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Molecular Endocrinology 21 (3): 635-650
Copyright © 2007 by The Endocrine Society

R5020 and RU486 Act as Progesterone Receptor Agonists to Enhance Sp1/Sp4-Dependent Gene Transcription by an Indirect Mechanism

Aymen Shatnawi, Thuyet Tran and Manohar Ratnam

Department of Biochemistry and Cancer Biology, Medical University of Ohio at Toledo, Toledo, Ohio 43614

Address all correspondence and requests for reprints to: Manohar Ratnam, Department of Biochemistry and Cancer Biology, Medical University of Ohio at Toledo, 3035 Arlington Avenue, Toledo, Ohio 43614. E-mail: mratnam{at}meduohio.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
It has been suggested that ligand-dependent gene activation by the progesterone receptor (PR) can result from recruitment of PR by the promoter bound Sp1. A detailed investigation of the Sp1-dependent agonistic activity of RU486 and R5020 on the folate receptor (FR) type {alpha}, p27, thymidine kinase 1 and p21 genes reveals a different mechanism. The FR-{alpha} P4 promoter and the endogenous FR-{alpha} gene were up-regulated by the PR agonist R5020 through either PR-A or PR-B. The classical antagonist RU486 also activated the promoter but only through PR-B. The most proximal (essential) G/C-rich (Sp1 binding) element and the initiator region constituted the minimal promoter responsive to PR regulation; substitution with a stronger cluster of G/C-rich elements enhanced the magnitude of the PR response. In contrast, substitution of the G/C-rich element with a TATA box resulted in the loss of regulation by PR. Overexpression of Sp1 and Sp4 but not Sp3 enhanced activation of the FR-{alpha} promoter by PR, knocking down Sp1 decreased the activation in a manner that was reversed by ectopic Sp1 or Sp4. The ligand-dependent action of PR on the promoter was delayed compared with its activation of a classical glucocorticoid response element-driven promoter and activation of both the promoter and the endogenous FR-{alpha} gene by PR required new protein synthesis. Activation by PR paralleled RNA polymerase II recruitment but was not accompanied by either association of PR or a change in the association of Sp1 with the endogenous FR-{alpha} P4 promoter. Similar observations were made for PR regulation of the genes encoding p27, thymidine kinase 1, and p21. The results contradict the current view of Sp1-dependent gene regulation by PR and point to the existence of one or more PR target genes whose promoter and cell context(s) must thus be key determinants of the agonistic activity of RU486 on a large group of important Sp1-dependent downstream target genes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE PHYSIOLOGICAL EFFECTS of progesterone and its antagonists are principally mediated by the transcriptional activity of the progesterone receptor (PR) (reviewed in Ref. 1). The two subtypes of PR, PR-A (94 kDa) and PR-B (120 kDa), are differentially expressed in a tissue-specific manner (2, 3, 4, 5). PR-A is a truncated form of PR-B lacking its N-terminal 164 amino acids (6, 7). The PR isoforms differentially regulate progesterone-responsive genes, and the transcriptional activation of a given promoter could be unequal (8, 9, 10, 11, 12, 13, 14).

Ligands that modulate PR action have clinical application in treating endocrine disorders, as anticancer agents and in terminating pregnancy (reviewed in Refs. 15, 16, 17, 18). Pure PR antagonists such as ZK98299 (onapristone) completely abrogate the transcriptional activity of PR, whereas selective PR modulators such as RU486 (mifepristone) have mixed effects acting as either PR agonists or antagonists in different cell and target gene contexts. Selective PR modulators do not affect homodimerization of PR or its binding to its cognate response element in the target gene (19, 20) but produce conformational changes in the AF-2 region of PR to block the association of coactivators and promote corepressor recruitment (reviewed in Refs. 21, 22, 23). The A and B subtypes of PR respond differently to RU486, which binds to PR-B to function as a partial agonist under certain conditions; in contrast, RU486 can only act as an antagonist of PR-A (8, 24). It has been proposed that the partial agonistic activity of RU486 is dependent on the presence of an intact AF-1 region in its receptor (8) as well as the N-terminal domain of PR-B (25). The molecular mechanism by which RU486 works as agonist is not fully understood. Treating cells with the PKA activator 8-bromo-cAMP converted RU486 from an antagonist into an agonist (26, 27, 28). The ratio of coactivator to corepressor in the cell was also found to influence the partial-agonistic activity of RU486 (29).

PR can regulate target promoters including the natural promoters for the p21, p27, glycodelin, and thymidine kinase (TK) genes in the absence of a functional classical response element [progesterone response element (PRE)/glucocorticoid response element (GRE)] through G/C-rich (Sp1 binding) elements (24, 30, 31, 32, 33). Some observations appeared to suggest that this regulation occurs by direct association of PR with DNA-bound Sp1 (31, 32). Additional support for this suggestion is derived from the well-established role of Sp proteins in mediating promoter activation by the estrogen receptor (ER) by a nonclassical mechanism; it has been demonstrated that ER is recruited by Sp1, Sp3, or Sp4 in the absence of classical estrogen response elements to exert its genotropic effects in a variety of gene promoters in the phyisiologic context (34, 35). Transactivation by PR independent of a PRE is of particular interest because this may underlie the agonistic activity of RU486 on promoter activation by PR-B (24).

Our initial interest in discovering and studying the regulation of the folate receptor (FR) type {alpha} gene by PR ligands was based on the well-established potential of FR-{alpha} as a tumor target in gynecological cancers for the selective delivery of therapeutic agents and the possibility of using innocuous PR ligands to selectively induce the expression of FR-{alpha} in the tumors (reviewed in Refs. 36 and 37). We discovered that the FR-{alpha} gene does not contain a functional hormone response element for PR and that the PR action is mediated by Sp1/Sp4 and the essential G/C-rich (Sp binding) elements within the TATA-less basal P4 promoter of the FR-{alpha} gene. The detailed mechanistic studies described in this report for the FR-{alpha} gene were extended to the genes encoding p27, thymidine kinase 1, and p21. These studies contradict the prevalent view that genes regulated by PR through their Sp1 elements are direct targets of PR action and demonstrate that they are in fact downstream targets of PR. It follows that the key event in the agonistic activity of both R5020 and RU486 on this important and potentially large class of PR target genes is an unusual regulation by PR of an upstream target gene(s) whose product(s) activates other genes through Sp1 or Sp4.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The FR-{alpha} Promoter and the Endogenous FR-{alpha} Gene Are Activated by PR in a Ligand-Dependent Manner
In HeLa cells transiently transfected with PR-A or PR-B expression vectors, the potent PR agonist R5020 (promegestone) increased FR-{alpha} promoter-luciferase reporter activity in a dose-dependent manner. The maximal activation produced by PR-A was comparable to that produced by PR-B (Fig. 1AGo). Under similar conditions, the control GRE2e1b-luciferase that is driven by the classical PRE/GRE was preferentially activated by PR-B (Fig. 1BGo). Under the conditions of transfection and treatment, the expression levels of PR-A and PR-B remained constant (Fig. 1CGo). The results demonstrate specific ligand- and receptor-mediated activation of the FR-{alpha} promoter by PR which differs from that of a classical PR target promoter in receptor subtype specificity.


Figure 1
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  		Fig. 1. Ligand-Dependent Regulation of the FR-{alpha} Promoter by PR

HeLa cells grown to 80% confluence were transfected with full-length FR-{alpha} promoter-luc (A) or (B) GRE2e1b promoter-luc (B) and with expression plasmid for either PR-A or PR-B or with empty vector; the cells were treated with the range of concentrations of R5020 or with vehicle and harvested 48 h later to measure luciferase activity. The values represent fold increase relative to the vehicle-treated control. C, Western blots of total cell lysates from the HeLa cells transfected in A with either PR-A or PR-B probed with antibody to PR-A (top panels) and PR-B (bottom panels), respectively, and reprobed with antibody to tubulin. D, HeLa cell cells were grown to 80% confluence and transfected with either FR-{alpha} promoter-luc or GRE2e1b promoter-luc and cotransfected with PR-A or PR-B; the cells were treated as indicated with R5020 and/or RU486 and/or vehicle and harvested to measure luciferase activity at 48 h. The values are represented as percent of maximum activity of either FR-{alpha} promoter-luc or GRE2e1b promoter-luc. All of the determinations were made in triplicate, and the SDs are indicated. A, P < 0.0001 for a vs. c, e, g, or i; b vs. d, f, h, or j. B, P < 0.001 for c vs. a or d; b vs. d; f vs. e; h vs. g and j vs. i. C, P < 0.0001 for g vs. a, b, d, or f; c vs. e or h.

 
HeLa cells transiently transfected with PR-B showed an increase in FR-{alpha} promoter-luciferase activity upon treatment with RU486 either alone or in combination with R5020 (Fig. 1DGo). Under similar conditions, RU486 acted as an antagonist of PR-A/R5020 on the FR-{alpha} promoter (Fig. 1DGo). Both PR-A/R5020 and PR-B/R5020 failed to activate RU486-treated cells when transfected with the GRE2e1b-luciferase reporter construct (Fig. 1DGo). The result shows selective induction of the FR-{alpha} promoter by RU486 under conditions in which it antagonizes ligand-dependent activation of a classical GRE-dependent promoter.

In FR-{alpha}-positive recombinant T47D breast cancer cells that express either PR-A (T47D-A cells) or PR-B (T47D-B cells), the transfected FR-{alpha} promoter-luc (Fig. 2AGo) was up-regulated by R5020. In contrast, RU486 acted as an antagonist of R5020 in T47D-A cells but as an agonist in T47D-B cells in regulating the FR-{alpha} promoter activity (Fig. 2AGo). Consistent with these observations, R5020 increased endogenous FR-{alpha} mRNA in both T47D-A and T47D-B cells and RU486 increased the FR-{alpha} mRNA in T47D-B cells alone (Fig. 2BGo). Thus transcription of the FR-{alpha} gene is positively regulated by R5020 through both PR-A and PR-B, whereas RU486 antagonizes the activation of the FR-{alpha} gene by R5020/PR-A but positively regulates the gene through PR-B.


Figure 2
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  		Fig. 2. Regulation of the FR-{alpha} Promoter and Induction of Endogenous FR-{alpha} mRNA in T47D-A and T47D-B Cells by PR Ligands

A, T47D-A or T47D-B cells were transfected with FR-{alpha} promoter-luc and treated with 50 nM of each ligand as indicated. After 48 h, the cells were lysed to measure luciferase activity. The values represent the relative luciferase activity unit (B) T47D-A or T47D-B cells were treated with vehicle or with 50 nM of either R5020 or RU486, and 48 h later the cells were harvested and total RNA extracted. The RNA was subjected to real-time RT-PCR to measure mRNA for FR-{alpha} or GAPDH. The values represent fold induction of FR-{alpha} mRNA compared with vehicle-treated controls and normalized to GAPDH. A, P < 0.0001 for a vs. b; c vs. d, e or f. B, P < 0.0001 for a vs. b; c vs. d or e.

 
The Target Site of PR Action Is the G/C-Rich Element in the Minimal P4 Promoter
Figure 3AGo shows a variety of deletion and chimeric constructs of the FR-{alpha} promoter region that were used to identify the target site for the agonistic action of R5020/PRA, R5020/PR-B, and RU486/PR-B in the FR-{alpha} gene. The full-length promoter construct spanning both the P1 and P4 promoters [–3394 to +33 nucleotides (nt)] could be deleted from the 5' end up to the most proximal and essential G/C-rich (noncanonical Sp1 binding) element of the P4 promoter without the loss of its typical PR-A (Fig. 3BGo) or PR-B (Fig. 3CGo)-dependent regulation by R5020 or RU486 albeit to a lesser magnitude; however, the magnitude of the PR ligand effects of the full-length promoter construct was restored if the additional noncanonical Sp1 elements of the P4 promoter were included (Fig. 3Go, B and C). To test the requirement for a G/C-rich element in mediating the PR effects, these elements in the P4 promoter (–272 to –35 nt) were replaced with a TATA box; the chimeric TATA box-driven promoter lost the regulation by both R5020 and RU486 through either PR-A (Fig. 3BGo) or PR-B (Fig. 3CGo), suggesting the requirement for at least one G/C-rich element. A role for G/C-rich elements in the regulation of the P4 promoter by PR ligands was also evident from the observation that replacing the noncanonical Sp1 elements in the P4 promoter with a strong cluster of six canonical Sp1 elements derived from the simian virus 40 (SV40) promoter significantly increased the magnitude of the induction of the promoter by R5020 or RU486 through PR-A (Fig. 3BGo) or PR-B (Fig. 3CGo). The above results demonstrate that PR ligands regulate the FR-{alpha} gene through its minimal P4 promoter and also collectively confirm the role of Sp1 binding G/C-rich elements in mediating the ligand-dependent trans-activation of the promoter by PR.


Figure 3
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  		Fig. 3. Mapping and Characterizing the Target Site of PR Action in the FR-{alpha} Promoter

A, Schematic representation of various promoter-luciferase reporter constructs. The figure shows the full-length FR-{alpha} promoter including both the P1 and P4 basal promoters (FR{alpha} –3394 to +33 nt) and its 5' deleted versions (FR{alpha} –272 to +33 nt), (FR{alpha} –116 to +33 nt) and (FR{alpha} –49 to +33 nt). The [FR{alpha} –3394 to –47 nt/ SV40(GC)6] construct represents a chimeric FR-{alpha}/SV40 promoter in which the G/C-rich region of the FR-{alpha} promoter was replaced with six tandem G/C-rich elements from the SV40 promoter. In (TATA-FR{alpha} –35 to +33 nt), the Sp1 binding site in construct (FR{alpha} –49 to +33 nt) is replaced by a TATA box element. In B and C, HeLa cells were transfected with the various promoter-luciferase constructs and cotransfected with PR-A (B) or with PR-B (C) and treated as indicated with vehicle, 50 nM R5020 or 50 nM RU486 alone or in combination. After 48 h of treatment, the cells were lysed to measure luciferase activity. The values for luciferase activity are expressed as the ratio to that for the corresponding vehicle-treated control. B, P < 0.001 for b vs. a or c; d vs. c or e. C, P < 0.001 for d vs. a or g; g vs. m; e vs. b or h; k vs. h or n; f vs. c or i; l vs. i or o.

 
Certain Sp Family Proteins Support the Regulation of the FR-{alpha} Promoter by PR
Because functional G/C-rich cis elements are known to bind the Sp family transcription factors, the potential roles of the Sp family proteins, Sp1, Sp3, and Sp4, in mediating PR regulation of the FR-{alpha} promoter was examined. Overexpression of Sp1 and Sp4 but not Sp3 enhanced the agonistic action of R5020/PR-A, R5020/PR-B and RU486/PR-B on the promoter (Fig. 4Go, A and B). Overexpression of each Sp protein was confirmed by Western blot (data not shown). The functionality of all three overexpressed proteins was evident from the fact that the basal P4 promoter activity was increased by the transfected Sp1, Sp3, and Sp4 by 35, 26, and 103%, respectively (not shown in Fig. 4Go).


Figure 4
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  		Fig. 4. Effect of Sp Family Proteins on Ligand-Dependent Activation of the FR-{alpha} P4 Promoter by PR

HeLa cells were cotransfected with FR{alpha} –272 to +33 nt-luc and expression plasmid for Sp1, Sp3, or Sp4 or with the empty vector and with expression plasmid for PR-A (A) or PR-B (B). The cells were then treated with vehicle or with R5020 (50 nM) or RU486 (50 nM) alone or in combination as indicated. The cells were harvested to measure luciferase activity 48 h after transfection. The data represent fold increase in the luciferase activity compared with the corresponding vehicle control. T47D-A cells (C) and T47D-B cells (D) were nucleofected with FR{alpha} –272 to +33nt-luc and expression plasmid for Sp1, Sp4 or small interfering RNA (siRNA) for Sp1 in the indicated combinations. Twelve hours later, the cells were treated with vehicle, R5020 (50 nM) or RU486 (50 nM). After 48 h of treatment, the cells were harvested and assayed for luciferase activity. The data represent relative luciferase units. E, Western blot of total cell lysates from the T47D-B cells nucleofected with expression vectors for Sp1, Sp4, Sp1 siRNA or a scrambled siRNA control and probed with antibody to Sp1 (top panel) and Sp4 (middle panel) and reprobed with antibody to GAPDH (bottom panel). A, P < 0.0001 for a vs. b or c. B, P < 0.0001 for a vs. d or g; b vs. e or h; c vs. f or i. C, P < 0.0001 for a vs. c, e, or g; d vs. b, f, or h. D, P < 0.0001 for a vs. d, g, or j; b vs. e; c vs. f; e vs. h or k; f vs. i or l.

 
When the endogenous Sp1 in T47D-A (Fig. 4CGo) or T47D-B (Fig. 4DGo) cells was knocked down, the activation of the FR-{alpha} promoter by R5020 or RU486 was greatly decreased. The activation by PR ligands in both cell lines was restored upon simultaneous ectopic expression of either Sp1 or Sp4 (Fig. 4Go, C and D). In this experiment, knockdown or overexpression of Sp proteins was confirmed by Western blot (Fig. 4EGo).

The results show that both Sp1 and Sp4 but not Sp3 must mediate the regulation of the FR-{alpha} gene through the G/C-rich elements of the P4 promoter.

PR Ligand Action on the FR-{alpha} Promoter Is Delayed
The time course of action of PR ligands on the promoter was determined in relation to the GRE2e1b promoter, a known direct target of PR action. Figure 5AGo shows that the GRE2e1b promoter was activated by R5020/PR-B close to the optimal level within 3 h-6 h. In contrast, under identical conditions, activation of the FR-{alpha} promoter by R5020/PR-A (Fig. 5BGo), R5020/PR-B (Fig. 5CGo) was only discernible at 12 h and showed a progressive increase up to 48 h. This delayed response of the FR-{alpha} promoter suggested that this gene may not be a direct target of PR action.


Figure 5
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  		Fig. 5. Time Course of R5020-Dependent Promoter Activation by PR

HeLa cells were transfected with GRE2e1b promoter-luc (A) or FR{alpha} –272 to +33 nt-luc (B and C). The cells were cotransfected with expression plasmid for PR-A (B) or PR-B (A and C). Twenty-four hours after transfection, the cells were treated with either vehicle or R5020 (50 nM) and harvested at the indicated times. The luciferase activities measured in the cell lysates are expressed as ratios to the corresponding vehicle-treated controls. A, P < 0.001 for b vs. a, c or d; d vs. f. B, P < 0.01 for b vs. a or c; c vs. d. C, P < 0.001 for b vs. a or c; c vs. d.

 
New Protein Synthesis Is Required for the Regulation of the FR-{alpha} Promoter by PR
The relatively slow time course of activation of the FR-{alpha} promoter by PR noted above suggested that the promoter may be an indirect or downstream target of PR action and that the protein product of one or more upstream or direct target genes of PR may be required to mediate this indirect regulation. To test this possibility, the effect of blocking de novo protein synthesis using cycloheximide (CHX) during a 12-h period of treatment with PR ligands on the activity of the FR-{alpha} promoter was examined (Fig. 6Go). In HeLa cells expressing PR-A, CHX effectively blocked the R5020-induced increase in the reporter mRNA generated from the FR-{alpha} P4 promoter plasmid (Fig. 6AGo). Similarly CHX also blocked the promoter activity induced by either R5020 or RU486 in cells expressing PR-B (Fig. 6BGo). In contrast, CHX did not affect the activity of the control GRE2e1b promoter, a known direct target promoter of PR (Fig. 6CGo). In the presence of CHX, there was no significant increase in luciferase enzyme activity, indicating complete inhibition of protein synthesis (data not shown). The results demonstrate that the ligand-dependent action of PR on the FR-{alpha} promoter is indirect and requires intermediate protein synthesis.


Figure 6
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  		Fig. 6. Effect of Cycloheximide on Activation of the FR-{alpha} Promoter by PR Ligands

HeLa cells were transfected with (A) FR{alpha} –272 to +33nt-luc (B) FR{alpha} –272 to +33nt-luc and (C) GRE2e1b-promoter-luc. The cells were cotransfected with PR-A (A) or PR-B (B and C). Forty-eight hours later, the cells were pretreated with either vehicle or cycloheximide (10 µM) for 2 h. At the end of the 2-h pretreatment, R5020 (50 nM) or RU486 (50 nM) was introduced alone or in combination with cycloheximide and the incubation continued for 12 h. The cells were then harvested and total RNA was extracted. The mRNAs for luciferase and GAPDH were measured by real-time RT-PCR. The values for the reporter were normalized to GAPDH. A, P < 0.0001 for b vs. a or c. B, P < 0.001 for a vs. b or c; b vs. d; c vs. e. C, P < 0.0001 for a vs. b or c.

 
The PR Ligand Effects on the Endogenous Genes for FR-{alpha}, TK1, and p27 Are Similar and also Require New Protein Synthesis
The preceding experiments indicate that the regulation of the FR-{alpha} promoter by PR is indirect and that this is mediated by G/C-rich cis elements in the promoter and their cognate trans-factors Sp1 or Sp4. It was therefore of interest to examine whether such an indirect mechanism was relevant to PR regulation of the endogenous FR-{alpha} gene and as well as other target genes of PR including TK1 and p27 in which it has been previously shown that Sp1 elements rather than classical response elements mediate induction by progestin.

Unlike TK1, the optimal increase in p27 reportedly occurs in the second phase of the cell’s biphasic response to progestin (38). After 48 h of treatment, both R5020 and RU486 substantially (by >5-fold) up-regulated the endogenous mRNA for p27 in T47D-B cells (Fig. 7Go); R5020 also increased the p27 mRNA by ~8-fold in T47D-A cells but RU486 was an antagonist of this effect (Fig. 7Go).


Figure 7
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  		Fig. 7. Induction of Endogenous p27 in T47D-A and T47D-B Cells by PR Ligands

TA7D-A or T47D-B cells were treated with vehicle, R5020 (50 nM) or RU486 (50 nM). Forty-eight hours later, the cells were harvested and the total RNA was extracted from the cells and subjected to real-time RT-PCR to measure the mRNAs for p27 and GAPDH. The values for p27 mRNA are normalized to those for GAPDH. P < 0.0001 for a vs. b; c vs. d or e.

 
In both T47D-A and T47D-B cells, treatment with CHX under the experimental conditions used in Fig. 8Go virtually completely blocked protein synthesis as seen by the loss of the p21 protein either in the absence or in the presence of R5020 or RU486 (Fig. 8AGo). In both T47D-A and T47D-B cells (Fig. 8BGo), a 12-h treatment with the appropriate PR ligand resulted in an increase in the endogenous FR-{alpha} mRNA in cells harvested 12 h after the treatment. However, treatment with CHX beginning 2 h before and during the 12 h treatment with PR ligands blocked this increase in the FR-{alpha} mRNA (Fig. 8BGo). A similar experiment was performed for TK1 with the exception that because TK1 is down-regulated in the later phase of the progestin response, the mRNA was measured at the end of the 12-h treatment with ligand (Fig. 8CGo); CHX blocked the induction of the TK1 mRNA by R5020 or RU486. Similar results were obtained for the endogenous mRNA for p27 (Fig. 8DGo). In Fig. 8Go, the c-myc gene, which is a known direct target for PR, was used as a control. CHX did not block the induction of c-myc mRNA by R5020 (Fig. 8EGo). It is thus evident that the endogenous genes for FR{alpha}, TK1, and p27 are all regulated by PR indirectly in a manner that requires new protein synthesis.


Figure 8
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  		Fig. 8. Effect of CHX on Activation of the Endogenous Genes for FR-{alpha}, TK1, and p27

A, T47D-A (left panel) or T47D-B cells (right panel) were pretreated with vehicle or with CHX (20 µmol/liter) for 2 h followed by the inclusion of vehicle, R5020 (50 nM) or RU486 (50 nM) as indicated and incubated for a further 12 h. The cells were then harvested and the lysates subjected to Western blot analyses to probe for p21 and the blots reprobed for tubulin. In B–E, T47D-A or T47D-B were pretreated with either CHX (20 µmol/liter) or vehicle (V) for 2 h followed by the addition of vehicle, R5020 (50 nM) (R) or RU486 (50 nM) (RU) as indicated. Where indicated, 12 h later after the treatment, CHX, R5020, and RU486 were removed by replacing with fresh media. The cells were harvested at the indicated times and total RNA extracted. The mRNAs for FR-{alpha} (B), TK1 (C), p27 (D), or c-myc (E) were measured by real-time RT-PCR together with the mRNA for GAPDH. The mRNA values are normalized to those for GAPDH. B, P < 0.001 for b vs. a or c; e vs. d or f. C, P < 0.001 for a vs. c; b vs. d or e; d vs. g; c vs. f; e vs. h. D, P < 0.0005 for a vs. b or c; b vs. d; c vs. e. In panel E, P < 0.0001 for a vs. b or c.

 
PR Does Not Associate with Its Target Elements in the Endogenous FR-{alpha}, TK1, or p27 Genes Either Basally or in Response to Ligand
The preceding experiments suggested that, in contrast to the c-myc gene, the genes encoding FR-{alpha}, TK1, and p27 were regulated by PR ligands although the product(s) of an upstream direct target gene(s). Chromatin immunoprecipitation (ChIP) assays were designed to further test whether PR was recruited to the promoters in the endogenous genes for FR-{alpha}, TK1, and p27 either in the absence or in response to treatment of cells with PR ligands (Fig. 9Go). Quantitative analysis by real-time PCR of ChIP signals from immunoprecipitated chromatin fragments using antibody to PR showed strong ligand-induced signals for the PRE region in the c-myc gene that was well above the background signal obtained from PCR amplification of a distal sequence from a coding exon chosen as the irrelevant target (Fig. 9Go). In contrast, the ChIP signals obtained for the G/C-rich promoter regions in the FR-{alpha}, p27, and TK1 genes corresponded to the background levels for their respective irrelevant targets chosen within distal coding exons (Fig. 9Go). Nonimmune IgG (negative control) did not give a significant ChIP signal above the background in any case (data not shown). It was separately confirmed that all of the real-time PCR primers and probes used in this experiment were capable of efficiently amplifying their target DNA sequences (Table 1Go). The results indicate that PR does not specifically associate with the promoter regions of the endogenous genes encoding FR-{alpha}, p27, or TK1 in contrast to c-myc promoter either in the absence or in the presence of ligand.


Figure 9
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  		Fig. 9. Quantitative ChIP Assays to Detect Association of PR with Endogenous Genes

T47D-B cells were treated with vehicle, R5020 (50 nM) or RU486 (50 nM) for 1.5 h. The cells were subjected to ChIP assays as described in Materials and Methods using antibody against PR or normal IgG (negative control). The target sequences for measurement of ChIP signals by real-time PCR include the PRE region in the c-myc promoter, Sp1 elements in the basal promoters of the genes encoding FR-{alpha}, p27, and TK1 and as well as sequences (irrelevant targets) distal to the promoters of the c-myc, FR-{alpha}, p27, and TK1 genes (i.e. within their coding exons). The ChIP signals are expressed as fold difference in relation to the corresponding irrelevant targets in the vehicle-treated controls. The normal IgG control did not produce ChIP signals (data not shown). P < 0.0001 for b vs. a, c, or d.

 

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  		Table 1. Amplification of Target Genomic DNA by the Real-Time PCR Primers and TaqMan Probes1

 
PR Ligands Neither Alter the Expression of Sp Proteins or Change Their Association with the Endogenous Genes Encoding FR-{alpha}, TK1, and p27
Because Sp1 and Sp4 mediated the induction of the FR-{alpha} promoter by PR, the possibility that PR ligands increased either the expression of Sp1/Sp4 or their association with the promoters for FR-{alpha}, TK1, or p27 was tested. Treating T47D-A or T47D-B cells with R5020 or RU486 did not significantly alter the expression of either Sp1 or Sp4 (data not shown). ChIP assays designed to detect a possible change in the association of Sp1 or Sp4 with the functional G/C-rich elements within the basal promoters for the endogenous genes encoding FR-{alpha}, p27, and TK1 showed a specific association of both these proteins with the promoters; however, the Sp1/Sp4 interactions with the promoters were not significantly different in groups treated for 30, 90, or 180 min with vehicle, R5020, or RU486 (data not shown). This result indicates that PR ligands can neither increase the association of Sp1 or Sp4 with their cognate functional cis elements in the FR-{alpha}, p27, or TK1 genes nor induce any other DNA binding protein that could displace them.

The Delayed Effect of R5020 on the Expression of FR-{alpha}, p27, TK1, and p21 Genes Is Temporally Related to Recruitment of RNA Polymerase II (Pol II) to the G/C-Rich Regions in the Basal Promoters of the Endogenous Genes
To confirm the delayed activation of transcription by PR agonist, ChIP assays were used to measure the association of RNA Pol II with the basal promoters of the endogenous genes for FR-{alpha}, p27, TK1, and p21 as a function of time of treatment with R5020 (Fig. 10Go). The C-myc gene promoter, which is a direct target of PR regulation, was used as a positive control. In T47D-B cells, there was a basal association of Pol II with the promoter region and R5020 greatly increased the specific association of Pol II within 1 h (Fig. 10AGo). In contrast, in the FR-{alpha} (Fig. 10BGo), p27 (Fig. 10CGo), and TK1 (Fig. 10DGo) gene promoters, ligand-induced association of Pol II only occurred between 6 and 12 h of treatment. The p21 gene, which also lacks a classical response element for PR and is dependent on G/C-rich elements for PR regulation, was also analyzed in this manner (Fig. 10EGo). Although R5020-induced association of Pol II with the p21 gene promoter was delayed compared with the C-myc gene promoter, it occurred earlier (by 6 h) than in the FR-{alpha}, p27, and TK1 gene promoters (Fig. 10EGo). This result suggests that, whereas the PR ligand-dependent induction of the p21 gene may be indirect, cis elements and trans-factors unique to the p21 gene may also play a role in mediating regulation by PR ligands.


Figure 10
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  		Fig. 10. Time Course of Induction of RNA Polymerase II Recruitment to Various Gene Promoters by R5020

T47D-B cells were treated with vehicle or R5020 (50 nM) for 1, 3, 6, 12, and 24 h. The cells were subjected to ChIP assays as described in Materials and Methods using antibody against Pol II or normal IgG (negative control). The target sequences for measurement of ChIP signals by real-time PCR include the PRE elements in the basal promoter of the c-myc gene (A) or Sp1 elements in the basal promoters of the genes encoding FR-{alpha} (B), p27 (C), TK1 (D), and p21 (E) and as well as coding exon sequences (irrelevant targets) distal to the promoters of the c-myc, FR-{alpha}, p27, TK1, and p21 genes. The ChIP signals are expressed as percent of the signal for the corresponding input. A, P < 0.0001 for b vs. a or c; d vs. c or e; e vs. f. B, P < 0.0001 for a vs. d or e; b vs. d or e; c vs. d. C, P < 0.0001 for a vs. d or e; b vs. d or e; c vs. d. D, P < 0.0001 for d vs. a, b or c. E, P < 0.0001 for a vs. d, e, or f; b vs. d, e, or f; c vs. d.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Agonists and antagonists of PR produce profound physiological effects, principally by altering global gene expression profiles in the target tissues. The altered gene expression profiles presumably not only reflect changes in the activities of the direct target genes of PR but also a host of downstream genes whose expression is altered as a consequence of the changes in expression of the direct PR target genes. A clinically significant aspect of PR action is that agents such as RU486 that are used to antagonize PR action have the undesirable quality of acting as PR agonists in several situations (39). In addition to understanding the means by which PR agonists influence global gene expression profiles, it is therefore also important to fully understand the underlying principles in the agonistic activity of RU486.

One aspect of significance in the present study is that the FR-{alpha} gene, whose product is potentially of great value in targeted drug delivery in cancer and as a diagnostic and imaging marker, may be up-regulated in the tumors using innocuous PR ligands. Inducing FR-{alpha} in this manner is potentially of value in combination therapies using the targeted drugs and in enhancing the sensitivity of the diagnostic and imaging agents (36, 37). It may also be noted that genes such as FR-{alpha} that are dependent on the binding of Sp family transcription factors for their basal promoter activities represent a large class of genes, several important members of which have been shown to be regulated by PR ligands (30, 31, 32, 38, 40). The nature of RU486 action and PR subtype specificity of Sp1-dependent gene regulation by PR are both distinct from those of classical PRE-mediated regulation. It is believed that this unique Sp1-dependent gene regulation by PR occurs by direct interaction of PR with Sp1 in the target promoters. Therefore, the broader significance of the mechanistic studies of FR-{alpha} regulation by PR is demonstrated here by extension to the TK1, p27, and p21 genes, leading to a more accurate understanding of how PR ligands regulate genes through Sp1 and the agonistic action of RU486 on this regulation.

In contrast to a typical GRE-driven promoter that is activated by R5020 predominantly through PR-B, both PR-A and PR-B showed a comparable ability to mediate activation of the FR-{alpha} P4 promoter and the endogenous FR-{alpha} gene by R5020. RU486, on the other hand, behaved as an antagonist of the activation of the FR-{alpha} promoter and the FR-{alpha} gene by R5020/PR-A but as an agonist through PR-B in contrast to its antagonistic activity on a GRE-driven promoter both through PR-A and PR-B. These observations also apply to the induction of the genes encoding TK1, p27, and p21 that do not have the classical response elements for PR but whose basal promoters are Sp1 dependent. The requirement for Sp1 or Sp4 and their cognate G/C-rich cis elements in mediating PR regulation of the FR-{alpha} promoter is clear from the following observations: 1) the entire DNA sequence upstream of the functional Sp1 (G/C rich) elements in the core P4 promoter could be deleted without affecting the nature or magnitude of PR regulation; 2) each of the three noncanonical Sp1 elements in the P4 promoter contributed to the magnitude of the PR effect; 3) substituting the natural Sp1 elements of the P4 promoter with a stronger cluster of six Sp1 elements derived from the SV40 promoter increased the magnitude of the PR effect; 4) substituting the Sp1 elements of the P4 promoter with a TATA-box element abrogated the regulation by PR; and 5) overexpression of Sp1 or Sp4 but not Sp3 increased the magnitude of the PR effect and conversely, knocking down Sp1 decreased the PR response in a manner that was reversed by ectopic overexpression of either Sp1 or Sp4.

This study undertook a reexamination of the literature data suggesting that the distinctive activation of several Sp1-dependent genes by R5020 as well as RU486 could be mediated by ligand-dependent association of PR with Sp1 in their basal promoters. Complementary lines of evidence used in this study indicate that the regulation of the natural FR-{alpha} promoter by PR is actually indirect. They include the observation that the action of PR ligands on the promoter including the increase in RNA polymerase II recruitment is considerably delayed in comparison with their action on a GRE-driven promoter and that intermediate new protein synthesis is required for the delayed induction of the promoter or of endogenous FR-{alpha} mRNA. The inability of PR to associate with the endogenous promoter examined by ChIP either in the absence or in the presence of PR ligands in contrast to the c-myc promoter further confirms that the action of PR on the FR-{alpha} gene is indirect. This study has also extended this conclusion to the TK1, p27, and p21 genes. The physiologic relevance of this indirect gene regulation by PR is clearly exemplified in both phases of the reported (38) biphasic response of breast cancer cells to progesterone in which there is first a stimulation of the cell cycle accompanied by an induction of TK1 followed by a delayed induction of p27 associated with G1 phase arrest. The time period for the delayed response of the p21 gene, however, was shorter than that of the TK1 or the p27 genes, suggesting a possible influence of additional target gene-specific factors in the PR ligand response.

The detailed studies reported here contradict the suggestion supported by limited observations (31, 32) that Sp1 can directly recruit PR to its target promoters to mediate ligand-dependent trans-activation. In addition to the role of Sp1 binding DNA elements in promoter regulation by PR, the basis for the suggestion included in vitro coimmunoprecipitation of PR and Sp1 (32) an observation whose relevance could be limited to physiologic situations that would mimic the overexpression systems used. The second study (31) used ChIP assays to show an apparent association of PR with the target promoter but did not establish ligand dependence for this association, was not quantitative, and was not supported by complementary data. The more extensive studies in the present report of the delayed Sp-dependent response of several target promoters to PR ligands showed that in no case did PR associate even basally with the promoter in contrast to the c-myc promoter.

The Sp-mediated regulation of genes by PR is clearly distinct from the nonclassical mechanisms by which transcription factors such as Sp proteins (34) and AP-1 (41) mediate direct transcriptional regulation by ER independent of a classical estrogen-response element. Sp1, Sp3, and Sp4 are known to recruit ER to G/C-rich elements by interacting with specific ER domains to transactivate genes in a cell type, ER subtype and promoter context-dependent manner (34, 35, 42, 43, 44). Indeed, ER also regulates the FR-{alpha} gene; however, this regulation is repressive consistent with the frequently opposing physiologic effects of estrogen and progesterone (45).

PR ligands altered neither the expression levels of several Sp proteins nor the association of Sp1 or Sp4 with the promoter regions of the endogenous FR-{alpha}, TK1, or p27 genes observed by ChIP. Therefore, it is unlikely that they induced some other DNA binding transcription factor that could bind to G/C-rich elements to increase transcriptional activity. The simplest explanation consistent with the observed nature of the indirect Sp1-dependent regulation of genes by PR is that the action of PR ligands results in the generation of a coactivator of Sp1-dependent trans-activation of the target genes. The gene encoding the putative coactivator may itself be either a direct or an indirect target of PR. In any event, the promoter and cell context of the direct target gene of PR in this pathway must allow RU486 to act as a PR agonist and its activation by RU486/PR-B must be the key step in the positive regulation of a variety of Sp1-dependent genes by RU486. This direct target gene of PR must also mediate the indirect Sp1-dependent gene regulation by R5020. The broad applicability of the results in this study to a variety of indirect target genes of PR that are important in regulating cell growth and physiology presents a compelling case to attempt the identification of the putative Sp1 coactivator and as well as the relevant direct target gene of PR in future studies.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Chemicals and Reagents
DMEM, MEM, LipofectAMINE 2000, Opti-MEM reduced serum medium, Geneticin, and the penicillin/streptomycin/L-glutamine stock mix were purchased from Invitrogen (Carlsbad, CA). Fetal bovine serum (FBS) was purchased from Irvine Scientific (Santa Ana, CA). FuGENE 6 was purchased from Roche Diagnostics (Indianapolis, IN). Promegestone (R5020) was from PerkinElmer (Shelton, CT). Luciferase assay reagents were from Promega (Madison, WI). Mifepristone (RU486), 100x nonessential amino acids, CHX, and mouse anti-tubulin clone B-5-1-2 antibody were from Sigma (St. Louis, MO). Protein A-coated magnetic beads and Vent DNA polymerase were from New England Biolabs (Beverly, MA). Affinity-purified rabbit antihuman antibodies to RNA polymerase II [Pol II (H-224)], Sp1 (PEP2), Sp4 (V-20), rabbit polyclonal IgG against PR (C-20) and mouse antihuman antibody to for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Custom oligonucleotide primers were from Invitrogen. The reagents for real-time RT-PCR were from Applied Biosystems (Branchburg, NJ).

Cell Culture and Transfection
HeLa cells were purchased from American Type Culture Collection (Manassas, VA). Recombinant T47D cells that express either PR-A (T47D-A) or PR-B (T47D-B) were a generous gift from Dr. Kathryn B. Horwitz (University of Colorado, Denver, CO). Cells were grown in 10-cm tissue culture plates at 37 C in 5% CO2 in the appropriate cell culture media. HeLa cells were routinely cultured in DMEM supplemented with FBS (10%), 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mmol/liter L-glutamine. T47D-A and T47D-B cells were cultured in MEM supplemented with 5% FBS, 1x of nonessential amino acids, 6 ng/ml insulin, and 200 µg/ml Geneticin. For treatment with various agents (R5020, RU486, CHX) and for transfection, cells were grown in phenol red-free DMEM supplemented with charcoal-stripped FBS (5% vol/vol), penicillin (100 U/ml), streptomycin (100 mg/ml), L-glutamine (2 mmol/liter), insulin (2 µg/ml), and transferrin (40 µg/ml). Transfections with various plasmid constructs were carried out in six-well plates (Corning, New York, NY) using FuGENE 6 (Roche Diagnostics) for HeLa cells and lipofectAMINE 2000 for T47D cells. The transfection method followed the manufacturers’ suggested protocols. Uniformity of transfection with promoter constructs attached to the firefly luciferase reporter was ensured by cotransfection with a Renilla luciferase expression plasmid and monitoring the activity of Renilla luciferase.

Promoter Constructs, Expression Plasmids, and Sp1 Knockdown
Construct design made use of either natural restriction sites or restriction sites created by the addition of the appropriate sequences to upstream and downstream PCR primers. The PCR products were first digested at both ends with the appropriate restriction enzymes and cloned into the PGL3-basic plasmid (Promega) or subcloned into the FR-{alpha} promoter construct (–3394 to +33 nt, relative to the transcription initiation site at +1 nt) in the PGL3 basic plasmid. Construction of the FR-{alpha} –3394 to –47 nt/SV40(GC)6 construct is described elsewhere (46). The 5' deletion constructs of the FR-{alpha} promoter, i.e. –272 to +33 nt, –116 to +33 nt, and –49 to +33 nt were all constructed by PCR using the appropriate primers and subcloned at MluI (upstream) and XhoI (downstream) sites in the pGL3 basic plasmid. In the TATA+ FR-{alpha} –35 to +33 nt construct, a TATA-box element (5'-AATAATTAA-3') was placed upstream of the most proximal Sp1 element in the FR-{alpha} promoter, i.e. upstream of FR-{alpha} –35 to +33 nt by the PCR method. The recombinant plasmids were amplified in Escherichia coli strain XL1Blue and purified using the QIAGEN plasmid kit (QIAGEN, Chatsworth, CA). The entire cloned DNA sequence in each construct was verified by automated DNA sequence analysis.

The GRE2e1b promoter-luciferase plasmid and the expression plasmids for PR-A and PR-B were kindly provided by Dr. Brian Rowan (Tulane University Health Sciences Center). The expression plasmids for Sp1 and Sp3 were provided by Dr. Sumudra Periyasamy (Medical University of Ohio). The expression plasmid for Sp4 was provided by Dr. Guntram Suske (Institut for Molekularbiologie und Tumorforschung Philipps-Universitat Marburg). The short hairpin RNA expression plasmid for knocking down Sp1 was purchased from Sigma. For the knockdown experiments the Amaxa Nucleofection System (Amaxa Biosystems, Gaithersburg, MD) was used to deliver the short hairpin RNA to T47D cells according to the vendor’s protocol.

Preparation of Cell Lysates and Luciferase Assay
Cells in each well of a six-well tissue culture plate were washed once with PBS of pH 7.5 (2 mmol/liter KH2PO4, 2.7 mmol/liter KCl, 10 mmol/liter Na2HPO4, and 137 mmol/liter NaCl) and lysed in 400 µl of reporter lysis buffer provided with the luciferase assay system (Promega). The samples were centrifuged at 12,000 x g for 2 min at room temperature. The supernatant was assayed for luciferase activity in a luminometer (Lumat LB9501; Berthold, Wildbad, Germany) using the luciferase substrate from Promega. All luciferase experiments were done at least in triplicate.

Real-Time RT-PCR and Analyses
Total RNA for real-time RT-PCR was prepared using the RNeasy Mini kit purchased from QIAGEN. Real-time RT-PCR was used to measure the mRNA for luciferase, FR-{alpha}, TK1, p27, or c-myc. The mRNA GAPDH was also measured by real-time RT-PCR in the same samples. For the real-time RT-PCR, the reverse transcription step was carried out following standard procedures. Essentially, 200 ng of total RNA were mixed with random hexamer primers (5 x 10–4 absorbance units/µl), ribonuclease inhibitor (1 U/µl), Moloney murine leukemia virus reverse transcriptase (5 U/µl), and deoxynucleotide triphosphates (1.0 mmol/liter each) in reverse transcriptase buffer [50 mmol/liter potassium chloride and 10 mmol/liter Tris-HCl (pH 8.3)]. The 10-µl reaction mixture was first incubated at 25 C for 10 min, then at 42 C for 15 min and finally at 99 C for 6.5 min. The subsequent real-time PCR step was carried out in the presence of 12.5 µl of PCR Mastermix (Applied Biosystems). A quantity of 0.5 µl each of the forward primer and reverse primer and 0.5 µl of the TaqMan probe were used in the reaction. The sequences of the forward and reverse probes and the appropriate TaqMan probe for each target are listed in Table 2Go. The primers and the TaqMan probe for the control GAPDH gene were purchased from Applied Biosystems. The PCR conditions were 2 min at 50 C, then 10 min at 95 C, followed by 40 cycles each of 15 sec at 90 C and 1 min at 60 C. Fluorescence data generated were monitored and recorded on a 7500 real-time PCR sequence detection system (Applied Biosystems). All samples were assayed in triplicate and normalized to GAPDH values.


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  		Table 2. DNA Sequences of Primers and TaqMan Probes for Real-Time PCR Assays

 
Western Blot Analysis
Cells were harvested after washing two times with cold PBS. The cells were lysed with high-salt buffer (400 mM NaCl; 10 mM Tris, pH 8.0; 1 mM EDTA; 1 mM EGTA; ß-mercaptoethanol; and 0.1% Triton X-100) containing a protease inhibitor cocktail (1 mM phenyl methyl sulfonyl fluoride and 5 µg/ml each of aprotinin, leupeptin, and pepstatin A). Protein samples (20–50 µg) were resolved by electrophoresis on 8% SDS-PAGE and electrophoretically transferred to nitrocellulose membranes. The blots were probed with the appropriate primary antibodies followed by goat antirabbit IgG or goat antimouse IgG conjugated to horseradish peroxidase and visualized using the enhanced chemiluminescence method. The same membrane was then similarly reprobed with a primary mouse anti-tubulin antibody and secondary goat antimouse IgG conjugated to horseradish peroxidase. The membranes were also stained using Coomassie blue to ensure uniform sample loading.

Quantitative ChIP Assay
T47D-A cells or T47D-B were grown in 10-mm plates and treated as appropriate for each experiment. For the ChIP assays, the cells were then washed twice with ice-cold PBS and fixed with formaldehyde (1% final concentration) for 15 min at room temperature. Then the cells were washed with ice-cold PBS twice and collected in 100 mM Tris-HCl (pH 9.0) and 10 mM dithiothreitol. After a 15-min incubation at 30 C, the cells were washed twice with ice-cold PBS and lysed in ChIP lysis buffer [1% sodium dodecyl sulfate, 10 mM EDTA, 50 mM Tris-HCl (pH 8.1) plus protease inhibitor cocktail (1 mM phenylmethylsulfonyl fluoride; and 5 µg/ml each of aprotinin, leupeptin, and pepstatin A)]and incubated on ice. 10 min later the samples were sonicated on ice with a sonic dismembrator (Fisher Scientific Co., Pittsburgh, PA) at output 3 for 10 sec pulse-on time followed by 40 sec pulse-off time, and this procedure was repeated three times resulting in chromatin fragmented to an average length of about 500 bp. The samples were then centrifuged at 16,000 x g for 10 min. The supernatant was diluted 10-fold in ChIP dilution buffer (1% Triton X-100; 2 mM EDTA; 150 mM NaCl; 20 mM Tris-HCl, pH 8.1; plus the protease inhibitor cocktail) and precleared with 2 µg of normal rabbit IgG and 5 µl of Protein-A-coated magnetic beads. The beads were then separated on a magnetic rack. Ten percent of the supernatant was set aside for input measurements. Two micrograms of the appropriate antibody [anti-Sp1 (sc-59), anti-PR (sc-539X), anti-Pol II (sc-9001) or normal rabbit IgG from Santa Cruz Biotechnology] were added to the supernatant and incubated overnight on a rotary shaker at 4 C. Then the samples were mixed with sonicated salmon sperm DNA (100 µg/ml) and 5 µl of Protein A magnetic beads for a 6 h incubation at 4 C. The beads were then washed for 5 min each, first with low-salt buffer [0.1% sodium dodecyl sulfate (SDS); 1% Triton X-100; 2 mM EDTA; 20 mM Tris-HCl, pH 8.1; and 150 mM NaCl] followed by high-salt buffer (0.1% SDS; 1% Triton X-100; 2 mM EDTA; 20 mM Tris-HCl, pH 8.1; and 500 mM NaCl) and finally with LiCl buffer (0.25 M LiCl; 1% Nonidet P-40; 1% deoxycholate; 1 mM EDTA; and 10 mM Tris-HCl, pH 8.1). The beads were washed with TE buffer (10 mM Tris-HCl, pH 8.0; and 1 mM EDTA) three times for 5 min each time. The immunoprecipitated chromatin complexes were removed from the beads and de-cross-linked by incubation for 6 h at 65 C in 100 µl of 1% SDS/0.1 M NaHCO3 with intermittent vortexing. DNA was purified from the samples using the QIAquick Spin Kit (QIAGEN) according to the manufacturer’s protocol. Ten microliters of the extracted DNA were used in real-time PCR assays to measure the appropriate ChIP target DNA. All of the forward and reverse primers and TaqMan probes used for the various target sequences in the ChIP assays are shown in Table 2Go. The ability of the various PCR primers and TaqMan probes to amplify the target sequences is shown in Table 1Go. Every sample was assayed in triplicate.

Statistical Analysis
Results are presented as mean ± SE. The significance of statistical differences (P values) between the indicated groups in each experiment was determined using ANOVA.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Kathryn B. Horwitz (University of Colorado, Denver, CO) for her generosity in providing the T47D-A and T47D-B cells. We thank Dr. Brian Rowan (Tulane University, New Orleans, LA), Dr. Sumudra Periyasamy (Medical University of Ohio, Toledo, OH), and Dr. Guntram Suske (Philipps-Universitat Marburg, Germany) for sharing various expression plasmids. We thank Mariana Stoeva for technical assistance.


   FOOTNOTES
 
This work was supported by National Institutes of Health Grant CA 103964 (to M.R.).

Disclosure: Some of the studies described here are covered by a pending institutional patent application. The authors have no relevant commercial affiliations to declare.

First Published Online December 27, 2006

Abbreviations: ChIP, Chromatin immunoprecipitation; CHX, cycloheximide; ER, estrogen receptor; FBS, fetal bovine serum; FR, folate receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GRE, glucocorticoid response element; nt, nucleotides; Pol II, polymerase II; PR, progesterone receptor; PRE, progesterone response element; SDS, sodium dodecyl sulfate; SV40, simian virus 40; TK, thymidine kinase.

Received for publication July 5, 2006. Accepted for publication December 22, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Graham JD, Clarke CL 1997 Physiological action of progesterone in target tissues. Endocr Rev 18:502–519[Abstract/Free Full Text]
  2. Rao BR, Wiest WG 1974 Receptors for progesterone. Gynecol Oncol 2:239–248[CrossRef][Medline]
  3. Rao BR, Wiest WG, Allen WM 1974 Progesterone "receptor" in human endometrium. Endocrinology 95:1275–1281[Medline]
  4. Lessey BA, Alexander PS, Horwitz KB 1983 The subunit structure of human breast cancer progesterone receptors: characterization by chromatography and photoaffinity labeling. Endocrinology 112:1267–1274[Medline]
  5. Attia GR, Zeitoun K, Edwards D, Johns A, Carr BR, Bulun SE 2000 Progesterone receptor isoform A but not B is expressed in endometriosis. J Clin Endocrinol Metab 85:2897–2902[Abstract/Free Full Text]
  6. Giangrande PH, McDonnell DP 1999 The A and B isoforms of the human progesterone receptor: two functionally different transcription factors encoded by a single gene. Recent Prog Horm Res 54:291–313; discussion 313–314[Medline]
  7. Kastner P, Krust A, Turcotte B, Stropp U, Tora L, Gronemeyer H, Chambon P 1990 Two distinct estrogen-regulated promoters generate transcripts encoding the two functionally different human progesterone receptor forms A and B. EMBO J 9:1603–1614[Medline]
  8. Meyer ME, Pornon A, Ji JW, Bocquel MT, Chambon P, Gronemeyer H 1990 Agonistic and antagonistic activities of RU486 on the functions of the human progesterone receptor. EMBO J 9:3923–3932[Medline]
  9. Meyer ME, Quirin-Stricker C, Lerouge T, Bocquel MT, Gronemeyer H 1992 A limiting factor mediates the differential activation of promoters by the human progesterone receptor isoforms. J Biol Chem 267:10882–10887[Abstract/Free Full Text]
  10. Tora L, Gronemeyer H, Turcotte B, Gaub MP, Chambon P 1988 The N-terminal region of the chicken progesterone receptor specifies target gene activation. Nature 333:185–188[CrossRef][Medline]
  11. Richer JK, Jacobsen BM, Manning NG, Abel MG, Wolf DM, Horwitz KB 2002 Differential gene regulation by the two progesterone receptor isoforms in human breast cancer cells. J Biol Chem 277:5209–5218[Abstract/Free Full Text]
  12. Giangrande PH, Pollio G, McDonnell DP 1997 Mapping and characterization of the functional domains responsible for the differential activity of the A and B isoforms of the human progesterone receptor. J Biol Chem 272:32889–32900[Abstract/Free Full Text]
  13. Conneely OM, Lydon JP 2000 Progesterone receptors in reproduction: functional impact of the A and B isoforms. Steroids 65:571–577[CrossRef][Medline]
  14. Vegeto E, Shahbaz MM, Wen DX, Goldman ME, O’Malley BW, McDonnell DP 1993 Human progesterone receptor A form is a cell- and promoter-specific repressor of human progesterone receptor B function. Mol Endocrinol 7:1244–1255[Abstract]
  15. Schindler AE, Campagnoli C, Druckmann R, Huber J, Pasqualini JR, Schweppe KW, Thijssen JH 2003 Classification and pharmacology of progestins. Maturitas 46(Suppl 1):S7–S16
  16. Cadepond F, Ulmann A, Baulieu EE 1997 RU486 (mifepristone): mechanisms of action and clinical uses. Annu Rev Med 48:129–156[CrossRef][Medline]
  17. Spitz IM 2003 Progesterone antagonists and progesterone receptor modulators: an overview. Steroids 68:981–993[CrossRef][Medline]
  18. Mahajan DK, London SN 1997 Mifepristone (RU486): a review. Fertil Steril 68:967–976[CrossRef][Medline]
  19. DeMarzo AM, Onate SA, Nordeen SK, Edwards DP 1992 Effects of the steroid antagonist RU486 on dimerization of the human progesterone receptor. Biochemistry 31:10491–10501[CrossRef][Medline]
  20. Skafar DF 1993 Dimerization of the RU486-bound calf uterine progesterone receptor. J Steroid Biochem Mol Biol 44:39–43[CrossRef][Medline]
  21. Glass CK, Rosenfeld MG 2000 The coregulator exchange in transcriptional functions of nuclear receptors. Genes Dev 14:121–141[Free Full Text]
  22. Edwards DP 2000 The role of coactivators and corepressors in the biology and mechanism of action of steroid hormone receptors. J Mammary Gland Biol Neoplasia 5:307–324[CrossRef][Medline]
  23. Leo C, Chen JD 2000 The SRC family of nuclear receptor coactivators. Gene 245:1–11[CrossRef][Medline]
  24. Tung L, Mohamed MK, Hoeffler JP, Takimoto GS, Horwitz KB 1993 Antagonist-occupied human progesterone B-receptors activate transcription without binding to progesterone response elements and are dominantly inhibited by A-receptors. Mol Endocrinol 7:1256–1265[Abstract]
  25. Wardell SE, Boonyaratanakornkit V, Adelman JS, Aronheim A, Edwards DP 2002 Jun dimerization protein 2 functions as a progesterone receptor N-terminal domain coactivator. Mol Cell Biol 22:5451–5466[Abstract/Free Full Text]
  26. Beck CA, Weigel NL, Moyer ML, Nordeen SK, Edwards DP 1993 The progesterone antagonist RU486 acquires agonist activity upon stimulation of cAMP signaling pathways. Proc Natl Acad Sci USA 90:4441–4445[Abstract/Free Full Text]
  27. Kahmann S, Vassen L, Klein-Hitpass L 1998 Synergistic enhancement of PRB-mediated RU486 and R5020 agonist activities through cyclic adenosine 3',5'-monophosphate represents a delayed primary response. Mol Endocrinol 12:278–289[Abstract/Free Full Text]
  28. Sartorius CA, Tung L, Takimoto GS, Horwitz KB 1993 Antagonist-occupied human progesterone receptors bound to DNA are functionally switched to transcriptional agonists by cAMP. J Biol Chem 268:9262–9266[Abstract/Free Full Text]
  29. Liu Z, Auboeuf D, Wong J, Chen JD, Tsai SY, Tsai MJ, O’Malley BW 2002 Coactivator/corepressor ratios modulate PR-mediated transcription by the selective receptor modulator RU486. Proc Natl Acad Sci USA 99:7940–7944[Abstract/Free Full Text]
  30. Gao J, Mazella J, Seppala M, Tseng L 2001 Ligand activated hPR modulates the glycodelin promoter activity through the Sp1 sites in human endometrial adenocarcinoma cells. Mol Cell Endocrinol 176:97–102[CrossRef][Medline]
  31. Gizard F, Robillard R, Gervois P, Faucompre A, Revillion F, Peyrat JP, Hum WD, Staels B 2005 Progesterone inhibits human breast cancer cell growth through transcriptional upregulation of the cyclin-dependent kinase inhibitor p27Kip1 gene. FEBS Lett 579:5535–5541[CrossRef][Medline]
  32. Owen GI, Richer JK, Tung L, Takimoto G, Horwitz KB 1998 Progesterone regulates transcription of the p21(WAF1) cyclin-dependent kinase inhibitor gene through Sp1 and CBP/p300. J Biol Chem 273:10696–10701[Abstract/Free Full Text]
  33. Thomson AA, Ham J, Bakker O, Parker MG 1990 The progesterone receptor can regulate transcription in the absence of a functional TATA box element. J Biol Chem 265:16709–16712[Abstract/Free Full Text]
  34. Safe S, Kim K 2004 Nuclear receptor-mediated transactivation through interaction with Sp proteins. Prog Nucleic Acid Res Mol Biol 77:1–36[Medline]
  35. Higgins KJ, Liu S, Abdelrahim M, Yoon K, Vanderlaag K, Porter W, Metz RP, Safe S 2006 Vascular endothelial growth factor receptor-2 expression is induced by 17ß-estradiol in ZR-75 breast cancer cells by estrogen receptor {alpha}/Sp proteins. Endocrinology 147:3285–3295[Abstract/Free Full Text]
  36. Elnakat H, Ratnam M 2006 Role of folate receptor genes in reproduction and related cancers. Front Biosci 11:506–519[CrossRef][Medline]
  37. Elnakat H, Ratnam M 2004 Distribution, functionality and gene regulation of folate receptor isoforms: implications in targeted therapy. Adv Drug Deliv Rev 56:1067–1084[CrossRef][Medline]
  38. Groshong SD, Owen GI, Grimison B, Schauer IE, Todd MC, Langan TA, Sclafani RA, Lange CA, Horwitz KB 1997 Biphasic regulation of breast cancer cell growth by progesterone: role of the cyclin-dependent kinase inhibitors, p21 and p27(Kip1). Mol Endocrinol 11:1593–1607[Abstract/Free Full Text]
  39. Bowden RT, Hissom JR, Moore MR 1989 Growth stimulation of T47D human breast cancer cells by the anti-progestin RU486. Endocrinology 124:2642–2644[Abstract]
  40. Tang M, Mazella J, Gao J, Tseng L 2002 Progesterone receptor activates its promoter activity in human endometrial stromal cells. Mol Cell Endocrinol 192:45–53[CrossRef][Medline]
  41. Jakacka M, Ito M, Weiss J, Chien PY, Gehm BD, Jameson JL 2001 Estrogen receptor binding to DNA is not required for its activity through the nonclassical AP1 pathway. J Biol Chem 276:13615–13621[Abstract/Free Full Text]
  42. Kim K, Thu N, Saville B, Safe S 2003 Domains of estrogen receptor {alpha} (ER{alpha}) required for ER{alpha}/Sp1-mediated activation of GC-rich promoters by estrogens and antiestrogens in breast cancer cells. Mol Endocrinol 17:804–817[Abstract/Free Full Text]
  43. Castro-Rivera E, Safe S 2003 17ß-Estradiol- and 4-hydroxytamoxifen-induced transactivation in breast, endometrial and liver cancer cells is dependent on ER-subtype, cell and promoter context. J Steroid Biochem Mol Biol 84:23–31[CrossRef][Medline]
  44. Stoner M, Wormke M, Saville B, Samudio I, Qin C, Abdelrahim M, Safe S 2004 Estrogen regulation of vascular endothelial growth factor gene expression in ZR-75 breast cancer cells through interaction of estrogen receptor {alpha} and SP proteins. Oncogene 23:1052–1063