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. 2008 May 15;22(7):1579–1595. doi: 10.1210/me.2007-0253

Estrogen Receptor β Isoform-Specific Induction of Transforming Growth Factor β-Inducible Early Gene-1 in Human Osteoblast Cells: An Essential Role for the Activation Function 1 Domain

John R Hawse 1, Malayannan Subramaniam 1, David G Monroe 1, Amanda H Hemmingsen 1, James N Ingle 1, Sundeep Khosla 1, Merry Jo Oursler 1, Thomas C Spelsberg 1
PMCID: PMC2453604  PMID: 18483178

Abstract

The estrogen receptors (ER) α and β are important ligand-mediated transcription factors known to play significant biological roles in numerous tissues including bone. Despite the high homology shared by these receptors, recent studies have suggested that their function is largely unique. Although these receptors have been studied in detail for more than a decade, little data exist concerning the mechanisms by which these two proteins regulate distinct sets of genes. Using the TGFβ-inducible early gene-1 (TIEG) as a model, we demonstrate that TIEG is rapidly induced in response to estrogen in osteoblasts by ERβ, but not ERα. We have identified the regulatory elements utilized by ERβ and have demonstrated that ERβ recruits steroid receptor coactivator (SRC)1 and SRC2 to this regulatory region. Additionally, deletion of the ERβ-activation function 1 (AF1) domain drastically decreases the estrogen induction of TIEG. Through the use of chimeric receptors, we have demonstrated that the AF1 domain of ERβ is responsible for recruiting SRC1 and SRC2 and inducing the expression of TIEG in osteoblasts. Finally, SRC1, but not SRC2, is essential for TIEG induction by ERβ. Overall, these data demonstrate that the estrogen induction of TIEG is ERβ specific and that the AF1 domain of ERβ confers this specificity. Finally, a novel and important role for ERβ’s AF1 is implicated in the recruitment of specific coactivators, suggesting that the AF1 may play a significant role in conferring the differences in regulation of gene expression by these two receptors.

ESTROGENS, INCLUDING 17β-ESTRADIOL (E2), are known to exert a wide variety of cellular effects and regulate numerous physiological conditions including cell growth, development, differentiation, and gene expression (1,2). Estrogens exert their genomic effects by binding to one of two specific estrogen receptor (ER) isoforms, ERα or ERβ, or their variants. ERα and ERβ are encoded by two separate genes and are members of the nuclear receptor superfamily that function as signal transducers and transcription factors (2). Upon ligand binding, these receptors undergo a conformational change, dimerize to form an activated receptor, and subsequently bind to specific DNA sequences to regulate target gene expression. ERs directly bind DNA through estrogen response elements (EREs) via their zinc finger domain, or indirectly interact with DNA through protein-protein interactions with other transcription factors (3).

ERα and ERβ are each encoded by eight to nine exons and have six protein domains designated as A–F (4). Four of these domains constitute the major functional domains of the ERs and include a highly conserved DNA-binding domain (DBD; C domain) that contains the zinc fingers, a ligand-binding domain (E domain), a highly conserved E/F domain containing the activation function 2 (AF2), and a highly divergent N-terminal domain (A/B domain) containing the activation function 1 (AF1) (5,6,7,8). The D domain comprises the hinge region, which separates the DBD and the ligand-binding domain and contains sequences necessary for receptor dimerization (9,10) and nuclear localization (11,12). The AF2 core domain is highly homologous between the ERα and ERβ isoforms, and this domain is known to be involved in the recruitment of nuclear coregulators to estrogen-responsive promoter and -enhancer regions (13,14,15,16). However, less is known about the biological role of the A/B domain, which contains the AF1. To date, the known functions of the A/B domain mainly involve the interaction with specific coregulators. These interactions are largely specified by the AF1 and therefore, the A/B domain of the ERs is referred to as the AF1 domain throughout this manuscript. The AF1 domain of ERβ is approximately 80 amino acids shorter than the AF1 domain of ERα and shares little sequence homology. Interestingly, the AF1 domain of ERβ is highly conserved between species, suggesting a functional importance that could denote isoform-specific functions (8).

As mentioned previously, ERα and ERβ bind specific DNA sequences known as EREs with high affinity. An ERE consists of a minimal palindromic inverted repeat: 5′-GGTCAnnnTGACC-3′, where n is any nucleotide (17). The majority of E2-regulated genes contain imperfect and nonpalindromic EREs that are still able to be activated by the ERs (18,19). In addition to EREs, ERs can also regulate gene expression through tethering to other transcription factors such as activating protein 1 (AP1) and stimulating protein 1 (Sp1). It has been shown that ERα exhibits E2-dependent activation of transcription when acting through AP1 sites, whereas ERβ has no effect (3). It is known that both ERα and ERβ specifically interact with Sp1 and that both agonists and antagonists activate the ERα-Sp1 or ERβ-Sp1 complexes (20,21,22,23).

After the discovery of ERβ in the mid-1990s (24), it was generally believed that its primary role was to serve as a modulator of ERα action. However, early studies from our laboratory analyzing selected genes (25,26), and more recent microarray data from our laboratory and others (27,28,29,30,31), have demonstrated that the actions of ERα and ERβ are largely different at the level of gene expression in osteoblasts and breast cancer cells. In fact, only about 20% of all genes regulated by ERα or ERβ are regulated by both isoforms of the receptor. The exact mechanisms responsible for regulation of specific genes by ERα and ERβ are currently unknown. Because the AF1 domains of ERα and ERβ are nonhomologous, differences in regulation of gene expression between the two receptors may be specified, in large part, by this domain (32,33).

This paper demonstrates that the TGFβ-inducible early gene 1 (TIEG), which was originally identified in osteoblasts (34), is specifically regulated by ERβ, and not ERα. Through the use of transient transfections, we have identified the regulatory elements likely responsible for this induction and have demonstrated that the AF1 domain of the ERs plays a critical role in conferring this isoform specificity. We have also shown that the coactivators, steroid receptor coactivator (SRC)1 and SRC2, and the transcription factor, Sp1, are a part of the transcriptionally competent activation complex. However, SRC2 does not appear to be necessary for TIEG expression in response to E2. Taken together, these data reveal that TIEG is specifically regulated by ERβ and demonstrate that this isoform-dependent regulation is specified by the AF1 domain. This model supports a novel and important role for the AF1 domain of the ERs in the recruitment of specific coactivators, resulting in isoform-specific regulation of genes.

RESULTS

Regulation of TIEG by E2

Based on the recent findings by our laboratory concerning the differential regulation of gene expression by ERα and ERβ in osteoblasts (28), we sought to characterize the induction of TIEG by E2. As a first step, we analyzed the transcriptional regulation of TIEG after E2 treatment from 0–24 h in U2OS-ERα and U2OS-ERβ expressing cell lines. These studies revealed that TIEG is rapidly, but transiently, induced by E2 in ERβ-expressing cells but not ERα-expressing cells (Fig. 1). After 2 h of E2 treatment in ERβ-expressing cells, TIEG mRNA is induced approximately 5-fold. By the 8-h time point, TIEG mRNA levels return to basal levels. A significant induction of TIEG is observed again at the 24-h time point. There was no significant induction of TIEG mRNA at any of the time points analyzed in ERα-expressing cells. To determine whether this phenomenon was cell type specific, we also examined TIEG expression in human breast cancer cells (Hs578T) expressing either ERα or ERβ. These cell lines were developed in our laboratory as previously described (31). Interestingly, the same patterns of TIEG gene expression were observed, confirming that TIEG is a primary response gene after E2 treatment and indicating that this induction likely occurs through the actions of ERβ and not ERα in multiple cell types (data not shown).

Figure 1.

Figure 1

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TIEG Is Regulated by E2 in an ERβ-Specific Manner in Osteoblasts

U2OS-ERα- and -ERβ-expressing cell lines were treated with 10 nm E2 for the indicated time points. Total RNA was harvested and subjected to real time RT-PCR analysis using TIEG and β-actin-specific primers. Data are expressed as TIEG mRNA abundance relative to β-actin levels. Asterisks denote significance at the P < 0.05 level (ANOVA) compared with control treatment (0 h). The experiment was conducted in triplicate, and a representative analysis is shown.

The TIEG Intron 1 Contains an Enhancer Region that Responds to E2 Stimulation

In an effort to identify the region of DNA through which the E2 regulation of TIEG occurs, the promoter region and genomic sequence of TIEG were scanned for potential EREs. As is depicted in Fig. 2A, this analysis revealed an ERE-like sequence and a consensus half-ERE in the first intron of the TIEG gene. In addition to these elements, a near-consensus Sp1 site was also identified. To determine whether this region of the TIEG gene was responsible for the ERβ-specific induction of TIEG, we cloned an approximately 800-bp fragment of intron 1, containing these candidate regulatory elements, into a luciferase reporter vector. Transient transfection of this construct, in combination with either ERα or ERβ, into parent U2OS cells (ER negative), treated with and without E2, revealed that this fragment of the TIEG gene was induced specifically by ERβ (Fig. 3A). Consistent with the E2 induction of the endogenous TIEG gene, no activation was observed by ERα (Fig. 3A). Additionally, activation of the TIEG intron 1 significantly decreased when the ERβ DBD mutant (DBD Mut) receptor was used, indicating that at least partial activation of TIEG expression is dependent on DNA binding (Fig. 3A). No differences were observed when the ERα DBD Mut receptor was used (Fig. 3A).

Figure 2.

Figure 2

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The First Intron of the Human TIEG Gene Contains ERE- and Sp1-Binding Sites

A, Schematic diagram of the human TIEG gene and the sequence of the enhancer region of intron 1 spanning from +4051 to +4827. Numbering is based on the gene accession number AF050110. Potential ERE- and Sp1-binding sites are indicated. B, The ERE-like, half-ERE, and Sp1 sites contained in the TIEG intron 1 enhancer region are depicted relative to the consensus binding sites.

Figure 3.

Figure 3

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Transient Transfection Assays Demonstrating Activation of a Consensus ERE and the TIEG Intron 1 Enhancer Region by ERα, ERβ, ERα DBD Mut, and ERβ DBD Mut Receptors in U2OS cells

A, Indicated ERα, ERβ, ERα DBD Mut, or ERβ DBD Mut expression vectors, and a consensus ERE or the TIEG intron 1 enhancer region fused to a luciferase reporter were transiently transfected into parent U2OS cells and treated with either ethanol vehicle or 10 nm E2 for 24 h. Luciferase values are reported as relative fold change compared with ethanol controls. Asterisks denote significance at the P < 0.05 level (ANOVA) compared with control treatment. δ denotes significance at the P < 0.05 level (ANOVA) between intact receptors and the DBD Muts. The experiment was conducted in triplicate, with six replicates per treatment, and a representative analysis is shown. B, Western blot analysis depicting protein expression of ERα, ERβ, ERα DBD Mut, and ERβ DBD Mut receptors after transient transfection of parent U2OS cells using a Flag-specific primary antibody.

As a control, a consensus ERE reporter construct was induced to similar levels by both ERα and ERβ in the parental U2OS cell line, and this induction was either completely lost, or significantly reduced, when using ERα and ERβ DBD Mut receptors, respectively (Fig. 3A). Western blot analysis was performed in parent U2OS cells transfected with ERα, ERβ, ERα DBD Mut, or ERβ DBD Mut receptors to confirm the relative expression levels and integrity of these receptors (Fig. 3B). These data support that this region of the TIEG gene likely contains the regulatory element(s) responsible for the ERβ-specific induction of TIEG.

Identification of the Enhancer Elements Responsible for E2 Induction of TIEG

To determine the role of the ERE-like sequences and/or the Sp1 site in the E2 induction of TIEG, deletion constructs of the TIEG intron 1 were created. Deletion of a fragment containing the EREs resulted in a significant, but incomplete, reduction in luciferase activity by ERβ (Fig. 4A). These results are in agreement with the ERβ DBD Mut receptor studies (Fig. 3A), in which a significant but incomplete reduction in luciferase activity was observed, indicating that a portion of TIEG induction results from direct binding of ERβ to the ERE-like sequences in the first intron of the TIEG gene. Deletion of an additional 130 bp had no effect on luciferase activity. However, deletion of a 39-bp fragment (+4403 to +4442) containing a near consensus Sp1 site nearly abolished the activation of this reporter construct by ERβ (Fig. 4A). Further deletions of this fragment had no additional effect on activation by ERβ (Fig. 4A). No activation of any of the deletion constructs was observed by ERα (Fig. 4A). The minimal activity that remains on the +4442 to +4827 and on the +4603 to +4827 reporter constructs in response to E2 may be due to partial activation of other weak and imperfect ERE, Sp1, or AP1 binding sites that have yet to be identified.

Figure 4.

Figure 4

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Deletion Analysis of the TIEG Intron 1 Enhancer Region Reveals Binding Sites Necessary for ERβ Induction of TIEG

ERα or ERβ expression vectors (A) or ERα DBD Mut or ERβ DBD Mut. expression vectors (B), and the indicated enhancer constructs fused to a luciferase reporter were transiently transfected into parent U2OS cells and treated with ethanol vehicle or 10 nm E2 for 24 h. Luciferase values are reported as relative fold change compared with ethanol controls. Asterisks denote significance at the P < 0.05 level (ANOVA) relative to wild-type receptor regulation of the full-length (+4051 to +4827) reporter construct. δ, Significance at the P < 0.05 level (ANOVA) between indicated reporter constructs. The experiment was conducted in triplicate, with six replicates per treatment, and a representative analysis is shown.

To further delineate the potential role of the near-consensus Sp1 site, we examined the activation of the full-length reporter construct and the +4403 to +4827 reporter construct by the ERα DBD Mut receptor and the ERβ DBD Mut receptor (Fig. 4B). These studies demonstrate that the ERβ DBD Mut receptor has less activity on both the full-length TIEG intron 1 construct, as well as the +4403 to +4827 construct, relative to wild-type receptor activation of the full-length construct (compare Fig. 4A with Fig. 4B). However, there is no significant difference between the activation of the full-length construct and the +4403 to +4827 construct by the ERβ DBD Mut receptor (Fig. 4B). These data suggest that the ERβ-specific induction of TIEG occurs through both the ERE-like sequences and the Sp1 site located in the first intron of the TIEG gene.

Chromatin Immunoprecipitation (ChIP) Analysis Reveals Differences between the ERα and ERβ Recruitment of Coactivators to the TIEG Enhancer Region

In an effort to delineate why ERβ induces the expression of TIEG whereas ERα does not, we sought to determine whether both receptor isoforms bound to the region of the TIEG intron containing the ERE and Sp1 sites after E2 stimulation. ChIP assays were performed in flag-tagged ERα- and ERβ-expressing U2OS cells treated with E2 for 1 h using a monoclonal flag antibody. As indicated in Fig. 5, both receptors bind to this region of the TIEG gene in vivo after E2 stimulation. Because both receptors bind to the TIEG intron in response to E2, but ERα is incapable of inducing the expression of TIEG, we next sought to investigate the possible role of a differential SRC recruitment. Interestingly, after 1 h of E2 treatment, SRC1 and SRC2 are enriched on the TIEG intron in ERβ-expressing cells whereas they are not enriched on this enhancer region in ERα-expressing cells (Fig. 5). We did not examine the involvement of SRC3 because it exhibits low expression levels and fails to enhance E2-dependent transcription in osteoblasts (35).

Figure 5.

Figure 5

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ChIP Analysis of the TIEG Intron 1 Enhancer Region

ChIP assays were performed in Flag-tagged U2OS-ERα- and -ERβ-expressing cell lines treated with either ethanol vehicle or 10 nm E2 for 1 or 2 h. Chromatin was prepared, immunoprecipitated with the indicated antibodies, and amplified by both real-time PCR (A and C) and semiquantitative PCR (B and D). Real-time PCR analysis was used for quantitation purposes, and the data are expressed as the abundance of the TIEG intron 1 enhancer region in E2-treated cells after immunoprecipitation with indicated antibodies relative to vehicle control-treated cells. All data were normalized using input samples. Asterisks denote significance at the P < 0.05 level (ANOVA) compared with vehicle control-treated cells. The resultant products obtained by semiquantitative PCR were separated using agarose gel electrophoresis. Experiments were conducted in triplicate, and a representative analysis is shown. IP, Immunoprecipitation; V, vehicle; Veh., vehicle.

Because the near-consensus Sp1 site also plays an important role in the regulation of TIEG expression after E2 treatment, we also examined the binding of Sp1 protein to the TIEG intron. Interestingly, Sp1 was not further enriched on the TIEG intron after 1 h E2 stimulation of either ERα- or ERβ-expressing cells (Fig. 5). This observation is most likely explained by the fact that Sp1 protein is either continuously bound to this particular site or that the recruitment of Sp1 to this site after E2 treatment is masked due to Sp1 binding to other elements that are located within this intronic region. Nevertheless, as demonstrated in Fig. 4, the near-consensus Sp1 site located in the first intron of the TIEG gene is necessary for complete activation of this enhancer region by ERβ in response to E2 stimulation. These results suggest that ERα is not able to induce the expression of TIEG due to its inability to recruit the common coactivators, SRC1 and/or SRC2, to the TIEG intron in response to E2.

Because the induction of TIEG by ERβ is transient, we performed the same ChIP assays after 2 h of E2 treatment in U2OS-ERα- and -ERβ-expressing cells. As was observed at the 1-h time point, both ERα and ERβ bind to this region of the TIEG gene after 2 h of E2 stimulation (Fig. 5). Interestingly, at this time point, both SRC1 and SRC2 exhibit decreased binding to this enhancer region in response to E2 in both ERα- and ERβ-expressing cells (Fig. 5). Once again, no differences in Sp1 binding were observed in either cell line. These data are in agreement with the real-time RT-PCR data described in Fig. 1 and likely explain why the induction of TIEG expression by ERβ is not maintained. The fact that TIEG mRNA does not return to basal levels in ERβ-expressing cells until 8 h, but the activation complex consisting of ERβ, SRC1, SRC2, and possibly Sp1, is being disassembled at the 2-h time point, is likely explained by mRNA stability and/or half-life.

The ERβ AF1 Domain Is Essential for Activation of TIEG Expression

It is well documented that the major differences between the two ERs lie in the AF1 domain, because this region is the least conserved of all domains (8). Because the other domains of the ER are highly conserved, we sought to determine the role of the AF1 domain in the ERβ-specific induction of TIEG. To test this hypothesis, N-terminal deletions of both ER isoforms were created, cloned into an expression vector, and stably transfected into U2OS cells as previously described (23). E2 time course studies from 0–24 h were performed in U2OS-ERα-ΔAF1 and U2OS-ERβ-ΔAF1 cell lines, as well as in cell lines expressing wild-type receptors for comparison purposes. Real-time RT-PCR analysis revealed that the E2 induction of TIEG by ERβ is essentially lost when the AF1 domain is deleted (Fig. 6B). There is no induction of TIEG in either cell line after 2 h of E2 treatment (Fig. 6B). However, there is a slight, but insignificant, induction of TIEG by E2 at the 8-h (P value = 0.051) and 24-h (P value = 0.060) time points in the U2OS-ERα-ΔAF1 cell line (Fig. 6B). These data are in contrast to the significant induction of TIEG observed in ERβ-expressing cells after 2 h of E2 treatment (Figs. 1 and Fig. 6A) and indicate that the AF1 domain of ERβ is essential in the regulation of TIEG expression. Western blot analysis was performed to confirm the expression of intact wild-type ERα, ERα-ΔAF1, wild-type ERβ, and ERβ-ΔAF1 receptors (Fig. 6C).

Figure 6.

Figure 6

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Deletion of the AF1 Domain Results in Loss of TIEG Induction by ERβ

Stably transfected U2OS-ERα- or ERβ-expressing cells (A), and stably transfected U2OS-ERα-ΔAF1 or -ERβ-ΔAF1 (encoding amino acids 1–173 and 1–129, respectively) expressing cells (B) were treated with either ethanol vehicle or 10 nm E2 for the indicated time points. Total RNA was harvested and subjected to real time RT-PCR analysis using TIEG and β-actin-specific primers. Data are expressed as TIEG mRNA abundance relative to β-actin levels. Asterisks denote significance at the P < 0.05 level (ANOVA) compared with control treatment (0 h) for each cell line. The experiment was conducted in triplicate, and a representative analysis is shown. C, Western blot analysis depicting protein expression of indicated ERs in stably transfected U2OS cells using a Flag-specific primary antibody. LBD, Ligand-binding domain.

As an extension of these studies, we used receptor constructs in which the AF1 domains have been exchanged between the two receptors (ERα with ERβAF1 and ERβ with ERαAF1) (20). These constructs were kindly provided by Dr. Stephen Safe of Texas A & M University and were subsequently PCR amplified and recloned into the pcDNA4/TO expression vector to maintain consistency with the other expression constructs used in these studies. Interestingly, when the ERα/βAF1 expression vector was transiently transfected into U2OS cells with the full-length TIEG intron 1 reporter construct, a significant regulation in response to 24 h E2 treatment was observed (Fig. 7A). This regulation was very similar to the regulation observed by ERβ on the full-length TIEG intron 1 reporter construct (Fig. 7A and Figs. 3 and 4). No activation of the TIEG intron was observed by the ERβ/αAF1 receptor (Fig. 7A). Also, very little to no activation of this reporter construct was observed with either of the ΔAF1 expression constructs (Fig. 7A). These data confirm the results observed in Fig. 6 and suggest that the ERβ AF1 domain confers a gain of function for ERα when exchanged for ERα’s AF1 domain. It does not appear that there is much of an inhibitory component to ERα’s AF1 domain because deletion of this domain from ERα does not result in significant transcriptional activation of TIEG expression in response to E2 (Figs. 6B and 7A). Nevertheless, these data indicate that the AF1 domain of ERβ is responsible for the E2 induction of TIEG in osteoblasts. Western blot analysis was performed to confirm the expression and integrity of all receptors (Fig. 7C). Although not all of the receptors are expressed at the same level, the data are reported as the relative fold change elicited by E2 treatment compared with vehicle-treated cells transfected in the same manner. The fold changes are not calculated by comparing cells transfected with different ER expression constructs. Therefore, it is not essential that each ER construct is expressed at the exact same level.

Figure 7.

Figure 7

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The ERβ AF1 Domain Is Essential for the Activation of TIEG Expression

A, U2OS cells were transiently transfected with wild-type ERα, wild-type ERβ, ERα-Δ AF1, ERβ-Δ AF1, ERα/βAF1, or ERβ/αAF1 expression constructs and the TIEG intron 1 enhancer region fused to a luciferase reporter. Cells were treated with ethanol vehicle or 10 nm E2 for 24 h. Luciferase values are reported as relative fold change compared with ethanol controls. Asterisks denote significance at the P < 0.05 level (ANOVA) compared with ethanol controls. The experiment was conducted in triplicate, with six replicates per treatment, and a representative analysis is shown. B, Depiction of the ERs used in this experiment. C, Western blot analysis depicting protein expression of the ERα (1), ERβ (2), ERα-ΔAF1 (3), ERβ-ΔAF1 (4), ERα/βAF1 (5), or ERβ/αAF1 (6) expression constructs after transient transfection of U2OS cells using a Flag-specific primary antibody. LBD, Ligand-binding domain.

Because the ERα/βAF1 receptor mimics the activation of TIEG by wild-type ERβ, and because we have shown that SRC1 and SRC2 are enriched on the first intron of the TIEG gene in ERβ-expressing cells stimulated with E2 for 1 h, but not in ERα-expressing cells, it was of interest to determine whether the ERα/βAF1 receptor was also capable of recruiting these coactivators. To test this hypothesis, we performed transient ChIP assays in parental U2OS cells transfected with the ERα/βAF1 or the ERβ/αAF1 receptor and examined the binding of SRC1, SRC2, and Sp1 in response to 1 h of E2 treatment. For comparison purposes, we also performed transient ChIP assays in parental U2OS cells transfected with the wild-type receptors. Both the ERα/βAF1 and the ERβ/αAF1 receptors bind to the TIEG intron after 1 h of E2 stimulation (Fig. 8, C and D). These data are in agreement with the binding of the wild-type receptors to this site (Fig. 5 and Fig. 8, A and B). Intriguingly, SRC1 and SRC2 are enriched on the TIEG intron in cells transfected with the ERα/βAF1 receptor after E2 treatment, but not in cells expressing the ERβ/αAF1 receptor (Fig. 8, C and D). As with the stable cell lines (Fig. 5), no differences in Sp1 binding were observed. It is interesting to note that the binding profiles of SRC1, SRC2, and Sp1 are nearly identical between the wild-type ERα receptor and the ERβ/αAF1 receptor, as well as between the wild-type ERβ receptor and the ERα/βAF1 receptor, indicating that the AF1 domain plays a major role in recruiting these coactivators to the TIEG intron 1 enhancer region in response to E2. These results further confirm that the AF1 domain of ERβ is responsible for recruiting the components necessary for formation of the activation complex, which ultimately results in the induction of TIEG expression by E2.

Figure 8.

Figure 8

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Transient ChIP Analysis of the TIEG Intron 1 Enhancer Region

U2OS cells were transiently transfected with either Flag-tagged ERα, ERβ, ERα/βAF1, or ERβ/αAF1 expression constructs and the TIEG intron 1 enhancer region and treated with ethanol vehicle or 10 nm E2 for 1 h. Chromatin was prepared, immunoprecipitated with the indicated antibodies, and amplified by both real-time PCR (A and C) and semiquantitative PCR (B and D). Real-time PCR analysis was used for quantitation purposes and the data are expressed as the abundance of the TIEG intron 1 enhancer region in E2-treated cells after immunoprecipitation with indicated antibodies relative to vehicle control-treated cells. All data were normalized using input samples. Asterisks denote significance at the P < 0.05 level (ANOVA) compared with vehicle control-treated cells. The resultant products obtained by semiquantitative PCR were separated using agarose gel electrophoresis. Experiments were conducted in triplicate and a representative analysis is shown. IP, Immunoprecipitation; V, vehicle; Veh., vehicle.

SRC1, But Not SRC2, Is Essential for TIEG Induction by ERβ

Because SRC1 and SRC2 were enriched on the first intron of the TIEG gene after E2 stimulation of ERβ-expressing cells, we wanted to further delineate their role in the induction of TIEG expression. To address this issue, short interfering RNAs (siRNAs) specific for SRC1 and SRC2 were used. The ability of these SRC-specific siRNAs to decrease the levels of SRC1 and SRC2 mRNA in parental U2OS cells was determined. Treatment of U2OS cells with 50 pmol of either SRC1 or SRC2 siRNA significantly reduced the levels of SRC1 and SRC2 mRNA at 24, 48, and 72 h after transfection (Fig. 9A). These results were confirmed in U2OS-ERα- and -ERβ-expressing cell lines (Fig. 9B). As shown in Fig. 9B, the siRNA targeting SRC1 is specific for SRC1 and does not alter the levels of SRC2 (Fig. 9B). The same was found for the siRNA targeting SRC2 (Fig. 9B). Combined treatment of SRC1 and SRC2 siRNAs significantly reduced the mRNA levels for both genes in U2OS-ERα and -ERβ cell lines (Fig. 9B).

Figure 9.

Figure 9

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Validation of SRC1- and SRC2-Specific siRNAs

A, Parent U2OS cells were transfected with 50 pmol of SRC1- or SRC2-specific siRNA for indicated times, and total RNA was harvested and subjected to real time RT-PCR analysis using SRC1- or SRC2-specific primers. Data are expressed as SRC1 or SRC2 mRNA abundance after indicated treatments relative to β-actin levels. Asterisks denote significance at the P < 0.05 level (ANOVA) compared with control treatment (0 h). The experiment was conducted in triplicate, and a representative analysis is shown. B, U2OS-ERα- or -ERβ-expressing cells were transfected with 50 pmol of either scrambled siRNA, SRC1 siRNA, SRC2 siRNA, or both siRNAs for 24 h. Total RNA was harvested and subjected to real-time RT-PCR analysis using both SRC1- and SRC2-specific primers. Data are expressed as SRC1 or SRC2 mRNA abundance after transfection with indicated siRNAs relative to β-actin levels. Asterisks denote significance at the P < 0.05 level (ANOVA) compared with scrambled siRNA treatment. The experiment was conducted in triplicate, and a representative analysis is shown.

Treatment of U2OS-ERα cells transfected with scrambled siRNA or siRNA targeting SRC1, SRC2, or both for 24 h followed by E2 stimulation for 0, 1, 2, 8, or 24 h had no effect on TIEG expression except at the 1- and 2-h treatment times in cells transfected with both siRNA constructs (Fig. 10A). At these treatment times, a slight, but significant reduction in TIEG expression was observed. Treatment of U2OS-ERβ cells with scrambled siRNA did not significantly alter the levels or pattern of TIEG expression in response to E2 treatment (Fig. 10B) relative to untransfected ERβ cells (Figs. 1 and 6A). Interestingly, transfection of U2OS-ERβ cells with SRC1-specific siRNA abolished the E2 induction of TIEG (Fig. 10B). However, no significant differences were observed in cells transfected with SRC2-specific siRNA (Fig. 10B). As expected, cells transfected with both siRNAs mimicked the results observed in cells transfected with SRC1-specific siRNA (Fig. 10B). These results demonstrate that SRC1 is required for the induction of TIEG expression by ERβ in response to E2 treatment. Although SRC2 binding is enriched on the first intron of the TIEG gene after 1 h E2 treatment, as determined by ChIP assays, this coregulator does not appear to be an active component of the transcriptionally competent activation complex.

Figure 10.

Figure 10

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SRC1 Is Necessary for TIEG Expression after Estrogen Treatment of U2OS-ERβ-Expressing Cells

U2OS-ERα (A) and U2OS-ERβ expressing cells (B) were transfected with 50 pmol of scrambled siRNA, SRC1 siRNA, SRC2 siRNA, or both siRNAs for 24 h. Cells were then treated with 10 nm E2 for indicated times, and total RNA was harvested and subjected to real time RT-PCR analysis using TIEG-specific primers. Data are expressed as TIEG mRNA abundance after indicated treatments relative to β-actin levels. Asterisks denote significance at the P < 0.05 level (ANOVA) compared with control treatments (0 h). The experiment was conducted in triplicate, and a representative analysis is shown.

DISCUSSION

This paper demonstrates that TIEG is specifically induced by ERβ, and not ERα, and provides evidence that this specificity is conferred by the AF1 domain of ERβ in osteoblasts. To our knowledge, this is one of the first reports demonstrating a critical role for the AF1 domain of the ERs in conferring ER isoform-specific regulation of a gene. Through the use of deletion constructs, we have determined that the ERE and Sp1 elements, located in the first intron of the TIEG gene, are both necessary for complete activation of TIEG expression by E2. In addition, ChIP assays reveal that both ERα and ERβ bind to this region of the TIEG gene in response to E2 treatment, but enrichment of the common coactivators, SRC1 and SRC2, and the enhancement of transcription, is only observed in ERβ-expressing cell lines. Interestingly, deletion of the AF1 domain of ERβ renders this receptor incapable of inducing TIEG expression. Furthermore, exchanging the AF1 domains between the two ERs results in induction of TIEG expression solely by the chimeric receptor, ERα/βAF1, but not by ERβ/αAF1. ChIP analysis of the chimeric receptors revealed that the AF1 domain of ERβ is likely responsible for enrichment of the coactivator complex on this enhancer region ultimately resulting in transcriptional activation of TIEG. Finally, SRC1, but not SRC2, is essential for the E2 induction of TIEG expression by ERβ.

Upon the discovery of ERβ in 1996 (24), it was generally thought that this newly identified receptor functioned as a modulator of ERα. However, recent studies have demonstrated that these two receptors function differently at least at the level of transcriptional control. As mentioned above, our laboratory has shown, in both U2OS osteosarcoma cells and Hs578T breast cancer cells, that there is only about a 20% overlap in the genes that are regulated by ERα and ERβ in response to E2 treatment, whereas the other 80% are unique to one receptor or the other (28,29,31). These observations have been independently confirmed in osteoblasts by other laboratories (27,30). To date, the mechanisms by which these two receptors regulate unique gene expression patterns are largely unknown. It is possible that the DNA elements, through which the receptors interact, are responsible for specifically recruiting either ERα or ERβ. However, this does not seem to be the case for the TIEG gene because both ERα and ERβ are recruited to, and bind to, the first intron of TIEG in response to E2 stimulation. It also appears that this specificity is conferred by the AF1 domains of the ERs. This is not totally surprising because this is the least conserved region between ERα and ERβ.

As mentioned previously, ERs can regulate gene expression through several modes of action including direct DNA binding to EREs or through tethering to other transcription factors including Sp1. The N-terminal region of the ERs, containing the AF1 domain, is known to physically interact with the C-terminal zinc finger domain of Sp1 (20,36,37). This physical interaction between ERα and Sp1 has been shown to be important for the transcriptional regulation of numerous genes (20,23,36,38,39,40,41,42,43,44,45,46,47). However, less is known concerning the transcriptional control of gene expression by ERβ and Sp1. Saville et al. (20) has demonstrated that, although ERβ and Sp1 interact in a ligand-dependent manner, this complex is transcriptionally incompetent with regard to activation of a synthetic Sp1 reporter construct. In contrast, other studies have demonstrated that ERβ and Sp1 can activate the transcription of an epidermal growth factor receptor promoter construct (48). Studies in our laboratory have also demonstrated that ERβ functions through a Sp1 site to activate a retinoblastoma-binding protein 1 RBBP1 (ARID4A) enhancer element (23). The present data also indicate that the induction of TIEG expression by E2 is ERβ specific and partially occurs through an Sp1 site located in the first intron of the TIEG gene. The apparent discrepancy in the literature concerning the effects of ERβ/Sp1 interactions, and their subsequent effects on transcription, could be explained by differences in the cell type and/or context of the specific promoters and enhancer elements used in these experiments. Nevertheless, it appears that the interaction of ERβ and Sp1 is capable of inducing the expression of specific genes.

In addition to DNA elements in target genes, ER isoform-specific regulation of gene expression could also be conferred by the AF domains of the receptor. In particular, the AF1 domain is highly divergent between the two ER isoforms and, in fact, some have questioned whether ERβ actually contains a functional AF1. The AF1 domain of the ERs, primarily ERα, is known to interact with a number of coregulators (49,50,51,52,53,54,55,56,57) and therefore is likely to play an important role in regulating gene expression. A comparison of the AF1 domains has indicated that this domain is highly active in ERα on a number of estrogen-responsive promoters whereas the ERβ AF1 domain plays a minimal role under identical conditions (58). However, recent data from our laboratory have indicated an important role for the AF1 domain of both ERα and ERβ in mediating the isoform-specific regulation of the retinoblastoma-binding protein 1 gene (23). Additionally, the present manuscript demonstrates that the AF1 domain of ERβ is essential for activating the transcription of TIEG in response to E2 in osteoblasts. It is becoming evident that the AF1 domains of both ERα and ERβ play important roles in regulating the expression of specific genes, and it is possible that these highly divergent domains are responsible, at least in part, for conferring ER isoform-specific regulation of gene expression.

In order for ERs to regulate transcription, recruitment of specific coregulators to the site of ER-DNA interaction is necessary. The p160 family members, including SRC1 and SRC2, are classic coactivators of numerous nuclear hormone receptors, including the ERs. ERs have been shown to interact with SRC1 and SRC2 through the AF2 and the ligand-binding domains (reviewed in Ref. 16). SRC1 is also known to interact directly with the ERα AF1 domain (50); however, less is known about the interaction of SRC1 and the ERβ AF1 domain. It has been shown that SRC2 interacts with the AF1 domain of ERα (50), but not ERβ (59). Interestingly, the present data demonstrate that the AF1 domain of ERβ is critical for the enrichment of both SRC1 and SRC2 on the first intron of the TIEG gene after E2 stimulation in osteoblasts. Whereas ERα is capable of interacting with both SRC1 and SRC2, they are not enriched on this specific enhancer region in response to E2. It is possible that additional DNA elements, surrounding the site of ER interaction, are involved in conferring this specificity, or that the AF1 domain of ERβ recruits other unique coactivators to this site that are necessary for SRC1 and/or SRC2 binding.

ERs are expressed in numerous cell types and tissues and are known to regulate multiple biological functions. Estrogen plays a critical role in breast cancer because approximately 70% of all diagnosed primary breast cancers in the United States are ER positive (60,61). Estrogen is generally thought to promote breast cancer cell proliferation; however, the majority of the cell models used in these studies express ERα and not ERβ. Using an Hs578T breast cancer cell line that only expresses ERβ, our laboratory has demonstrated that these cells exhibit decreased proliferation in response to E2 treatment (31) and exhibit increased expression of TIEG after 2 h of E2 stimulation. We have previously demonstrated that overexpression of TIEG results in decreased cell proliferation (62) and that TIEG expression levels decrease with the progression of breast cancer (63). It is interesting to speculate that the decreased proliferation observed in ERβ-expressing Hs578T breast cancer cells treated with E2 is either directly or indirectly a result of increased TIEG expression. Although these preliminary observations require more study, it is possible that the expression levels of TIEG and the presence or absence of ERβ could have a significant impact on the progression and outcome of this devastating disease.

In addition to breast tissue, ERs are also highly expressed and play important roles in regulating bone metabolism and homeostasis in both men and women (64). Osteoblasts express ERα and ERβ, and interestingly, both receptors are differentially expressed during osteoblast maturation. The expression of ERα increases dramatically during the differentiation of bone marrow stromal cells into mature osteoblasts whereas ERβ levels increase only slightly (65). It has been suggested that ERα is the major mediator of E2 effects on bone (66); however, ERβ also plays a significant role, especially in trabecular bone (66,67). Our laboratory has demonstrated that TIEG null mice display significant defects in both cortical and trabecular bone resulting in an osteopenic phenotype (68). Interestingly, only female animals are affected, because males show no changes in any of the bone parameters analyzed to date (69). Based on these observations, it is possible that the induction of TIEG by E2 in an ER isoform-specific manner could have significant effects on the differentiation of osteoblasts and the maintenance of bone quality, particularly in females.

These studies have demonstrated that TIEG is specifically induced by ERβ and have provided evidence that this induction occurs through an ERE and Sp1 site located in the first intron of the TIEG gene. We have also shown that this isoform specificity is likely determined by the AF1 domain of the ERs. Deletion of the ERβ AF1 domain results in the inability of this receptor to induce the expression of TIEG. Exchanging the AF1 domains between the two ERs results in the induction of TIEG by the chimeric ERα/βAF1 receptor, but not by the ERβ/αAF1 receptor. Additionally, ERs with the ERβ AF1 domain cause enrichment of SRC1 and SRC2 binding to the first intron of the TIEG gene after E2 treatment. These data demonstrate that the AF1 domain of ERβ is a critical component of the activation complex and, at least in part, explains the ER isoform-specific induction of TIEG expression. These data also demonstrate that SRC1, but not SRC2, is essential for the E2 induction of TIEG by ERβ. The ER isoform-specific induction of TIEG expression could significantly impact the field of bone and breast cancer research due to the important roles for both the ERs and TIEG in these tissues.

MATERIALS AND METHODS

Development of Expression and Reporter Constructs

The development of flag-tagged human ERα and ERβ expression constructs has been described previously (35). All of the ERα and ERβ expression constructs and cell lines used throughout this study are human, and the ERβ expression constructs reflect the full-length ERβ as described by Moore et al. (70) and not the originally described shorter version of ERβ as identified by Kuiper et al. (24). ERα and ERβ DBD Mut receptors, in which the P-Box region of the first zinc finger has been mutated, were developed in our laboratory as previously described (23). Construction and development of the ERα-ΔAF1 and ERβ-ΔAF1 expression plasmids and cell lines have also been described (23). The ΔAF1 constructs were Flag tagged for validation of protein expression and cloned into the pcDNA4/TO expression vector (Invitrogen, Carlsbad, CA). These constructs were used to create the Dox-inducible U2OS-ER-ΔAF1 cell lines using the T-Rex system (Invitrogen). Selected U2OS-ER-ΔAF1 cell lines were analyzed by Western blotting to confirm expression of the ERα-ΔAF1 and ERβ-ΔAF1 proteins using Flag antibody. Expression constructs for ERα with the ERβ AF1 domain (ERα/βAF1) and ERβ with the ERα AF1 domain (ERβ/αAF1) were kindly provided by Dr. Stephen Safe (20) and were subcloned into the pcDNA4/TO expression vector. The TIEG intron 1 enhancer region, containing an ERE-like element, a consensus half-ERE, and a Sp1 site, was PCR amplified using specific primers containing NheI and XhoI restriction sites for cloning into the pGL3-promoter luciferase vector (Promega Corp., Madison, WI). The PCRs were performed using 100 ng of human genomic DNA as template and Platinum Pfx DNA polymerase (Invitrogen) to decrease amplification errors. All of the TIEG intron deletion constructs were created in the same manner using the TIEG intron 1 enhancer region plasmid as template. The resulting constructs were sequenced to ensure fidelity.

Cell Culture

The U2OS and Hs578T -ERα- and -ERβ expressing cell lines were developed by our laboratory as previously described (29,31). Cells were cultured in phenol red-free DMEM/F12 medium containing 10% (vol/vol) fetal bovine serum (FBS), 1× antibiotic-antimycotic solution containing penicillin, streptomycin, and amphotericin B (Invitrogen), 5 mg/liter blasticidin S (Roche Applied Science, Indianapolis, IN), and 500 mg/liter zeocin (Invitrogen). The U2OS-ERα and -ERβ ΔAF1 cell lines were cultured in the same medium. The parental U2OS cells were cultured in DMEM/F12 containing 10% (vol/vol) FBS and 1× antibiotic-antimycotic solution. Steroid treatments were performed in FBS and antibiotic-antimycotic free DMEM. Doxycycline (Dox) and E2 were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO).

Real-Time RT-PCR Analysis

The U2OS and Hs578T -ERα and -ERβ cells and the U2OS-ERα and -ERβ ΔAF1 cells were plated at a density of 50% in six-well tissue culture plates and pretreated with 100 ng/ml Dox for 24 h in serum free medium. The cells were then treated with 100 ng/ml Dox plus either ethanol control or 10 nm E2 for the indicated times. Total RNA was isolated using Trizol reagent (Invitrogen) as specified by the manufacturer. Total RNA (4 μg) was denatured at 68 C for 15 min in a reverse transcription reaction buffer containing 1× first-strand buffer (50 mm Tris-HCl, 75 mm KCl, 3 mm MgCl2), 50 mm dithiothreitol, 1 μm deoxynucleotide triphosphates, and 500 ng of oligo deoxythymidine primer. After heat denaturation, 1 U of mouse Moloney leukemia virus-reverse transcriptase (Invitrogen) was added, and the mixture was incubated at 37 C for 45 min followed by a 68 C incubation for 15 min. The reaction was diluted to 50 μl with water. Real-time PCR was performed as described previously (35). Primers specific for TIEG were as follows: human TIEG forward, 5′-GCCAACCATGCTCAACTTCG-3′ and human TIEG reverse, 5′-TGCAGTTTTGTTCCAGGAATACAT-3′. Control primers specific for β-actin were as follows: human β-actin forward, 5′-TCACCCACACTGTGCCCATCTACGA-3′ and human β-actin reverse, 5′-CAGCGGAACCGCTCATTGCCAATGG-3′.

Transient Transfection Assays

U2OS cells were plated at a density of 50% in 12-well tissue culture plates. All ER expression constructs and the TIEG intron 1 enhancer construct or a consensus ERE reporter construct were transiently transfected in replicates of six using FuGENE 6 transfection reagent (Roche Diagnostics, Indianapolis, IN) at a concentration of 250 ng/well in serum free and antibiotic/antimycotic free medium. The next day, medium was removed and replaced with serum and antibiotic/antimycotic free medium containing either ethanol control or 10 nm E2 for 24 h. Cells were harvested and 5 μl of extract was assayed using luciferase assay reagent (Promega Corp.). Values were normalized to total protein concentration. All experiments were repeated at least three times.

Western Blotting

To validate the expression and integrity of all of the receptors used throughout these studies, Western blot analysis was used. Briefly, stable cell lines or transiently transfected parent U2OS cells were lysed and total protein was harvested. Protein was quantitated using standard Bradford assays, and equal amounts of cell lysate were loaded onto a 10% SDS-PAGE gel and separated by electrophoresis. Protein was transferred to polyvinylidene fluoride, and expression of the ERs was monitored using Flag-specific antibodies and chemiluminescence.

ChIP Assays

U2OS-ERα and -ERβ cells were plated in 10-cm plates at a density of 50% and treated with Dox for 24 h in serum and antibiotic/antimycotic free medium. Cells were then treated with either ethanol control or 10 nm E2 for 1 or 2 h in FBS and antibiotic/antimycotic free medium. Chip assays were performed using a modified version of the procedure previously described by Lambert and Nordeen (71). Briefly, cells were fixed in collection buffer, pelleted, and resuspended in 400 μl of lysis buffer. Extracts were incubated on ice for 10 min and sonicated four times for 10 sec using a Heat Systems-Ultrasonics Cell Disruptor Model W-220F (Ultrasonic Instruments, Plainview, NY). Samples were diluted to 8.5 ml in dilution buffer and aliquoted into 1-ml samples. Each sample was immunoprecipitated overnight at 4 C with 1 μg of Flag-specific antibody (Sigma-Aldrich, St. Louis, MO), SRC1-specific antibody (05-522; Upstate Biotechnology, Charlottesville, VA) SRC2-specific antibody (developed in our laboratory), or a Sp1-specific antibody (H-225; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Complexes were recovered by a 2-h incubation with Protein A/G Sepharose beads (Pierce Biotechnology Inc., Rockford, IL). Precipitates were serially washed with 1 ml of low-salt wash buffer, 1 ml of high-salt wash buffer, 1 ml of LiCl wash buffer, and twice with 1 ml of Tris-EDTA. Precipitated chromatin complexes were recovered with two 250-μl washes with elution buffer, and combined. Salt concentrations were increased to 200 mm with NaCl and heated at 65 C for 4 h. Samples were then treated with 5 μl of 10 mg/ml proteinase K for 1 h at 45 C, phenol/chloroform extracted, and ethanol precipitated. Chromatin pellets were resuspended in 50 μl of water and used in subsequent semiquantitative and real-time PCRs. Inputs were generated as above excluding the antibody immunoprecipitation. Primers used in the PCRs were designed to surround the ERE and Sp1 elements in the first intron of the TIEG gene and were as follows: TIEG enhancer region forward, 5′-CTTTACCATAATCAAGTGATCAAATTT-3′ and TIEG enhancer region reverse, 5′-ATATAGACTTTCCAGAGTAGCTACCT-3′.

For transient ChIP assays, U2OS cells were plated in 10-cm tissue culture plates and transfected with 5 μg of either the wild-type ERα receptor, the wild-type ERβ receptor, the ERα/βAF1 receptor, or the ERβ/αAF1 receptor (pcDNA4/TO; Invitrogen) and 5 μg of the TIEG intron 1 reporter construct (pGL3-promoter vector; Promega Corp.). Cells were treated with ethanol control or 10 nm E2 for 1 h, and ChIP assays were performed as described above.

siRNA Treatments

Parental U2OS cells, U2OS-ERα, or U2OS-ERβ cell lines were plated in six-well plates at a density of 50%. U2OS-ERα and -ERβ cells were treated with 100 ng/ml Dox for 24 h. All cells were transfected with 50 pmol/well of either scrambled siRNA, SRC1-specific siRNA, SRC2-specific siRNA, or both siRNAs using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s protocol. Cells were allowed to grow for the indicated times. For E2 treatments, cells were plated and transfected as above. Twenty-four hours after transfection, cells were treated with ethanol control or 10 nm E2 for an additional 24 h. Total RNA was harvested using Trizol reagent and used for analyzing the expression of SRC1, SRC2, and TIEG by real-time RT-PCR as described above. Scrambled control siRNA was purchased from Invitrogen (catalog no. 12935–300). The SRC1- and SRC2-specific siRNAs were also purchased from Invitrogen (catalog nos. 1299001 oligo identification no. HSS112681 and 1299001 oligo identification no. HSS116117, respectively). Primers specific for SRC1 were as follows: human SRC1 forward, 5′-TGCCTCCGGGTATCAGTCACCAG-3′ and human SRC1 reverse, 5′-AGGCGTGGGCTGGTTCTGGACAG-3′. Primers specific for SRC2 were as follows: human SRC2 forward, 5′-GTGGTATGCCAGCAACTATGAGC-3′ and human SRC2 reverse, 5′-TGGATCAGGTTGCTGACTTATTCCG-3′.

Acknowledgments

We thank Sarah Grygo and Kay Rasmussen for their excellent technical support, Dr. Frank Secreto for his numerous scientific discussions, and Jacquelyn House for her outstanding secretarial assistance. We also thank Dr. Stephen Safe, Texas A & M University, for providing us with the ERα/βAF1 and ERβ/αAF1 expression constructs.

This work was supported by National Institutes of Health Grants DE14036 (to T.C.S), AG04875 (to T.C.S.), and AR52004 (to M.J.O.); a Breast Cancer Research Foundation grant (to T.C.S. and J.N.I.); a generous gift from Bruce and Martha Atwater (to T.C.S. and J.R.H.); and the Mayo Foundation. Additionally, Dr. John Hawse was supported by a National Institutes of Health Kirschstein Traning Grant (AR53983) and currently holds a Kendall-Mayo Fellowship grant from the Mayo Clinic.

Footnotes

Disclosure Statement: J.R.H., M.S., D.G.M., A.H.H., M.J.O., and T.C.S. have nothing to declare. J.N.I. has received consulting fees and lecture fees from Novartis. S.K. has received consulting fees from Novartis.

First Published Online May 15, 2008

Abbreviations: AF, Activation function; AP1, activating protein 1; ChIP, chromatin immunoprecipitation; DBD, DNA-binding domain; DBD Mut, DBD mutant; Dox, doxycycline; E2, 17β-estradiol; ER, estrogen receptor; ERE, estrogen response element; FBS, fetal bovine serum; siRNA, short interfering RNA; Sp1, stimulating protein 1; SRC, steroid receptor coactivator; TIEG, TGFβ-inducible early gene.

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