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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
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIAL AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIAL AND METHODSREFERENCES |
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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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIAL AND METHODSREFERENCES |
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). First we incubated in
vitro transcribed and translated ER with full-length GST-HET/SAF-B
bound to glutathione-sepharose beads (Fig. 1A). 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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) 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. 1C, 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. 2A, 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. 2A, right
panel).
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).
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. 2C, 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. 2D, 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. 2D, 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. 2D, 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. 2E). 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. 2E, 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. 2, 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. 3A). The results in Fig. 3A
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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). 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. 3C.
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 3D 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. 4) to exclude a nonspecific repressor
effect of HET/SAF-B. As shown in Fig. 3, 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. 4A, left bars). In the same experiment the ERE-tk-promoter
was inhibited by overexpression of HET/SAF-B (Fig. 4A, right
bars), as shown previously (Fig. 3). 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. 4B), 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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(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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). 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. 5A, 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. 5B, 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. 5C). Constitutive expression of this
reporter construct was inhibited by coexpression of ER (Fig. 5C). 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.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIAL AND METHODSREFERENCES |
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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.
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MATERIAL AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIAL AND METHODSREFERENCES |
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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.).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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. 
Received for publication May 28, 1999. Revision received November 19, 1999. Accepted for publication December 9, 1999.
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