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Molecular Endocrinology, doi:10.1210/me.2007-0082
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Molecular Endocrinology 21 (11): 2651-2662
Copyright © 2007 by The Endocrine Society

Long-Range Activation of GREB1 by Estrogen Receptor via Three Distal Consensus Estrogen-Responsive Elements in Breast Cancer Cells

Jun Sun, Zafar Nawaz and Joyce M. Slingerland

Braman Family Breast Cancer Institute (J.S., Z.N., J.M.S.), University of Miami Sylvester Comprehensive Cancer Center, and Department of Biochemistry and Molecular Biology (Z.N., J.M.S.), University of Miami Miller School of Medicine, Miami, Florida 33136

Address correspondence to: Jun Sun or Joyce Slingerland, Braman Family Breast Cancer Institute, 1580 NW 10 Avenue (M-877), University of Miami, Miami, Florida 33136. E-mail: jsun{at}med.miami.edu or jslingerland{at}med.miami.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The estrogen receptor (ER) binds to estrogen-responsive elements (EREs) to activate gene transcription. The best characterized EREs are located in proximal gene promoters, but recent data indicate that only a minority of ER binding sites lie within proximal promoter regions. GREB1 (gene regulated by estrogen in breast cancer 1) is an ER target gene that regulates estrogen-induced proliferation in breast cancer cells. We identified three consensus EREs, located at –21.2, –9.5, and –1.6 kb upstream of the closest GREB1a transcription start site that appear to mediate long-range GREB1 gene activation by ER. All three ERE sites nucleate ER, steroid receptor coactivator-3 (SRC-3), and RNA polymerase II (Pol II) and undergo histone acetylation in response to estradiol. Estrogen-stimulated ER binding at all three EREs was cyclic and synchronous. SRC-3 and Pol II recruitment to all three EREs was activated by estrogen but not tamoxifen. In contrast, estrogen stimulated only Pol II and not ER or SRC-3 recruitment to the GREB1 core promoter regions. Long-range histone acetylation, centered on the three ERE motifs and the GREB1 core promoters, was observed in response to estrogen but not to tamoxifen. These data suggest that estrogen-stimulated GREB1 transcription may involve coordinated ER binding to all three distal consensus ERE motifs. Long-range activation by ER acting at multiple EREs may be more common than previously appreciated.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ESTROGEN PLAYS IMPORTANT roles in cell growth and differentiation as well as in the progression of breast cancer (1). Most actions of estrogen are mediated through the estrogen receptors (ERs), which are members of the nuclear receptor superfamily (2). There are two subtypes of ER, ER{alpha} (3, 4) and ERß (5, 6, 7). They share similar domain organization and have overlapping but nonidentical tissue distributions (8, 9). Here we focus on ER{alpha} and refer hereafter to ER{alpha} as ER. The ligand-binding domain (LBD), located at the C terminus of the ER, binds to estrogen and is the target of selective ER modulators such as tamoxifen (10). The DNA-binding domain, located in the central region of the receptor, binds to the estrogen response elements (EREs) in ER target genes (11, 12).

Upon ligand binding, the LBD of ER undergoes a structural change to provide a binding surface for cofactors (13). The p160 coactivator family includes three homologous steroid receptor coactivator (SRC) molecules, SRC-1, SRC-2, and SRC-3. They are recruited to a hydrophobic groove formed on the surface of the agonist-bound LBD through direct contact of their LXXLL motifs. ER binding to antagonists prevents this interaction (14). Although p160 coactivators have been shown to possess intrinsic histone acetyltransferase (HAT) activity, they also recruit other chromatin-modifying proteins including cAMP response element-binding protein-binding protein (CBP)/p300, p300/CBP-associated factor (pCAF), coactivator-associated arginine methyltransferase-1 (CARM1), and protein arginine N-methyltransferase (PRMT) (15). The combinatorial modification of histones dictates the modulation of gene expression (16). Histone acetylation occurs in response to hormone and serves to open the chromatin structure, facilitating receptor-coactivator complex assembly at hormone response elements. Recent evidence suggests that ER and its cofactors cycle on and off the promoter of target genes (17, 18). However, the mechanisms whereby the ER interacts with the basic transcriptional machinery to activate gene transcription are not completely understood.

One mechanism whereby ER activates gene transcription is through direct ER binding to EREs in target gene promoters (19). The ERE consensus sequence, first identified in the Xenopus A2 gene promoter, is a 5-bp inverted repeat separated by any three base pairs, GGTCAnnnTGACC, to which ER binds as a symmetrical dimer (20). Most of the well studied EREs from estrogen-responsive genes are located at the proximal promoter regions, deviate from the consensus sequence by one to three base pairs, and have reduced binding affinity for ER (19).

Microarray experiments have been used to identify ER target genes on a global scale (21). However, this type of assay does not distinguish genes that are regulated by direct ER binding to EREs from those activated by indirect tethering of ER to target promoters via other transcription factors. With the completion of the human genome project, in silico genome-wide screening has been used to identify EREs in the ER target genes with focus on the regions close to the transcriptional start sites (22, 23). Enhancers, however, can be located much farther way from transcriptional start sites, from tens of kilobases up to a megabase (24).

GREB1 (gene regulated by estrogen in breast cancer 1) was first identified as an estrogen-regulated gene expressed in breast cancer in a subtractive hybridization screen in MCF-7 cells. The three cDNAs of GREB1 (GREB1a, GREB1b, and GREB1c) have distinct 5'-untranslated regions but share extensive coding sequences, indicating different promoter usage (25). In our search for EREs, we found three consensus ERE motifs that are located within a 20-kb region on human chromosome 2, upstream of the GREB1 gene. In this study, we analyzed GREB1 gene expression and employed chromatin immunoprecipitation (ChIP) assays to monitor the binding of ER, SRC-3, and RNA polymerase II (Pol II) and the level of histone H3 acetylation at the GREB1 locus in response to 17ß-estradiol (E2) or 4-hydroxyl tamoxifen (OHT) in MCF-7 breast cancer cells. We found that E2 stimulates ER, SRC-3, and Pol II binding to all three EREs within the distal GREB1 promoter region. Our data suggest that coordinated ER binding to all three distal EREs may contribute to the regulation of estrogen-induced GREB1 gene expression. These data support a model in which the coordinated binding of ER to distal sites interacts with Pol II to activate gene transcription from core promoters located at a considerable distance.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The GREB1 Gene Locus Contains Three Consensus ERE Motifs
During a genome-wide search for ERE motifs in the human genome, we identified three consensus ERE motifs located within a 20-kb region upstream of the transcriptional start sites of the GREB1 gene on chromosome 2. The GREB1 locus has been shown to encode at least three different transcripts, GREB1a, GREB1b, and GREB1c (25) with distinct 5'-untranslated regions, indicating the presence of multiple promoters. The transcriptional start sites for GREB1c and GREB1b are 6 and 9 kb downstream of that for GREB1a (25). Figure 1Go shows a schematic representation of the three consensus ERE motifs identified herein (referred to as ERE1, ERE2, and ERE3, respectively) and the three known transcriptional start sites for the GREB1 gene. Because GREB1 expression is strongly up-regulated by 17ß-estradiol, we postulated that these three consensus ERE motifs located in the distant upstream promoter region might contribute to ER-mediated GREB1 gene activation.


Figure 1
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  		Fig. 1. The Genomic Structure of the Human GREB1 Gene

A, A schematic representation of human GREB1 gene locus on chromosome 2. The three transcriptional start sites are labeled as a, c, and b, respectively. The positions of three consensus ERE motifs are marked by black boxes. The locations of the fragments used for PCR in the ChIP analysis are indicated by black bars, and their positions are listed as the distance to the transcriptional start site of the GREB1a promoter. B, The DNA sequences of the three ERE motifs within the GREB1 locus.

 
GREB1 Expression Is Induced by Estrogen in ER-Positive Breast Cancer Cells
We assayed estrogen-induced GREB1 gene expression in ER-positive breast cancer cells. Total RNA was extracted from estrogen-deprived MCF-7 cells and from cells subsequently exposed to 17ß-estradiol (E2) for either 4 or 8 h. A region common to all the three GREB1 transcripts was amplified by real-time RT-PCR. E2 increased GREB1 mRNA levels by approximately 20- and 40-fold after 4 and 8 h, respectively (Fig. 2AGo). The estrogen antagonist, OHT, alone had no effect on GREB1 expression, and it abolished E2-induced GREB1 expression after 8-h treatments (Fig. 2BGo). GREB1 was also induced by E2 in two other ER-positive breast cancer cell lines, ZR-75 and BT-474 but not in the ER-negative MDA-MB-231 cells (data not shown).


Figure 2
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  		Fig. 2. Estrogen Stimulates GREB1 Gene Expression in MCF-7 Breast Cancer Cells

A, MCF-7 cells were harvested for RNA extraction after hormone deprivation for 3 d (time = 0 h) or after treatment with 10 nM E2 for 4 or 8 h, respectively. The expression of the total GREB1 transcripts was analyzed by real-time RT-PCR. GAPDH was used as an internal control. The data are plotted as fold induction over the control level. B, Estrogen-deprived MCF-7 cells (control) were treated for 8 h with 10 nM E2, 1 µM OHT, or combinations of E2 and OHT as indicated, and the expression of total GREB1 mRNA was analyzed as in A. GREB1 mRNA levels are presented as percentages of the maximal level when treated with E2. C, The three species of GREB1 transcripts a, b, and c were analyzed separately using transcript-specific primer sets in real-time RT-PCR using samples in B. The mRNA level of each GREB1 transcript (a–c) is presented as percentages of its level when treated with E2. The data are from three separate experiments (mean ± SE). In some cases, the very low levels of GREB1b and GREB1c preclude detection on this graph. *, GREB1c expression level differed significantly from that of GREB1a or GREB1b transcripts, P < 0.05 (Student’s t test).

 
We then assayed individual GREB1 transcripts, GREB1a, -b, or -c, using different primer pairs in real-time RT-PCR. The 5' primer was specific to each transcript and the 3' primer was common to all three transcripts. Both GREB1b and GREB1c were strongly induced after E2 treatment. E2-stimulated GREB1a expression was lower than that of either GREB1b or GREB1c (not shown), and its relative induction by E2 was also lower (data presented as percent maximal induction, Fig. 2CGo). These data suggest that when activated by ER, the GREB1b and GREB1c promoters are used preferentially over that of GREB1a. Interestingly, the inhibitory effect of OHT was slightly less effective on GREB1c at both concentrations of OHT tested. This may indicate that different transcriptional regulators act to modulate ER-driven transcription from the different GREB1 core promoters.

The GREB1 EREs Synergistically Mediate E2-Induced Transcription in Vitro
Although GREB1 is a known ER target gene, the relative abilities of its different ERE motifs to drive estrogen-induced transcription have not been demonstrated. The ERE1 motif is 1.6 kb upstream of the transcription start site for GREB1a. To demonstrate ERE1-mediated estrogen-responsive transcription via the GREB1a promoter, a 1.7-kb fragment that included ERE1 and the GREB1a promoter region was cloned into the pGL3-basic reporter plasmid. Because ERE2 and ERE3 are too far apart to clone as a single fragment, we synthesized 50-bp oligos centered on each of the ERE2 and ERE3 motifs and including their surrounding sequences, respectively, to prepare reporter constructs. Constructs containing ERE1 alone, ERE1 and ERE2, and all three EREs were used in reported assays. As controls, reporter constructs containing mutant ERE motifs were also prepared (see Fig. 3AGo for schematics of these reporter constructs). These reporter constructs were transfected into MCF-7 cells, which were then treated with either E2 or OHT for 24 h. Luciferase activity was measured, and values were corrected for transfection efficiency using renilla luciferase activity from a cotransfected reference plasmid. For the construct bearing the ERE1 motif alone, a 2.2-fold increase in luciferase activity was observed when treated with E2 over the basal level, whereas OHT did not increase luciferase activity. Mutation of the ERE1 motif in the reporter construct abolished E2-induced luciferase activity. Thus, ERE1 is capable of mediating estrogen-responsive GREB1a promoter activity in vitro. When ERE2 was inserted upstream of ERE1, E2 treatment increased luciferase activity by approximately 36-fold over its basal level. For the reporter with all three ERE motifs, E2-induced luciferase activity went up to about 170-fold over its basal level. These data indicate that all three of the EREs are functional and can act synergistically to module E2-dependent ER activity in these assays. There was no E2-induced luciferase activity from reporters with mutant ERE motifs (Fig. 3BGo).


Figure 3
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  		Fig. 3. ERE Motifs from the GREB1 Locus Are Functional in a Reporter Gene Assay

A, The 1.7-kb region of the GREB1a promoter containing ERE1 or mutated ERE1 was inserted upstream of the luciferase gene in the pGL3-basic vector. DNA fragments containing ERE2, ERE3, or their mutant forms were inserted upstream of ERE1. The schematics of these constructs are shown with mutant ERE motifs represented in crossed boxes. B, These luciferase reporter constructs were cotransfected with a control reporter vector pRL-SV40 encoding for renilla luciferase into MCF-7 cells. Luciferase activity was measured for samples either with no hormone (control) or after treatment of E2 deprived cells with 10 nM E2 or 100 nM OHT for 24 h. The data are plotted as fold induction of luciferase activity over the control level from the ERE1-containing reporter and are from three separate experiments (mean ± SE). The values of fold induction for wild-type ERE-containing reporters are shown at the top of each treatment.

 
E2 Induces Cyclic ER Binding to All Three ERE Motifs
E2-driven ER binding to the ERE motif of the pS2 promoter has been shown to be maximal at 45 min (17, 18). The GREB1 ERE2 and ERE3 motifs are located approximately 8 and 19.5 kb upstream of ERE1, respectively. To test the potential relevance of the three putative EREs to E2-stimulated GREB1 expression in vivo, we assayed E2-stimulated ER binding to different sites within a 35-kb region of the GREB1 locus after 45 min E2 treatment of MCF-7 cells using ChIP assays. As shown in Fig. 4AGo, in a nonquantitative PCR assay, both E2 and OHT induced ER binding to the ERE1 region, whereas no amplification signal was observed in the ChIP using nonspecific IgG. Quantification of ER binding to various sites at the GREB1 locus in response to E2 is shown in Fig. 4BGo. It was not possible to assay the ER binding to the GREB1a core promoter because this was obscured by the strong ER binding to the adjacent ERE1. Among the nine regions tested, ER bound only to the three regions containing the consensus ERE motifs, and ERE1 was the strongest ER-binding sites of the three. ER binding to regions containing the most distal ERE (ERE3) at –21.2 kb and the middle ERE (ERE2) at –9.5 kb was similar. There was no ER binding at –24.5-, –17-, –13-, and –5-kb regions and the regions containing the core promoters for GREB1b and GREB1c, respectively (Fig. 4BGo). Unliganded ER also bound to all three ERE-containing regions but to a much lesser extent than did liganded ER. ERE1 showed the greatest binding to unliganded ER (Fig. 4BGo).


Figure 4
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  		Fig. 4. ER Is Selectively Recruited to the ERE Motifs in the GREB1 Promoter

A, MCF-7 cells were hormone deprived for 3 d (control) followed by treatment with 10 nM E2 or 100 nM OHT for 45 min. ChIP assays were then performed using anti-ER antibody (middle panel) or normal IgG (bottom panel) as a control. The ER-bound DNA was amplified by regular PCR for 35 cycles using a pair of primers that encompasses the ERE1 region. The top panel shows 10% chromosome input in the same PCR. B, The binding of ER to different upstream GREB1 promoter regions after 45 min of E2 treatment was measured by real-time PCR. In addition to assays of ER binding to the three EREs, ChIP assays also tested nonspecific ER binding to control sequences located –24.5, –17, –13, and –5 kb from the GREB1a transcriptional start site as well as GREB1c and GREB1b core promoter sites. The levels of ER binding to each region were expressed as percentages of total chromosome input before immunoprecipitation. The data are derived from three separate experiments (mean ± SE). *, ER binding to ERE1 was statistically different from that to ERE2 and ERE3, P < 0.05 (Student’s t test). C, The dynamics of ER binding to each ERE-containing region was measure in real-time PCR at the intervals shown between 15 and 165 min after E2 treatment. The data are plotted as percentages of total chromosome input before immunoprecipitation and are from an experiment with triplicate samples (mean ± SD).

 
Previous studies have indicated that ER cycles on and off the estrogen-responsive promoter of the pS2 gene (17, 18). To compare the kinetics of ER binding to all three ERE motifs at the GREB1 locus, we assayed ER binding to these ERE motifs at intervals after E2 addition. As previously observed for the pS2 promoter (17, 18), ER cycled on and off all three ERE motifs of the GREB1 locus in a synchronized manner, with an approximately 60-min periodicity (Fig. 4CGo). The coordinated kinetics of ligand-stimulated ER binding to all three EREs suggests that GREB1 gene activation may be mediated by a cooperative action of ER at these ERE motifs. The second peak of ER binding was higher than the first peak and higher than that observed at any subsequent time point assayed. The extent of ER binding to ERE1 was greater than to either ERE2 or ERE3 (Fig. 4CGo). Thus, ERE1 may play a more important role in mediating E2-activated GREB1 gene expression than ERE2 or ERE3.

Coactivator SRC-3 Associates with Three ERE Motifs
To test further E2 regulation of the GREB1 promoters, we tested the association of the p160 coactivator, SRC-3, using quantitative ChIP assays, with regions at the GREB1 locus after E2 treatment. E2, but not OHT, induced SRC-3 binding to all three ERE motifs but not to the GREB1b or GREB1c core promoters (Fig. 5AGo). There was also a low level of SRC-3 association with all three ERE motifs in the absence of E2. As was the case for ER binding, both ligand-dependent and ligand-independent SRC-3 binding to ERE1 was greater than to either ERE2 or ERE3. The estrogen-induced binding of SRC-3 to ERE2 was less than that to ERE3. This might reflect a conformational constraint at ERE2, because SRC-3 association at ERE2 was also lower in the unliganded state.


Figure 5
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  		Fig. 5. SRC-3 Is Associated with All Three ERE Motifs, and Histone H3 Acetylation Is Stimulated by E2 at ERE Motifs and Core Promoters

ChIP was performed with an antibody against SRC-3 or acetylated histone H3 from MCF-7 cell samples that were E2 deprived for 3 d (control) or treated with either 10 nM E2 or 100 nM OHT for 45 min. The binding of SRC-3 (A) and the histone H3 acetylation level at the regions upstream of the promoter (B) and at the core promoter sites as well as at an internal site within the GREB1 transcript (C) were measured by real-time PCR using the primer pairs that cover the regions indicated. The data are plotted as percentage of total chromosome input before immunoprecipitation and are from three separate experiments (mean ± SE). *, Test results with E2 differed significantly from those of controls, P < 0.05 (Student’s t test).

 
Estrogen Induces Long-Range Histone H3 Acetylation at the GREB1 Gene Locus
One of the functions of agonist-bound ER is to recruit the coactivator complex, which, in turn, modifies chromatin structure through histone acetylation to facilitate gene transcription. Histone H3 acetylation at the GREB1 gene locus was monitored by ChIP assay. E2 induced histone H3 hyperacetylation in much broader regions at the GREB1 locus than those specifically bound by ER (Fig. 5Go, B and C). In addition to the three ERE regions to which ER and SRC-3 bound, several intervening regions that failed to bind ER were hyperacetylated in response to E2. However, peak acetylation was observed in the regions close to the EREs. Histone H3 acetylation after E2 stimulation was higher in the regions around ERE1 than in the vicinities of ERE2 or ERE3 (Fig. 5BGo), consistent with ER and SRC-3 binding patterns.

Of the three GREB1 core promoters, the greatest E2-induced histone H3 acetylation was seen at GREB1b and GREB1c promoters. The reduced GREB1a promoter acetylation may reflect its weaker promoter activity. The histone H3 acetylation of a coding region assayed was much lower than at the promoters (Fig. 5CGo). In contrast, OHT did not increase the histone H3 acetylation at any region assayed within the GREB1 gene locus.

Pol II Binds to GREB1 Transcription Start Sites and to All Three ERE Motifs
The core promoter is classically defined as the region around the transcription start site where RNA polymerase II (Pol II) initiates transcription. ChIP assay was used to monitor Pol II binding at ERE-containing regions and at the regions containing GREB1 transcription start sites (Fig. 6AGo). As expected, E2 induced strong Pol II binding to the GREB1b and GREB1c core promoters and very weak binding to the GREB1a promoter, consistent with the observed strength of each promoter based on the extent of E2 induction of their respective transcripts. ChIP failed to capture the transient Pol II interaction with a GREB1 coding region that would occur during active gene transcription.


Figure 6
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  		Fig. 6. The Recruitment of Pol II to Both ERE-Containing Sites and Core Promoter Regions in Response to E2

MCF-7 cells were treated with 10 nM E2 or 100 nM OHT or vehicle (control) for 45 min. A, Chromatin samples were immunoprecipitated with anti-Pol II antibody, and DNA fragments bound to Pol II were quantified by real-time PCR. B, After the first ChIP using an antibody to ER, the products were subjected to re-ChIP analysis using an antibody to Pol II. The bound DNA fragments were quantified by real-time PCR. C, After the first ChIP using an antibody to Pol II, the products were subjected to re-ChIP analysis using an antibody to ER. The bound DNA fragments were quantified by real-time PCR. A cDNA region was included in the analysis as a control. The data are plotted as percentages of total chromosome input before immunoprecipitation and are from three independent experiments (mean ± SE). *, Test results with E2 differed significantly from those of controls, P < 0.05 (Student’s t test).

 
In addition to its stimulation of ER and SRC3 binding and histone acetylation at these sites, E2 also induced strong association of Pol II with all three ERE motifs. The strength of Pol II binding showed the same pattern as did ER binding to these EREs, with greater Pol II binding to ERE1 than to ERE2 or ERE3. OHT again failed to recruit Pol II to any of the core promoter or ERE enhancer regions.

In contrast to our observation with ER, which bound only ERE sites, Pol II bound to both the EREs and the core promoters. To confirm that the Pol II complex bound to the core promoters differed from the Pol II complex at the ERE sites, we used ChIP and re-ChIP assay to test Pol II association with ER. In the ER/Pol II ChIP assay where the antibody to ER was used in the first ChIP and the antibody to Pol II was used in the re-ChIP, we detected E2-induced Pol II association with ER only at the EREs but not at the GREB1b and GREB1c core promoters (data shown for ER-Pol II binding to the EREs in Fig. 6BGo). Because there was no positive amplification from ChIP assays with the GREB1b and -c core promoters, they were not plotted (Fig. 6BGo). Similarly, in the Pol II/ER ChIP assay where the antibody to Pol II was used in the first ChIP and the antibody to ER was used in the re-ChIP, E2-induced ER/Pol II complex was detected only at the EREs (Fig. 6CGo) but not at the GREB1b and GREB1c core promoters. The latter were not plotted due to the negative real-time PCR. Thus, Pol II bound to the EREs was in a different complex from that at the core promoters.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
GREB1 was discovered as an E2-induced gene in ER-positive breast cancer cells. Its expression is strongly correlated with ER protein expression in breast cancer tumors and cell lines (25, 26). Recent data indicate that GREB1 is required for estrogen-stimulated proliferation of cultured ER-positive breast cancer cells (26). The mechanisms regulating estrogen-induced GREB1 expression have not been fully elucidated.

Through an in silico search for the consensus EREs in the whole human genome, we found a 20-kb region containing three consensus ERE motifs upstream of the human GREB1 transcription start sites. Here we investigated their potential involvement in E2-stimulated GREB1 gene expression. Estrogen stimulated ER binding to each of the three GREB1 EREs, and the ER cycled on and off in a synchronized fashion. In addition, the coactivator SRC-3 and Pol II were brought to the ERE sites with activated ER, accompanied by long-range histone acetylation centered on the ERE motifs and core promoters. Among the three previously identified GREB1 cDNA transcripts, induction of GREB1b and GREB1c transcripts by E2 was more robust than that of GREB1a in MCF-7 cells. Thus, the GREB1b and GREB1c core promoters, although more distant from the ERE sites, appear to be activated to a greater extent by E2 than the GREB1a core promoter.

Other reports that identified functional EREs based on in silico screening have mainly focused on regions proximal to transcription start sites. ER binding to both the proximal and middle EREs of the human GREB1 gene was reported by others during the course of the present study (22, 23). Lin et al. (23) characterized ER binding only to the proximal ERE using ChIP. Bourdeau et al. (22) assayed ER binding at all the three ERE sites in a simple nonquantitative PCR assay but initially observed only ER:ERE1 and ER:ERE2 interaction and failed to demonstrated ER binding to the most distal ERE3 site. However, at a recent meeting, this group reported ER binding to all three ERE motifs at the GREB1 locus (27). Our study extends previous observations by using quantitative analysis to demonstrate cyclic dynamics of E2-stimulated ER binding at all three ERE sites and by demonstrating coactivator and Pol II recruitment to these EREs. Our data suggest that all three EREs of the GREB1 promoter may contribute to E2-stimulated GREB1 expression. The notion that all three distantly spaced ERE motifs may contribute to E2-dependent ER regulation of the GREB1 gene expression is supported by their phylogenetic conservation. All three consensus ERE motifs within the GREB1 locus are conserved in chimpanzees and canines.

There is prior evidence that nuclear receptor target gene promoters contain multiple functional receptor binding sites (28). In the Xenopus vitellogenin B1 and B2 genes, two copies of adjacent nonconsensus EREs act synergistically to activate transcription induced by estrogen (29). Based on this observation, ER reporter plasmids have made use of multiple consensus EREs to achieve synergistic activation by ER (30). Earlier studies identified many ER target genes that contain functional EREs, most of which were located close to the core promoters and are nonconsensus (19). Nonconsensus ERE motifs usually deviate one to three nucleotides from the consensus and have been shown to have lower ER binding affinities in vitro compared with the consensus ERE. ER binding to EREs in vivo appears to be more complicated. Although all three GREB1 locus EREs are perfect consensus motifs, they exhibit different degrees of ER, coactivator, and Pol II binding, with ERE1 used more frequently than ERE2 and ERE3. Moreover, we have observed that not all the consensus ERE motifs in the genome are capable of binding to ER in vivo when cells are treated with E2 (Sun, J., and J. M. Slingerland, unpublished data). Thus, other factors may regulate the ER-ERE interaction and its functional importance in vivo. Interestingly, the GREB1 gene locus contains multiple FOXA1 binding motifs located close to all three ERE motifs. Thus, this forkhead factor may play a role to modulate ER-ERE interaction and estrogen-induced GREB1 gene expression (31, 32).

ER is thought to stimulate the assembly of an initiation complex and regulate the frequency at which new Pol II molecules reinitiate transcription (33). In the present study, we found that E2 induced Pol II association not only at the GREB1 core promoters but also at the distant ERE sites. In contrast, both the ER and the coactivator SRC-3 associated only with the ERE sites and not with the core promoters. The re-ChIP studies also confirmed that Pol II complexes at the core promoters and at the EREs are distinct, with the former lacking and the latter containing associated ER. This is in agreement with other studies that reported androgen-induced Pol II association with both the PSA promoter and enhancer regions. The androgen receptor and Pol II were present together at the enhancer but not at the promoter (34, 35). Similar findings were also recently reported by Carroll et al. (31) for the ER target gene XBP-1.

We observed E2-induced histone hyperacetylation at the GREB1 core promoters and at distant upstream regions centered on all of the three ERE motifs. Our data indicate strong association of Pol II with these enhancers. Together with recent findings in other gene contexts (31, 36, 37), these data support a looping model in which ER binding may stimulate GREB1 gene activation by bringing combinations of either two or three enhancer regions and core promoters transiently into close proximity. This would facilitate transfer of Pol II from the enhancer complexes to the basal transcription machinery at the core promoters and increase the potential for fine tuning of promoter activity. This model of GREB1 activation by ER is supported by a recent analysis of ER binding in the human genome, in which a majority of ER binding sites were located at significant distances, with many over 100 kb from transcription start sites of ER target genes (31, 38). Moreover, multiple distal ER binding sites were present in a number of ER target genes. Thus, activation by coordinate ER binding at multiple distal enhancer regions within a single gene, as we have observed for GREB1, may be a common feature of ER-regulated genes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Reagents and Antibodies
E2, OHT, formaldehyde, and protein A-Sepharose beads were purchased from Sigma Chemical Co. (St. Louis, MO). Antibodies against ER (HC20) and SRC-3 (C20) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody against acetylated histone H3 (06-599) was from Upstate (Charlottesville, VA). Anti-RNA Pol II antibody (8WG16) was from Covance (Berkeley, CA).

Cell Culture
MCF-7, T47D, ZR-75, and MDA-MB-231 cells were maintained in Improved MEM (IMEM) (Invitrogen, Carlsbad, CA) supplemented with 5% fetal bovine serum in a humidified incubator with 5% CO2. Before ligand treatment, the cells were maintained in phenol red-free IMEM supplemented with 5% charcoal dextran-treated fetal bovine serum for 3 d.

RNA Extraction and Real-Time RT-PCR
The total RNA from MCF-7 cells was isolated using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. cDNA was synthesized from 1 µg total RNA as template using iScript cDNA synthesis kit (Bio-Rad, Hercules, CA). Real-time PCR was performed using an icycleriQ PCR detection system (Bio-Rad) using 10 ng cDNA sample in iQ SyberGreen supermix (Bio-Rad). PCR conditions were 95 C for 30 sec, followed by 60 C for 60 sec for 40 cycles. The GREB1 cDNA was amplified with the following primers: forward 5'-ATCAGCTGCTCGGACTTGCTG-3' and reverse 5'-TGAGCTCCGGTCCTGACAGATG-3'. For amplification of GREB1 cDNA derived from each of three transcriptional start sites, we used promoter-specific forward primers (5'-TGTGGAAGGACATGGCTTTTA-3' for GREB1a, 5'-GGCTTTGTTTGGAGCAGAAAA-3' for GREB1b, and 5'-GTCTGTGGAGTGCCTGAAGTG-3' for GREB1c) and one reverse primer (5'-GTCTGTGGAGTGCCTGAAGTG-3') common for all three transcripts. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which was used as an internal control, was amplified with the following primers: forward 5'-GAAGGTGAAGGTCGGAGTC-3' and reverse 5'-GAAGATGGTGATGGGATTTC-3'. The comparative threshold cycle method was used to determine the relative expression level of GREB1 mRNA. Melt-curve analysis was performed to monitor the quality of the PCR amplification.

Luciferase Reporter Gene Assay
A 1.7-kb region from the GREB1a promoter containing the ERE1 motif was amplified from MCF-7 genomic DNA with the following primers: forward 5'-GGGGTACCagtgtggcaactgggtcatt-3' and reverse 5'-GCCCAAGCTTgcaagccttccattgaaaaa-3' (underlined sequences indicate the restriction sites added). The PCR-amplified product was sequenced and cloned into the pGL3-basic vector (Promega, Madison, WI) at KpnI/HindIII sites to generate reporter plasmid pGREB-ERE1. To generate a reporter construct containing mutated ERE1, site-directed mutagenesis was performed based on the QuikChange method (Stratagene, La Jolla, CA) using the primers 5'-GTACCAGTGTGGCAACTGGATCCTTCTGACCTAGAAGCAAC-3' and 5'-GTTGCTTCTAGGTCAGAAGGATCCAGTTGCCACACTGGTA C-3' to generate pGREB1-ERE1 mutant. To add ERE2 and ERE3 motifs or their mutant forms to the reporter plasmids, two complementary oligos covering each motif as well as its adjacent genomic sequence were synthesized. Each pair of oligos was phosphorylated by T4 polynucleotide kinase and annealed in vitro. The double-strand DNA was cloned into the parental report plasmid with according restriction enzyme sites. The oligos for ERE2 motif are 5'-CGAGCTCTTCTTCTCAAAAGGTCATCATGACCTTATTGTCTGGGAGTAC-3' and 5'-TCCCAGACAATAAGGTCATGATGACCTTTTGAGAAGAAGAGCTCGGTAC-3'; the ones for mutant ERE2 motif are 5'-CGAGCTCTTCTTCTCAAAACCTGATCACGTGGTTATTGTCTGGGAGTAC-3' and 5'-TCCCAGACAATAACCACGTGATCAGGTTTTGAGAAGAAGAGCTCGGTAC-3'. The oligos for ERE3 motif are 5'-CTTATAGACAAAGATAATCAGGTCAAAATGACCTTCTTTCAGTTGTTGTAC-3' and 5'-AACAACTGAAAGAAGGTCATTTTGACCTGATTATCTTTGTCTATAAGGTAC-3'; the ones for mutant ERE3 motif are 5'-CTTATAGACAAAGATAATCACGTGAAAAGTGAATTCTTTCAGTTGTTGTAC-3' and 5'-AACAACTGAAAGAATTCACTTTTCACGTGATTATCTTTGTCTATAAGGTAC-3'.

These reporter constructs were cotransfected, respectively, with the phRL-SV40 plasmid from Promega, which encodes renilla luciferase as an internal control, into MCF-7 cells using Lipofectin (Invitrogen). Cells were treated with 10 nM E2 or 100 nM OHT for 24 h before harvest. The luciferase activity was measure with a dual-luciferase reporter assay system (Promega) using a luminometer from Thermo Labsystems (Needham, MA).

ChIP Assay and Re-ChIP Assay
MCF-7 cells were grown in phenol red-free IMEM with 5% charcoal-dextran stripped serum for 3 d to 80–90% confluence before the treatment with 10 nM E2 or 100 nM OHT for specified times. ChIP assays were performed as described previously with minor modifications (17). To identify other protein components in protein-DNA complexes, re-ChIP assays were performed in which the cross-linked immunocomplex was eluted from the first ChIP with 10 mM dithiothreitol at 37 C for 30 min, and then the product was diluted 50-fold in 1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl (pH 8.1). Re-ChIP assay was performed with a different antibody. The primers used for the real-time PCR detection of GREB1 locus are listed in Table 1Go.


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  		Table 1. Genomic PCR Primer Sequences Used for Detection at the Human GREB1 Locus

 
Statistical Analysis
Significant changes in GREB1 gene expression (Fig. 2CGo) and in ER binding to ERE1 vs. ERE2 and ERE3 (Fig. 4BGo) and in results of histone acetylation and ChIP assays shown in Figs. 5Go and 6Go were calculated using the Student’s t test.


   ACKNOWLEDGMENTS
 
We thank Dr. S. Elliot of University of Miami Miller School of Medicine for assistance in the initiation of real-time PCR analysis.


   FOOTNOTES
 
This work was supported by the Braman Family Breast Cancer Institute of the Sylvester Comprehensive Cancer Center.

Disclosure Statement: The authors have nothing to disclose.

First Published Online July 31, 2007

Abbreviations: ChIP, Chromatin immunoprecipitation; E2, 17ß-estradiol; ER, estrogen receptor; ERE, estrogen response element; LBD, ligand-binding domain; OHT, 4-hydroxyl tamoxifen; Pol II, RNA polymerase II; SRC-3, steroid receptor coactivator-3.

Received for publication February 14, 2007. Accepted for publication July 24, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Nilsson S, Gustafsson JA 2002 Biological role of estrogen and estrogen receptors. Crit Rev Biochem Mol Biol 37:1–28[CrossRef][Medline]
  2. Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P, Evans RM 1995 The nuclear receptor superfamily: the second decade. Cell 83:835–839[CrossRef][Medline]
  3. Green S, Walter P, Kumar V, Krust A, Bornert JM, Argos P, Chambon P 1986 Human oestrogen receptor cDNA: sequence, expression and homology to v-erb-A. Nature 320:134–139[CrossRef][Medline]
  4. Greene GL, Gilna P, Waterfield M, Baker A, Hort Y, Shine J 1986 Sequence and expression of human estrogen receptor complementary DNA. Science 231:1150–1154[Abstract/Free Full Text]
  5. Mosselman S, Polman J, Dijkema R 1996 ERß: identification and characterization of a novel human estrogen receptor. FEBS Lett 392:49–53[CrossRef][Medline]
  6. Enmark E, Pelto-Huikko M, Grandien K, Lagercrantz S, Lagercrantz J, Fried G, Nordenskjold M, Gustafsson JA 1997 Human estrogen receptor ß-gene structure, chromosomal localization, and expression pattern. J Clin Endocrinol Metab 82:4258–4265[Abstract/Free Full Text]
  7. Ogawa S, Inoue S, Watanabe T, Hiroi H, Orimo A, Hosoi T, Ouchi Y, Muramatsu M 1998 The complete primary structure of human estrogen receptor ß (hERß) and its heterodimerization with ER{alpha} in vivo and in vitro. Biochem Biophys Res Commun 243:122–126[CrossRef][Medline]
  8. Kuiper GG, Carlsson B, Grandien K, Enmark E, Haggblad J, Nilsson S, Gustafsson JA 1997 Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors {alpha} and ß. Endocrinology 138:863–870[Abstract/Free Full Text]
  9. Couse JF, Lindzey J, Grandien K, Gustafsson JA, Korach KS 1997 Tissue distribution and quantitative analysis of estrogen receptor-{alpha} (ER{alpha}) and estrogen receptor-ß (ERß) messenger ribonucleic acid in the wild-type and ER{alpha}-knockout mouse. Endocrinology 138:4613–4621[Abstract/Free Full Text]
  10. Katzenellenbogen BS, Choi I, Delage-Mourroux R, Ediger TR, Martini PG, Montano M, Sun J, Weis K, Katzenellenbogen JA 2000 Molecular mechanisms of estrogen action: selective ligands and receptor pharmacology. J Steroid Biochem Mol Biol 74:279–285[CrossRef][Medline]
  11. Kumar V, Green S, Staub A, Chambon P 1986 Localisation of the oestradiol-binding and putative DNA-binding domains of the human oestrogen receptor. EMBO J 5:2231–2236[Medline]
  12. Kumar V, Green S, Stack G, Berry M, Jin JR, Chambon P 1987 Functional domains of the human estrogen receptor. Cell 51:941–951[CrossRef][Medline]
  13. Brzozowski AM, Pike AC, Dauter Z, Hubbard RE, Bonn T, Engstrom O, Ohman L, Greene GL, Gustafsson JA, Carlquist M 1997 Molecular basis of agonism and antagonism in the oestrogen receptor. Nature 389:753–758[CrossRef][Medline]
  14. Shiau AK, Barstad D, Loria PM, Cheng L, Kushner PJ, Agard DA, Greene GL 1998 The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen. Cell 95:927–937[CrossRef][Medline]
  15. McKenna NJ, O’Malley BW 2002 Combinatorial control of gene expression by nuclear receptors and coregulators. Cell 108:465–474[CrossRef][Medline]
  16. Jenuwein T, Allis CD 2001 Translating the histone code. Science 293:1074–1080[Abstract/Free Full Text]
  17. Shang Y, Hu X, DiRenzo J, Lazar MA, Brown M 2000 Cofactor dynamics and sufficiency in estrogen receptor-regulated transcription. Cell 103:843–852[CrossRef][Medline]
  18. Metivier R, Penot G, Hubner MR, Reid G, Brand H, Kos M, Gannon F 2003 Estrogen receptor-{alpha} directs ordered, cyclical, and combinatorial recruitment of cofactors on a natural target promoter. Cell 115:751–763[CrossRef][Medline]
  19. Klinge CM 2001 Estrogen receptor interaction with estrogen response elements. Nucleic Acids Res 29:2905–2919[Abstract/Free Full Text]
  20. Schwabe JW, Chapman L, Finch JT, Rhodes D 1993 The crystal structure of the estrogen receptor DNA-binding domain bound to DNA: how receptors discriminate between their response elements. Cell 75:567–578[CrossRef][Medline]
  21. Frasor J, Danes JM, Komm B, Chang KC, Lyttle CR, Katzenellenbogen BS 2003 Profiling of estrogen up- and down-regulated gene expression in human breast cancer cells: insights into gene networks and pathways underlying estrogenic control of proliferation and cell phenotype. Endocrinology 144:4562–4574[CrossRef][Medline]
  22. Bourdeau V, Deschenes J, Metivier R, Nagai Y, Nguyen D, Bretschneider N, Gannon F, White JH, Mader S 2004 Genome-wide identification of high-affinity estrogen response elements in human and mouse. Mol Endocrinol 18:1411–1427[Abstract/Free Full Text]
  23. Lin CY, Strom A, Vega VB, Kong SL, Yeo AL, Thomsen JS, Chan WC, Doray B, Bangarusamy DK, Ramasamy A, Vergara LA, Tang S, Chong A, Bajic VB, Miller LD, Gustafsson JA, Liu ET 2004 Discovery of estrogen receptor {alpha} target genes and response elements in breast tumor cells. Genome Biol 5:R66
  24. West AG, Fraser P 2005 Remote control of gene transcription. Hum Mol Genet 14 Spec No 1:R101–R111
  25. Ghosh MG, Thompson DA, Weigel RJ 2000 PDZK1 and GREB1 are estrogen-regulated genes expressed in hormone-responsive breast cancer. Cancer Res 60:6367–6375[Abstract/Free Full Text]
  26. Rae JM, Johnson MD, Scheys JO, Cordero KE, Larios JM, Lippman ME 2005 GREB1 is a critical regulator of hormone dependent breast cancer growth. Breast Cancer Res Treat 92:141–149[CrossRef][Medline]
  27. Deschenes J, Bourdeau V, White JH, Mader S 2006 Long-range regulation of the GREB1 gene by estrogen receptor {alpha} in breast cancer cell line MCF-7. Nuclear receptors: bench to bedside. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 104
  28. Sinkkonen L, Malinen M, Saavalainen K, Vaisanen S, Carlberg C 2005 Regulation of the human cyclin C gene via multiple vitamin D3-responsive regions in its promoter. Nucleic Acids Res 33:2440–2451[Abstract/Free Full Text]
  29. Klein-Hitpass L, Kaling M, Ryffel GU 1988 Synergism of closely adjacent estrogen-responsive elements increases their regulatory potential. J Mol Biol 201:537–544[CrossRef][Medline]
  30. Sathya G, Li W, Klinge CM, Anolik JH, Hilf R, Bambara RA 1997 Effects of multiple estrogen responsive elements, their spacing, and location on estrogen response of reporter genes. Mol Endocrinol 11:1994–2003[Abstract/Free Full Text]
  31. Carroll JS, Liu XS, Brodsky AS, Li W, Meyer CA, Szary AJ, Eeckhoute J, Shao W, Hestermann EV, Geistlinger TR, Fox EA, Silver PA, Brown M 2005 Chromosome-wide mapping of estrogen receptor binding reveals long-range regulation requiring the forkhead protein FoxA1. Cell 122:33–43[CrossRef][Medline]
  32. Laganiere J, Deblois G, Giguere V 2005 Functional genomics identifies a mechanism for estrogen activation of the retinoic acid receptor {alpha}1 gene in breast cancer cells. Mol Endocrinol 19:1584–1592[Abstract/Free Full Text]
  33. Kraus WL, Kadonaga JT 1998 p300 and estrogen receptor cooperatively activate transcription via differential enhancement of initiation and reinitiation. Genes Dev 12:331–342[Abstract/Free Full Text]
  34. Shang Y, Myers M, Brown M 2002 Formation of the androgen receptor transcription complex. Mol Cell 9:601–610[CrossRef][Medline]
  35. Louie MC, Yang HQ, Ma AH, Xu W, Zou JX, Kung HJ, Chen HW 2003 Androgen-induced recruitment of RNA polymerase II to a nuclear receptor-p160 coactivator complex. Proc Natl Acad Sci USA 100:2226–2230[Abstract/Free Full Text]
  36. Cullen KE, Kladde MP, Seyfred MA 1993 Interaction between transcription regulatory regions of prolactin chromatin. Science 261:203–206[Abstract/Free Full Text]
  37. Gothard LQ, Hibbard JC, Seyfred MA 1996 Estrogen-mediated induction of rat prolactin gene transcription requires the formation of a chromatin loop between the distal enhancer and proximal promoter regions. Mol Endocrinol 10:185–195[Abstract/Free Full Text]
  38. Carroll JS, Meyer CA, Song J, Li W, Geistlinger TR, Eeckhoute J, Brodsky AS, Keeton EK, Fertuck KC, Hall GF, Wang Q, Bekiranov S, Sementchenko V, Fox EA, Silver PA, Gingeras TR, Liu XS, Brown M 2006 Genome-wide analysis of estrogen receptor binding sites. Nat Genet 38:1289–1297[CrossRef][Medline]

NURSA Molecule Pages Link:

Nuclear Receptors:   ERα
Coregulators:   AIB1
Ligands:   17β-Estradiol  |  4-Hydroxytamoxifen



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