kais, JDP, unpublished observations).
Combining the functional analysis described in this study with genetic and clinical analysis of point mutants (3, 4) will undoubtedly be important for counseling women who carry missense mutations of BRCA1. As the data become more complete, such results will likely become an excellent resource for guiding clinical decisions.
We thank members of the Parvin lab for invaluable assistance, including Z. Kais for photographing the cells used in figure 1B. We thank K. Sweet, L. Senter, R. Pilarski and R. Nagy from OSU Clinical Cancer Genetics Program for ascertainment of family history information for individuals with BRCA1 missense mutations. This project was supported by start-up funds provided by the OSU Comprehensive Cancer Center (J.D.P. and A.E.T.) and the Department of Biomedical Informatics (J.D.P.).