Tamoxifen-Bound Estrogen Receptor (ER) Strongly Interacts with the Nuclear Matrix Protein HET/SAF-B, a Novel Inhibitor of ER-Mediated Transactivation

Steffi Oesterreich1, Qingping Zhang1, Torsten Hopp1, Suzanne A. W. Fuqua1, Marten Michaelis, Holly H. Zhao, James R. Davie, C. Kent Osborne1 and Adrian V. Lee1

Department of Medicine (S.O., Q.Z., T.H., S.A.W.F., M.M., H.H.Z., C.K.O., A.V.L.) Division of Oncology University of Texas Health Science Center San Antonio, Texas 78284
Department of Biochemistry and Molecular Biology (J.R.D.) University of Manitoba Winnipeg, Manitoba, Canada R3E 0W3


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIAL AND METHODS
 REFERENCES
 
The estrogen receptor (ER) is a ligand-dependent transcription factor that acts in a cell- and promoter-specific manner. Evidence suggests that the activity of the ER can be regulated by a number of other stimuli (e.g. growth factors) and that the effects of the ER are modulated by nuclear factors termed coregulators. While the interplay among these factors may in part explain the pleiotropic effects elicited by the ER, there are several other less well described mechanisms of control, such as interactions with the nuclear matrix. Here we report that the nuclear matrix protein/scaffold attachment factor HET/SAF-B is an ER-interacting protein. ER and HET/SAF-B interact in in vitro binding assays, with HET binding to both the ER DNA-binding domain and the hinge region. Coimmunoprecipitation experiments reveal that HET/SAF-B and ER associate in cell lines in the presence or absence of estradiol, but binding is increased by the antiestrogen tamoxifen. HET/SAF-B enhances tamoxifen antagonism of estrogen-induced ER-mediated transactivation, but at high concentrations can inhibit both estrogen and tamoxifen-induced ER activity. HET/SAF-B-mediated repression of ER activity is dependent upon interaction with the ER-DBD. While the existence of high-affinity binding sites for the ER in the nuclear matrix has been known for some time, we now provide evidence of a specific nuclear matrix protein binding to the ER. Furthermore, our data showing that HET/SAF-B binds to ER particularly strongly in the presence of tamoxifen suggests that it may be important for the antagonist effect of tamoxifen.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIAL AND METHODS
 REFERENCES
 
The estrogen receptor (ER) is a member of a superfamily of nuclear transcription factors. When the ER binds estrogen it undergoes a conformational change that results in dimerization, binding to specific elements of DNA, and finally altered gene transcription (1, 2). While this model of ER action has held true for the last 30 yr, a more complete understanding has revealed that activation of the ER is extremely complex, with regulation by a diverse set of signals and nuclear factors. ER action can be altered by: 1) interaction with other nuclear transcription factors such as AP1 (3), SP1 (4, 5, 6), and members of the basal transcription machinery (1); 2) cross-talk with growth factor systems (7); and 3) associations with nuclear receptor coactivators and corepressors (8).

The existence of cofactors that can regulate the transcriptional activity of nuclear hormone receptors was first suggested by transcriptional squelching between ER and progesterone receptor (9, 10). A number of cofactors capable of increasing hormone receptor action, termed coactivators, have been identified (reviewed in Refs. 8, 11). The family of corepressors is smaller, the best characterized being nuclear receptor corepressor (N-CoR) (8, 12) and silencing mediator of retinoid and thyroid receptors (SMRT) (13, 14). Recently, a corepressor termed REA, which is specific for ER, has been identified (15). Many cofactors seem to regulate receptor activity by modulating chromatin structure. Coactivators such as p300/CBP (16, 17), PCAF (18, 19), and SRC-1 (20) have intrinsic histone acetyltransferase activity, which results in the destabilization of nucleosomes, creating a permissive state for promoter activation. In contrast, the corepressors N-CoR (21) and SMRT (22) associate with histone deacetylases, leading to a repressive chromatin state.

Another modulator of hormone action is the nuclear matrix, which is a dynamic structure involved in DNA replication, transcription, repair, and RNA processing (23). A role for the nuclear matrix in hormone receptor action was postulated many years ago (24, 25, 26, 27, 28), but only recently have specific nuclear matrix proteins been characterized that directly bind to hormone receptors and modulate their activity (29). Most recently, the glucocorticoid receptor-interacting protein GRIP 120 has been identified as the nuclear matrix protein hnRNPU (30).

HET was originally cloned in our laboratory as a nuclear matrix protein binding to the promoter of the estrogen-regulated heat shock protein hsp27 (31). Renz and Fackelmayer (32) cloned the same protein based on its ability to bind to scaffold/matrix attachment regions (S/MAR’s), and hence called it scaffold attachment factor B (SAF-B). Scaffold attachment factors are a specific subset of nuclear matrix proteins that are thought to mediate the attachment of chromatin to nuclear protein structures (33, 34). A specific role for scaffold attachment factors in hormone receptor action has not been described.

HET/SAF-B has recently been shown to bind to the C-terminal domain of RNA polymerase II (RNA pol II) and to a subset of serine-/arginine-rich RNA processing factors (SR proteins) (35). This suggests that HET/SAF-B is involved in the formation of a transcriptosomal complex, bringing transcription and pre-mRNA processing together. These macromolecular complexes have previously been shown to be associated with the nuclear matrix (36, 37).

Given the recent identification of nuclear matrix factors in hormone receptor action, we asked whether the nuclear matrix protein HET/SAF-B might be involved in ER action. In this report we describe the in vitro and in vivo association of ER with HET/SAF-B, with HET/SAF-B binding the ER in both the DBD and the hinge region. The association of ER with HET/SAF-B occurs in the absence of ligand but is increased by the antiestrogen tamoxifen (Tam). HET/SAF-B can enhance the antiestrogenic effect of Tam, but when overexpressed at high levels can also repress both estrogen and Tam agonist activity on the ER. Finally, we have shown that the ER DBD is critical for the repressive activity of HET/SAF-B on ER, as HET/SAF-B does not repress activity of an ER-GAL4DBD chimera and can cause transcriptional repression of an ER DBD fused to a heterologous transcription factor. We are currently performing further studies to identify the mechanism of transcriptional repression and whether this is dependent upon the nuclear matrix properties of HET/SAF-B.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIAL AND METHODS
 REFERENCES
 
HET/SAF-B Binds to the ER
To analyze whether HET/SAF-B could bind to ER, we performed glutathione-S- transferase (GST)-pulldown experiments (Fig. 1Go). First we incubated in vitro transcribed and translated ER with full-length GST-HET/SAF-B bound to glutathione-sepharose beads (Fig. 1AGo). There was no signal when ER was incubated with GST only, in the absence of hormone or in the presence of estradiol (E2). In contrast, ER interacted with GST-HET/SAF-B in the absence of hormone, in the presence of E2, and especially in the presence of Tam. We consistently saw increased binding of ER to HET/SAF-B in the presence of Tam compared with no ligand.



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Figure 1. HET/SAF-B Directly Interacts with ER in Vitro (GST-Pull-Down Experiments)

A, ER was labeled with 35S-methionine by in vitro transcription/translation and tested for interaction with GST alone and GST-HET/SAF-B in the absence of ligands, or in the presence of 10-6 M E2 or 10-6 M Tam (as indicated). The input lane contains 20% input of the in vitro transcribed/translated ER. B, Schematic presentation of GST-tagged ER domains. The numbers indicate amino acids in ER’s open reading frame. C, HET/SAF-B was labeled with 35S-methionine by in vitro transcription/translation and tested for interaction with GST alone and a number of GST-ER domain fusion proteins (as indicated). The input lane contains 20% input of the in vitro transcribed/translated HET/SAF-B.

 
Next we examined the ability of HET/SAF-B to interact with different domains of ER (represented graphically in Fig. 1BGo) in GST-pulldown assays. The different GST-ER domain fusion proteins were separated on SDS-PAGE and Coomassie stained, to ensure that the input of immobilized GST-fusion proteins was equal (data not shown). We examined HET/SAF-B interaction with the AF1, AF1/DNA-binding domain (DBD), DBD/Hinge, DBD, Hinge, and AF2/Hinge domains. All incubations were performed in the absence of hormone. As shown in Fig. 1CGo, HET/SAF-B consistently interacted strongly with the DBD/Hinge, AF2/Hinge, and AF1/DBD domains and weakly with the Hinge or DBD only. In contrast, we could not detect an interaction between HET/SAF-B and AF1. Thus, there are at least two HET/SAF-B binding sites in the ER protein, one in the DBD and the other one in the Hinge region. The HET/SAF-B interaction with AF2/Hinge was stronger than the interaction with Hinge only, suggesting that there may be another interaction domain within AF2. Thus, as described for the interaction between other cofactors and steroid receptors (38), HET/SAF-B potentially interacts with multiple regions within ER.

We next asked whether we could detect an interaction between HET/SAF-B and ER within cells. Therefore, we transiently transfected COS-7 cells with expression plasmids for HET/SAF-B and hemagglutinin (HA)-tagged ER. Immunoprecipitation of HET/SAF-B followed by immunoblotting for HA revealed a band with the molecular mass of ER (~68 kDa) that was only seen when cells were transfected with both ER and HET/SAF-B, but not in cells transfected with HET/SAF-B only (Fig. 2AGo, left panel). A similar experiment, but in a reciprocal manner, was performed using an HA-antibody to immunoprecipitate and the HET/SAF-B antibody for immunoblotting. As expected, a band at the molecular mass of HET/SAF-B (~130 kDa) was detected in cells transfected with HET/SAF-B and ER, but not in cells transfected with HET/SAF-B only (Fig. 2AGo, right panel).



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Figure 2. HET/SAF-B and ER Interact in Cells (Coimmunoprecipitations)

A, COS-7 cells were transiently transfected with expression constructs for HA-tagged ER (ER/HA) and HET/SAF-B as indicated, and lysed in low stringency buffer. The cell lysates were immunoprecipitated with an anti-HET/SAF-B antibody, subjected to electrophoresis, and immunoblotted with an anti-HA antibody (left panel). The cell lysates were immunoprecipitated with an anti-HA antibody, subjected to electrophoresis, and immunoblotted with an anti-HET/SAF-B antibody (right panel). B, MCF-7 cells were lysed in LS and HS buffer, the lysates from duplicate plates were immunoprecipitated with an anti-HET/SAF-B antibody, and the immunoprecipitates were subjected to electrophoresis. For the immunoblot anti-HET/SAF-B and anti-ER antibody were used. C, MCF-7 LS lysates were immunoprecipitated with an HET/SAF-B antibody, with HET/SAF-B antibodies preincubated with HET/SAF-B peptide, or without antibody. The immunoblots were performed with HET/SAF-B antibody. D, MCF-7 cells were incubated without ligand, with 10-9 M E2, or with 10-9 M Tam for 24 h. The immunoblots were performed with HET/SAF-B antibody (left top panel) and ER antibody (left bottom panel). The lysates were also immunoblotted with an ER antibody (right top panel). The bar graph represents intensity ratios of immunoprecipiated ER to immunoblotted ER in the lysates (see Materials and Methods). E, COS-7 cells were transfected with expression plasmids for HET/SAF-B and ER-HA. Treatment of the cells, immunoprecipitation, and immunoblotting were performed as described in panel C with the exception that HA antibody was used instead of ER antibody.

 
The next set of coimmunoprecipitation experiments was performed to see whether endogenous HET/SAF-B and ER indeed interact and whether this interaction was altered by E2 or Tam. First, we immunoprecipitated HET/SAF-B from MCF-7 breast cancer cells lysed under low stringency (LS) and high stringency (HS) conditions (Fig. 2BGo). Under low-stringency conditions we observed coimmunoprecipitation of ER and HET/SAF-B, whereas under high-stringency conditions more HET/SAF-B was immunoprecipitated, but ER was dissociated from the complex. To demonstrate that the bands on the immunoblot are indeed antibody specific, we repeated the HET/SAF-B immunoprecipitation with HET/SAF-B antibodies preincubated with HET/SAF-B peptide, or without antibody. As shown in Fig. 2CGo, only the immunoprecipitation with HET/SAF-B antibody resulted in a detectable band at the molecular mass of HET/SAF-B (~130 kDa), whereas no bands were detected using a peptide-preincubated antibody or no antibody. Thus, in breast cancer cells endogenous HET/SAF-B and ER interact, and this interaction can be detected when the cells are lysed under low-stringency conditions.

To investigate the ligand dependency of this interaction, we incubated MCF-7 cells in the absence of ligand and in the presence of E2 or Tam, and lysed them in LS buffer. After immunoprecipitation with HET/SAF-B antibodies, the membrane was immunoblotted with HET/SAF-B antibodies (Fig. 2DGo, left top panel) and ER antibodies (left bottom panel). While HET/SAF-B levels remained constant, coimmunoprecipitated ER levels changed. ER was detectable in the absence of ligand, low levels were detectable in the presence of E2, but much higher levels of ER were coimmunoprecipitated in the presence of Tam. Since ER itself is known to be down-regulated by E2 via ubiquitin-mediated degradation (39, 40), as a control we also measured ER levels in the lysate (Fig. 2DGo, right top panel). In contrast to HET/SAF-B, which did not change with E2 and Tam treatment (data not shown), ER levels decreased dramatically after E2 treatment but were unaffected by Tam. To account for the differences in ER levels within the actual lysates, we measured the amount of ER in the immunoprecipitate and the lysate by densitometry and presented the results as the ratio of ER levels immunoprecipiated with HET/SAF-B antibodies to ER levels in the lysate (Fig. 2DGo, bar graph). While the changes in ER levels complicate an exact quantitative analysis of the coimmunoprecipitation in the E2-treated samples, Tam did not affect ER levels, and it can be clearly seen that ER binds more strongly to ER in the presence of Tam than in its absence (i.e. no ligand).

Finally, we confirmed that HET/SAF-B is strongly bound to ER in the presence of Tam by transfecting COS-7 cells with HET/SAF-B and an HA-tagged ER construct (Fig. 2EGo). As in MCF-7 cells, there was an association between HET/SAF-B and ER in the absence of ligand or in the presence of E2, but again association was greater in the presence of Tam (left bottom panel). In cell lysates, HET/SAF-B levels did not change as a result of E2 or Tam treatment (data not shown). However, as seen with endogenous ER in MCF-7 cells, ER levels were reduced in COS-7 cells after E2 treatment (right top panel). When we corrected the changes in immunoprecipitated ER for the changes in endogenous ER levels, we were again able to detect a significant increase in the binding of HET/SAF-B to ER in the presence of Tam (Fig. 2EGo, bar graph). Thus, we conclude from our coimmunoprecipitation experiments that HET/SAF-B and ER interact, and that this interaction is stronger in the presence of the antiestrogen Tam.

HET/SAF-B Overexpression Decreases ER Activity
As shown in Fig. 2Go, the association between HET/SAF-B and ER is stronger in the presence of Tam as compared with no ligand. This observation prompted us to study the effect of HET/SAF-B on the antagonist activity of Tam. To do this we performed transient transfection assays in ER-negative HepG2 cells using a single estrogen response element (ERE)-tk-luciferase construct as the reporter gene (Fig. 3AGo). The results in Fig. 3AGo represent the effect of HET/SAF-B on Tam acting as an antagonist of E2-occupied ER; i.e. cells were incubated in the presence of both E2 and Tam. As expected, increasing concentrations of Tam resulted in a dose-dependent inhibition of E2-mediated ER activity (pcDNAI curve). Cotransfection of 10 ng HET/SAF-B vector did not affect E2-mediated activation of the ER in the absence of Tam, or when Tam was added at a low concentration that does not have an antagonistic effect (10-10 M). In contrast, at higher concentrations of Tam (10-9 to 10-6 M) which antagonize E2 activation of ER, coexpression of HET/SAF-B (10 ng) significantly enhanced the antagonism by Tam.



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Figure 3. Overexpression of HET/SAF-B Inhibits ER Activity

Cotransfection of the ERE-tk-luc (1 µg) reporter gene with expression vectors coding for HET/SAF-B and ER as indicated in HepG2 cells. Values are the mean ± SEM of triplicate wells, and the graphs are representative of at least three experiments each. A, Cells were transiently transfected with ER (25 ng) and 10 ng of pcDNAI or HET/SAF-B plasmids and incubated in the presence of 10-9 M E2 and increasing amounts of Tam, as indicated (*, P < 0.05, t test). B, Cells were transfected with ER and HET/SAF-B plasmids as indicated. Open bars represent incubation in the absence of E2, and black bars represent incubation in the presence of 10-9 M E2. C, Cells were transiently transfected with ER (25 ng) and HET/SAF-B (100 and 250 ng) plasmids and incubated in the presence of 10-9 M E2 and increasing amounts of Tam, as indicated. D, Cells were incubated in the absence of ligand (open bar) and in the presence of 10-8 M Tam (black bars), and transfected with ER (25 ng) and the indicated amounts of HET/SAF-B.

 
We next addressed how increased overexpression of HET/SAF-B could affect the transcriptional activity of E2-occupied ER. In the absence of ER, the addition of E2 did not result in a significant change in basal activity of the construct, and HET/SAF-B had no effect on this basal activity. As expected, transfection of ER led to an approximately 6-fold increase of transcriptional activity in the presence of E2. The coexpression of increasing amounts of HET/SAF-B (0–150 ng) led to a significant dose-dependent decrease in ER activity (Fig. 3BGo). A similar HET/SAF-B-mediated repression was also seen in Saos-2 cells transfected with ER (data not shown). Increasing concentrations of HET/SAF-B (100 and 250 ng) were also able to further enhance the antagonist activity of Tam, as shown in Fig. 3CGo.

Using the same transfection system in HepG2 cells, but incubating the cells in the presence of Tam alone, Tam acts as an agonist and can activate the ER. We therefore tested whether HET/SAF-B overexpression could affect Tam agonist activity. Figure 3DGo shows that Tam (10-8 M) caused a 2- to 2.5-fold increase in ER activity. Cotransfection with HET (100 ng) reduced this increase by 58%, while 250 ng HET completely abolished Tam agonist activity.

We next performed a series of additional control experiments (Fig. 4Go) to exclude a nonspecific repressor effect of HET/SAF-B. As shown in Fig. 3Go, A and B, basal activity of the ERE-tk-promoter was not inhibited by overexpression of HET/SAF-B. As expected, the deletion of the ERE sequence led to a tk-promoter construct that was also not affected by HET/SAF-B overexpression (Fig. 4AGo, left bars). In the same experiment the ERE-tk-promoter was inhibited by overexpression of HET/SAF-B (Fig. 4AGo, right bars), as shown previously (Fig. 3Go). As is common for transient transfection assays, the luciferase values were corrected for the values of a second cotransfected gene, which is, in our case, an SV40-promoter-driven ß-galactosidase (ß-gal) construct. Overexpression of HET/SAF-B did not change ß-gal expression (Fig. 4BGo), thus representing another internal negative control. In several other experiments using other transcription factors and other reporter constructs, we again did not see a nonspecific repression by HET/SAF-B (detailed later).



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Figure 4. HET/SAF-B-Mediated Repression of ER’s Transcriptional Activity Is Not the Result of a General Repression Mechanism

Cotransfection of ERE-tk-luc and tk-luc reporter genes with expression vectors coding for HET/SAF-B and ER as indicated. Bars are the mean ± SEM of triplicate wells and each graph is representative of at least three experiments. A, HepG2 cells were transfected with expression plasmids for ER and HET/SAF-B, as indicated, and with the reporter constructs tk-luc and ERE-tk-luc, respectively. Cells were incubated in the presence of 10-9 M E2. B, ß-Gal values measured from the experiment shown in panel A.

 
The ER-DBD Is Necessary for the Repressive Effects of HET/SAF-B
Most nuclear receptors including ER share a typical domain structure: a Zn finger DBD is flanked by an N-terminal region that displays a constitutive activator function domain 1 (AF-1) and the C terminus containing the ligand-binding domain, heterodimerization domain, and ligand-dependent activation function domain 2 (AF-2). To delineate the importance of the DBD of ER on HET/SAF-B-mediated repression, we made use of chimeric constructs in which the ER-DBD (aa 178–257) was replaced by a GAL4-DBD and tested reporter activity on four copies of a gal4-responsive element upstream of luciferase (gal4-luc). As a negative control we included the GAL4DBD alone (GAL4DBD). As a positive control we transfected wild-type ER, HET/SAF-B, and the ERE-tk-luc. All constructs were cotransfected with HET/SAF-B into HepG2 cells. The data are presented in Fig. 5AGo (left panel) as fold over control of each construct, since the activity of the different GAL4DBD constructs varied over magnitudes. The relative luciferase units for the controls (no estrogen and no HET/SAF-B) were ER = 1437.0, gal4DBD = 1.2, and ER-gal4DBD = 26.1.



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Figure 5. ER-DBD Is Involved in HET/SAF-B’s Mediated Repression of ER’s Activity

HepG2 cells were transiently transfected with expression vectors as indicated. Bars are the mean ± SEM of triplicate wells and the graphs are representative of at least three experiments each. A, Cells were transiently transfected with various ER constructs (25 ng) and HET/SAF-B (200 ng), as indicated, and ERE-tk-luc and the Gal4-responsive promoter construct Gal4-luc, respectively. Since the activity of the different Gal4DBD constructs varied over magnitudes, the data are presented as fold over control calculated in relation to the activity of each construct seen in the absence of ligand. The right panel shows the result from an immunoprecipitation using lysates from COS-7 cells which were transiently transfected with expression plasmids for HET/SAF-B and ER-Gal4DBD. The cell lysates were immunoprecipitated with an anti-HET/SAF-B antibody, subjected to electrophoresis, and immunoblotted with an anti-ER antibody. B, Cells were transiently transfected with HET/SAF-B (as indicated), with 25 ng VP16-gal4DBD and VP16-ERDBD, and with the Gal4-responsive promoter construct Gal4-luc and ERE-tk-luc, respectively. C, Cells were transfected with 1 µg CMV-ERE-CAT, and ER, and HET/SAF-B as indicated. Cells were incubated in the absence of ligand (white bars), in the presence of 10-9 M E2 (gray bars), and 10-7 M Tam (black bars).

 
As expected, E2 increased transcriptional activity from the ERE-tk-luc reporter construct, and the induction was repressed by coexpression of HET/SAF-B (Fig. 5AGo). Coexpression of the gal4DBD with the gal4-luc reporter construct resulted in basal activity that was not affected by E2 treatment and was also not affected by coexpression of HET/SAF-B. Expression of both the AF-1 and the AF-2 domains fused to GAL4DBD (ER-GAL4DBD) behaved like wild-type ER with strong E2 inducibility. However, HET/SAF-B did not repress activity while it did repress wild-type ER activity. In addition, HET/SAF-B was not able to repress activity of either AF-1 fused to GAL4DBD, or AF-2 fused to GAL4DBD (data not shown). Interestingly, while HET/SAF-B was not able to repress activity of the ER-GAL4DBD chimera, HET/SAF-B was still able to bind to this chimera as shown by coimmunoprecipitation (Fig. 5AGo, right panel). The binding of HET/SAF-B to ER GAL4DBD substantiates the earlier in vitro GST binding experiments indicating that HET/SAF-B can bind ER not only in the DBD, but also in the Hinge/AF2 region. Thus, while HET/SAF-B can bind ER-GAL4DBD, it cannot repress its activity, suggesting that the ERE-DBD is required for transcriptional repression.

To directly assess the importance of the ER-DBD in HET/SAF-B-mediated repression, we examined the effect of HET/SAF-B on the ER-DBD fused with a heterologous transcription factor (VP16). As a control we examined the effect of HET/SAF-B on VP-16 with a GAL4-DBD. As shown in Fig. 5BGo, the addition of increasing amounts of HET/SAF-B (50, 100, and 250 ng) did not affect the activity of VP16-GAL4 DBD on a GAL4 reporter construct. In contrast, HET/SAF-B caused a dose-dependent decrease of VP16-ER-DBD activity on a ERE-Luc reporter construct. Thus we can conclude that the ER-DBD can mediate the HET/SAF-B transcriptional repression effect.

A simple explanation for the repressive effect of HET/SAF-B would be if HET/SAF-B bound to the ERE-DBD and blocked ER binding to DNA. To examine this possibility, we asked whether HET/SAF-B had an effect on the DNA binding properties of ER. First we confirmed that HET/SAF-B could not bind directly to an ERE sequence using gel-shift assays and in vitro transcribed and translated HET/SAF-B (data not shown). To then examine whether HET/SAF-B could inhibit ER binding to DNA, we used a promoter interference assay originally described by Reese and Katzenellenbogen (41) in which an ERE is inserted between the cytomegalovirus (CMV) promoter (containing the TATA box) and the start site of transcription of the chloramphenicol acetyl transferase reporter gene (CMV-ERE-CAT) (Fig. 5CGo). Constitutive expression of this reporter construct was inhibited by coexpression of ER (Fig. 5CGo). This inhibition occurs in the absence of ligand, but is enhanced by addition of E2 or Tam, as previously shown by Reese and Katzenellenbogen. Coexpression of HET/SAF-B (10 ng) did not affect the activity of the reporter construct in the absence of ER and did not alter the ability of ER to inhibit reporter activity either in the absence or in presence of ligand. Overexpression of HET (250 ng) again did not affect the constitutive expression of the reporter construct in the absence of ER. However, this high concentration of HET/SAF-B actually increased the ability of ER to reduce reporter activity. This would suggest that binding of HET/SAF-B does not block the ability of ER to bind DNA, but rather that in the presence of high concentrations of HET/SAF-B more interference occurs.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIAL AND METHODS
 REFERENCES
 
Nuclear receptors are divided into three groups: steroid receptors, retinoic acid/thyroid receptors, and orphan receptors. A fundamental difference between steroid receptors and retinoic acid/thyroid receptors is that the latter are DNA-bound active repressors in the absence of ligand. An active role for corepressors such as N-CoR (8, 12) and SMRT (14) for the silencing activity of unliganded retinoic acid/thyroid receptors has been well established. Recent findings of disturbed corepressor interaction with mutated thyroid receptors in patients with resistance to thyroid hormone support the importance of corepressors for the normal action of agonist- and antagonist-bound receptors (42, 43, 44).

In contrast to retinoid/thyroid receptors, steroid receptors show little DNA binding activity in the absence of ligand and thus are thought to have no silencing ability. However, recently it has become clear that steroid receptors are also found in repressor complexes, particularly when the receptor is bound to antagonists, and that the antagonist function may in part be mediated by corepressors (15, 45, 46).

In the presence of the antiestrogen Tam, ER can still dissociate from heat shock proteins and bind to DNA, but its AF-2 domain activity is inhibited (47). It has been shown that ER can bind the corepressors N-CoR and SMRT (45, 46). While binding of these corepressors is constitutive under in vitro conditions (48), coimmunoprecipitation experiments have indicated that N-CoR binds to ER only in the presence of Tam (46). The specific role of N-CoR and SMRT in the antagonist effect of Tam is unclear, but more detailed studies have been performed concerning the agonist role of Tam. It has been shown that overexpression of N-CoR or SMRT can inhibit Tam’s agonist activity (45, 46, 48). Additionally, reduction of N-CoR by microinjection of N-CoR-specific antibodies can convert Tam into a full ER agonist displaying activity similar to estrogen (46). However, no data at present confirm that N-CoR or SMRT are actually responsible or necessary for the antagonist activity of Tam. More recently a novel ER-specific corepressor, termed REA, has been discovered (15). REA can potentiate the antiestrogenic effect of Tam, but when overexpressed at high levels also inhibits estrogen activation of the ER.

In the present paper we describe another protein association with ER, that of the nuclear matrix protein HET/SAF-B, which also has properties consistent with its being an ER corepressor. Under in vitro conditions, HET/SAF-B interacts with ER in the absence of ligand, although the association is increased by Tam. Furthermore, coimmunoprecipitation experiments show that the interaction between HET/SAF-B and ER is stronger in the presence of Tam. The ability of Tam to recruit HET/SAF-B, as well as N-CoR and REA, to ER suggests an active corepression mechanism, although this remains to be specifically proven. Due to the ability of N-CoR and REA to alter the agonist/antagonist activity of Tam, it has been proposed that the ratio of corepressor to coactivator levels can alter the response of the ER to estrogen or Tam (46). Our studies with HET/SAF-B certainly fit this model. We show that HET/SAF-B potentiates Tam’s antagonist activity, while overexpression of HET/SAF-B at high levels inhibits E2 and Tam agonist activities. Although HET/SAF-B’s interaction with ER is weaker in the presence of E2 as compared with Tam, we were able to detect repression of E2-activated ER, just as described for N-CoR (45) and REA (15). This repression probably represents inappropriate binding between ER and HET/SAF-B in the presence of E2 resulting from transient overexpression of HET/SAF-B. Under normal conditions we believe that Tam recruits HET/SAF-B to ER and that this association may be responsible, in part, for the antagonist effect of Tam.

Over the last couple of years it has become clear that transcriptional repression is an important strategy for fine regulation of growth, development, and differentiation. Despite the identification of corepressors, repressor motifs, and their targets, little is known about specific mechanisms of repression. Models that have been proposed include 1) interference with the formation or activity of the basal transcriptional machinery, 2) quenching of a transcriptional activator, and 3) induction of an inactive chromatin structure (reviewed in Ref. 49). It is likely that repression mediated by a corepressor like N-CoR is the result of a combination of these mechanisms. N-CoR is a large protein (270 kDa) that interacts with mSin3 and recruits histone deacetylase (21). Deacetylation results in conformational changes of the nucleosome structure, thereby limiting the accessibility of chromatin to the transcriptional machinery. In addition to its interaction with chromatin remodeling factors, Muscat et al. (50) have recently shown that N-CoR directly interacts with the basal transcription factors TFIIB, TAFII32, and TAFII70.

HET/SAF-B is a nuclear matrix protein with several recently described characteristics (31, 32, 35), which could be involved in repressive mechanisms. Like N-CoR, which interacts with multiple factors, HET/SAF-B is probably part of a multiprotein complex regulating ER activity. While the work described here does not directly address the mechanism of HET/SAF-B-mediated repression, some potential mechanisms can be considered.

First, HET/SAF-B has recently been shown to bind to the C-terminal domain of RNA pol II (35) in yeast two-hybrid systems. As hypothesized for the interaction of N-CoR and basal transcription factors (50), it is conceivable that HET/SAF-B locks the transcriptional initiation complex into a nonfunctional state. Second, HET/SAF-B-mediated repression might also involve changes in histone acetylation, since in our own preliminary experiments treatment with the histone deacetylase inhibitor trichostatin A (51) relieves HET/SAF-B-mediated repression (S. Oesterreich, unpublished results). Third, it is possible that the RNA-binding domain of HET/SAF-B is involved in repression. In addition to HET/SAF-B, other RNA-binding proteins, such as L7/SPA (45), hnRNP U (30), and more recently RNA itself, SRA (52), have been described as coregulators of nuclear hormone receptor action.

While HET/SAF-B shares some of the characteristics of other coregulators, it is possible that its repressive action results from its ability to associate with the nuclear matrix. The presence of specific binding sites for ER, also called "acceptor proteins", in the nuclear matrix was postulated several years ago after in vitro reconstitution experiments showed binding of the ER to the nuclear matrix to be saturable and of high affinity (53, 54). We have shown previously that 1) HET/SAF-B is associated with the nuclear matrix in biochemical fractionations (31); 2) HET/SAF-B and ER can both be cross-linked to scaffold attachment regions (55); and 3) ER can associate with the nuclear matrix as shown by direct visualization with a green fluorescent protein-tagged ER (56). In this paper we have described HET/SAF-B as a nuclear matrix protein/scaffold attachment factor that associates with the ER.

Finally, it is important to define the domains of the steroid receptors that are involved in the interaction with the nuclear matrix. Eggert et al. (30) demonstrated that the C terminus of the glucocorticoid receptor was sufficient for hnRNP U-mediated repression. In contrast, Tang et al. (57, 58) and van Steensel et al. (59) have shown that the DBD of the glucocorticoid receptor is required for interaction with the nuclear matrix, and that replacement of the GR-DBD by a Gal4DBD resulted in loss of hnRNPU-mediated repression. In a similar way, HET/SAF-B-mediated repression is lost when the ER-DBD is substituted by a Gal4DBD. Indeed, the ER-DBD is sufficient for HET/SAF-B-mediated repression. However, the repression is not a result of inhibiting ER’s ability to bind to DNA.

In summary, our study has revealed that the nuclear matrix protein/scaffold attachment factor HET/SAF-B directly binds to ER and inhibits its activity. The binding is stronger in the presence of Tam, as compared with no ligand, suggesting that HET/SAF-B-mediated corepression may be involved in the antiestrogenic effects of Tam.


    MATERIAL AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIAL AND METHODS
 REFERENCES
 
Plasmid Constructs and Chemicals
The cloning of the HET/SAF-B expression construct (31) and of the mammalian expression vector for full-length ER has been previously described (60). To generate an HA-tagged full length ER construct, ER was PCR-amplified (61) using the following primers: sense 5'-GCGAATTCATGGCTTACCCCTACGACGTC-CCCGACTACGCCATGACCATGACCCTCCAC-3' comprising the HA-tag, and nucleotides 1–18 coding for the ER, and the antisense primer was 5'-GATGAATTCCTCAGACTGTGGC-AGGGAA-3' comprising nucleotides 1770–1789 of the ER. The PCR product was cloned into pcDNA3.1/V5/His-TOPO (Invitrogen, Carlsbad, CA). To generate a GST-fusion protein, the full-length HET/SAF-B clone (31) was cloned into EcoRI sites of the pGEX-2TK gene fusion vector (Pharmacia Biotech, Piscataway, NJ). Bacterial expression vectors for GST-ER fusion proteins containing the AF1, DBD/Hinge, DBD, Hinge, and AF2/Hinge domains were generated by performing ligation reactions with the appropriate PCR products and EcoRI/BamHI-digested pGEX-2TK. The positions of the PCR primers (linked to EcoRI or BamHI sites) within the ER{alpha} cDNA (61) are: AF1 –sense (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) and antisense (519–540); DBD/Hinge –sense (519–540) and antisense (849–869); DBD –sense (513–533) and antisense (730–746); Hinge –sense (750–771) and antisense (844–863); AF2/Hinge –sense (756–775) and antisense (1769–1788). The AF1/DBD construct was a kind gift of Dr. S. Kato (62, 63). The gal4-luc construct (pfrluc) was purchased from Stratagene (La Jolla, CA). Constructs containing the activation domains (AF-1 and AF-2) of the ER fused to the gal4 DBD (amino acids 1–94) were a kind gift of Dr. O’Malley, and have been previously described (60). Briefly, the AF-1 domain of the ER was cloned upstream of the gal4DBD in pABgal94 (64) to create AF-1 gal4. The AF-2 domain of ER was cloned downstream of the gal4DBD to create AF2-gal4. Finally the AF-1 and AF-2 domains were cloned upstream and downstream, respectively, of the gal4DBD to create ER-gal4DBD. A construct containing a chimeric activator with the ER DBD and the activation region of VP16 was provided by Dr. P. Chambon and has been previously described (65). Finally, the ER-dependent promoter interference reporter plasmid was provided by Dr. B. Katzenellenbogen and has been described by Reese and Katzenellenbogen (41). The antiestrogen 4-hydroxytamoxifen (Tam) was a gift from Zeneca Pharmaceuticals (Macclesfield, UK). All other chemicals were purchased from Sigma (St. Louis, MO) unless stated otherwise.

Cell Culture and Transient Transfection
Human breast cancer cells (MCF-7), human hepatocyte carcinoma cells (HepG2) cells, and human osteosarcoma cells (Saos-2) were maintained in improved MEM (IMEM) supplemented with 5% FBS (Life Technologies, Gaithersburg, MD), 200 U/ml penicillin, 200 µg/ml streptomycin, 6 ng/ml insulin. COS-7 cells were maintained in DMEM +10% FBS, 200 U/ml penicillin, 200 µg/ml streptomycin, 6 ng/ml insulin. For reporter assays, cells were transiently transfected using Fugene (Roche Clinical Laboratories, Indianapolis, IN) following the manufacturer’s protocol. One day before transfection cells were plated at 8 x 105 in six-well plates. For E2 induction experiments the cells were plated in serum-free medium which consisted of phenol red-free IMEM + 10 mM HEPES, pH 7.4 + 1 µg/ml fibronectin (Life Technologies) + trace elements (Biofluids, Rockville, MD) + 1 µg/ml transferrin (Life Technologies). Cotransfections were performed using 1 µg reporter plasmid, 100 ng ß-galactosidase (ß-gal) expression vector, and HET/SAF-B and ER plasmids as indicated in the figure legends for each experiment. Twenty-four hours after transfection, the medium was replaced with serum free medium containing the appropriate ligand. Forty-eight hours later cells were washed twice with PBS, and luciferase activity was measured using the Luciferase kit from Promega Corp. (Madison, WI). ß-gal activity was measured as described (31), and the luciferase activities were normalized by dividing by the ß-gal activity to give relative luciferase units. For determining CAT activity, we used a CAT enzyme-linked immunosorbent assay from Roche Clinical Laboratories and followed the manufacturer’s instructions. Values were corrected for protein concentrations and are presented as relative CAT activity. For transient transfections, triplicate samples were measured in each experiment, and the data are presented as the average ± SEM and are representative of at least three independent experiments. For coimmunoprecipitation experiments, COS-7 cells were plated at 0.6 x 106 into 10- cm dishes, and transiently transfected with 5 µg expression plasmids for HET/SAF-B and ER-HA. Twelve hours later the medium was replaced with phenol red-free IMEM + 5% charcoal-stripped serum and ligands as indicated in the figure legends. The cells were lysed 24 h later.

In Vitro Protein-Protein Interaction (GST Pull-Down)
Overnight cultures of Escherichia coli BL21 expressing the appropriate fusion constructs were diluted 1:10 in LB medium and incubated for 1 h. GST only or GST-fusion proteins were induced for 2.5 h with 0.1 mM isopropyl-ß-D-thiogalactoside, followed by centrifugation, and resuspended at 1:100 in cell suspension buffer (1x PBS, 100 mM EDTA, pH 8.0, 0.1 mM phenylmethylsulfonyl fluoride, 0.2 µg/ml pepstatin, 0.2 µg/ml leupeptin, 0.2 µg/ml aprotinin, 0.2 µg/ml antipain). Cells were sonicated and then centrifuged for 10 min at 4 C, and 400 µg of crude E. coli bacterial extract proteins were incubated with 60 µl glutathione Sepharose 4B beads (50% slurry, Pharmacia Biotech) (1 h, 4 C). For the binding assay, the beads were incubated in IPAB buffer (150 mM KCl, 0.1% Triton X-100, 0.1% NP40, 5 mM MgCl2, 20 mM HEPES, 20 µg/ml BSA, protease inhibitors), and ligand was added as indicated in the experiments. In vitro transcription-translation mixture (TNT kit, Promega Corp.) containing 35S-methionine was programmed with HET/SAF-B and ER expression plasmids. Lysates (10 µl) were incubated with 60 µl equivalent amounts of GST proteins (as assessed by Coomassie staining) at 4 C for 1 h. The beads were washed three times with IPAB buffer without BSA. Bound proteins were eluted in SDS sample buffer, resolved by SDS-PAGE, and visualized by fluorography.

Generation of Anti-HET/SAF-B Monoclonal Antibodies
The peptide used for generation of a monoclonal antibody (mAb) to HET-SAF/B was identical to the peptide used to generate a polyclonal antibody described previously (31). The mAb was generated at the UTHSCSA Institutional Hybridoma Facility following methods described by Kohler (66) and Oi and Herzenberg (67). Briefly, spleen cells from two BALB/c female mice immunized subcutaneously three times with 50 µg keyhole limpet hemocyanin-coupled peptide in Freund’s adjuvant were fused with NS-1 myeloma cell line. A 50% PEG solution was added in a drop-wise manner. The subsequent dilution was performed in selection media (hypoxanthine, aminopterin, thymidine-containing medium), and 10 days later supernatants were screened for relevant antibody using the A156 HET/SAF-B peptide coupled to an alternative carrier (BSA). Culture supernatant from clone 6F7 was purified using the ImmunoPure (A/G) IgG purification kit (Pierce Chemical Co., Rockford, IL).

Coimmunoprecipitation
MCF-7 cells were plated at 2 x 106 cells in 10-cm dishes. The next day the media was changed to media containing 5% charcoal-stripped FCS and ligand as indicated in the figure legends. Twenty-four hours later the cells were lysed in low-stringency (LS) buffer (PBS, 0.1% NP40, protease inhibitors), and HS buffer (20 mM Tris, pH 7.4, 50 mM NaCl, 1 mM EDTA, 0.5% NP40, 0.5% SDS, 0.5% deoxycholate, and protease inhibitors), followed by sonication. Sodium tetrathionate (50 µM) was added to the lysis buffer since it is known to selectively stabilize interactions between hormone receptors and the nuclear matrix (68); however, its addition is not essential for coimmunoprecipitation of ER and HET/SAF-B. The lysate was precleared with 50 µl protein G-agarose for 30 min at 4 C, and then incubated overnight with 7 µl HET/SAF-B mAb at 4 C. Protein G agarose was added for another 4 h, and the beads were pelleted and washed three times with the indicated buffer. For immunoprecipitation of HA-ER we precleared the lysates with 20 µl protein A-agarose, incubated with 5 µl HA antibodies (Babco, Richmond, CA) overnight, and finally added 20 µl protein A-agarose. Bound proteins were eluted in SDS sample buffer, subjected to SDS-PAGE, and analyzed by immunoblotting (see below). For quantification, the scanned image was analyzed using NIH Image 2.0. The background intensity was subtracted from the intensity of the ER band in the immunoprecipitation, and this arbitrary number was divided by the intensity for the ER band in the immunoblot. The result is represented as an arbitrary number of the intensity ratio of immunoprecipitated ER to immunoblotted ER in the lysates.

Immunoblotting
Proteins were resolved on 8% SDS-PAGE and electrophoretically transferred to nitrocellulose. The membrane was blocked in PBS/0.1% Tween 20 (PBST) + 5% milk for 1 h at room temperature. HET/SAF-B, ER (6F11, Novacastra, Newcastle upon Tyne, UK), and HA-(Babco, Richmond, CA) antibodies were diluted at 1:1000, 1:100, and 1:1000, respectively, in PBST + 5% milk. After incubation for 1 h, the membrane was washed six times for 5 min each time with PBST, the membrane was incubated with horseradish peroxidase-linked anti-mouse IgG at 1:1000 (Amersham Pharmacia Biotech, Arlington Heights, IL) in PBST + 5% milk, washed six times for 5 min each time, and the signal was developed using enhanced chemiluminescence according to the manufacturers instructions (Pierce Chemical Co.).


    ACKNOWLEDGMENTS
 
We would like to thank Drs. M. Gottardis, B.W. O’Malley, B. Katzenellenbogen, P. Chambon, and S. Kato for providing constructs [ERE-tk-luc (M.C.), ER-gal4 (B.O’M.), pCMV(ERE)2CAT (B.K.), GalVP16/pSG5 (P.C.), ER(C)-VP16 (P.C.), pGEX2T-AF1/DBD (S.K.)]. The authors are grateful to L. Hernandez for excellent technical assistance and to Dr. G. Chamness for critical reading of the manuscript. We also would like to thank Dr. C. Smith for providing access to laboratory facilities at Baylor College of Medicine.


    FOOTNOTES
 
Address requests for reprints to: Steffi Oesterreich, Baylor College of Medicine, Breast Center, Alkek MS:600, One Baylor Plaza, Houston, Texas 77030. E-mail: steffio{at}bcm.tmc.edu

This work was supported by an NIH Howard Temin Award (KO1 CA-77674) and a Department of Defense Grant (DAMD17–98-1–8340) to S.O., a Breast Cancer Specialized Program of Research Excellence (PHS P50 CA-58183) and 5P01 CA30195 to C.K.O., a Susan G. Komen Breast Cancer Foundation Award to A.V.L., a NIH Cancer Center Support Grant (P30 CA-54174), and the Medical Research Council of Canada and Manitoba Health Research Council (J.R.D.). T.H. was supported by a Department of Defense Grant (DAMD 17–945-4112).

1 Present Address: Breast Center/Department of Medicine and Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030. Back

Received for publication May 28, 1999. Revision received November 19, 1999. Accepted for publication December 9, 1999.


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