Estrogen Receptor Isoform-specific Regulation of the Retinoblastoma-binding Protein 1 (RBBP1) Gene

The transcriptional effects of estrogens (e.g. 17β-estradiol or E2)² are largely mediated by two related, but distinct, estrogen receptor (ER) isoforms ERα and ERβ (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 ERα and ERβ 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 ERα and ERβ homodimers and ERα/ERβ 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 ERα and ERβ 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 ERα and ERβ in a U2OS model system. Using deletion and mutational analysis, we demonstrate that ERα utilizes a consensus Sp1 sequence, whereas ERβ functions through both the Sp1 and a nearby ERE sequence. Deletion of the ERα AF1 in a stable U2OS transfectant cell line causes the temporal profile to mimic that of wild type ERβ. These data indicate that differential transcriptional activation of RBBP1 by ERα and ERβ and transcription factor binding site utilization are AF1-dependent.

EXPERIMENTAL PROCEDURES

Cell Culture and Chemicals—The U2OS-ERα and -ERβ 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), 1× antibiotic/antimycotic (Invitrogen), 5 mg/liter blasticidin S (Roche Applied Science), and 500 mg/liter zeocin (Invitrogen). The U2OS-ER-ΔAF1 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 1× 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 17β-estradiol (E2) were purchased from Sigma.

Reverse Transcription PCR (RT-PCR) Analysis —The U2OS-ERα and -ERβ 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 (1× 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 β-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′. β-actin primers are described previously (26).

Chromatin Immunoprecipitation (ChIP) Assay—U2OS-ERα and -ERβ cells (1 × 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 ERα-specific antibody (HC20; Santa Cruz Biotechnology, Santa Cruz, CA), ERβ-specific antibody (P2571; Invitrogen), SRC1-specific antibody (05–522; 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 ERα and ERβ P-box mutation expression plasmids was performed using two-step PCR. Briefly, primers were designed to mutate Glu²⁰³ and Gly²⁰⁴ to alanines for ERα and Glu¹⁶⁷ and Gly¹⁶⁸ to alanines for ERβ. The ERα double mutation was as reported previously (34). Because ERα and ERβ share 97% identify within the DNA-binding domain, the same mutation was made in ERβ. 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 ERα and ERβ P-box mutations, respectively.

Development of the ER-ΔAF1 Expression Constructs and the U2OS-ER-ΔAF1 Cell Lines—Construction of the ERα-ΔAF1 and ERβ-ΔAF1 expression plasmids was performed using PCR. Briefly, primers were designed to amplify amino acids 174–595 of human ERα and amino acids 130–530 of human ERβ. 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 ERα-ΔAF1 and ERβ-ΔAF1, respectively. The generation of the U2OS-ER-ΔAF1 cell lines was performed using the T-REx system (Invitrogen) as described previously (26). The ER-ΔAF1-pcDNA4/TO vectors were linearized using a PvuI restriction digest and transfected into the U2OS-Tet^R 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 ERα-ΔAF1 and ERβ-ΔAF1. The resulting cell lines are termed U2OS-ERα-ΔAF1 and -ERβ-ΔAF1.

Transient Transfection Analyses—U2OS-ERα and U2OS-ERβ 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 1× 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.

Western Blot Analysis—The U2OS-ER-ΔAF1 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 1× protease inhibitor mixture (Roche Diagnostics) in 1× phosphate-buffered saline) and the protein concentrations determined using a standard Bradford assay. ER-ΔAF1 protein levels were analyzed by Western blot using a FLAG-M2 antibody (Sigma) and visualized using enhanced chemiluminescence (Amersham Biosciences).

RESULTS

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-ERα cell line, whereas no regulation was detected in the U2OS-ERβ cell line (27). In contrast, at 2 h of E2 treatment, RBBP1 was regulated 3.74- and 2.55-fold in the U2OS-ERα and -ERβ 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-ERα cell line. However, in the U2OS-ERβ 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 ERα and ERβ—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 ERα and ERβ is observed following 1 h of E2 treatment. More specifically, ERα exhibits an E2-dependent binding to the RBBP1 intron 1 enhancer region throughout the 24-h time course, wherein ERβ displays E2-dependent binding only at 1 h of E2 treatment. It is interesting to note that ERβ 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-ERα cells and the temporary increase of RBBP1 expression in U2OS-ERβ 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 ERα and ERβ 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-ERα and -ERβ 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 ERα-dependent activation, whereas only a slight effect was observed on ERβ-dependent activation. This suggests that the Sp1 site within this sequence is critically important for ERα- but not ERβ-dependent transcriptional activation.

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-ERα 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-ERα cell line. Mutation of either the Sp1 or ERE decreased ERβ-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-ERβ cell line. These data suggest differential mechanisms for ERα- versus ERβ-dependent activation, wherein ERα exclusively utilizes the Sp1 site and ERβ utilizes both the Sp1 and ERE sites for maximal activation.

ERα-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 ERα 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 ERα and ERβ 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 ERα and ERβ P-box mutations significantly inhibit activation through a consensus ERE element when compared with the wild type receptor. Interestingly, the ERα P-box mutation had no effect on transcriptional activation of the RBBP1 +851 to +1140 construct, whereas the ERβ 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 ERα is ERE-independent, whereas activation through ERβ is at least partially ERE-dependent.

The ERα 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 ERα-ΔAF1 construct, starting at Met¹⁷⁴ of ERα, matches the previously described 46-kDa form of ERα, which is expressed in normal osteoblasts (36, 37). Therefore, wild type and ER-ΔAF1 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 ERα is purely Sp1-dependent (ERE mutation has no effect), and maximal activity of ERβ is dependent on both the ERE and Sp1 sequences. Coexpression of ERα-ΔAF1 with the wild type RBBP1 +851 to +1140 construct (Fig. 6C) leads to an increased -fold activation by E2 over wild type ERα (4.96-versus 1.99-fold), indicating that the ERα AF1 may play a suppressor role in E2-dependent activation of RBBP1. Interestingly, the ERα-ΔAF1 construct is now sensitive to the RBBP1 ERE mutation as is normally observed with wild type ERβ (Fig. 6, compare ERβ WT (B) with ERα-ΔAF1 (C)). No significant change was observed with the ERβ-ΔAF1 compared with wild type ERβ with any of the RBBP1 constructs. These data suggest that the ERα 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 ERα AF1 now allows signaling through the ERE.

Because the data in Fig. 6 suggest that the deletion of the ERα AF1 results in an “ERβ-like” pattern of E2-inducible expression, we determined the effect of ERα-ΔAF1 on endogenous RBBP1 expression. Therefore, we constructed stable U2OS cells lines expressing either the ERα-or ERβ-ΔAF1 construct. As seen in Fig. 7A, the U2OS-ERα-ΔAF1 and -ERβ-ΔAF1 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-ERα-ΔAF1 and -ERβ-ΔAF1 cell lines. The U2OS-ERα-ΔAF1 cell line exhibited a transient increase in RBBP1 expression peaking at 2 h of E2 treatment, whereas the U2OS-ERα cell line exhibited a sustained increase in RBBP1 expression (Fig. 7B) as observed previously in Fig. 1. Interestingly, the U2OS-ERα-ΔAF1 expression pattern is similar to that of U2OS-ERβ. The U2OS-ERβ-ΔAF1 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 ERα AF1 confers ERα-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.

DISCUSSION

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 ERα and ERβ 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 ERα, whereas only a transient induction is seen with ERβ. 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. ERα required the presence of the Sp1 site, whereas ERβ was capable of E2 induction through either the ERE or Sp1 sites. Interestingly, deletion of the ERα AF1 domain elicited a temporal transcription profile similar to that of ERβ, suggesting that differences in the ER AF1 domains play a critical role in E2-dependent activation of the RBBP1 gene.

There are numerous reports describing the functional interactions of ERα and Sp1 in the E2-dependent regulation of gene expression (39–50). Furthermore, the N-terminal region of ERα 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 ERα-specific transcription of the RBBP1 intron 1 enhancer region. However, comparatively little is known concerning ERβ 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 ERα-Sp1 interaction is transcriptionally competent on a synthetic Sp1 reporter construct, whereas the ERβ-Sp1 interaction fails to activate transcription. In contrast, Salvatori et al. (52) demonstrated that ERβ-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 ERβ can support E2-inducible transcription through both the ERE and Sp1 sites. The apparent discrepancy in the literature of the effects ERβ-Sp1 interactions on transcription may be because of cell type and promoter/enhancer context specificity.

Our laboratory has previously reported that in our U2OS-ER cell models, ERα and ERβ homodimers as well as ERα/ERβ 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, ERα activity is largely influenced by AF1. Our data support this model because deletion of ERα AF1 caused an ERβ-like temporal pattern in the E2-dependent activation of the RBBP1 gene.

Previous studies have reported the existence of a natural splice variant of ERα that lacks the first 173 amino acids of ERα (termed hERα46) and is consequently identical to the ERα-ΔAF1 receptor described in this study (36). hERα46 is found to be an effective E2-inducible transcription factor and exhibits a strong inhibitory effect on wild type ERα (hERα66) transactivation, possibly because of interference with coregulator binding to the hERα46/hERα66 heterodimer (36, 53). Furthermore, hERα46 form is demonstrated to be naturally expressed in human primary trabecular osteoblasts (37). Data from this study suggest that ERα-ΔAF1 (e.g. hERα46) is capable of RBBP1 activation in U2OS cells, however, in a manner reminiscent of wild type ERβ and not wild type ERα. Because the ER-ΔAF1 cell system in this paper expresses only ER-ΔAF1, future studies are needed to determine the interactions between ER-ΔAF1 mutants and wild type ERs in the same cell on RBBP1 regulation.

We previously reported decreased E2-dependent cell proliferation in the U2OS-ERα cell line, whereas no effect was observed in the U2OS-ERβ 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 ERα AF1 is involved in imparting ERα-specific patterns to E2-dependent RBBP1 activation. Further experimentation is required to expand this hypothesis to other ERα-regulated genes.