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-Mediated Transcriptional Activation by Altering the Recruitment of c-Fos and c-Jun to Estrogen-Responsive Promoters
Department of Biosciences at Novum (J.M., B.W., M.T., J.W., A.S., J.-Å.G.), Karolinska Institutet, Novum, S-14157 Huddinge, Sweden; and Department of Medical Nutrition (J.-Å.G.), Karolinska Institutet, Novum, S-14186 Huddinge, Sweden
Address all correspondence and requests for reprints to: Jason Matthews, Ph.D., Department of Biosciences at Novum, Karolinska Institutet, Huddinge 14157, Sweden. E-mail: jason.matthews{at}biosci.ki.se.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-expressing T47D cell line containing an inducible tet-off FLAG-ERß was used to examine the influence of ERß on ER activity. Real-time PCR analysis of mRNA levels of two well-studied estrogen-responsive genes, pS2 and progesterone receptor (PR), showed that the expression levels of both genes were reduced in the presence of ERß. Chromatin immunoprecipitation assays showed that the 17ß-estradiol (E2)-induced recruitment patterns to the pS2 and PR promoters were similar for both ER and ERß. ERß expression did not significantly influence the kinetic recruitment profile of ER to the pS2 promoter, but it was evident that ER occupancy at the PR promoter was reduced. The E2-induced recruitment of c-Fos to a 12-O-tetradecanoylphorbol-13-acetate response element site in the PR promoter was significantly reduced in the presence of ERß, whereas only a slight reduction in the recruitment of c-Fos to the pS2 promoter was observed. ERß expression resulted in a significant reduction in the E2-induced expression of c-Fos mRNA. The recruitment pattern of c-Jun was also altered by ERß, although the expression levels of c-Jun were not. Expression of ERß caused a further 30–50% decrease of the E2-induced reduction in ER protein after 3 h of E2 treatment, showing that ERß influences ER protein levels. The altered recruitment of the activating protein-1 complex, combined with the reduction in ER protein levels, may partly explain the antagonistic effect of ERß on ER-mediated transcription. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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or ERß, which belong to the nuclear receptor (NR) superfamily, a family of ligand-regulated transcription factors (1, 2). Ligand binding induces conformational changes in the receptor leading to dimerization, protein-DNA interaction, recruitment of coregulator proteins and other transcription factors, and ultimately the formation of the preinitiation complex (3). ERs regulate gene expression by binding to their cognate response element or through protein-protein interactions with other transcription factors (4). The discovery of ERß (5) has caused a shift in our understanding of estrogen action.
Characterization of mice lacking either ER, or ERß, or both (ERKO, ßERKO, and ßERKO, respectively) has demonstrated that each subtype has similar but also unique roles in estrogen action in vivo. Both ERs are widely distributed throughout the body, displaying distinct but overlapping expression patterns in a variety of tissues (2, 6). Cell types that simultaneously express both ER and ERß, such as neurons and thymocytes (7, 8), are targets for potential interplay between the two receptors.
The transactivating activities of ER and ERß are mediated by two separate but not mutually exclusive transcription activation functions (AFs) that allow the receptors to stimulate the transcription of estrogen-regulated genes: an N-terminal ligand-independent AF-1, and a C-terminal ligand-dependent AF-2 located within the ligand binding domain (9). Although ER and ERß share similar mechanisms of action, several differences in the transcriptional abilities of each receptor have been described (10). Both ER subtypes regulate gene expression in two ways; via the classical pathway through direct DNA-binding via estrogen response elements (EREs) or via the nonclassical pathway through protein-protein interactions with other transcription factors, such as activating protein-1 (AP-1) (4), as well as nuclear factor-
B, and stimulating protein-1 (Sp1) (11). ERß has been suggested to act as a dominant regulator of estrogen signaling, and when coexpressed with ER, ERß causes a concentration-dependent reduction in ER-mediated transcriptional activation (4, 12, 13). Gene expression profiling using high-density microarray analysis of bone and liver tissue isolated from ERKO, ßERKO, or ßERKO animals support the notion that ERß represses ER-mediated transcriptional activity (13). Collectively, data from cell-based assays, gene expression studies, and analysis of ER null animals suggest that ERß attenuates ER activity.
The mammalian AP-1 transcription factors are homo- and heterodimers composed of basic-leucine zipper proteins that belong to the Jun (c-Jun, JunB, JunD), Fos (c-Fos, FosB, Fra-1, and Fra-2), Maf (c-Maf, MafB, MafA, MafG/F/K, and Nr1) and ATF (ATF2, LRF1/ATF3, B-ATF, JDP1, JDP2) subfamilies. AP-1 factors recognize either 12-O-tetradecanoylphorbol-13-acetate (TPA) response elements (TRE) or cAMP response elements (CRE) (14). c-Jun can also function as a homodimer and is the most potent transcriptional activator in the AP-1 protein family. Its activity can, however, be attenuated by heterodimerization with JunB (15). The Fos proteins on the other hand, do not form stable homodimers, but rather form heterodimers with Jun proteins, which enhance their DNA binding activity (16). The activities of preexisting and newly synthesized AP-1 factors are modulated through phosphorylation by different types of MAPKs.
Several studies have shown that ER induces c-Fos expression in an E2-dependent manner (17, 18, 19, 20, 21). The E2-responsive sequence has been localized to a 240-bp region in the human promoter (–1300 to –1060), which contains an imperfect palindromic ERE that ER binds to in gel mobility shift assays but is not sufficient to activate transcription (17). Subsequent studies have shown that the induction of c-Fos expression by E2 is dependent on the formation of a transcriptionally active ER/Sp1 complex that binds to a GC-rich site downstream of the ERE element (21).
In this study, an ER-expressing T47D cell line containing an inducible tet-off FLAG-ERß was used to examine the influence of ERß on ER activity. The original human ERß clone encoded a protein of 485 amino acids (22); however, cloning of an additional N-terminal sequence has extended the N terminus, resulting in a 530-amino-acid (a.a.) protein (23). These proteins have been designated ERß long (530 a.a.) and ERß short (485 a.a.), and although transfection studies suggest that both forms are functionally equivalent (24), the possible distinct roles of the variable N termini have yet to be fully explored. ERß short form was used for the studies described herein. Chromatin immunoprecipitation (ChIP) was used to study the coregulator dynamics of ER-mediated transcription to two estrogen-responsive promoters, the pS2 and progesterone receptor (PR) promoters, in the presence or absence of ERß after estrogen treatment. The expression of both pS2 and PR mRNA levels was significantly reduced in the presence of ERß. ER and ERß exhibited similar recruitment patterns to both promoters; however, the expression of ERß caused a marked reduction in the recruitment of AP-1 factors to both the pS2 and PR promoters. This was particularly evident for the recruitment of c-Fos to the PR promoter. ERß expression significantly reduced the E2-dependent induction of c-Fos mRNA supporting the ChIP results; however, no marked differences in the level of c-Fos protein were evident. Interestingly, ERß expression caused a further and significant reduction of the E2-dependent degradation of ER protein, which was observed after 3 and 8 h of E2 treatment. The altered recruitment patterns of the AP-1 proteins and reduction in ER protein levels may partly explain the antagonistic effect of ERß on ER-mediated transcription.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-mediated transcription, we generated the T47D tet-off ERß cell line expressing inducible FLAG-ERß (485 a.a.). The generation and characterization of the T47D tet-off ERß cells have been described previously (25). In these initial studies it was determined that 6 ng/liter of tetracycline in the media resulted in equal expression levels of endogenous ER and the inducible form of FLAG-ERß (25), and hence, this level was used throughout the current study to induce ERß expression. The cells were maintained in media containing 200 ng/liter doxycyclin, a tetracycline analog that is more potent and more stable than tetracycline, and this chemical had to be removed for appropriate expression of FLAG-ERß. Two estrogen-responsive promoters were selected for analysis: the pS2 and PR promoters. To check whether ERß affects the expression of E2-responsive and ER-regulated target genes, real-time PCR of RNA isolated from cells treated with E2 in the presence or absence of ERß using primer pairs that amplify pS2, and PR mRNA was performed. The data shown in Fig. 1
support previous studies and demonstrate that the expression levels of pS2 and PR mRNA are reduced when ERß is expressed. The expression patterns for pS2 and PR revealed that mRNAs were not detected until after 90 min of treatment with E2, and accumulated to a steady state that was maintained at a significantly higher level in the absence of ERß than in its presence. These data support previous findings that ERß antagonizes ER activity.
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and ERß to Estrogen-Responsive Promoters illustrates the E2-induced recruitment of ERß to the pS2 promoter, which was only observed in the absence of doxycyclin, whereas ER was recruited to pS2 both in the presence or absence of doxycyclin. No promoter enrichment of ER or ERß was observed to the downstream coding sequence of pS2, nor to the 5' regulatory region of the ß-actin promoter (Fig. 2B).
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-Mediated Recruitment of c-Fos and c-Jun to Estrogen-Responsive Promoters and ERß exhibit opposing actions at AP-1-regulated promoters, and it has been suggested that p300 and cAMP response element binding protein-binding protein (CBP) bridge the ER to the AP-1 factors, c-Fos and c-Jun (26). With this in mind, we investigated the effects of ERß on the recruitment of these proteins to the pS2 and PR promoters using ChIP assays. Figure 3A shows that ERß expression alters the temporal recruitment of these factors to the proximal promoter region of pS2. Equal amounts of the same chromatin preparation were incubated with each of the antibodies tested, and analyzed as described in Materials and Methods. ER displayed a time-dependent recruitment to the pS2 promoter, reaching a peak at 60–75 min and then showing a decreasing level of promoter occupancy for the remainder of the 150-min time course. The promoter occupancy by ER at pS2 was unaffected by the ERß expression. The recruitment of ERß to pS2 was similar to that of ER; ERß promoter occupancy was evident after 15 min of treatment, reaching a peak at 60–75 min and showing a decreasing level of promoter occupancy thereafter (Fig. 3A). No oscillatory recruitment was observed for either receptor subtype. Recruitment of ERß was not observed in the presence of doxycyclin. The recruitment patterns of p300 and CBP to pS2 closely followed that of the ERs, and the promoter occupancy by either p300 or CBP at pS2 was not affected by ERß expression. Promoter occupancy by c-Fos and c-Jun to pS2 exhibited distinct patterns from that of the ERs. Very little promoter occupancy was apparent until 105–120 min after E2 treatment, suggesting that ERs occupy the promoter before the AP-1 factors. It cannot be excluded, however, that other AP-1 factors are recruited to the promoter earlier during the time course. Although the overall recruitment patterns for c-Fos and c-Jun were similar in the presence or absence of ERß expression, a marked reduction in the recruitment of both AP-1 factors, particularly c-Fos, was observed in the presence of ERß, suggesting that ERß expression can alter the E2-dependent and ER-mediated recruitment of AP-1 factors to target promoters.
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and ERß to the TRE site was similar to that of the pS2 promoter (Fig. 3B
). Expression of ERß resulted in a small but reproducible reduction in the promoter occupancy of ER to the TRE site of the PR promoter. The promoter occupancy of both p300 and CBP was reduced when ERß was expressed, most evident for p300, which was reduced by 3- to 4-fold in three independent experiments. This was also supported by a small but reproducible reduction in the acetylation status of histone H3 (Fig. 3B). Furthermore, the expression of ERß significantly reduced the recruitment of the AP-1 factors to the PR promoter. Collectively, these data suggest that alterations in the recruitment patterns of AP-1 factors constitute one plausible mechanism for the ERß antagonism of ER activity.
Real-Time PCR Analysis of Promoter Occupancy of c-Fos and c-Jun at Estrogen-Responsive Promoters
Because PCR samples separated by electrophoresis do not accurately depict small changes among the different samples, the recruitment of c-Fos and c-Jun to estrogen-responsive promoters was examined by real-time PCR. This also allowed for quantitation of the relative fold enrichment of the AP-1 factors at each promoter. The real-time PCR data presented in Fig. 4 support the conventional PCR results in Fig. 3, but demonstrate more clearly the altered recruitment of both c-Fos and c-Jun when ERß was expressed (Fig. 4). The E2-induced promoter occupancy of c-Fos to pS2 was reduced from a maximum level of 11-fold in the absence of ERß to approximately 2.5-fold when ERß was expressed. The promoter occupancy of c-Fos at the PR promoter was reduced from levels of 15- and 25-fold to less than 2-fold in the presence and absence of ERß expression, respectively. Reductions in the promoter occupancy of c-Jun to both the pS2 and PR promoters, as well as a shifting of the peak of promoter occupancy were also apparent.
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), showing that the expression levels of c-Fos mRNA are induced much earlier than those for pS2 and PR. E2-induced c-Fos expression was significantly reduced when ERß was expressed, whereas the expression levels of c-Jun were not. This suggests that the ERß-dependent reduction in the recruitment of c-Fos may be due to reduced expression of c-Fos mRNA, revealing c-Fos to be a key regulator of the antagonistic effects of ERß on ER activity. However, no changes in the c-Fos or c-Jun protein levels were observed (data not shown). The discrepancy between the real-time PCR and protein analysis may be due to differences in sensitivity between expression and protein analysis, respectively, and that the changes in mRNA levels were not sufficient to alter total cellular levels of c-Fos protein.
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and c-Fos to the c-Fos Promoter, ChIP assays were done to examine the effect of ERß on the recruitment of ER to the c-Fos promoter. Figure 6A shows that ERß expression resulted in a small but significant reduction in the level of occupancy by ER of the c-Fos promoter. The lower promoter occupancy by ER was apparent as early as 30 min after E2 treatment and remained at a lower level throughout the time course examined. A lower level of promoter occupancy by ER at the PR promoter is observed in Fig. 3, but because those data were not analyzed by real-time PCR it is not as evident as in the case of c-Fos shown in Fig. 6B.
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show that significant recruitment of c-Fos to the c-Fos promoter was not observed until 90–105 min after E2 treatment, and that ERß expression significantly reduces the promoter occupancy by c-Fos of the c-Fos promoter. Because ERß expression inhibits the E2-dependent induction of c-Fos expression mediated by ER, the consequence of this altered c-Fos autoregulation is unknown.
ERß Increases E2-Dependent Degradation of ER
Expression of ERß caused a significant increase in the E2-dependent degradation of ER levels after 3 h treatment. Western blot analysis showed a 40–50% reduction in the levels of ER after E2 treatment, whereas 80–85% reduction was seen in the presence of ERß compared with solvent-treated controls (Fig. 7). Similar results were observed after 8 h E2 treatment (data not shown). ERß expression did not affect the basal levels of ER. No effect on the ER mRNA levels were observed (data not shown). These data suggest that some of the antagonistic effects of ERß are the result of a down-regulation of ER protein levels, and hence a reduced estrogen response.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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target genes including c-Fos, supporting the notion that ERß antagonizes ER activity. The altered recruitment patterns of both c-Fos and c-Jun to TRE sites in estrogen-responsive promoters suggests that ERß may preferentially antagonize the nonclassical AP-1-mediated pathway regulated by ER. This was evident by the ERß-dependent alteration in the recruitment patterns of c-Fos and c-Jun to PR, which is regulated by AP-1 proteins and does not contain an ERE-related sequence, whereas only a mild alteration in the recruitment of the AP-1 proteins to the pS2 promoter that contains both an ERE and TRE elements was observed (30). Because full activation of the pS2 requires both ERE and TRE elements, the reduction in the expression of pS2 that was observed may be a result of the reduction in the promoter occupancy of the AP-1 proteins.
The expression of PR is regulated by E2 in normal reproductive tissues and human cancer cell lines, but the PR promoter lacks an ERE (31). PR expression is dependent on ER (32), and the promoter is regulated by multiple transcription factors; important PR promoter elements include binding sites for Sp1 that are flanked by a putative ERE half-site at position +571 and the TRE at +745 (27, 33). Transient transfection experiments have shown that overexpression of c-Fos and c-Jun inhibits the estrogen-induced transcription of PR through the AP-1 element located at +745 (34). We observed, however, that the expression of ERß dramatically decreased the promoter occupancy by c-Fos and c-Jun at the AP-1 site. This suggests that the ERß-mediated reduction in transcriptional activation of PR mRNA levels observed using real-time PCR was related to the lack of recruitment of c-Fos and c-Jun to this AP-1 element.
The precise mechanism of ER action at AP-1 elements is unknown but requires both the AP-1 element and AP-1 proteins (35). Because both ER subtypes can directly interact with c-Jun, but not with c-Fos (36), the regulation may occur via direct protein-protein interaction. An alternative hypothesis is that the ER-dependent regulation at AP-1 sites occurs via interactions with p300/CBP and the p160 coactivators, forming a large protein complex that is recruited to target promoters (35). Our ChIP studies on the PR promoter demonstrate that ERß expression reduces the recruitment of c-Fos and c-Jun, as well as p300 to the TRE site, suggesting that ERß can affect both AP-1 factor and p300 recruitment. Whether ERß directly inhibits the recruitment of c-Fos or c-Jun to the TRE sites in the pS2 and PR promoters or whether this inhibition is due to the reduced promoter occupancy of p300/CBP is unclear.
ERß expression not only altered the recruitment of c-Fos to pS2, PR, and the c-Fos promoters, but also inhibited the E2-induced expression of c-Fos at the mRNA level. Members of the Jun family have also been reported to be responsive to E2 (37), but no changes in c-Jun levels were observed in the current study. The results from the ChIP assay show that although no apparent changes in the total c-Fos protein levels were seen, significant effects were observed at the level of occupancy by c-Fos at the PR and c-Fos promoters, suggesting that ERß expression influences c-Fos activity. Previous studies have shown that the E2-dependent activation of the c-Fos is dependent on the formation of a transcriptionally active ER/Sp1 complex (21). ER and ERß physically interact with the C-terminal region of Sp1, but only ER/Sp1 and not ERß/Sp1 is active in the presence of E2 (11). The difference is attributable to the N-terminal AF-1 region of ER because the inability of ERß to function on chromatin templates was overcome by swapping its AF-1 region with that of ER (11). Expression of ERß may reduce the activity of the ER/Sp1 complex thus inhibiting induction of c-Fos mRNA.
One of the intriguing findings in this study is that the promoter occupancy by the ERs preceded that of c-Fos and c-Jun. Real-time PCR revealed that small increases in promoter occupancy were apparent after 15–30 min, but reached maximum levels at 105–120 min. The recruitment of the ERs before that of the AP-1 proteins is in agreement with the observations that ERß can prevent the proper recruitment of these factors. The ERs may be recruited to the promoter before the AP-1 proteins by an additional unidentified transcription factor or through their well-characterized interactions with the basal transcriptional machinery, such as the tata-binding protein (38). Alternatively, the TRE site could be occupied by one of the other AP-1 proteins that were not included in this study.
Whether the alteration in the recruitment of AP-1 factors caused by ERß is due to its ability to form heterodimers with ER or if it is the ERß homodimer that mediates the effect is unclear. The results from the ChIP assay might suggest that both receptors occupy the promoter simultaneously but do not provide evidence for heterodimerization. ER mutants that prevent heterodimerization of ER and ERß but permit receptor homodimerization would be very helpful in resolving this issue. The strong influence of ERß on the recruitment of c-Fos and c-Jun certainly suggests that ERß can inhibit the signals mediated by ER, preventing the recruitment of the AP-1 proteins.
Expression of ERß also had a direct effect on ER activity, causing a reduction in the recruitment of ER to the c-Fos promoter and to a lesser extent the PR promoter, as well as an increase in the E2-induced degradation of ER protein. The molecular mechanisms for this inhibition are unknown and could be due to formation of heterodimers, which in turn causes an increase in degradation of ER. Nonetheless, the lower levels of total ER demonstrate that coexpression of ERß reduces ER-mediated activity by reducing the level of ER protein. The reduced level of ER could cause the dissociation of weakly bound ER to low affinity response elements such as the c-Fos ERE, and subsequently reduce transcription activity.
Collectively, our results support the previous notion of the inhibitory effects of ERß on ER-mediated activities. ERß expression alters the activities of AP-1 factors, both in terms of expression of c-Fos as well as E2-dependent recruitment to estrogen-responsive target genes that are regulated by an AP-1-dependent mechanism. This may be due to competition for the AP-1 proteins between E2-ER, which activates transcription at AP-1 response elements and E2-ERß, which fails to activate transcription of AP-1-regulated genes (4). ERß expression also resulted in a significant decrease in ER protein levels, thus possibly shifting the ratio of active E2-ER to more E2-ERß complexes and reducing the overall estrogen response in these cells. Interestingly, in the presence of an antagonist such as the selective ER modulator, tamoxifen and the pure antagonist ICI 182,780, ERß, but not ER is a potent activator of AP-1-mediated transcription (12). Current studies are focused on investigating the effects of ERß on estrogen-responsive and AP-1-regulated genes in the presence of these ligands. The role that ERß plays in normal breast development and malignant breast tissue is still unresolved (39); however, the studies described herein provide further evidence for antagonistic effects of ERß on ER-mediated activity.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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, H-184; for c-Fos, N; for c-Jun, H-125; for p300, N-15; and CBP, A-22 (all from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-FLAG (M5) was from Sigma (St. Louis, MO); and anti-acetyl-histone H3, 06-599 was from Upstate Biotechnology (Lake Placid, NY). Doxycyclin was purchased from Clontech (Mountain View, CA). 17ß-Estradiol and -amanitin were from Sigma. DMEM, F12 medium, fetal calf serum (FCS), L-glutamine, and penicillin/streptomycin were all from Invitrogen Corp. (Carlsbad, CA). All other chemicals and biochemicals were of the highest quality available from commercial suppliers.
Cell Culture and Transient Transfection
The generation and characterization of T47D human breast carcinoma cells expressing ERß (T47D tet-off ERß) have been described elsewhere (25). T47D tet-off ERß cells were cultured in 1:1 mixture of DMEM, and F12 medium supplemented with 5% FCS, 2 mM L-glutamine, 1% penicillin/streptomycin, and 0.2 µg/liter doxycycline. Cells were maintained at 37 C in a humidified mixture of 5% CO2 and 95% air and subcultured at approximately 80% confluency.
ChIP
T47D tet-off ERß cells were seeded in 150-mm dishes and grown for 3 d in phenol red-free DMEM supplemented with 5% dextran-coated charcoal (DCC)-treated serum. Cells were treated with 10 nM E2 and the protein-DNA complexes were cross-linked with 1% formaldehyde for 10 min. For the -amanitin studies, T47D tet-off ERß cells were pretreated for 2 h with 2.5 µM -amanitin, after which the medium was replaced, and the cells were treated with 10 nM E2 before cross-linking with 1% formaldehyde. Cross-linking was quenched by adding 125 mM glycine and cells were washed with PBS, harvested and resuspended in lysis buffer [50 mM Tris-HCl (pH 8.0); 150 mM NaCl; 1 mM EDTA; 1% Triton X-100; 0.1% Na-deoxycholate] containing protease inhibitors (Roche, Mannheim, Germany) and sonicated ten times 10 sec. The soluble chromatin was collected by centrifugation and the supernatants were incubated with 30 µl protein A/G Sepharose (50% slurry; GE Healthcare Bio-Sciences Corp., Uppsala, Sweden) under gentle agitation for 2 h at 4 C. The supernatant was transferred to a new microcentrifuge tube, and 0.5–1 µg of antibody was added and incubated overnight at 4 C. Protein A/G Sepharose (20 µl of a 50% slurry) was then added and incubated for 1.5 h. The pellets were successively washed for 10 min in 1 ml buffer 1 [20 mM Tris-HCl (pH 8.0); 150 mM NaCl; 2 mM EDTA; 1% Triton X-100; 0.1% sodium dodecyl sulfate (SDS)], 1 ml buffer 2 [20 mM Tris-HCl (pH 8.0); 500 mM NaCl; 2 mM EDTA; 1% Triton X-100; 0.1% SDS], 1 ml LiCl buffer [20 mM Tris-HCl(pH 8.0); 250 mM LiCl; 1 mM EDTA; 1% Nonidet P-40; 1% Na-deoxycholate] and 2x 1 ml TE [10 mM Tris-HCl (pH 8.0); 1 mM EDTA]. Protein:DNA complexes were eluted in 120 µl elution buffer [TE; 1% SDS] for 30 min, and the cross-links were reversed by overnight incubation at 65 C. DNA was purified using a PCR purification kit (QIAGEN, Valencia, CA) and eluted in 50 µl. ChIP DNA (5 µl) was amplified by PCR with 5'-GGCCATCTCTCACTATGAATCACT-3' and 5'-GGATTTGCTGATAGACAGAGACGA-3' for pS2, and 5'-GGCGACACAGCAGTGGGGAT-3' and 5'-TCTCCTCCCTCTGCCCCTATATTC-3' for PR. All samples were amplified with the same number of cycles, 29 cycles for the amplification of pS2 and 30 cycles for the amplification of the PR promoter fragment. Equal amounts of PCR product were analyzed by gel electrophoresis and visualized by ethidium bromide staining. For real-time PCR, Platinum SYBR green quantitative PCR supermix uracil DNA glycosylase (Invitrogen) was used to amplify 1 µl of precipitated DNA using primer pairs 5'-CCGGCCATCTCTCACTATGAA-3' and 5'-CCTCCCGCCAGGGTAAATAC-3' for pS2, and 5'-CCCCGAGTTAGGAGACGAGAT-3' and 5'-GGGAACTGTGGCTGTCGTTT-3' for PR.
RNA Isolation and Real-Time PCR
T47D tet-off ERß cells were seeded in six-well plates and grown in phenol red-free DMEM supplemented with 5% DCC-treated FCS for 48 h before treatment with ligands. Real-time PCR was performed as described previously (40). For the pS2 mRNA, the PCR primer pairs were 5'-CATCGACGTCCCTCCAGAAGAG-3' and 5'-CTCTGGGACTAATCACCGTGCTG-3', for PR mRNA the primer pairs were 5'-CGCGCTCTACCCTGCACTC-3', for c-Fos mRNA the primer pairs were 5'-TGAATCCGGCTCAGGTAGTT-3', and for c-Jun mRNA the primers pairs 5'-AACGACCTTCTATGACGATGCCCTC-3' and 5'-GCGAACCCCTCCTGCTCATCTGTC-3'. All target gene transcripts were normalized to the 18S rRNA (PE Applied Biosystems, Foster City, CA) content and to the time zero sample.
Western Blot
T47D tet-off ERß cells were seeded in six-well plates and grown for 2 d in phenol red-free DMEM-Ham’s F12 1:1 (Invitrogen), 10% DCC-FBS, 0.2 µg/liter doxycyclin, then treated with doxycyclin or vehicle alone for 15 h before addition of 10 nM E2 or vehicle alone. Cells were harvested at 0, 3, or 8 h after estrogenic treatment and whole cell extracts were prepared by freeze-thaw cycles in 10 mM HEPES-KOH, 10 mM KCl, 1 mM EDTA, 1x Complete, EDTA-free protease inhibitor cocktail (Roche Applied Science). Equal amounts (5 µg) of protein extract were resolved by SDS-PAGE, transferred to Hybond-P nylon membrane (GE Healthcare Bio-Sciences Corp.) and the immunodetection was performed with ECL Advance western blotting detection kit (Amersham Biosciences) according to the manufacturer’s instructions. ER was detected using H-184 rabbit polyclonal antibody (Santa Cruz Biotechnology) at 1:10,000 and ECL antirabbit IgG, horseradish peroxidase-linked (Amersham Biosciences) at 1:100,000. The Image J software (Research Services Branch, National Institute of Mental Health, Bethesda, MD) was used for densitometry of the autoradiographs.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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First Published Online November 17, 2005
Abbreviations: a.a., Amino acid; AF, activation function; AP-1, activating protein-1; CBP, cAMP response element binding protein-binding protein; ChIP, Chromatin immunoprecipitation; DCC, dextran-coated charcoal; E2, 17ß-estradiol; ER, estrogen receptor; ERE, estrogen response element; ERKO, ßERKO, and ßERKO, mice lacking either ER, or ERß, or both, respectively; FCS, fetal calf serum; PR, progesterone receptor; SDS, sodium dodecyl sulfate; Sp1, stimulating protein-1; TRE, 12-O-tetradecanoylphorbol-13-acetate (TPA) response elements.
Received for publication March 29, 2005. Accepted for publication November 9, 2005.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and ERß at AP1 sites. Science 277:1508–1510 and progestin receptor proteins in the female rat forebrain: effects of estradiol treatment. Endocrinology 142:5172–5181/ß)-dependent activation at GC-rich (Sp1) promoter elements. J Biol Chem 275:5379–5387 and ß on cyclin D1 gene expression. J Biol Chem 277:24353–24360-regulated gene transcription, supporting a "ying yang" relationship between ER and ERß in mice. Mol Endocrinol 17:203–208 in vivo and in vitro. Biochem Biophys Res Commun 243:122–126[CrossRef][Medline] transcriptional activity and is a key regulator of the cellular response to estrogens and antiestrogens. Endocrinology 140:5566–5578 and activating protein-1 mediate estrogen responsiveness of the progesterone receptor gene in MCF-7 breast cancer cells. Endocrinology 143:4583–4591 depends on the coactivator subtype, the type of estrogen response element, and the promoter context. Mol Endocrinol 16:2571–2581ERKO mouse. Steroids 65:551–557[CrossRef][Medline] and JUN proteins. J Biol Chem 276:36361–36369 and ß are unstructured in vitro and show different TBP binding properties. J Biol Chem 276:45939–45944NURSA Molecule Pages Link:
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 Y. Liu, H. Gao, T. T. Marstrand, A. Strom, E. Valen, A. Sandelin, J.-A. Gustafsson, and K. Dahlman-Wright The genome landscape of ER{alpha}- and ER{beta}-binding DNA regions PNAS, February 19, 2008; 105(7): 2604 - 2609. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. G.P. Andrews, M. W. Kennedy, C. M. Popadiuk, and K. R. Kao Oncogenic Activation of the Human Pygopus2 Promoter by E74-Like Factor-1 Mol. Cancer Res., February 1, 2008; 6(2): 259 - 266. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Kawagoe, M. Ohmichi, S. Tsutsumi, T. Ohta, K. Takahashi, and H. Kurachi Mechanism of the Divergent Effects of Estrogen on the Cell Proliferation of Human Umbilical Endothelial Versus Aortic Smooth Muscle Cells Endocrinology, December 1, 2007; 148(12): 6092 - 6099. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Heldring, A. Pike, S. Andersson, J. Matthews, G. Cheng, J. Hartman, M. Tujague, A. Strom, E. Treuter, M. Warner, et al. Estrogen Receptors: How Do They Signal and What Are Their Targets Physiol Rev, July 1, 2007; 87(3): 905 - 931. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Shao, B. Weijdegard, J. Fernandez-Rodriguez, E. Egecioglu, C. Zhu, N. Andersson, A. Thurin-Kjellberg, C. Bergh, and H. Billig Ciliated epithelial-specific and regional-specific expression and regulation of the estrogen receptor-beta2 in the fallopian tubes of immature rats: a possible mechanism for estrogen-mediated transport process in vivo Am J Physiol Endocrinol Metab, July 1, 2007; 293(1): E147 - E158. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Zhao, J. Matthews, M. Tujague, J. Wan, A. Strom, G. Toresson, E. W-F. Lam, G. Cheng, J.-A. Gustafsson, and K. Dahlman-Wright Estrogen Receptor {beta}2 Negatively Regulates the Transactivation of Estrogen Receptor {alpha} in Human Breast Cancer Cells Cancer Res., April 15, 2007; 67(8): 3955 - 3962. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. L. Cho, S. H. Javid, A. M. Carothers, M. Redston, and M. M. Bertagnolli Estrogen Receptors {alpha} and {beta} Are Inhibitory Modifiers of Apc-Dependent Tumorigenesis in the Proximal Colon of Min/+ Mice Cancer Res., March 1, 2007; 67(5): 2366 - 2372. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Howell Pure oestrogen antagonists for the treatment of advanced breast cancer. Endocr. Relat. Cancer, September 1, 2006; 13(3): 689 - 706. [Abstract] [Full Text] [PDF]
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