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Endocrinology |
Department of Radiation Oncology, Long Island Jewish Medical Center, New Hyde Park, New York 11040 [S. F., Y. X. M. R-Q. Y., Q. M., J-A. W., I. D. G., E. M. R.]; Departments of Developmental and Molecular Biology and Medicine, Albert Einstein College of Medicine, Bronx, New York 10461 [C. W., R. G. P.]; Genetics and Molecular Biology Branch, National Human Genome Research Institute, NIH, Bethesda, Maryland 20892 [M. E.]; and Metabolic Research Unit, University of California-San Francisco, University of California School of Medicine, San Francisco, California 94143-0540 [P. W., P. J. K.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) in human breast and prostate cancer cell lines but only weakly inhibits ER- activity in cervical cancer cells (S. Fan et al., Science (Wash. DC), 284: 1354–1356, 1999). We now report that the ability of BRCA1 to repress ER- activity correlates with its ability to induce down-regulation of the cellular levels of the transcriptional coactivator p300 in breast and prostate, but not in cervical cancer cells. On the other hand, BRCA1 failed to alter the expression of the CREB binding protein (CBP), the structural and functional homologue of p300, in any of these cell types. Ectopic expression of either p300 or CBP "rescued" (i.e., reversed) the BRCA1 inhibition of ER- activity, whereas two other nuclear receptor coactivators, the p300/CBP-associated factor (PCAF) and the glucocorticoid receptor-interacting protein-1 (GRIP1), failed to rescue the ER- activity. The rescue function mapped to the cysteine-histidine rich domain CH3, a region of p300/CBP that we found to interact directly with the conserved COOH-terminal activation domain (AF-2) of ER-. p300 and ER- were also found to interact in vivo and to colocalize within the nucleus in breast cancer cells. These findings suggest that the cofactors p300 and CBP modulate the ability of the BRCA1 protein to inhibit ER- signaling. They further suggest that the BRCA1 inhibition of ER- activity may be attributable, at least in part, to the down-regulation of p300. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recently, we demonstrated that the overexpression of a wild-type BRCA1 gene (wtBRCA1) inhibits signaling by the liganded ER- in various human breast and prostate cancer cell lines (13)
. It was postulated that the inhibition of estrogenic stimulation of mammary epithelial cell growth by BRCA1 may contribute to breast cancer suppression and that the loss of this inhibitory activity may promote mammary carcinogenesis. This idea would be consistent with a dual role for BRCA1 in the development of mammary cancers: (a) the loss of the caretaker activities of BRCA1 because of mutational inactivation or other mechanisms is an initiation event leading to the accumulation of gene mutations; and (b) the loss of the ability of BRCA1 to suppress ER- transcriptional activity contributes to or promotes the proliferation of previously genetically damaged mammary epithelial cells.
This hypothesis is consistent with the long-established observation that breast cancer is an estrogen-dependent tumor type and with the recent findings that antiestrogen therapy can prevent the development of breast cancer in a high-risk population of women (14
, 15)
. However, it does not explain the linkage of BRCA1 mutations to ovarian cancer, because the growth regulation of ovarian epithelial cells is not primarily regulated by estrogenic steroids. Nor does it explain why BRCA1 mutations have not been linked to other estrogen-dependent tumor types, including endometrial and cervical cancers. It is now understood that a variety of transcriptional coactivators and corepressors, some general and some receptor-specific or -selective, may regulate the transcriptional activity of nuclear receptors such as ER- (16)
. Tissue-specific expression of these cofactors could contribute to tissue-specific regulation of nuclear receptor activity.
In this report, we demonstrate a role for p300 and its functional homologue CBP,3
each of which can function as transcriptional coactivators for ER- in modulating the ability of BRCA1 to repress ER- transcriptional activity. Our studies further indicate that the ability of these proteins to reverse (or "rescue") the BRCA1 inhibition of ER- activity may be distinct from their transcriptional coactivator function. These findings suggest that the BRCA1 repression of ER- activity is, in part, cofactor dependent.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Expression Vectors and Reporters
BRCA1.
The wild-type BRCA1 expression plasmid (wtBRCA1) was created by cloning the BRCA1 cDNA into the pcDNA3 vector (Invitrogen), using artificially engineered 5' HindIII and 3' NotI sites. Mutant BRCA1 vectors were created by the modification of the BRCA1 cDNA in the pcDNA3 vector. BRCA1 mutants 5382 insC and C5365G were a gift from Dr. Barbara Weber (School of Medicine, University of Pennsylvania, Philadelphia, PA). The proteins encoded by the various BRCA1 expression vectors are illustrated schematically in Fig. 1
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shows schematic diagrams of the proteins encoded by the p300 expression vectors used in this study. The wild-type expression vector pCMV-ER- (ER-) was use to express ER-.
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Transient Transfections
Subconfuent proliferating cultures in 100-mm plastic Petri dishes were incubated overnight with 15 µg of each vector in 5.0 ml of serum-free DMEM containing LipofectAMINE. Cultures were washed to remove the excess vector and LipofectAMINE and then postincubated for 24 h in fresh complete culture medium to allow the expression of the transfected gene(s).
RT-PCR Analysis
Semiquantitative RT-PCR assays were performed as described previously (8
, 19
, 20)
. The PCR reaction conditions and cycle numbers were rigorously adjusted for each product, so that all reactions occurred within the linear range of product amplification. The primer sequences (5' 
3') and the locations within the cDNA sequences were as shown in Table 1
. The identity of each of PCR product was confirmed by complete sequencing of the purified product. PCR products were analyzed by electrophoresis through 0.8% agarose gels containing 0.1 mg/ml of ethidium bromide; and gels were photographed under UV light. p300 and BRCA1 mRNA levels were quantitated by densitometry of the cDNA bands and were expressed relative to the control gene (ß-actin).
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transcriptional activity was measured by assessment of the estrogen-stimulated, ER--mediated activation of the ERE-TK-Luc (see above), as described previously (13)
. Briefly, proliferating cells at about 50–70% of confluency in 24-well plastic dishes were incubated overnight with 0.5 µg of each vector in serum-free DMEM containing LipofectAMINE (Life Technologies, Inc.). The total transfected DNA was kept constant by addition of an appropriate amount of the control vector. The transfected cells were washed to remove the excess vector and LipofectAMINE; incubated in serum-free, phenolphthalein-free DMEM (0.2 ml per well) ± 17ß-estradiol (E2, 1 µM) for 24 h; and then harvested for luciferase assays. Luciferase activities were expressed relative to the no-estrogen (-E2) controls. Each experimental condition was tested in four replicate wells; and the experiments shown are representative of three independent experiments.
Assay of ER- AF-2 Activity
AF-2 activity was determined as described previously (13)
. Cells were transfected, under the same conditions described above, with expression plasmid pCMV-gal4-ER(EF) [gal4-ER(AF-2)], which encodes a fusion protein of the yeast gal4 DNA-binding domain (DBD) and EF boxes of ER- (containing the ligand-binding domain and AF-2), and the gal4-responsive reporter gal4-Luc. Gal4-Luc is composed of a gal4 site (pentameric yeast upstream activating sequence) controlling a minimal adenovirus E1B promoter (including the TATA box) and luciferase (18)
. The transcriptional activity of gal4-ER(AF-2) is estrogen dependent. AF-2 activity was determined as the E2-stimulated gal4-ER(AF-2)/gal4-Luc activity relative to the (-E2) control.
Western Blotting
Protein extraction and Western blot analyses were performed essentially as described previously (8
, 19)
. Equal aliquots of total protein (50 µg per lane) were electrophoresed on a 4–13% SDS-polyacrylamide gradient gel, transferred to nitrocellulose membranes (Millipore), and blotted using as primary antibodies: BRCA1 (C-20, rabbit polyclonal, 1:200 dilution; Santa Cruz Biotechnology); p300 (05–267, mouse monoclonal, 4 µg/ml; Upstate Biotechnology), CBP (C-20, rabbit polyclonal, 1:400) Santa Cruz Biotechnology; ER- (H-184, rabbit polyclonal, 1:1000; Santa Cruz Biotechnology); and -actin (I-19, goat polyclonal, 1:500; Santa Cruz Biotechnology), as a control for loading and transfer. The blotted proteins were visualized using the enhanced chemiluminescence detection system (Amersham), with colored size markers (Bio-Rad).
IP
Subconfluent proliferating cells in 150-cm2 dishes were harvested, and nuclear extracts were prepared, as described previously (21)
. Each IP was carried out using 6 µg of antibody or antibody combination and 1000 µg of nuclear extract. Precipitated proteins were collected using protein G beads, washed, eluted in boiling Laemmli sample buffer, and subjected to Western blotting. The IP antibodies were as follows: BRCA1 (Ab-1 + Ab-2 + Ab-3, mouse monoclonals; Oncogene Research Products), p300 (AB-1; Santa Cruz Biotechnology), ER- (C-314, mouse monoclonal, and HC20, mouse monoclonal; Santa Cruz Biotechnology), and control (normal mouse IgG; Santa Cruz Biotechnology). Western blotting was carried out as described above.
GST Capture Assays
GST bead assays were performed essentially as described previously (22)
. [35S]methionine-labeled proteins were prepared by in vitro transcription (using the T7 promoter of pcDNA3) and translation. The GST fusion proteins were generated from cDNAs cloned into the p-GEX vector, expressed in Escherichia coli, and purified by affinity chromatography (23)
. Labeled proteins were incubated with GST (control) or GST fusion proteins for 4 h at 4°C, recovered using glutathione agarose beads, eluted in boiling sample buffer, and analyzed by SDS-PAGE autoradiography. GST fusion proteins were visualized by Western blotting, using an anti-GST mouse monoclonal (B-14, 1:10,000; Santa Cruz Biotechnology). GST binding competition assays were performed as described above, except that GST-ER 282–420 was preincubated for 3 h at 4°C with different quantities of the IVT competing protein.
Confocal Laser Scanning Microscope Analysis
MCF-7 cells were seeded at 1 x 105/well in an 8-well Lab-Tek chamber slide (Nunc, Naperville, IL) in 0.5 ml of culture medium. The cells were fixed for 8 min at 37°C with freshly prepared 3.7% paraformaldehyde in fixation buffer (274 mM NaCl, 10 mM PIPES, 4 mM EGTA, and 11 mM glucose in phosphate buffer). The fixation was followed by two rinses with TBS/glycine. The cells were permeabilized at room temperature with 0.5% Triton X-100 in fixation buffer for 20 min and then washed three times with TBS for 5 min each. Samples were blocked with 1% goat serum and 2% BSA in TBS at room temperature for 30 min. Cells were incubated with primary antibody (see below), diluted with block solution in a humidity chamber at 37°C for 20 min, or room temperature for 60 min, rinsed and then incubated with second antibody conjugated with Alexa Fluor using the same conditions as the primary antibody-binding reaction.
The primary antibodies were to p300 (C-20, rabbit polyclonal; Santa Cruz Biotechnology) and to ER- (C-314, mouse monoclonal; Santa Cruz Biotechnology). The secondary antibodies were goat antimouse IgG conjugated to Alexa Fluor 488 (green) and goat antirabbit IgG conjugated to Alexa Fluor 546 (red; Molecular Probes Corporation). The cells were then washed twice with PBS; and coverslips were applied using Gel/Mount (Biomeda, Foster City, CA). After immunocytochemical staining, the cells were examined with a NIKON confocal laser fluorescence inverted microscope with objective lenses (x60 with oil; LSM 410, Carl Zeiss, Oberkochen, Germany) using simultaneous lasers with excitation wavelengths of 543 nm and 488 nm for red and green, respectively, and detection using red and green narrow band filters. Images were collected through the specimens every 2 µm in the vertical plane, overlaid to generate focus composite images, and stored as TIFF files. Figures were assembled from the TIFF files using Adobe Photoshop software.
Statistical Analysis
When appropriate, statistical analyses were performed using the two-tailed Student’s t test. For example, the estrogen-responsive reporter activities (relative luciferase activity values) determined in cells transfected with wtBRCA1 and without or with wild-type or mutant forms of p300 were compared using two-tailed t tests.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, as compared with untransfected or empty vector (neo) transfected control cell lines (8)
. At that time, the significance of this finding was unclear. In this study, semiquantitative RT-PCR assays, performed within the linear range of product amplification (8
, 19, 20, 21)
, revealed that the same wtBRCA1 or 5677insA clones that showed reduced levels of p300 protein also showed significantly decreased quantities of p300 mRNA, as compared with control (neo) clones (Fig. 2A)
. On the basis of quantitation by densitometry and normalization relative to ß-actin, the p300 levels in the BRCA1-transfected cell clones were reduced by a factor of three or more; and they were significantly lower than in the control (neo) clones (P < 0.01, two-tailed t test). At the same time, the BRCA1-transfected cell clones showed increased levels of BRCA1 mRNA (P < 0.01) and no change in control gene (ß-actin). The RT-PCR assay does not distinguish between endogenous wild-type BRCA1 and the 5677insA mutant, so that the 285-bp amplified BRCA1 cDNA band reflects the total of endogenous plus transgenic transcript.
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. On the other hand, in our experience, proteasomal inhibitor treatment under similar conditions (i.e., 10 µM inhibitor for 6 h) resulted in significant increases in the levels of two proteins known to be regulated by the ubiquitin-26S proteasome degradation pathway: p21WAF1/Cip1 and Mdm-2 (25)
. Although these findings do not rule out the possibility that BRCA1 regulates p300, in part, through other proteolytic pathways, taken together, the findings suggest that BRCA1 regulation of p300 expression in DU-145 cells occurs, at least in part, at the mRNA level.
Role of Limiting Levels of the Coactivator p300 in BRCA1 Inhibition of ER- Activity
BRCA1 Down-Regulates Expression of p300 but not CBP in Prostate and Breast Cancer Cells.
Previously, we tested the ability of various BRCA1 genes to inhibit ER- transcriptional activity, including: wtBRCA1, five breast cancer associated mutations (185delAG, T300G, 5382 insC, 5677insA, and C5365G), an engineered mutation of the consensus Rb binding motif LXCXE (BRCA1-RXRXH), and three engineered protein truncating mutations (BRCA1-
EcoRI, - KpnI, and - BamHI; 21
). The expression of the different BRCA1 genes under transient transfection assay conditions was verified previously (21)
. Of these genes, only wtBRCA1 and 5677insA caused a significant inhibition of ER- activity, as determined using the transient transfection ER-/ERE-TK-Luc reporter assay. Because both p300 and CBP are components of ER- coactivator complexes (26, 27, 28, 29)
, we compared the abilities of the different BRCA1 genes to regulate the protein levels of p300 and CBP under transient transfection conditions parallel to those used in the reporter assays.
Significant down-regulation of the p300 protein levels was observed at 24 h posttransfection of wtBRCA1 and 5677insA, in both prostate cancer (DU-145) and breast cancer (T47D) cell types (Fig. 3, A and B)
, suggesting that stable expression is not required for p300 regulation by BRCA1. On the other hand, neither the empty BRCA1 vector (pcDNA3; negative control) nor any of the other mutant BRCA1 cDNAs significantly altered the p300 protein levels in several independent experiments.
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activity, although they do not prove a causative relationship (see "Discussion").
BRCA1 Fails to Down-Regulate p300 or CBP in Human Cervical Cancer Cell Lines.
We reported previously that transient expression of wtBRCA1 in various human prostate and breast cancer cell lines (including DU-145 and T47D, respectively) strongly inhibited ER- transcriptional activity in an estrogen-dependent reporter assay (ERE-TK-Luc), whereas wtBRCA1 gave only relatively weak or no inhibition of ER- activity in three different human cervical cancer cell lines (C33A, CaSki, and SiHa; Ref. 13
). To determine whether the failure of wtBRCA1 to strongly inhibit ER- correlates with its inability to regulate p300 in this cell type, we performed transient transfection assays, as described above, using these cell lines.
In all three cervical cancer lines, wtBRCA1 failed to cause any obvious change in p300 or CBP protein levels, as compared with the empty pcDNA3 vector (Fig. 3C)
. The BRCA1 protein levels in the wtBRCA1-transfected cervical cancer cell lines were as high or higher than those achieved in DU-145 and T47D. Fig. 3D
shows the basal protein levels of p300 and CBP in DU-145, T47D and in the three cervical cancer cell lines. There were no significant differences in the endogenous (untransfected) levels of p300 or CBP and any of these cell types. Thus, it does not appear that the inability of wtBRCA1 to inhibit ER- activity in this cell type could be attributed either to failure of wtBRCA1 transgene expression or to excessively high basal levels of p300/CBP, in comparison with DU-145 and T47D cells.
p300 Rescues the BRCA1 Inhibition of ER- Transcriptional Activity through Its CH3 Domain.
Because BRCA1 down-regulates p300 protein levels, we next tested the ability of wild-type (wt-p300) and mutant p300 expression vectors (illustrated in Fig. 4A
) to reverse (or "rescue") the wtBRCA1-mediated inhibition of ER- activity. ER- transcriptional activity was measured as described previously, using the ER-/ERE-TK-Luc reporter assay (13)
. DU-145 cells lack ER-, whereas T47D express a modest level of endogenous ER-. To ensure high-level expression in both cell types, an ER- expression vector was cotransfected along with the ERE-TK-Luc reporter. As observed previously, estradiol (E2) induced a large increase in reporter activity (ranging from several hundred- to several thousand-fold), whereas the cotransfection of wtBRCA1 nearly completely abolished the E2-induced increase in luciferase activity (Fig. 4B)
. As expected, the empty pcDNA3 vector had little or no effect on reporter activity. To determine the effect of p300, each p300 expression vector was cotransfected in either the absence or the presence of wtBRCA1.
In the absence of wtBRCA1, wt-p300 and several mutant p300 genes caused a moderate (up to 2- to 3-fold) increase in E2-stimulated ER-/ERE-TK-Luc reporter activity. These increases in reporter activity appear to be relatively modest when the data are plotted on a logarithmic scale, as in Fig. 4B
. In contrast to these relatively small changes in the basal E2-stimulated reporter activity, cotransfection of wt-p300 along with wtBRCA1 fully rescued the wtBRCA1 inhibition of reporter activity in both DU-145 and T47D cells. In addition to wt-p300, p300 mutants missing the HAT or Bromo domain also fully rescued the wtBRCA1 inhibition of ER- activity. However, a p300 mutant missing the cysteine/histidine rich domain CH3, a region containing binding sites for viral oncoproteins, transcription factors, and the nuclear receptor cofactor PCAF (30)
, failed to rescue the inhibition of ER- activity.
Somewhat surprisingly, a p300 mutant containing only CH3 and surrounding sequences (p300 1514–1922) fully rescued the wtBRCA1 inhibition of ER- activity, suggesting that the CH3 region of p300 may be both necessary and sufficient for the p300 rescue function. Because neither the CREB domain, located NH2-terminally, nor the SRC-1 binding domains are present in the p300 1514–1922 fragment, these regions appear to be dispensable for the rescue function.
When wtBRCA1 and wt-p300 genes were cotransfected into DU-145 cells, the levels of each protein were similar to those observed when either wtBRCA1 or wt-p300 were individually transfected (Fig. 4C)
. Furthermore, the cotransfection of p300 1514–1922 did not block the wtBRCA1-mediated down-regulation of endogenous p300 protein levels, nor did it significantly alter the levels of the BRCA1 protein (Fig. 4C)
. The finding that p300 1514–1922 rescues the wtBRCA1 inhibition of ER- activity but does not restore the levels of endogenous p300 suggests that the p300 rescue function may be qualitatively distinct from its coactivator function (see "Discussion").
CBP Also Rescues the BRCA1-Mediated Inhibition of ER- Activity.
Although the p300 homologue CBP was not down-regulated by BRCA1, like p300, cotransfection of a wild-type CBP (wt-CBP) expression vector along with wtBRCA1 blocked the BRCA1-mediated inhibition of ER- activity in both DU-145 and T47D cells (Fig. 5)
. This finding suggests that CBP, which contains a CH3 domain closely related to that of p300, functions similarly to p300 with regard to the rescue of BRCA1 inhibition of ER- activity. In contrast to p300 and CBP, expression vectors for two other nuclear receptor coactivators, PCAF (31)
and GRIP1 (32)
, a member of the p160/SRC-1 class of coactivators, failed to rescue the wtBRCA1 inhibition of ER- activity (data not shown).
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in Vitro, Independently of Estrogen, through Its CH3 Domain. in vivo and are components of ER- coactivator complexes (26, 27, 28, 29)
. We performed GST capture assays to further delineate the interaction between p300/CBP and ER-. GST peptide linked to the AF-2/LBD region of ER- was used as bait, because the AF-2/LBD regions of nuclear receptors are major sites for interactions with coactivators (33
, 34)
. These assays revealed an E2-independent interaction between the AF-2/LBD region of ER- and the COOH-terminal region of CBP (aa 1680–2441) but not the NH2-terminal region (aa 1–1286; Fig. 6A
). As a positive control, GRIP1 exhibited E2-dependent binding to the ER- AF-2/LBD, as described previously (32)
. ICI, tamoxifen, and raloxifene are selective ER- antagonists.
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shows the binding of IVT ER- and other proteins to a series of overlapping GST-linked fragments of the COOH-terminal region of CBP. The ER- binding site of CBP mapped to the CH3 domain, the region of p300 that was found to be essential for rescue of the wtBRCA1 inhibition of ER- activity. In particular, our results suggest the presence of an ER- binding site within the region aa 1801–1880 of CBP. The CH3 domain of CBP also interacted with c-Fos and transcription factor IIB (TFIIB; positive controls) but not with progesterone receptor-B or GRIP1 (negative controls). As expected, GRIP bound to CBP within the p160 coactivator binding site.
Finally, the binding of IVT ER- to the two overlapping CBP fragments containing the CH3 region (GST-CBP-A and GST-CBP-B) was found to be E2-independent (Fig. 6C)
. Thus, the quantity of ER- captured by each CBP peptide was unaffected by the presence of E2 or of various ER- antagonists.
p300 Fragment Containing CH3 Binds ER- and Blocks the BRCA1:ER Interaction in Vitro.
Consistent with the results obtained for CBP, a GST-ER fusion protein containing the AF-2/LBD region of human ER- (aa 282–420) captured IVT p300 1514–1922, which contains the CH3 region of p300 plus the immediately flanking amino acid sequences, in the absence of estrogen (Fig. 7)
. In another study, we found that ER- associates with the BRCA1 protein in vivo as well as in vitro; and we mapped the BRCA1-interacting region of ER- to the same region that interacts with p300 (aa 282–420; Ref. 21
). The capture of IVT wtBRCA1 protein by GST-ER 282–420 is illustrated in Fig. 7
.
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, we performed GST binding competition assays to determine whether these interactions are mutually exclusive. In these assays, GST-ER 282–420 was preincubated for 3 h at 4°C with increasing quantities of the competing protein [either wtBRCA1 (Fig. 7
, top panel) or IVT p300 1514–1922 (Fig. 7
, bottom panel)]; and then a fixed quantity of the other protein was added. Preincubation of GST-ER 282–420 with IVT wtBRCA1 or p300 1514–1922 competed out the binding of IVT p300 1514–1922 or wtBRCA1, respectively (Fig. 7)
.
Note that the protein quantities shown in Fig. 7
represent the amount of plasmid DNA used in the IVT reaction. As a negative control, IVT reaction product was generated using equivalent quantities of empty pcDNA3 vector; and these products failed to inhibit the pull-down of IVT p300 1514–1922 by GST-ER 282–420. These findings suggest that the binding of BRCA1 and the CH3 domain of p300/CBP to ER- may be mutually exclusive.
Endogenous p300 and ER- Interact in Breast Cancer Cells in Vivo.
T47D and MCF-7 breast cancer cells contain endogenous ER-. To demonstrate in vivo protein association, cells cultured in the absence of added estrogen were subjected to IP-Western blotting. For both cell types, ER- was detected in the p300 IPs but not in a control IP using an equivalent quantity of normal mouse IgG (Fig. 8A)
. Conversely, p300 could be detected in ER- IPs of MCF-7 cells, using two different ER- IP antibodies, but not in an IP of MCF-7 cells using normal mouse IgG (Fig. 8B)
. As expected, there was no ER- or p300 detected in an ER- IP of DU-145 cells, which lack endogenous ER-.
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IP of MCF-7 cells but not in the control IPs (Fig. 8B)
. In the converse experiment, ER-, p300, and CBP were detected in the BRCA1 IP of MCF-7 cells but not in the control (normal mouse IgG) IP of the same cells. The in vivo association of BRCA1 and ER- is consistent with our previously published findings (21)
. It should be noted that these IP assays can only demonstrate two-way protein associations, e.g., ER- and p300 or ER- and BRCA1. They cannot tell whether ER-, p300, and BRCA1 coexist within the same protein complex or whether the ER- interactions with p300 and BRCA1 are mutually exclusive. The significance of these additional findings is considered in the "Discussion."
To further assess the in vivo ER-:p300, confocal laser scanning microscopy was performed to compare immunolocalization of ER- with that of p300. In confocal immunofluorescence studies of MCF-7 cells grown in the absence of added estrogen, ER- showed nuclear as well as cytoplasmic localization, whereas the subcellular localization of p300 was essentially entirely nuclear but extranucleolar (Fig. 9)
. The merged images showed significant colocalization (yellow color) of ER- and p300 within nuclear granules of cells containing nuclear ER-. On the other hand, there is very little colocalization of cytoplasmic ER- and p300. We have also demonstrated that BRCA1 and ER- colocalize within the nucleus of MCF-7 cells via confocal laser microscopy (data not shown). Taken together with the in vitro (GST) and in vivo (IP) assays, these findings suggest that some portion of the endogenous ER- and p300 interact with each other, independently of estrogen.
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AF-2 Activity in Cervical Cancer Cell Lines. activity in three human cervical cancer cell lines (Caski, SiHa, and C33a; Ref. 13
). Consistent with these findings, wtBRCA1 caused little or no inhibition of ER- activity in Caski or SiHa cervical cancer cells, using the standard ER-/ERE-TK-Luc assay (Fig. 10A)
. Both Caski and SiHa are ER--positive cell lines therefore, there is significant E2-stimulated activation of ER- activation in the presence or absence of transfected ER- expression vector. In this experiment, wtBRCA1 failed to suppress E2-stimulated ERE-TK-Luc activity in these cell lines, whether or not exogenous ER- was provided.
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, we used the gal4-ER(AF-2)/gal4-Luc assay system (13)
. This assay measures the ability of a fusion protein of the yeast gal4 DNA-binding domain and EF boxes of ER- [which contain the ligand-binding domain and AF-2; gal4-ER(AF-2)] to activate the gal4-responsive reporter gal4-Luc (see "Materials and Methods"). As expected, the gal4-ER(AF-2) activity was E2-dependent in both prostate (DU-145) and cervical cancer (Caski and SiHa) cell types, because the gal4-ER(AF-2) fusion protein contains the LBD of ER- (Fig. 10B)
.
In DU-145, the gal4-ER(AF-2)/gal4-Luc reporter activity was reduced to near background levels by wtBRCA1 but was unaffected by the empty pcDNA3 vector. Cotransfection of p300 blocked most of the wtBRCA1-mediated inhibition of AF-2 activity in DU-145 cells, in agreement with the finding that p300 rescued the wtBRCA1-inhibition of ER-/ERE-TK-Luc activity in this cell line (Fig. 4B)
. On the other hand, wtBRCA1 had little or no effect on the AF-2 activity in Caski or SiHa cells. A control reporter (TATA-Luc) missing the gal4-binding DNA elements showed little or no basal or E2-stimulated lucfierase activity (data not shown).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several findings indicate a correlation between the down-regulation of p300 and inhibition of ER- transcriptional activity by BRCA1. First, the wtBRCA1 gene, which only very weakly inhibits ER- activity in human cervical cancer cell lines, failed to down-regulate p300 in three of three cervical cancer cell lines. Secondly, the ability of different mutant BRCA1 genes to inhibit ER- activity in prostate and breast cancer cells correlated with their ability to down-regulate p300. Among 11 different BRCA1 genes tested, only wtBRCA1 and the 5677insA mutant down-regulated p300. Among the same group of BRCA1 genes, wtBRCA1 and 5677insA were the only two BRCA1 genes to significantly inhibit ER- activity in transcriptional assays (21)
.
The correlation between down-regulation of p300 and ER- inhibition by BRCA1 does not itself prove a causal relationship. In fact, we found that in all of the cell types tested, BRCA1 failed to down-regulate the levels of CBP, a structural and functional homologue of p300 that shares its activity as a nuclear receptor coactivator (35)
. Because either p300 or CBP can rescue the BRCA1 inhibition of ER-, it could be asked why the endogenous CBP fails to block the ER- inhibition by BRCA1. It is possible that the inhibition of p300 expression, by itself, is insufficient to block ER- activity, and an additional activity of BRCA1 is required, e.g., the direct interaction between the BRCA1 and ER- proteins, as demonstrated recently (21)
. Alternatively, in BRCA1-transfected cells, the total (p300 + CBP) protein levels may be insufficient to support ER- activity because of the substantial reduction of p300 levels. The latter possibility seems especially reasonable, because several studies suggest that limiting levels of the CBP/p300, which are coactivators for a variety of transcriptional pathways, including AP-1 dependent transcription, may physiologically regulate the relative activities of competing transcriptional pathways (17
, 35
, 36)
. Thus, it has been suggested that the p300/CBP class of coactivators integrate a variety of extracellular and intracellular signals and thus determine the net cellular consequences of these signals.
At the least, our findings indicate that p300 and CBP can strongly modulate the ability of BRCA1 to inhibit ER- activity. Thus, tissue-specific differences in the levels of BRCA1, p300, and CBP could provide a basis for tissue-specific differences in ER activity. The rescue experiments suggest that the CH3 domain of p300 is necessary and sufficient for the p300-mediated rescue of BRCA1 inhibition of ER- activity. This was a surprising finding, because other domains, including the HAT domain and the SRC-1 binding domain are thought to contribute to the function of p300/CBP as nuclear receptor coactivators (37
, 38)
. Histone acetylation has been implicated in the modification of the chromatin template to a configuration suitable for active gene transcription (reviewed in Ref. 16
), whereas SRC-1 interacts with the AF-2/LBD region of the liganded ER- and stimulates its transcriptional activity (18
, 27
, 38)
. Because these domains are not required for the p300 rescue function, we postulate that the rescue function of p300 and, by extension, CBP may be qualitatively distinct from their classic coactivator functions. However, this hypothesis remains to be established.
Clues to the function of p300/CBP in the rescue of BRCA1 inhibition come from the findings that: (a) the ER- binding site on these proteins maps to the same domain found to be essential for the rescue function, i.e., the CH3 domain; (b) BRCA1 and p300/CBP interact with the AF-2/LBD region of ER-; and (c) a fragment of p300 (aa 1514–1922) that contains the CH3 domain is able to compete out the binding of the BRCA1 protein to ER-. Previously, we had identified a BRCA1:ER- interaction in vivo and in vitro; and we mapped the interacting sites to the NH2 terminus of BRCA1 (aa 1–302) and the AF-2/LBD region of ER- (aa 282–420; Ref. 18
). However, because of the size of BRCA1 and p300/CBP and the complexity of their in vivo interactions, we cannot postulate a simple "bumping model" in which p300 or CBP displace BRCA1 from the ER- transcriptional complex based on these in vitro studies.
Thus, in a recent study, BRCA1 was found to associate in vivo, and interact directly, with CBP/p300 in vitro (39)
. The binding regions were mapped to the COOH terminus of BRCA1 (aa 1314–1863) and the CREB domain of CBP (aa 451–721 of murine CBP). Our findings confirm the ability of BRCA1 to associate with p300 and CBP in vivo and document the in vivo interaction of p300 and CBP with ER- in human breast cancer cells. Thus, theoretically, the full-length CBP or p300 protein could simultaneously interact with BRCA1 through the CREB domain and with ER- through the CH3 domain. Alternatively, BRCA1 could simultaneously interact with ER- through its NH2 terminus and with CBP/p300 through its COOH terminus. These considerations raise the possibility that BRCA1 could modulate the functional activity of p300 or CBP via a direct protein interaction independently of its ability to down-regulate p300 expression. This possibility is strengthened by the observations that: (a) exogenous wtBRCA1 can virtually ablate ER- activity, although it has little or no effect on the expression of CBP; and (b) CBP rescues the wtBRCA1 inhibition of ER- activity as efficiently as does p300. Here, the residual CBP rescue activity could be functionally blocked by BRCA1.
In other studies, BRCA1 was found to interact physiologically with the BRG-1 (Brahma related gene-1) protein, a component of the SWI-SNF transcriptional complex with ATPase activity (40) . And the acidic COOH-terminal transcriptional activation domain of BRCA1 was found to bind several proteins implicated in transcriptional repression [retinoblastoma (Rb) protein, COOH-terminal interacting protein (CtIP), and histone deacetylases (HDACs-1, -2, and -3); Refs. 41 , 42 ] as well as components of the RNA polymerase holoenzyme (43) . Thus, the roles of BRCA1 in transcriptional regulation, positive and negative, are likely to be very complex.
It should be emphasized that the BRCA1 inhibition of ER- activity is not caused by nonspecific transcriptional repression, because under similar conditions to those under which wtBRCA1 blocked ER- activity, it failed to alter the activity of several other transcription factors, including several cell cycle-dependent transcription factors (E2F-1 and Sp1) and c-Jun- and AP-1-dependent transcriptional activity (13)
. The issue of cytotoxicity has been addressed previously: under both our stable and transient transfection conditions, the overexpression of wtBRCA1 did not induce apoptosis or any other measurable evidence of toxicity (8
, 13)
. However, wtBRCA1 expression did render cells more susceptible to apoptosis induced by DNA-damaging agents (13)
. Furthermore, BRCA1 functioned to activate the p21WAF1/Cip1 gene promoter (6)
. In preliminary studies using several different reporter assay systems (including ARE-TK-Luc and MMTV-Luc), wtBRCA1 failed to alter the transcriptional activity of the androgen receptor,4
suggesting that even within the nuclear receptor class, the transcriptional inhibitory activity of BRCA1 is selective. E2F-1, c-Jun, AP-1, and androgen receptor pathways are all stimulated by the p300/CBP coactivators but not inhibited by BRCA1, further attesting to the complexity of the role played by the BRCA1 gene product in transcriptional regulation.
It was noted that with regard to the ability to down-regulate p300 levels (this study and Ref, 8
), repress ER- activity (18)
, and render prostate cancer cells more susceptible to apoptosis induced by DNA-damaging agents (8)
, the BRCA1–5677insA mutant behave similarly to wtBRCA1. In contrast, multiple other tumor-associated or truncated BRCA1 mutants lacked these activities. The retention of these wild-type activities by the 5677insA mutant gene, which encodes a near full-length BRCA1 protein missing only the last 11 amino acids, appears to conflict with the finding that this mutation, although quite rare, has been linked to familial breast cancer (44)
. Although the 5677insA mutant retained wild-type BRCA1 activities under standard transient transfection assay conditions, plasmid dose-response studies revealed that when lower doses of plasmid were transfected into both DU-145 and T47D cells, a 5-fold higher dose of 5677insA was required to produce the same percentage of inhibition of ER- transcriptional activity as wtBRCA1 (21)
. Similar results were obtained in assays of the ability of BRCA1 to induce chemosensitivity.4
Thus, it appears that the 5677insA mutant is defective with regard to these functional activities of BRCA1.
Finally, the issue arises as to how a highly potent inhibitor of ER- activity such as wild-type BRCA1 is able to accommodate the ER- activation under physiological conditions in normal estrogen responsive breast tissue. Our previous studies suggest that under endogenous conditions, there may be insufficient BRCA1 to fully repress ER activity. Thus, a fragment of BRCA1 ( EcoRI, corresponding to aa 1–302), which includes the ER- binding region but is missing an essential COOH-terminal repression domain, not only rescued the repression of ER- activity by exogenous wtBRCA1 but also increased the ER- activity in the absence of exogenous wtBRCA1 by 2- to 3-fold (21)
. The increase in ER- activity induced by the EcoRI fragment was not observed in a cell line that lacked endogenous functional wild-type BRCA1 (HCC1937). These findings suggest that under endogenous conditions, wild-type BRCA1 partially inhibits ER- activity, and when exogenous wtBRCA1 is supplied, the BRCA1 protein levels become sufficient to fully block the ER- activity. The mechanisms underlying these observations are not clear at present. However, because the BRCA1 protein has many binding partners and is present within high-molecular-weight transcriptional complexes (43)
, we can speculate that in the presence of exogenous wtBRCA1, the ER- is recruited into a BRCA1-positive, p300-negative, transcriptional inhibitory complex. Further study will be required to dissect the mechanism.
In conclusion, our findings suggest that the cofactors p300 and CBP modulate the ability of the BRCA1 protein to inhibit the transcriptional activity of ER-. The ability of these cofactors to modulate the BRCA1 inhibition of ER- appears to involve protein interactions mediated primarily by their conserved cysteine-histidine rich domain, CH3. Our results further suggest that the BRCA1 inhibition of ER- activity is attributable, in part, to the down-regulation of the expression of p300, which may reduce the total intracellular quantity of (p300 + CBP) to levels too low to stimulate the ER- transcriptional pathway.
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FOOTNOTES |
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1 Supported, in part, by the USPHS, Grants R01-ES09169, R01-82599, and RO1-80000 (to E. M. R.); the Elsa U. Pardee Cancer Foundation of Michigan (to E. M. R.); the New York State Department of Health (to E. M. R.); United States Army Breast Cancer Research Program Grant DAMD17-99-1-9254 (to E. M. R.); the Susan G. Komen Breast Cancer Foundation (to E. M. R. and R. G. P.); and the USPHS Grants RO1-CA75503 and R01-CA70897 (to R. G. P.). 
2 To whom requests for reprints should be addressed, at Department of Radiation Oncology, Long Island Jewish Medical Center, The Long Island Campus for the Albert Einstein College of Medicine, 270–05 76th Avenue, New Hyde Park, NY 11040. Phone: (718) 470-7386; Fax: (718) 470-9756; E-mail: erosen{at}lij.edu
3 The abbreviations used are: CBP, CREB binding protein; PCAF, p300/CBP-associated factor; GRIP1, glucocorticoid receptor-interacting protein-1; ER, estrogen receptor; ERE, estrogen-responsive enhancer; TK, thymidine kinase; RT-PCR, reverse transcription-PCR; IVT, in vitro translated; TBS, Tris-buffered saline; HAT, histone acetyl transferase (domain); SRC-1, steroid receptor coactivator-1; AF-2, activation function-2; LBD, ligand binding domain; IP, immunoprecipitation; GST, glutathione S-transferase.
Received 7/20/01. Accepted 11/ 1/01.
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