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Originally published In Press as doi:10.1074/jbc.M605226200 on July 26, 2006

J. Biol. Chem., Vol. 281, Issue 39, 28596-28604, September 29, 2006
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Estrogen Receptor Isoform-specific Regulation of the Retinoblastoma-binding Protein 1 (RBBP1) Gene

ROLES OF AF1 AND ENHANCER ELEMENTS*

David G. Monroe{ddagger}1, Frank J. Secreto{ddagger}, John R. Hawse{ddagger}, Malayannan Subramaniam{ddagger}, Sundeep Khosla§, and Thomas C. Spelsberg{ddagger}

From the {ddagger}Department of Biochemistry and Molecular Biology and the §Endocrine Research Unit, Mayo Clinic College of Medicine, Rochester, Minnesota 55905

Received for publication, May 31, 2006 , and in revised form, July 26, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Estrogen (E2) is involved in mediating many important functions relevant to osteoblast biology through the actions of the estrogen receptors (ER) {alpha} and beta. To further understand the mechanisms of ER-specific regulation, we used microarray and reverse transcription-PCR analyses of E2-treated U2OS-ER{alpha} or -ERbeta cells and identified retinoblastoma-binding protein 1 (RBBP1) as a major E2-regulated gene. RBBP1 is a retinoblastoma cofactor involved in the control of osteoblastic proliferation. Although RBBP1 mRNA levels rapidly increased after 2 h of E2 treatment in both U2OS-ER-expressing lines, a sustained induction was only observed in U2OS-ER{alpha} cells. Examination of the RBBP1 genomic sequence revealed an ER response element and a Sp1 site located within the first intron. Chromatin immunoprecipitation analyses demonstrated that E2-dependent ER{alpha} binding to the intron 1 enhancer region was constitutive, whereas ERbeta binding was transient, consistent with the mRNA time course. Interestingly, transient transfection and receptor mutational studies revealed that RBBP1 induction by ER{alpha} only requires the Sp1 site, whereas ERbeta utilizes both the Sp1 and estrogen response elements binding sites for maximal E2-dependent activation. Stable U2OS transfectants containing a deletion of the ER{alpha} activation function 1 (AF1) resulted in a temporal mRNA induction profile similar to that of wild type ERbeta. Further, overexpression and chromatin immunoprecipitation analyses also demonstrated that E2-dependent RBBP1 induction is SRC2-dependent for both ER isoforms. These results describe an E2-dependent, ER isoform-specific transcriptional activation of the RBBP1 gene, which in part, is explained by the differential activity of ER AF1 and enhancer element binding.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The transcriptional effects of estrogens (e.g. 17beta-estradiol or E2)2 are largely mediated by two related, but distinct, estrogen receptor (ER) isoforms ER{alpha} and ERbeta (1–7). The ERs are ligand-inducible transcription factors that bind E2 through a C-terminally located ligand-binding domain (or E/F domain). The ER isoforms contain a centrally located and highly conserved DNA-binding domain (or C domain) that associates directly with estrogen response elements (ERE) or indirectly through protein tethering to AP1 or Sp1 (8, 9). Both ER{alpha} and ERbeta contain two activation functions (AF); a highly divergent N-terminal A/B (AF1) domain and a more conserved C-terminal E/F (AF2) domain that overlaps with the ligand-binding domain (10–13). Activation of transcription is achieved through the recruitment of specific transcriptional coregulators resulting in chromatin remodeling and the recruitment of the basal transcriptional machinery (1, 9, 14–16). Although coregulator recruitment and activation is largely mediated through AF2, the A/B domain (AF1) is known to bind the p160 and p300/CBP (CREB-binding protein) families of coregulators (16–20) as well as p68/p72 (21, 22), steroid receptor RNA activator (23, 24), and MMS19 (25) proteins.

Recent data from our laboratory (26, 27) and others (28, 29) have demonstrated that significant differences exist in gene expression patterns, elicited by ER{alpha} and ERbeta homodimers and ER{alpha}/ERbeta heterodimers, in a human osteosarcoma (U2OS) model system. Specifically, only ~20% of all genes regulated through either ER isoform are regulated by both ER isoforms. The mechanism(s) of ER isoform-specific gene regulation are unknown; however because of the low level of homology between the ER{alpha} and ERbeta A/B (AF1) domains, differences in transcriptional activation may be largely due to AF1 function (30, 31).

In this study, we show that the RBBP1 gene, the protein product of which is involved in retinoblastoma-mediated suppression of cell proliferation, is differentially regulated by ER{alpha} and ERbeta in a U2OS model system. Using deletion and mutational analysis, we demonstrate that ER{alpha} utilizes a consensus Sp1 sequence, whereas ERbeta functions through both the Sp1 and a nearby ERE sequence. Deletion of the ER{alpha} AF1 in a stable U2OS transfectant cell line causes the temporal profile to mimic that of wild type ERbeta. These data indicate that differential transcriptional activation of RBBP1 by ER{alpha} and ERbeta and transcription factor binding site utilization are AF1-dependent.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Chemicals—The U2OS-ER{alpha} and -ERbeta cell lines, described previously (26), were cultured in phenol red-free Dulbecco's modified Eagle's medium/F12 medium containing 10% (v/v) fetal bovine serum (FBS), 1x antibiotic/antimycotic (Invitrogen), 5 mg/liter blasticidin S (Roche Applied Science), and 500 mg/liter zeocin (Invitrogen). The U2OS-ER-{Delta}AF1 cell lines, described in Fig. 6, were cultured in the same medium supplemented with 100 mg/liter hygromycin B (Invitrogen). Mouse calvarial osteoblasts were cultured in Dulbecco's modified Eagle's medium/F12 plus 10% (v/v) FBS and 1x antibiotic/antimycotic. Steroid treatments were performed either in charcoal-stripped fetal bovine serum (CS-FBS) containing Dulbecco's modified Eagle's medium/F12 medium (Hyclone Laboratories, Logan, UT) or in serum-free medium in the transient transfection assay. Doxycycline (Dox) and 17beta-estradiol (E2) were purchased from Sigma.

Reverse Transcription PCR (RT-PCR) Analysis —The U2OS-ER{alpha} and -ERbeta cell lines were plated in 6-well cell culture dishes at a cell density of 50% and pretreated with 100 ng/ml Dox for 24 h in CS-FBS medium. The cells were then treated with 100 ng/ml Dox plus ethanol control or 10 nM E2 for 1, 2, 4, 6, 8, or 24 h. Mouse calvarial osteoblasts were plated and treated with E2 in the same manner as the U2OS-ER cells. The RNA was harvested using TRIzol reagent (Invitrogen). Four µg of total RNA was heat-denatured at 68 °C for 15 min in a reverse transcription reaction buffer (1x first strand buffer (50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl2), 50 mM dithiothreitol, 1 µM dNTPs, 500 ng of oligo(dT) primer). Following heat denaturation, 1 unit 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 an additional 15 min. The cDNA products were diluted to 50 µl with PCR-grade water; 2 µl was used in a PCR reaction using primers specific for human RBBP1 and beta-actin; and products were separated on a 1.5% agarose gel as described previously (26). Real-time PCR was performed as described previously (32). Primers specific for RBBP1 were as follows: human RBBP1 forward, 5'-GCAAAAGCGTACCCCAAAGCGAAC-3'; human RBBP1 reverse, 5'-TCCCGCATGAGACACTTCCCTGTC-3'; mouse RBBP1 forward, 5'-GCAAAAGCGAACCCCAAAGCGGAC-3'; mouse RBBP1 reverse, 5'-TCCTGCGTGAGACACTTCTCTGTC-3'. beta-actin primers are described previously (26).

Chromatin Immunoprecipitation (ChIP) Assay—U2OS-ER{alpha} and -ERbeta cells (1 x 107) were seeded into 10-cm plates and treated with Dox for 24 h followed by either ethanol control or 10–8 E2 for 1, 2, 4, 8, and 24 h in CS-FBS medium. ChIPs were then performed using modifications of the procedure described by Lambert and Nordeen (33) and as described previously (27). Five µg of ER{alpha}-specific antibody (HC20; Santa Cruz Biotechnology, Santa Cruz, CA), ERbeta-specific antibody (P2571; Invitrogen), SRC1-specific antibody (05–522; Upstate%20Biotechnology">Upstate Biotechnology, Charlottesville, VA), or SRC2-specific antibody was used. Standard end point and real-time PCR reactions were performed as described previously using primers specific for the RBBP1 intron 1 enhancer region (27).

Production of the RBBP1 Intron 1 Enhancer Constructs—RBBP1 intron 1 enhancer constructs were produced by PCR amplification of the desired construct (see Fig. 4) using specific primers containing engineered XhoI restriction sites for cloning into the XhoI site of pGL3-promoter luciferase vector (Promega, Madison, WI). The PCR reactions were as described previously (32) using 100 ng of human genomic DNA as template. Platinum Pfx DNA polymerase (Invitrogen) was used to decrease amplification errors, and the resultant constructs were sequenced to ensure fidelity. The ERE and Sp1 mutations were introduced into the respective amplifying primers with the mutated sequence as seen in Fig. 5A.

Production of the DNA-binding ER Mutants—Construction of the ER{alpha} and ERbeta P-box mutation expression plasmids was performed using two-step PCR. Briefly, primers were designed to mutate Glu203 and Gly204 to alanines for ER{alpha} and Glu167 and Gly168 to alanines for ERbeta. The ER{alpha} double mutation was as reported previously (34). Because ER{alpha} and ERbeta share 97% identify within the DNA-binding domain, the same mutation was made in ERbeta. Resultant products were mixed and amplified using specific flanking primers containing an N-terminal FLAG epitope tag. The resulting PCR fragment was subcloned into the BamHI/XhoI and HindIII/XbaI sites of pcDNA4/TO for the ER{alpha} and ERbeta P-box mutations, respectively.

Development of the ER-{Delta}AF1 Expression Constructs and the U2OS-ER-{Delta}AF1 Cell Lines—Construction of the ER{alpha}-{Delta}AF1 and ERbeta-{Delta}AF1 expression plasmids was performed using PCR. Briefly, primers were designed to amplify amino acids 174–595 of human ER{alpha} and amino acids 130–530 of human ERbeta. An in-frame N-terminal FLAG epitope was included in the forward primers for validation of protein expression (32). The resulting PCR fragment was subcloned into the BamHI/XhoI and HindIII/XbaI sites of pcDNA4/TO for ER{alpha}-{Delta}AF1 and ERbeta-{Delta}AF1, respectively. The generation of the U2OS-ER-{Delta}AF1 cell lines was performed using the T-REx system (Invitrogen) as described previously (26). The ER-{Delta}AF1-pcDNA4/TO vectors were linearized using a PvuI restriction digest and transfected into the U2OS-TetR cells at a density of 50% using FuGENE 6 transfection reagent (Roche Diagnostics). 48 h later, the transfected cells were split and plated in an antibiotic-containing, selective medium. Individual cell clones were isolated and Western blot analysis performed to confirm expression of both ER{alpha}-{Delta}AF1 and ERbeta-{Delta}AF1. The resulting cell lines are termed U2OS-ER{alpha}-{Delta}AF1 and -ERbeta-{Delta}AF1.

Transient Transfection Analyses—U2OS-ER{alpha} and U2OS-ERbeta cells were plated at a density of 50% in 6-well plates. RBBP1 intron 1 enhancer constructs and ER mutant constructs were transiently transfected in triplicate using FuGENE 6 transfection reagent (Roche Diagnostics) at a concentration of 250 ng/well in CS-FBS-containing medium supplemented with 100 ng/ml Dox for ER induction. The next day, cells were washed twice with 1x phosphate-buffered saline and treated with 100 ng/ml Dox and either ethanol control or 10 nM E2 in serum-free medium for an additional 24 h. Cells were harvested, and 1 µlof protein extract was assayed using luciferase assay reagent (Promega). For the SRC overexpression, 250 ng/well of SRC1 or SRC2 was cotransfected using the same procedure.


Figure 1
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  		FIGURE 1.
RBBP1 is regulated by E2 in human osteoblasts. A, U2OS-ER{alpha}, U2OS-ERbeta, and mouse calvarial osteoblasts (OBs) were treated for the time points indicated as described under "Experimental Procedures." RNA was harvested and subjected to RT-PCR analysis using primers specific for either human or mouse RBBP1 and beta-actin. B, real-time RT-PCR was employed on the samples described in A with data expressed as mRNA abundance relative to beta-actin. Asterisks denote significance at the p < 0.01 level (ANOVA) compared with control treatment (0 h). The experiment was repeated twice, and a representative experiment is shown.

 
Western Blot Analysis—The U2OS-ER-{Delta}AF1 cell lines were plated at a density of 75% in 10-cm culture dishes and treated with 100 ng/ml Dox for 24 h. Total protein extracts were prepared in radioimmune precipitation assay buffer (1% (v/v) Nonidet P-40, 0.5% (v/v) sodium deoxycholate, 0.1% (v/v) sodium dodecyl sulfate, and 1x protease inhibitor mixture (Roche Diagnostics) in 1x phosphate-buffered saline) and the protein concentrations determined using a standard Bradford assay. ER-{Delta}AF1 protein levels were analyzed by Western blot using a FLAG-M2 antibody (Sigma) and visualized using enhanced chemiluminescence (Amersham Biosciences).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Regulation of RBBP1 by E2 in Osteoblastic Cells—Our previous microarray data demonstrated that RBBP1 was regulated 4.01-fold following 24 h of E2 treatment in the U2OS-ER{alpha} cell line, whereas no regulation was detected in the U2OS-ERbeta cell line (27). In contrast, at 2 h of E2 treatment, RBBP1 was regulated 3.74- and 2.55-fold in the U2OS-ER{alpha} and -ERbeta cell lines, respectively (data not shown). To further understand the complex kinetics of RBBP1 induction by both ER isoforms, we performed an E2 time course from 0 to 24 h of treatment and determined RBBP1 mRNA induction using conventional and real-time RT-PCR analyses. As shown in Fig. 1, RBBP1 expression is induced over control levels following 24 h of E2 treatment in the U2OS-ER{alpha} cell line. However, in the U2OS-ERbeta cell line, RBBP1 induction appears only transient, peaking at 2 h of E2 treatment and returning to near control levels by 8–24 h, supporting the microarray expression data. To assess whether RBBP1 is regulated in nontransformed osteoblasts, a similar analysis was conducted using mouse calvarial osteoblasts, which are known to contain significant amounts of both ER isoforms. Fig. 1A demonstrates that RBBP1 induction by E2 is sustained, indicating that RBBP1 regulation by E2 is not specific to the U2OS-ER model and is observed in normal, nontransformed osteoblasts.

The RBBP1 Intron 1 Enhancer Region Contains an ERE and Binds ER{alpha} and ERbeta—In an effort to understand the genetic components of E2 regulation of RBBP1 gene expression, the genomic sequence of the RBBP1 gene was scanned for potential ERE-like sequences. As depicted in Fig. 2, an ERE was found within intron 1 at nucleotide +899 (relative to the transcriptional start site), which closely matched the pure ERE consensus sequence (GGTCAXXXTGACC). Interestingly, a Sp1 transcription factor binding site was also found at nucleotide +949. The core Sp1 sequence and the extended sequence perfectly match the established Sp1 consensus, (G/T)GGGCGG(G/A)(G/A)(C/T) (35).

ChIP analysis was conducted to determine binding of the ER isoforms in the context of chromatin structure and the extent of binding over a period of time to the RBBP1 intron 1 enhancer region. Fig. 3 demonstrates that significant binding, in vivo, of both ER{alpha} and ERbeta is observed following 1 h of E2 treatment. More specifically, ER{alpha} exhibits an E2-dependent binding to the RBBP1 intron 1 enhancer region throughout the 24-h time course, wherein ERbeta displays E2-dependent binding only at 1 h of E2 treatment. It is interesting to note that ERbeta binding is minimal at 2 h following E2 treatment and that the RBBP1 mRNA (see Fig. 1) does not completely decrease to near control levels until 8 h of E2 treatment, suggesting that mRNA stability is playing a role in these differential chronologies. These data are in general agreement with the RT-PCR data (Fig. 1), which suggest that the sustained increase in the expression of RBBP1 in U2OS-ER{alpha} cells and the temporary increase of RBBP1 expression in U2OS-ERbeta cells correlate with and are probably dependent on ER binding to the RBBP1 intron 1 enhancer region.

Analysis of the RBBP1 Intron 1 Enhancer Region Delineates Different Requirements for ER{alpha} and ERbeta Binding and Promoter Activation—To determine the role of the ERE and neighboring trans-acting sites in E2 regulation of RBBP1 by the ER isoforms, various RBBP1 intron 1 enhancer constructs were PCR-amplified, cloned into a luciferase reporter vector, and transiently transfected in both U2OS-ER{alpha} and -ERbeta cells. Both ERs activate the RBBP1 +412 to +1140 construct, which contains the ERE and Sp1 sites (Fig. 4A). Loss of the +950 to +1140 sequences, including most of the Sp1 site, completely abolished ER{alpha}-dependent activation, whereas only a slight effect was observed on ERbeta-dependent activation. This suggests that the Sp1 site within this sequence is critically important for ER{alpha}- but not ERbeta-dependent transcriptional activation.


Figure 2
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  		FIGURE 2.
The RBBP1 intron 1 region contains both ERE and Sp1 binding sites. A, a schematic diagram of the RBBP1 intron 1 enhancer region (+400 to +1140, relative to the transcriptional start site) is depicted. B, the ERE (+899) and Sp1 (+949) sequences from the RBBP1 intron 1 enhancer is depicted with comparisons to the consensus binding sites.

 


Figure 3
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  		FIGURE 3.
ChIP analysis of the RBBP1 intron 1 enhancer region demonstrates ER binding. A, ChIP assays were performed in U2OS-ER{alpha} and -ERbeta cell lines treated with 10 nM E2 for the indicated times using antibodies specific for either ER{alpha} or ERbeta as described under "Experimental Procedures." The resultant products were separated using agarose gel electrophoresis. B, real-time PCR analysis was employed on samples from three independent ChIP experiments. Asterisks denote significance at the p < 0.01 level (ANOVA) compared with control treatment (0 h) within each cell line.

 
To specifically characterize the effects of the ERE and Sp1 sites in E2-dependent activation in the same context, mutations of the ERE and Sp1 sites, alone or in combination, were created in the +851 to +1140 context and transiently transfected in the U2OS-ER cell lines. Fig. 4B demonstrates that mutation of the Sp1 site completely abolishes E2-dependent activation in the U2OS-ER{alpha} cell line, consistent with the deletion of the Sp1 site in Fig. 4A. Interestingly, no significant effect was observed with the ERE mutation in the U2OS-ER{alpha} cell line. Mutation of either the Sp1 or ERE decreased ERbeta-specific activation by ~50% but still exhibited a statistically significant E2 induction, whereas mutation of both sites completely abolished E2-dependent activation in the U2OS-ERbeta cell line. These data suggest differential mechanisms for ER{alpha}- versus ERbeta-dependent activation, wherein ER{alpha} exclusively utilizes the Sp1 site and ERbeta utilizes both the Sp1 and ERE sites for maximal activation.

ER{alpha}-dependent Activation of the RBBP1 Intron 1 Enhancer Region Is Independent on ERE Binding—Because the RBBP1 intron 1 enhancer region contains both an ERE and Sp1 element, we determined the relative contribution of these elements in the RBBP1 wild type +851 to +1140 context. Jakacka et al. (34) created a mutation in the mouse ER{alpha} DNA-binding domain, which inhibited ER binding to an ERE but did not affect signaling through non-ERE sites. We therefore created the same mutation in the human ER{alpha} and ERbeta DNA-binding domains (Fig. 5A) to generate constructs in which signaling through the RBBP1 ERE was eliminated yet still maintained the ability to signal through the RBBP1 Sp1 site. Fig. 5B demonstrates that both the ER{alpha} and ERbeta P-box mutations significantly inhibit activation through a consensus ERE element when compared with the wild type receptor. Interestingly, the ER{alpha} P-box mutation had no effect on transcriptional activation of the RBBP1 +851 to +1140 construct, whereas the ERbeta P-box mutation decreased activation by ~60% (Fig. 5C). This is in agreement with Fig. 4B demonstrating that activation of the RBBP1 intron 1 enhancer region through ER{alpha} is ERE-independent, whereas activation through ERbeta is at least partially ERE-dependent.

The ER{alpha} AF1 Domain Is Important for Sustained Activation of RBBP1 Intron 1 Enhancer Activity—To test the hypothesis that the ER A/B domain (AF1) was involved in mediating ER isoform-specific activation of the RBBP1 gene, N-terminal deletions of both ER isoforms were created (Fig. 6A). The ER{alpha}-{Delta}AF1 construct, starting at Met174 of ER{alpha}, matches the previously described 46-kDa form of ER{alpha}, which is expressed in normal osteoblasts (36, 37). Therefore, wild type and ER-{Delta}AF1 constructs were transiently cotransfected with the RBBP1 +851 to +1140 reporter plasmids (Fig. 4B) in U2OS cells. The data presented in Fig. 6B closely resembles the transfection data from the U2OS-ER stable transfectants (Fig. 4B), wherein the activity of ER{alpha} is purely Sp1-dependent (ERE mutation has no effect), and maximal activity of ERbeta is dependent on both the ERE and Sp1 sequences. Coexpression of ER{alpha}-{Delta}AF1 with the wild type RBBP1 +851 to +1140 construct (Fig. 6C) leads to an increased -fold activation by E2 over wild type ER{alpha} (4.96-versus 1.99-fold), indicating that the ER{alpha} AF1 may play a suppressor role in E2-dependent activation of RBBP1. Interestingly, the ER{alpha}-{Delta}AF1 construct is now sensitive to the RBBP1 ERE mutation as is normally observed with wild type ERbeta (Fig. 6, compare ERbeta WT (B) with ER{alpha}-{Delta}AF1 (C)). No significant change was observed with the ERbeta-{Delta}AF1 compared with wild type ERbeta with any of the RBBP1 constructs. These data suggest that the ER{alpha} AF1 is involved in mediating specific interactions with the RBBP1 Sp1 site and inhibition of the use of the RBBP1 ERE sequence because deletion of the ER{alpha} AF1 now allows signaling through the ERE.


Figure 4
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  		FIGURE 4.
Mutational analysis of the RBBP1 intron 1 enhancer region results in ER isoform-specific effects. A, U2OS-ER{alpha} and -ERbeta cells were transiently transfected in triplicate with the indicated RBBP1 intron 1 enhancer region constructs as described under "Experimental Procedures." -Fold regulation relative to ethanol control was determined for each of the constructs, which were assayed in at least three independent experiments. Individual errors were less than 10% of the mean for each experiment. The black bars represent the mean ± S.D. of n number of trials. Asterisks denote significance at the p < 0.01 level (ANOVA) compared with the +412 to +1140 construct. B, cells were treated, transiently transfected, assayed, and analyzed with either ERE or Sp1 mutations in the +851 to +1140 context. Asterisks denote significance at the p < 0.01 level (ANOVA) compared with +851 to +1140 wild type construct. C, the wild type sequences of the ERE and Sp1 sites of the RBBP1 intron 1 enhancer region is depicted with the mutations listed in bold.

 
Because the data in Fig. 6 suggest that the deletion of the ER{alpha} AF1 results in an "ERbeta-like" pattern of E2-inducible expression, we determined the effect of ER{alpha}-{Delta}AF1 on endogenous RBBP1 expression. Therefore, we constructed stable U2OS cells lines expressing either the ER{alpha}-or ERbeta-{Delta}AF1 construct. As seen in Fig. 7A, the U2OS-ER{alpha}-{Delta}AF1 and -ERbeta-{Delta}AF1 cell lines express similar levels of ER to those observed in U2OS-ER wild type cell lines and correspond to the expected size (~46 kDa). We next examined the temporal E2 induction profile of endogenous RBBP1 in the U2OS-ER{alpha}-{Delta}AF1 and -ERbeta-{Delta}AF1 cell lines. The U2OS-ER{alpha}-{Delta}AF1 cell line exhibited a transient increase in RBBP1 expression peaking at 2 h of E2 treatment, whereas the U2OS-ER{alpha} cell line exhibited a sustained increase in RBBP1 expression (Fig. 7B) as observed previously in Fig. 1. Interestingly, the U2OS-ER{alpha}-{Delta}AF1 expression pattern is similar to that of U2OS-ERbeta. The U2OS-ERbeta-{Delta}AF1 cell line exhibits an overall decreased level of RBBP1 expression at 2 h of E2 treatment with minor induction observed at later time points. Collectively, these data suggest that the ER{alpha} AF1 confers ER{alpha}-specific patterns of regulation in both transient transfection analysis and endogenous RBBP1 expression.

SRC2 Preferentially Enhances E2-induced RBBP1 Intron 1 Enhancer Activity—To investigate the possible role of a differential SRC coactivator recruitment to explain the ER isoform-specific regulation of RBBP1, we overexpressed SRC1 and SRC2 with the +851 to +1140 construct in both U2OS-ER cell lines. SRC3 was not considered because a previous study demonstrated little or no SRC3 expression in osteoblasts (32). Fig. 8A demonstrates that SRC1 overexpression has no effect on E2-dependent transcriptional coactivation, whereas SRC2 has a significant effect in both U2OS-ER cell lines. In fact, SRC1 overexpression appears to inhibit the E2-dependent response to both ER isoforms. We repeated the experiment using the vitellogenin A2 ERE element to verify that our SRC overexpression was effective (38). Fig. 8B demonstrates that both SRC1 and SRC2 equally and effectively increase E2-dependent transcription with both ER isoforms with the vitellogenin A2 ERE. ChIP analysis was performed to determine SRC recruitment to the RBBP1 intron 1 enhancer region and demonstrated that no detectable SRC1 was recruited, whereas significant SRC2 protein was recruited in an E2-dependent manner (Fig. 8, C and D), which supports the transient transfection data (Fig. 8A). These results confirm that SRC2 is the preferred coactivator recruited to the RBBP1 intron 1 enhancer region. Interestingly, mutation of the RBBP1 ERE to the vitellogenin A2 consensus, in the RBBP1 +851 to +1140 context, does not rescue the ability of SRC1 to function in RBBP1 activation (data not shown). This demonstrates that not only the ERE sequence but also the surrounding DNA context is important for the induction of RBBP1 by E2.


Figure 5
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  		FIGURE 5.
ER{alpha}-dependent activation of the RBBP1 intron 1 enhancer region is independent of ERE binding. A, a schematic diagram of the P-box mutations made in ER{alpha} and ERbeta is shown. B, the WT or the P-box mutations (P-Box Mut) of ER{alpha} or ERbeta was cotransfected with a consensus ERE reporter construct (ERE-TK-LUC) into U2OS cells as described under "Experimental Procedures." The black bars represent the mean ± S.D. of n number of trials. Asterisks denote significance at the p < 0.01 level (ANOVA) compared with the wild type construct. The bars represent data from at least three independent experiments. C, identical to B, except it is for the RBBP1 +851 to +1140 reporter construct.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
This paper presents evidence that ER isoform-specific transcriptional activation of the RBBP1 gene is because of the AF1 activity. We chose the RBBP1 gene because previous microarray and RT-PCR analyses demonstrated that RBBP1 was a highly E2-regulated gene, and ER{alpha} and ERbeta exhibited a differential induction pattern. Furthermore, RBBP1 is a known Rb cofactor and may have an involvement in the control of osteoblastic proliferation and differentiation. We demonstrate that the RBBP1 gene displays a unique regulatory response by which a sustained induction is observed with ER{alpha}, whereas only a transient induction is seen with ERbeta. This pattern was paralleled by ChIP assays demonstrating that differential ER occupancy of the RBBP1 intron 1 enhancer region was most likely responsible for the observed expression time course. Mutational analysis demonstrated different cis-acting requirements for activation of RBBP1 by the ER isoforms. ER{alpha} required the presence of the Sp1 site, whereas ERbeta was capable of E2 induction through either the ERE or Sp1 sites. Interestingly, deletion of the ER{alpha} AF1 domain elicited a temporal transcription profile similar to that of ERbeta, suggesting that differences in the ER AF1 domains play a critical role in E2-dependent activation of the RBBP1 gene.


Figure 6
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  		FIGURE 6.
Deletion of the ER{alpha} AF1 domain results in modulation of RBBP1 transcriptional activation. A, AF1 deletions were made in ER{alpha} and ERbeta (encoding amino acids 174–595 and 130–530, respectively) that contained an N-terminal FLAG epitope tag (black bars). B, the ER{alpha} or ERbeta WT constructs were cotransfected with the indicated RBBP1 intron 1 enhancer region mutation constructs in U2OS cells, treated, and analyzed as in Fig. 4. Asterisks denote significance at the p < 0.01 level (ANOVA) compared with the wild type construct. The bars represent data from three independent experiments. C, idntical to B except with the ER{alpha}- or ERbeta-{Delta}AF1 expression constructs.

 
There are numerous reports describing the functional interactions of ER{alpha} and Sp1 in the E2-dependent regulation of gene expression (39–50). Furthermore, the N-terminal region of ER{alpha} is known to physically interact with the C-terminal zinc-finger region of Sp1 (47, 50, 51). This functional and physical evidence suggests that DNA-bound Sp1 recruits the ER, which is necessary for transcriptional activation of numerous genes. Indeed, our data suggest that the Sp1 site is necessary and sufficient for ER{alpha}-specific transcription of the RBBP1 intron 1 enhancer region. However, comparatively little is known concerning ERbeta and Sp1 interactions and their effects on transcriptional activity. Saville et al. (47) demonstrated that although both ER isoforms bind Sp1 in a ligand-independent manner, only the ER{alpha}-Sp1 interaction is transcriptionally competent on a synthetic Sp1 reporter construct, whereas the ERbeta-Sp1 interaction fails to activate transcription. In contrast, Salvatori et al. (52) demonstrated that ERbeta-Sp1 interactions result in transcriptional activation of an epidermal growth factor receptor promoter construct. Our data using the RBBP1 intron 1 ERE enhancer domain demonstrate that ERbeta can support E2-inducible transcription through both the ERE and Sp1 sites. The apparent discrepancy in the literature of the effects ERbeta-Sp1 interactions on transcription may be because of cell type and promoter/enhancer context specificity.


Figure 7
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  		FIGURE 7.
Deletion of the ER{alpha} AF1 domain results in modulation of endogenous RBBP1 mRNA expression. A, stable U2OS cell lines expressing the ER{alpha}- or ERbeta-{Delta}AF1 expression construct were generated as described under "Experimental Procedures." Western blot analysis of the U2OS-ER{alpha}, -ERbeta, -ER{alpha}-{Delta}AF1, and -ERbeta-{Delta}AF1 was performed using an anti-FLAG antibody. The observed bands migrate at the expected molecular size. B and C, E2 treatment time courses were performed in the U2OS-ER{alpha}, -ERbeta, -ER{alpha}-{Delta}AF1, and -ERbeta-{Delta}AF1 cell lines as described in the legend to Fig. 1B. Real-time RT-PCR was employed with data expressed as mRNA abundance relative to beta-actin. Asterisks denote significance at the p < 0.01 level (ANOVA) compared with control treatment (0 h) for each cell line. The bars represent data from three independent experiments.

 


Figure 8
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  		FIGURE 8.
SRC2 preferentially activates and binds to the RBBP1 intron 1 enhancer region. A and B, U2OS-ER{alpha} and -ERbeta cells were transfected in triplicate with either the RBBP1 intron 1 enhancer +851 to +1140 construct or the consensus vitellogenin A2 ERE construct, alone or in combination with SRC1 or SRC2. Cells were treated and assayed for luciferase activity as described in the legend to Fig. 4. A single asterisk denotes significance at the p < 0.01 level (ANOVA) compared with the control treatment for each cell line and construct, whereas a double asterisk denotes significance at the p < 0.01 level (ANOVA) compared with the E2 treatment in the absence of SRC overexpression for each cell line and construct. C, ChIP assays were performed in U2OS-ER{alpha} and -ERbeta cell lines treated with 10 nM E2 for 2 h using antibodies specific for either SRC1 or SRC2 as described under "Experimental Procedures." D, real-time PCR analysis was employed on samples from three independent ChIP experiments. An asterisk denotes significance at the p < 0.05 level (ANOVA) compared with ethanol control.

 
Our laboratory has previously reported that in our U2OS-ER cell models, ER{alpha} and ERbeta homodimers as well as ER{alpha}/ERbeta heterodimers appear to regulate distinct cadres of genes with only 20% of all E2-regulated genes being regulated by both ER isoforms (26, 27). These data have been confirmed by other laboratories (28, 29). Our laboratory reported a similar ER isoform specificity using the selective estrogen receptor modulator 4-hydroxytamoxifen and further showed that E2 and 4-hydroxytamoxifen regulated unique patterns of gene expression. The data presented in this paper begin to delineate the mechanism of these gene-specific inductions by the ER isoforms. Although ER isoform-specific interactions of coregulators cannot be ruled out, at least with respect to the RBBP1 gene, the differential interactions of the ERs with the ERE enhancer domains/elements, the differential time course of ER occupancy on these elements, and the ER AF1 domain appear to play a large role in the ER isoform-specific regulation. Merot et al. (31) previously demonstrated that in well differentiated E2-sensitive tissues/cell lines, ER{alpha} activity is largely influenced by AF1. Our data support this model because deletion of ER{alpha} AF1 caused an ERbeta-like temporal pattern in the E2-dependent activation of the RBBP1 gene.

Previous studies have reported the existence of a natural splice variant of ER{alpha} that lacks the first 173 amino acids of ER{alpha} (termed hER{alpha}46) and is consequently identical to the ER{alpha}-{Delta}AF1 receptor described in this study (36). hER{alpha}46 is found to be an effective E2-inducible transcription factor and exhibits a strong inhibitory effect on wild type ER{alpha} (hER{alpha}66) transactivation, possibly because of interference with coregulator binding to the hER{alpha}46/hER{alpha}66 heterodimer (36, 53). Furthermore, hER{alpha}46 form is demonstrated to be naturally expressed in human primary trabecular osteoblasts (37). Data from this study suggest that ER{alpha}-{Delta}AF1 (e.g. hER{alpha}46) is capable of RBBP1 activation in U2OS cells, however, in a manner reminiscent of wild type ERbeta and not wild type ER{alpha}. Because the ER-{Delta}AF1 cell system in this paper expresses only ER-{Delta}AF1, future studies are needed to determine the interactions between ER-{Delta}AF1 mutants and wild type ERs in the same cell on RBBP1 regulation.

We previously reported decreased E2-dependent cell proliferation in the U2OS-ER{alpha} cell line, whereas no effect was observed in the U2OS-ERbeta cell line (26, 27). The specific gene(s) involved in the suppression of osteoblastic proliferation are currently unknown. RBBP1, in concert with Rb, has been previously shown to decrease E2F1 promoter activity, to decrease cellular proliferation, and to be involved in transcriptional repression (54–56). Interactions of Rb with Runx2, a major regulator of osteogenesis, result in decreased cellular proliferation and increased differentiation (57). Whether RBBP1 has a role, if any, in E2-dependent regulation of cellular proliferation or differentiation requires further investigation.

In conclusion, the current study describes RBBP1 as a novel E2-regulated gene in osteosarcoma cells that exhibit a differential response to the ER isoforms. The ER isoform-specific regulation of RBBP1 expression appears to be caused by the differential occupancy of the ER isoforms on the RBBP1 intron 1 ERE enhancer domain, which in turn appears to involve ER isoform-specific interactions with specific enhancer elements (e.g. ERE, Sp1). Furthermore, it appears that the ER{alpha} AF1 is involved in imparting ER{alpha}-specific patterns to E2-dependent RBBP1 activation. Further experimentation is required to expand this hypothesis to other ER{alpha}-regulated genes.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant PO1-AG04875-21 and by grants from the Breast Cancer Research Foundation (New York) and the Mayo Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, 1601C Guggenheim, 200 1st St. S.W., Rochester, MN 55905. Tel.: 507-284-1926; Fax: 507-284-2053; E-mail: Monroe.David{at}mayo.edu.

2 The abbreviations used are: E2, 17beta-estradiol; ER, estrogen receptor; ERE, estrogen response element; RT, reverse transcription; ChIP, chromatin immunoprecipitation; RBBP1, retinoblastoma-binding protein 1; AF, activation function; WT, wild type; FBS, fetal bovine serum; CS-FBS, charcoal-stripped FBS; Dox, doxycycline; ANOVA, analysis of variance; SRC, steroid receptor coactivator. Back


   ACKNOWLEDGMENTS
 
We thank Barbara Getz, Amanda Hemmingson, and Grant Bender for their excellent technical assistance.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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