Molecular Biology Program (V.G.T., S.K.N.), Department of Pathology (S.K.N.), University of Colorado Health Sciences Center, Denver, Colorado 80262; and Department of Biochemistry and Molecular Biology (D.O.T.), Mayo Graduate School, Rochester, Minnesota 55905
Address all correspondence and requests for reprints to: Steven Nordeen, Department of Pathology B216, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, Colorado 80262. E-mail: Steve.Nordeen{at}UCHSC.edu.
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ABSTRACT |
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INTRODUCTION |
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PR regulates the expression of genes involved in the development and maintenance of tissues essential for female reproduction such as the brain, mammary gland, ovary, and uterus (3). In the absence of its ligand, PR is located predominantly in the nucleus of the cell where it is bound to a complex containing heat shock protein (hsp) chaperones and immunophilins (4, 5). The binding of progesterone to PR induces a conformational change that allows the ligand-bound receptor to dissociate from the chaperone complex, dimerize, and bind to DNA to modulate the transcription of specific target genes (6, 7, 8).
When PR is stripped of its hsp chaperone complex, it loses its ability to bind hormone (9, 10, 11). However, hormone binding activity can be restored by reconstitution of the PR-chaperone complex (12, 13, 14, 15, 16). Purified, recombinant hsp90 and hsp70 are sufficient to reactivate some of the receptor in vitro, although optimal reactivation requires the addition of cochaperones (hsp40, p23, and Hop) (17, 18, 19, 20). In addition to increasing the affinity of the receptor for ligand, chaperones may also regulate PR function subsequent to hormone binding (21, 22). For example, chaperone proteins may play a role in the recycling of steroid receptors because hsp90 and p23 have been shown to decrease the association of steroid receptors with DNA (23, 24). Moreover, geldanamycin, a specific hsp90 inhibitor, prevents the release of the glucocorticoid receptor from chromatin in vivo (25).
Biochemical and genetic studies have demonstrated that transcription is inhibited on chromatin templates (26). One way that chromatin can repress transcription is by blocking the access of regulatory proteins to DNA. Although PR binds to hormone response elements in vitro (27), it is not clear whether the receptor binds independently or whether it requires chromatin remodeling proteins to bind to progesterone response elements in the context of chromatin (28, 29, 30, 31). Receptor binding to the promoter recruits protein factors that alleviate chromatin repression by modifying and remodeling nucleosome structure. These factors include histone acetyltransferases and methyltransferases such as steroid receptor coactivator 1 (SRC-1), CREB binding protein (CBP)/p300, and coactivator-associated arginine methyltransferase 1 (CARM-1) (32, 33, 34, 35, 36) and ATP-driven switch defective/sucrose nonfermenter (SWI/SNF) chromatin remodeling complexes that disrupt promoter-proximal nucleosomes (37, 38). Chromatin modification and remodeling appears to be necessary for the recruitment of general transcription factors and the subsequent activation of transcription.
Initial studies of PR action in a cell-free transcription system employing chromatin templates used nuclear extracts from T47D mammary carcinoma cells or recombinant PR expressed and purified with ligand (39, 40, 41). To study the mechanism of hormone-dependent transactivation and the action of partial agonists/antagonists, we used recombinant PR that was purified in the absence of ligand. However, we found that the recombinant receptor was inactive on chromatin templates upon the addition of hormone. We show that PR transcriptional competence is restored upon incubation with rabbit reticulocyte lysate (RRL). Surprisingly, the recovery of hormone binding to the receptor, mediated by hsp chaperones, is not sufficient to promote PR-dependent transcription. Our studies reveal that incubation with RRL increases the ability of the receptor to bind to chromatin templates and, concomitantly, promotes coactivator recruitment. Based on these observations, we suggest that the receptor undergoes an additional activation step to bind its cognate hormone response element in the context of chromatin.
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RESULTS |
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To test whether RRL decreased the specificity of PR, we measured the amount of transcription on a chromatin template that was identical to PRE2S2CAT except that the progesterone response elements were replaced by two estrogen response elements (ERE2S2CAT) (Fig. 2B). RRL had a minimal effect on PR-dependent transcription with the ERE2S2CAT template. This result suggests that the specificity of the receptor for its response element is unchanged. We also measured the amount of transcription on naked and chromatin templates using a transcription factor containing the GAL4 DNA binding domain fused to the VP16 activation domain (GAL4-VP16). Recombinant GAL4-VP16 was active on both naked and chromatin templates containing two GAL4 DNA binding elements (GAL42S2CAT) (Fig. 2C
). The addition of RRL had no effect on basal transcription or on GAL4-VP16-dependent transcription, indicating that the effect of RRL was not due to a general effect on transcription.
Preliminary studies indicate that, like PR, recombinant glucocorticoid receptor is transcriptionally inactive on chromatin templates and that coincubation with RRL restores hormone-dependent transcriptional induction (Parrish, J., data not shown). Unlike PR, recombinant ligand-free estrogen receptor is capable of activating transcription in response to hormone (43). Nonetheless, coincubation of estrogen receptor with RRL increases the hormone-dependent transcriptional induction by estrogen receptor (Thackray, V. G., data not shown). Because unliganded PR and glucocorticoid receptor are significantly less stable than the estrogen receptor, refolding by chaperones in the RRL may play a role in the restoration of transcription. The role of chaperones is explored further below.
We also tested the possibility that the stimulatory effect of RRL was simply due to inhibition of chromatin assembly. The efficiency of chromatin assembly was monitored using micrococcal nuclease digestion of the template DNA. As indicated in Fig. 3, efficient chromatin assembly occurred in the presence of the Drosophila S190 assembly extract and Drosophila core histones. The inclusion of PR and/or hormone did not alter the pattern of chromatin assembly (data not shown). Because chromatin assembly was not disrupted by the addition of RRL to the S190 assembly extract, the restoration of transcription by RRL shown in Fig. 2A
cannot be attributed to relief of repression by chromatin.
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Regeneration of Hormone Binding to PR by Chaperones Is Necessary But Not Sufficient to Restore PR-Dependent Transcription on Chromatin
RRL is an abundant source of hsp chaperones that are necessary for high affinity binding of hormone to PR (10). As demonstrated in Fig. 5A, we determined the amount of specific hormone binding to purified PR in the absence or presence of RRL. RRL did not exhibit hormone binding activity on its own. However, incubation of PR with RRL in the presence of ATP and an ATP regeneration system substantially increased the amount of hormone binding compared with PR by itself, indicating that maximal hormone binding was dependent on ATP. The addition of 10 µg/ml of geldanamycin abrogated the ability of RRL to promote hormone binding to PR. Geldanamycin is a specific hsp90 inhibitor that binds to the adenine nucleotide binding site of hsp90 and blocks ATP-dependent regeneration of hormone binding by hsp90 (45). Thus, consistent with previous studies, RRL regenerated hormone binding to recombinant PR in an ATP-dependent manner involving chaperone mediated protein folding.
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Because hsp90, provided by RRL, was required for the restoration of hormone binding and transcription on chromatin templates, we investigated whether recombinant hsp90 and other chaperone proteins could replace RRL in the hormone binding and cell-free transcription assays. The incubation of PR with hsp90, hsp70, p23, Hop, and YDJ-1 promoted hormone binding to PR, although the recombinant chaperones were not as effective as RRL (Fig. 6A). Next, we measured the amount of PR-dependent transcription from chromatin templates in the presence of the recombinant chaperone proteins. We used two different concentrations of PR in the transcription assay to show the amount of transcription that would be seen if the recombinant proteins were one fifth as effective as RRL. However, there was no restoration of transcription when hsp90, hsp70, p23, Hop, and YDJ-1 were added to the chromatin assembly reaction (Fig. 6B
). Increasing or decreasing the concentration of the chaperone proteins also had no effect on the amount of transcription on chromatin templates (data not shown). In addition, PR-dependent transcription on naked templates was unaffected by the addition of the recombinant chaperones (data not shown). This result suggests that the lack of transcription on chromatin templates in the presence of the chaperone proteins was not simply attributable to an inhibitory effect due to the chaperone protein storage buffers. Together, these data indicate that the recombinant chaperone proteins are not sufficient to restore PR transcriptional activity on chromatin templates, even though they do enhance hormone binding to the receptor.
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Because PR transcriptional activity on chromatin templates was dependent on the addition of SRC-1 (Fig. 2A), we asked whether restoration of receptor binding to the chromatin templates by RRL resulted in the concomitant recruitment of SRC-1 to the promoter. A similar experiment to the one described above was performed except that SRC-1 was added to the reactions. In the absence of RRL, the small amount of receptor bound to the DNA template did not promote recruitment of SRC-1 to the promoter (Fig. 9
, A and B). However, SRC-1 recruitment to the promoter was enhanced 7-fold upon the addition of RRL. SRC-1 was not recruited to the promoter in the absence of hormone indicating that incubation of the liganded receptor with RRL was necessary to recruit SRC-1 to the promoter. Conversely, the presence of SRC-1 was not required for the receptor to bind to the promoter. Finally, we determined whether the effect of RRL on PR binding to chromatin templates and the concomitant recruitment of SRC-1 was specific to the progesterone response element. RRL did not enhance the recruitment of PR or SRC-1 to the promoter of the ERE2S2CAT template (Fig. 8
, A and B). These data suggest that the restoration of PR transcriptional activity on chromatin templates is attributable to a specific effect of RRL on the ability of PR to bind its hormone response element in the context of chromatin and, therefore, to recruit the coactivator SRC-1.
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DISCUSSION |
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A variety of approaches were used to address the mechanism of RRL action. RRL does not simply exert a general effect on transcription from chromatin templates. RRL does not alter the level of basal transcription from any of the three templates used in our studies (Fig. 2). RRL increases transcription mediated by PR and other chaperone-dependent steroid receptors but not the synthetic activator GAL4-VP16. In the presence of RRL, PR activity is sequence specific and hormone and coactivator dependent (Fig. 2
). RRL does not act as a nonspecific stabilizing agent because heat-inactivated RRL does not restore PR transcriptional activity on chromatin templates (Fig. 4
). RRL does not inhibit assembly of chromatin (Fig. 3
); thus, its transcriptional effect cannot be attributed merely to preventing the repression of transcription by nucleosome formation on the template.
Interestingly, addition of RRL to the assembly reaction slightly, but consistently, increased the length of the nucleosome repeat pattern after micrococcal nuclease digestion (Fig. 3). The repeat length can be affected by salt concentration, but because RRL dialyzed into a low salt buffer produced the same effect it is unlikely that the change in the nucleosome repeat length is a salt artifact (data not shown). It is possible that the increase of the nucleosome repeat represents histone H1 incorporation into the chromatin templates. However, H1 was not readily detected in RRL by Western blot analysis (data not shown). Despite the change in the length of the nucleosomal repeat length in the micrococcal nuclease assay, it is unlikely that the activity of RRL can be attributed to altered chromatin structure or active chromatin remodeling activity. The S190 extract used in the chromatin assembly already contains abundant chromatin remodeling activities; furthermore, the chromatin remodeling factors Brg-1 and Brm cannot be detected in RRL. Finally, RRL has a minimal effect on receptor-mediated transcription when receptor purified with hormone is used in the assay, suggesting that the effect of RRL is not a passive consequence of altered chromatin structure, but is instead directly on PR, perhaps a step in the maturation of PR to its transcriptionally active form.
Because a protein-mediated activity in RRL appears to be responsible for the restoration of PR transcriptional activity, we investigated whether hsp chaperones were involved in this process. The hsp90 inhibitor, geldanamycin, blocks the ATP-dependent restoration of hormone binding ability to PR (Fig. 5A). The inhibition of PR-dependent transcription by geldanamycin also demonstrates that the regeneration of hormone binding by hsp chaperones is necessary for PR transcriptional activity (Fig. 5B
). Although chaperones are necessary for the receptor to bind hormone, our evidence indicates that the binding of hormone to recombinant PR is not sufficient to restore PR-dependent transcription on chromatin templates. Both purified, recombinant chaperones and the Drosophila S190 extract restore the hormone binding capacity of PR purified in the absence of ligand. The chaperone activity of the S190 extract explains the ligand-dependent transcriptional activity of PR seen on naked DNA templates. Nonetheless, neither the S190 extract (which possesses abundant Drosophila SWI/SNF containing chromatin remodeling complexes) nor recombinant hsp chaperones can restore PR-dependent transcription on chromatin templates (Figs. 2A
and 6B
). Therefore, additional protein factor(s) present in RRL must be required.
There are several steps subsequent to hormone binding that could be involved in the acquisition of PR-dependent transcription on chromatin templates. Although capable of binding ligand, the insect ecdysone receptor fails to bind its hormone response element when it is expressed and purified as a recombinant protein. Incubation with RRL or recombinant hsp chaperones restores the ability of the ecdysone receptor/RXR heterodimer to bind DNA in a gel-shift assay (46). This example contrasts with the present work in which we show that recombinant PR can bind to naked DNA once hsp chaperones have restored the receptors ability to bind hormone and that RRL does not affect binding to naked DNA (Fig. 8A).
Unlike the situation with naked DNA, RRL dramatically increases the affinity of the ligand-bound receptor for chromatin templates (Fig. 8, B and C). This result suggests that exposure to RRL transforms the receptor so that it can now access its DNA response element in the context of chromatin. One functional consequence of the receptor binding to chromatin is the recruitment of the coactivator SRC-1 to the promoter (Fig. 9
, A and B). Because exogenous SRC-1 is absolutely required for robust PR transcriptional activity on this promoter, the recruitment of SRC-1 to the promoter provides an explanation for the 50-fold increase in PR-dependent transcription observed on chromatin templates in the presence of RRL.
Several possibilities exist for the necessary factor provided by RRL. The recombinant chaperone system tested here has been shown to restore hormone binding activity to PR. However, this is a minimal system that may leave PR in an inadequate state for binding to chromatin. Studies by the Toft laboratory have shown that although recombinant chaperone proteins can restore ligand-binding ability to PR, they fail to dissociate from PR upon hormone binding (data not shown). In contrast, chaperone proteins dissociate from PR upon hormone binding when RRL is used instead of recombinant chaperones (47). It is possible that refolded receptor remaining bound to chaperones can bind naked DNA but cannot bind chromatin until chaperones are dissociated. Thus, an activity in RRL that completes the receptor folding cycle could account for both chaperone dissociation and acquisition of the ability of PR to bind its target site in the context of chromatin. This may or may not involve another conformation change or folding step on the part of the receptor. Initial studies suggest that both chaperone dissociation and acquisition of chromatin binding are mediated by activities that are very labile upon fractionation of RRL
When PR complexes are assembled in RRL, chaperones and additional proteins of indeterminate function also associate with the receptor. For example, the protein Hip participates in an intermediate step in assembly (48). Furthermore, the mature receptor complex with hormone binding activity also contains immunophilins such as FKBP51, PKBP52, and CyP40 (4, 11, 49). Because both PR and hsp90 are phosphoproteins, the possibility that phosphorylation is required for PR activity on chromatin templates should also be considered. In this case, the necessary factor in RRL could be a protein kinase or an accessory factor needed for PR or hsp90 to be recognized by protein kinases.
In summary, our analysis of ligand-free PR in a cell-free transcription system demonstrates that incubation of the receptor with RRL is necessary to restore the binding of PR to chromatin. Although hsp chaperones promote hormone binding to the receptor, additional protein factor(s) in RRL are necessary to restore the binding of the receptor to chromatin templates. Important questions remain to be answered concerning the identity and the function of the protein factor(s) responsible for restoring the transcriptional competence of PR on chromatin templates. The present system provides an assay to identify the necessary factor(s) from RRL and study their mechanism of action.
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MATERIALS AND METHODS |
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Purification of Flag-Tagged PRB
Full-length, human PRB containing a Flag epitope tag on the amino terminus was overexpressed in Sf9 cells via a baculovirus expression system (Fig. 1). The Sf9 cells were inoculated with virus at a multiplicity of infection of 1.0 and allowed to grow for an additional 48 h at 27 C. Cells were harvested by centrifugation at 1500 rpm for 15 min, washed once in TG buffer [10 mM Tris-HCl (pH 8.0) and 10% glycerol] and frozen as pellets at -80 C. The Sf9 cells were lysed in a homogenization buffer [20 mM Tris-HCl (pH 7.5), 350 mM NaCl, 1 mM dithiothreitol (DTT), 10% glycerol, 0.5 µg/ml leupeptin, 10 µg/ml bacitracin, 2 µg/ml aprotinin, 1 µg/ml pepstatin]. All procedures were done at 04 C. The cell lysate was centrifuged at 40,000 rpm for 30 min and the supernatant was taken as a soluble whole cell extract. The whole cell extract was diluted 1:2 in a dilution buffer [20 mM Tris-HCl (pH 7.5), 350 mM NaCl, 1 mM DTT, and 0.5% Nonidet P-40 (NP-40)] and added to anti-Flag M2 resin equilibrated with dilution buffer. The whole cell extract was incubated with resin on a rotator for 4 h. The resin was washed three times in a high salt wash buffer [20 mM Tris-HCl (pH 7.5), 600 mM NaCl, 1 mM DTT, 10% glycerol, and 0.2% NP-40] and then washed two times in a low salt wash buffer [20 mM Tris-HCl (pH 7.5), 350 mM NaCl, 1 mM DTT, 10% glycerol and 0.2% NP-40]. The protein was eluted by incubating the resin with an elution buffer [20 mM Tris-HCl (pH 7.5), 350 mM NaCl, 1 mM DTT, 10% glycerol, 0.1% NP-40, 0.2 mg/ml Flag peptide, and 0.5 mg/ml insulin]. ZnCl2 (1 µM) and MgCl2 (1 mM) were added to the eluted protein and the protein was frozen in liquid nitrogen and stored at -80 C in aliquots. Protein concentration was estimated by comparison to BSA standards on a silver stained SDS-PAGE gel. Typical yield was 0.5 mg of protein/500 ml flask of Sf9 cells.
Purification of Recombinant Proteins
Full-length, human SRC-1a containing a Flag epitope tag on the carboxyl terminus was synthesized in Sf9 cells via a baculovirus expression system and was purified as recently described (Fig. 1) (50). We used two strategies to optimize the expression and recovery of full-length SRC-1. The use of a Flag epitope tag on the carboxyl terminus of SRC-1 increased the production of full-length protein. Coexpression of hormone-bound PR with the Flag-tagged coactivator further increased the amount of full-length SRC-1. GAL4-VP16 was synthesized in Escherichia coli and purified as described (51). GAL4-VP16 was a kind gift from Jim Kadonaga. Human hsp90ß and human hsp70 were synthesized in Sf9 cells with a baculovirus expression system and were purified as previously described (52, 53). Human Hop (p60), human p23, and yeast YDJ-1 (hsp40) were synthesized in bacteria and purified as described (52, 53).
DNA Templates
The PRE2S2CAT template contains two consensus progesterone response elements (AGAACAGTTTGTTCT) located upstream of the core promoter of the human pS2 gene linked to the chloramphenicol (CAT) gene whereas the ERE2S2CAT template contains two consensus estrogen response elements (AGGTCACAGTGACCT) from the Xenopus vitellogenin A2 gene and the GAL42S2CAT template contains two GAL4 binding elements (54). The ERE2S2CAT and GAL42S2CAT templates were generous gifts from W. Lee Kraus and Jim Kadonaga.
Assembly and Analysis of Chromatin
Chromatin assembly reactions were performed with an S190 extract derived from postblastoderm Drosophila embryos (06 h) as described previously (43, 55). S190 extract (18.8 µl) in buffer R [10 mM potassium HEPES (pH 7.6), 10 mM KCl, 1.5 mM MgCl2, 0.5 mM EGTA, 10% glycerol, 10 mM ß-glycerophosphate, 1 mM DTT, 0.2 mM phenylmethylsulfonyl fluoride] incubated with 25 µl RRL and purified Drosophila core histones (1 µg) for 30 min at room temperature. The RRL was added to the assembly reaction due to a volume constraint in the rest of the chromatin-based in vitro transcription reaction. In experiments where purified chaperones were employed, 5 µg hsp90, 5 µg hsp70, 1.25 µg Hop, 1.25 µg p23 and 1.25 µg YDJ1 replaced 25 µl of RRL. In a separate tube, the receptor, hormone and the plasmid DNA template (500 ng) were incubated on ice for 15 min. Then the S190-histone mix (45.5 µl) and the activator-template mix (18.8 µl) were combined with an ATP regeneration mix (10.7 µl) (20 mM ATP, 0.2 M creatine phosphate, 650 µg/ml creatine phosphokinase, and 30 mM MgCl2) and the mixture (75 µl) was incubated at 27 C for 4 h. SRC-1 was added to the chromatin after the assembly reaction was complete and incubated for 20 min at room temperature to allow interaction of the factors with the preassembled chromatin. In the experiments with naked templates, the entire assembly reaction was done without the addition of core histones. The chromatin assembly reaction was then used in in vitro transcription experiments or subjected to micrococcal nuclease digestion to monitor the efficiency of chromatin assembly (56).
In Vitro Transcription
In vitro transcription reactions were performed with ammonium sulfate precipitated HeLa cell nuclear extracts as described previously (43). HeLa cell pellets were obtained from the National Cell Culture Center (Minneapolis, MN). Each transcription reaction consisted of 15 µl of chromatin, 7.5 µl of Buffer H [66 mM HEPES (pH 8), 0.66 mM EDTA, 234 mM KCl, and 15 mM MgCl2], 12.5 µl of 10% polyvinyl alcohol and 10 µl of HeLa nuclear extract. The mixture was incubated for 15 min at room temperature to form preinitiation complexes. Transcription was initiated by the addition of ribonucleoside triphosphates and the templates transcribed at 30 C for 30 min. The resulting transcripts were detected by primer extension using a CAT primer (5'-GCCTCAAAATGTTCTTTACGATGCCAT-3') radiolabeled with [-32P]ATP. Except where indicated otherwise, the final concentration of PR, R5020, SRC-1 and Gal4-VP16 in the transcription reactions was 20 nM, 60 nM, 25 nM, and 7.5 nM, respectively. Each experiment was performed a minimum of two separate times to ensure reproducibility. The data were quantitated by PhosphorImager analysis (Storm Imager, Molecular Dynamics, Sunnyvale, CA). A representative background was subtracted from each sample and the amount of relative transcription was determined by dividing the sample with the amount of basal transcription.
Hormone Binding
One picomole of PR was incubated in TDG buffer [10 mM Tris-HCl (pH 7.5), 1 mM DTT, and 10% glycerol] at 30 C for 30 min in the presence or absence of an ATP regeneration system and 20 µl of RRL and then chilled on ice. In the experiments with purified chaperones, 4 µg hsp90, 4 µg hsp70, 1 µg Hop, 1 µg p23, and 1 µg YDJ1 were used to replace 20 µl of RRL. The 3H-R5020 binding activity was determined in a dextran-coated charcoal steroid binding assay in the presence of 2.5 pmol of 3H-R5020. The amount of specific 3H-R5020 bound was obtained by analyzing a second set of samples incubated in the presence of 100-fold excess of cold R5020. The reactions were incubated for 1 h at 4 C. Resuspended dextran-coated charcoal was added to the reactions and the reactions were agitated for 10 min, centrifuged at 3000 rpm for 10 min, and the unbound fraction was counted with scintillation fluid. Each experiment was performed a minimum of two separate times to ensure reproducibility. The error bars indicate the SD of several experiments.
EMSA
The ability of PR to bind an oligonucleotide was determined by EMSA as previously described (57) with several modifications. S190 extract (9 µl) was incubated at 30 C for 30 min with an ATP regeneration system (5 µl) in the absence or presence of 300 nM PR, excess R5020 and RRL. The S190-receptor mix (30 µl) was incubated at 4 C for 20 min in a DNA binding buffer [10 mM HEPES (pH 7.8), 50 mM KCl, 4 mM MgCl2, 5 mM DTT, 40 µg/ml ovalbumin, 40 µg/ml poly(deoxyinosine-deoxycytidine), 50 µg/ml salmon sperm DNA, and 7.5% glycerol]. The DNA binding reactions were then incubated at 4 C for 1 h with 0.6 ng of a 32P end-labeled 28-bp oligonucleotide containing a consensus progesterone response element. The oligonucleotide was end-labeled by filling in the 5' single-stranded ends with the Klenow fragment of DNA polymerase and [-32P]dCTP and dATP. After 1 h, the DNA binding reactions (40 µl) were electrophoresed on a 5% polyacrylamide gel (40:1 acrylamide: bisacrylamide) containing 2.5% glycerol at 4 C in a 0.25x TBE buffer [20 mM Tris-borate (pH 8), 20 mM boric acid; 0.5 mM EDTA].
In Vitro ChIP Assay
ChIP assay was performed as described (58) with a few modifications. The major modification was fragmentation of the chromatin template by micrococcal nuclease digestion before cross-linking with formaldehyde. Chromatin was assembled as described above with 1 µg template, 1 µg of PR, and excess R5020. Two micrograms of SRC-1 were added after chromatin assembly and incubated for 30 min at 30 C. Chromatin was fragmented to mono- and di-nucleosomes with micrococcal nuclease (0.7 U/ml) for 10 min at room temperature in the presence of 0.1 M CaCl2. The digestion was stopped by the addition of EDTA and incubation for 15 min at room temperature. One percent formaldehyde was added to the chromatin and the samples were rotated for 15 min at room temperature. After a brief spin, the samples were diluted with ChIP dilution buffer [0.01% sodium dodecyl sulfate (SDS), 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl (pH 8), 167 mM NaCl] to a total volume of 3.5 ml. The samples were precleared with Protein A agarose beads (Pierce Chemical Co., Rockford, IL) as described previously (59). One milliliter of the precleared sample (input) was incubated with no antibody, 5 µl mouse monoclonal antibody PR 1294, or 5 µl mouse monoclonal antibody SRC-1 1135/H4. Dean Edwards generously provided the antibodies. The samples were rotated overnight at 4 C. The immune complexes were recovered by addition of Protein A/G agarose beads (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and rotation for 1 h at 4 C. The beads were harvested, washed and eluted as described previously (59). The input (10%) and the samples were incubated for 4 h at 65 C in the presence of 0.2 M NaCl to reverse the formaldehyde cross-links. The samples were phenol/chloroform extracted and precipitated.
The DNA samples were denatured for 10 min in alkali at 37 C and slot blotted onto a positively charged nylon membrane (Hybond, Amersham Pharmacia, Piscataway, NJ). The nylon membrane was prehybridized for 30 min at 42 C with hybridization solution [50% formamide, 50 nM NaPO4 (pH 6.5), 1.6 M NaCl, 2 mM EDTA, 5x Denhardts, 0.5 mg/ml salmon sperm DNA, 0.2% SDS] and hybridized overnight at 42 C with a promoter-proximal probe. The probe was prepared by polymerase chain reaction amplification of a promoter-proximal piece of either the PRE2S2CAT or ERE2S2CAT template using a primer to the promoter of the pS2 gene (5'-TTTATTGCTTTATTCGG-3') and a CAT primer (5'-CCAGGTTTTCACCGTAACACG-3'). The amplified DNA was radiolabeled with [-32P]dCTP using Rediprime (Amersham Pharmacia). The membrane was washed with 1x saline sodium citrate, 0.1% SDS, for 15 min at 65 C and quantitated by phosphorimager analysis. Each experiment was performed a minimum of two separate times to ensure reproducibility.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Current address for V.G.T.: Department of Reproductive Medicine, University of California, San Diego, La Jolla, California 92093.
Abbreviations: CAT, Chloramphenicol; ChIP, chromatin immunoprecipitation; CREB, cAMP response element binding protein; DTT, dithiothreitol; GAL4-VP16, GAL4 DNA binding domain fused to the VP16 activation domain; hsp, heat shock protein; NP-40, Nonidet P-40; PR, progesterone receptor; RRL, rabbit reticulocyte lysate; SDS, sodium dodecyl sulfate; SRC, steroid receptor coactivator; SWI/SNF, switch defective/sucrose nonfermenter.
Received for publication May 30, 2003. Accepted for publication September 22, 2003.
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REFERENCES |
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