Identification of a Third Autonomous Activation Domain within the Human Estrogen Receptor
John D. Norris,
Daju Fan,
Sandra A. Kerner and
Donald P. McDonnell
Department of Pharmacology, Duke University Medical Center,
Durham, North Carolina 27710
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ABSTRACT
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Using a genetic selection system established in
the yeast Saccharomyces cerevisiae, we have isolated, by
random mutagenesis of the human estrogen receptor (ER), six mutants
that display constitutive transcriptional activity. All of the mutants
identified contained single base insertions or deletions leading to
frameshift mutations, resulting in receptor truncations within the
hormone-binding domain between amino acids (aa) 324351.
Interestingly, an ER mutant (aa 1282) was transcriptionally inactive
in yeast, suggesting that a domain important for transcriptional
activity lies between aa 282 and 351 within human ER. Deletions
representative of the mutants isolated in the yeast system were created
in mammalian expression vectors and examined for transcriptional
activity in animal cells to determine the physiological relevance of
this domain. Receptors truncated at aa 282 were either weakly active or
inactive; however, an ER deletion at aa 351 was approximately 50% as
active as wild type ER (induced with estrogen). Furthermore, a chimeric
receptor consisting of the DNA binding domain of GAL4 fused to aa
282351 of the human ER was transcriptionally active on a GAL4
reporter. We conclude, therefore, that an autonomous activation domain
(referred to as AF2a), functional in both yeast and mammalian cells,
lies between aa 282351 of the human ER.
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INTRODUCTION
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The human estrogen receptor (ER) is a member of the steroid
receptor family of ligand-activated nuclear transcription factors (1).
In the absence of hormone, ER resides in a transcriptionally inactive
form within the nucleus of target cells (2). Interaction with its
cognate ligand results in a conformational change within the receptor,
allowing the displacement of associated heat shock proteins and
permitting receptor homodimerization (3, 4). In this activated state,
ER can interact with target gene promoters directly, by binding to
specific estrogen response elements (5), or indirectly through its
association with other DNA-bound proteins (6, 7, 8, 9, 10). The biological
result of these interactions can lead to either up-regulation or
down-regulation of target genes, depending on the cell and promoter
context (11).
We and others have demonstrated that ER contains at least two distinct
activation functions (AFs) that determine its transcriptional activity
in target cells (11, 12). These ER activation sequences located within
the amino (AF-1) and carboxyl (AF-2) terminus of the receptor function
in a cell- and promoter-specific manner to manifest the activity of
bound ligands (13). Specifically, it has been shown that estradiol acts
as an ER agonist in contexts where either or both AFs are required. In
contexts where AF-1 alone is required, tamoxifen and other
triphenyl-ethylene-derived antiestrogens function as partial agonists,
whereas in contexts where AF-2 alone is required, tamoxifen behaves as
an ER antagonist (11, 13, 14, 15). The pure antiestrogen ICI 182,780
inhibits the activity of AF-1 and AF-2 and may also be involved in
receptor degradation (11, 16, 17). However, it has become apparent
recently that the ability to differentially regulate AF-1 and AF-2
cannot account for all of the biological effects of ER ligands.
Specifically, we have shown that a novel ER antagonist GW5638 does not
activate either AF-1 or AF-2, yet functions as a full agonist in the
bone and cardiovascular systems (T. M. Willson, J. D. Norris, B. L.
Wagner, I. Asplin, P. Baer, H. R. Brown, S. A. Jones, B. Henke, H.
Sauls, S. Wolfe, D. Morris, and D. P. McDonnell, submitted). This would
suggest that ER can operate in a nonclassic manner in these tissues or
alternately that additional sequences within ER contribute to its
transcriptional activity. Previous studies from our laboratory prompted
us to pursue the latter hypothesis. Specifically, we observed that in
the context of wild type ER (wtER), AF-2 was not required for AF-1
function. However, a construct containing the N-terminal AF-1 alone was
not functional as a transcriptional activator in yeast or mammalian
cells (11, 19). We hypothesized, therefore, that sequences in addition
to AF-1 and AF-2 may be involved in ER transcriptional activity.
Because similar findings were obtained in both yeast and mammalian
cells, we considered that a genetic screen in yeast would be
appropriate to examine this activity. Using a genetic selection system
developed in the yeast Saccharomyces cerevisiae, we have
identified a series of ER mutants that lead to the definition of a
third activation sequence which functions in an autonomous manner. The
discovery of an additional activation domain that functions in animal
cells may help to explain the tissue-specific agonist activity of
compounds that previously have been shown to inhibit AF-1 and AF-2.
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RESULTS
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Identification of ER Mutants That Function Independently of
AF-2
We have developed a genetic selection system in the yeast S.
cerevisiae to screen for mutations within ER that manifest
transcriptional activity in the absence of a functional AF-2. To
accomplish this, we created an ER-dependent survival system in which
the yeast HIS3 gene was placed under control of a chimeric estrogen
response element (ERE)/GAL1 promoter (Fig. 1
). This
construct permits 17ß-estradiol-dependent growth of the ER containing
his3 yeast strain YPH500 ER (20, 21). To permit a
quantitative estimation of ER responsiveness, an additional reporter
containing the Escherichia coli ß-galactosidase gene under
the control of the ERE/CYC1 promoter was also introduced. This tester
strain, containing both reporters, was then used to screen for ER
mutants that displayed AF-2-independent transcriptional activity.

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Figure 1. Identification of ER Mutants That Demonstrate
Transcriptional Activity in the Absence of a Functional AF-2
A library of ER mutants was created using the E. coli
mutator strain XL-1 red. Resultant DNA was transformed into the yeast
strain YPH500, and colonies that grew in the presence of raloxifene
(keoxifene) were selected for further analysis as described in
Materials and Methods.
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Libraries of ER mutants were created by random mutagenesis and
transformed into the yeast tester strain. To identify mutants that grew
in the absence of a functional AF-2, we added raloxifene (an AF-2
antagonist) to the selection media (Fig. 1
). Although ER antagonists do
not effectively inhibit agonist-bound receptor in yeast and most
exhibit partial agonist activity, we were able to use raloxifene in
these screens to activate ER as it does not appear to function as an
agonist in yeast. In addition, this compound was shown to be a full
AF-2 antagonist in mammalian cells. Aminotriazole (a competitive
inhibitor of the HIS3 enzyme) at a concentration of 1 mM
was also added to increase the stringency of the screen. Yeast colonies
that grew under these conditions within 5 days were selected for
further analysis. Two successive rounds of primary screening,
representing 100,000 individual transformants, were performed. A
secondary screen was performed under the same conditions resulting in
the selection of 23 individual colonies that demonstrated ER-dependent
growth in the presence of raloxifene. The transcriptional activities of
the resultant mutants were constitutive and thus unaffected by the
addition of ligand. The receptor expression plasmids from each of these
colonies were recovered, and the ER coding region was sequenced.
Characterization of the Mutants Identified in Yeast
DNA sequence analysis of the 23 mutants identified represented six
different alleles generated by frameshift mutations at the boundary of
the D (hinge) and E (ligand-binding) regions of the ER-coding sequence
(Table 1
). All of the mutations contained a single base
insertion or deletion within an 80-bp region. Interestingly, ER62 in
which a single C residue was deleted was isolated 13 times, and ER 65,
which contained an inserted C residue, was found four times in our
libraries. The six different alleles predicted receptor truncations
with translation terminating at stop codons in the shifted frame
yielding proteins of 339, 342, and 374 amino acids. Each of the six
mutants were then remade in the yeast expression vector so that stop
codons were introduced after the last authentic amino acid of ER (Table 1
) to avoid any inaccuracies caused by the extraneous sequences. We
confirmed that these mutations in the ER-DNA coding sequence gave rise
to proteins of the predicted molecular mass by performing Western
immunoblot analysis of protein extracts from representative isolated
yeast colonies (Fig. 2A
). The wtER migrates as an intact
species at 65 kDa, whereas all of the mutants migrated at their
predicted molecular masses (3842 kDa). The additional minor bands are
likely to be proteolytic fragments derived from the larger receptor
species. Additionally, we observed that the expression level of wtER
and that of the mutants were approximately the same in this strain.

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Figure 2. Characterization of the Biochemical Properties of
the AF-2-Independent Mutations
A, Expression vectors for each of the mutants M324-M351, wtER, and
ERN282g were transformed separately into BJ5457, a protease-deficient
strain of S. cerevisiae. Extracts were prepared from the
resultant strains and examined by Western immunoblot analysis using
anti-ER antibody H226. Extracts were also prepared from an empty
expression vector as control. B, Expression vectors for each of the
mutants (M324-M351), ERN282g, and wtER were transformed separately into
YPH500 together with the ERE-CYC1-ß-GAL reporter plasmid. The
transcriptional activity of wtER was assayed in both the presence and
absence of 10-6 M 17ß-estradiol. The data
shown for each of the mutants represent constitutive activity since
these proteins are unable to bind ligand. Experiments were performed
several times in triplicate and representative data are shown. The
percent error in all yeast transcriptional assays was less than 20%.
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We then measured the transcriptional activity of the ER mutants
on the ERE-ß-galactosidase reporter plasmid and compared their
activity to that of the wtER (induced with 17ß-estradiol) containing
both AFs and ERN282g (19) containing only AF-1. As shown in Fig. 2B
, wtER functions as an efficient hormone-dependent transcription factor
whereas ERN282g is transcriptionally inactive under identical
conditions. However, all six classes of ER truncations displayed
transcriptional activity that was approximate to or greater than wtER.
Thus, it appears that in yeast, sequences contributing to the
transcriptional activity of ER lie between aa 282 and 351. These data
are in agreement with the work of Pierrat et al. (22) who
also found that truncations within this region of ER resulted in
receptor mutants that displayed constitutive activity in a yeast
system. They referred to this sequence as AF2a, a nomenclature that we
have adopted.
In Vitro DNA-Binding Affinity of wtER and ER
Mutants
Before concluding that the region within ER between aa 282 and 351
was directly involved in transcriptional activity, we examined whether
this region was important for DNA binding. One possible explanation for
the inability of ERN282g to activate transcription, as opposed to the
ability of isolated truncation mutants to activate transcription, would
be that the region between aa 282 and 351 enhanced the ability of the
mutant to bind DNA, allowing for transcriptional activation by AF-1.
This was examined using in vitro gel shift analysis. Yeast
were transformed with expression vectors for wtER, ERN282g, or M351
(one of the most active mutants). Nuclear extracts were prepared from
these yeast, and the ability of the expressed wtER or ER mutant to
interact with a radiolabeled vitellogenin probe was measured. The
results of this analysis are shown in Fig. 3
. Extracts
containing wtER, but not control extract, were able to interact with
the vitellogenin probe. The specificity of this association was
confirmed by showing that the resultant band was not affected by the
addition of a 100-fold molar excess of unlabeled progesterone response
element (PRE). Additionally, it was shown that the resultant complex
could be supershifted by an anti-ER antibody (H226), indicating that
the complex contained ER. Under identical conditions, extracts
containing either ERN282g or M351 were not capable of high-affinity DNA
interactions with the labeled ERE, although ERN282g did display some
affinity for DNA. This may be due to the lack of more C-terminal
sequences that contain a strong dimerization domain (23). However, upon
the addition of anti-ER antibody, dimerization and high-affinity
interactions were established. Stabilization of DNA/ER interactions by
antibody are consistent with the work of others (17). Contrary to what
was expected, it appeared that ERN282g interacted with DNA in a more
efficient manner than did M351, yet they demonstrate dissimilar
transcriptional activities. Thus, it appears in this system that
DNA-binding affinities do not correlate well with transcriptional
activity. Moreover, it indicates that the dramatic difference between
the transcriptional activities between ERN282g and M351 does not appear
to be the result of an increased affinity for target DNA. These data
therefore suggest that the region between aa 282 and 351 is important
for efficient ER transcriptional activity.

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Figure 3. Characterization of the in Vitro
DNA-Binding Properties of wtER, ER-M351, and ERN282g
Recombinant proteins corresponding to wtER, ERN282g, and M351 were
obtained after transformation in yeast (BJ5457) and isolation of
protein extracts. The ability of these proteins to interact with a
radiolabeled vitellogenin ERE was determined by electrophoretic
mobility shift assay. Extracts from nontransformed yeast were used as
control. The specificities of the DNA/ER complexes were tested using
100-fold molar excess of a progesterone response element (PRE), and the
presence of ER within the complexes was ascertained using anti-ER
antibody H226 (Ab) to supershift the complex.
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The Sequence between aa 282 and 351 Contributes to ER
Transcriptional Activity in Mammalian Cells
We next sought to determine whether the activation sequence
identified in the yeast system also functions in a mammalian cell line.
To accomplish this, representative mutants isolated in yeast were
subcloned into the mammalian expression vector pRST7ER (20). Transient
transfections were performed in the human liver cell line HepG2. Cells
were cotransfected with both a receptor expression plasmid and a
reporter plasmid containing three EREs placed upstream of an
enhancerless luciferase reporter vector containing only a TATA sequence
element. The results of this analysis are shown in Fig. 4
. As expected, wtER functioned as a potent
transcriptional activator in the presence of 17ß-estradiol. Mutants
terminating at aa 282, 325, or 335 were either weakly active or
inactive compared with wtER in the absence of hormone. However, mutants
terminating at aa 343 and 351 showed considerable transcriptional
activity with the mutant terminating at aa 351 possessing more than
50% activity of wtER induced with ligand. These results support the
finding in yeast that an efficient transcriptional domain lies between
aa 282 and 351 (putative AF2a) in the human ER.

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Figure 4. The AF2a Sequence Identified in Yeast Is Also
Functional in Mammalian Cells
Plasmids expressing wtER (pRST7-ER) and ER mutants (indicated in the
figure) were cotransfected into the human liver cell line HepG2 along
with a reporter construct consisting of three EREs placed upstream of a
TATA sequence element initiating transcription of the luciferase gene.
Transcriptional activities of wtER are shown in the presence and
absence of 10-7 M 17ß-estradiol.
Transfections were normalized for efficiency using a constitutive
ß-galactosidase reporter plasmid (pCMV-ß-GAL) (20). Representative
assays are shown in which triplicate transfections were performed;
error bars represent the SEM.
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Amino Acid 282351 Exhibits Autonomous Transcriptional Activity in
Mammalian Cells
Having demonstrated that aa 282351 of the human ER is important
for the transcriptional activity of truncation mutants in both yeast
and mammalian cells, we next wanted to determine whether this domain
could act independently of AF-1 (N-terminal domain of ER). To define
this sequence within ER as an activation domain (or AF), we felt that
it was necessary to demonstrate that it can activate transcription in
an autonomous fashion. To accomplish this, we fused aa 282351 of the
human ER to the DNA-binding domain (DBD) of the yeast transcription
factor GAL4. This chimeric protein was then assayed for transcriptional
activity in a transient transfection system using a reporter plasmid
containing five GAL4 DNA-binding sites placed upstream of a TATA
sequence element initiating transcription of the luciferase gene. For
comparative purposes, the AF-1 sequence between ER aa 52149 (24) and
the hormone binding domain (AF-2) consisting of ER aa 312595 were
also fused to the DBD of GAL4. This analysis was performed using
several cell lines to determine whether there was any cell
specificity within the defined parameters of this experiment. In
all cell lines tested (Fig. 5
, A, B, and C), the fusion
containing the putative AF-2a sequence was transcriptionally active in
an autonomous manner. As expected, the ER-hormone-binding domain (HBD)
fusion protein (containing AF-2) functioned in a ligand-dependent
manner in all cells (Fig. 5
, A, B, and C). AF2a appeared to be most
active in the HepG2 and Hela cell lines where it induced expression of
the reporter 36- and 25-fold, respectively (Fig. 5
, A and B). The GAL4
DBD is included as control and shows no transcriptional activity on its
own. Interestingly, in HepG2 cells where tamoxifen functions as a
partial agonist (11, 15), ER-AF1 is clearly the dominant AF, and in
Hela cells where tamoxifen acts as a complete ER antagonist, ER-AF-2 is
dominant (Fig. 5
, A and B). AF-1 also appears to be the dominant AF in
CV-1 cells; however, tamoxifen shows no agonist activity in this cell
line (14). The transcriptional activity of AF2a in CV-1 cells was much
weaker than that of the other cell lines tested (Fig. 5C
). In light of
these results, it is interesting to speculate that in order for
tamoxifen to display agonist activity through AF-1, a fully active AF2a
is required. Regardless, we have shown for the first time that the
region marking the boundary between the D (hinge) and E (HBD) domains
of ER act as a bona fide transcriptional activation domain
in animal cells.

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Figure 5. Amino Acids 282351 Representing the ER AF2a
Sequence Function in an Autonomous Fashion in Mammalian Cells
Vectors expressing fusion proteins consisting of the DNA binding
domain (DBD) of GAL4 and ER fragments representing AF-1 (aa 51149),
AF-2 (aa 312595), and AF2a (aa 282351) were cotransfected into (A)
HepG2, (B) Hela, or (C) CV-1 cells along with a reporter construct
consisting of five GAL4 DNA-binding sites placed upstream of a tata
sequence element initiating transcription of the luciferase gene.
Addition of 17ß-estradiol was used to activate the GAL4 fusion
containing the HBD of ER. An expression vector containing only the GAL4
DBD was included as control. Induction values were calculated by
dividing the activity of individual ER-AF fusions by the activity of
the GAL4-DBD and are indicated in the figure within the corresponding
bar. Representative assays are shown in which triplicate transfections
were performed; error bars represent the SEM.
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DISCUSSION
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Several constitutive human ER mutants were obtained using a
phenotypic screen in yeast. Although the primary goal of the screen was
to isolate ER mutants that displayed AF-2-independent or
raloxifene-dependent activation, only mutants corresponding to a coding
sequence terminating at the boundary between the D and E regions of the
receptor were detected. Perhaps the method of mutagenesis or the
possibility that such raloxifene-dependent mutants are not viable
resulted in the selection of mutants that displayed hormone-independent
activity. Similar results were obtained by Pierrat et al.
(22) using a yeast-screening system. Interestingly, mutations truncated
at aa 282 appear to have little or no activity in yeast unless the
yeast transcriptional repressor complex SSN6/TUP1 (19) is
disrupted, indicating that AF-1 (N terminus) alone is not sufficient
for transactivation. However, extension of the receptor to aa 351
results in a more efficient transcription factor that no longer
requires AF-2. The reason for these differences in ER transcriptional
activities cannot be explained by differences in DNA binding or protein
expression levels, as their affinities for DNA and expression levels
both appear to be roughly equivalent. This would suggest that an
efficient transcriptional activation sequence lies between aa 282351
(AF2a) and that perhaps in yeast it is required in order for full
activation of the N-terminal AF-1. The fact that full transcriptional
activation (equivalent to that obtained by ligand-induced wtER) is
obtained by these truncated mutants in yeast would also suggest that
AF-1 and AF2a are the dominant AFs in this biological system.
Although yeast has proven to be a valuable tool in the dissection of
the functional domains of the nuclear receptors, including ER (4, 25),
any data collected there must be tempered by the fact that this is only
a model system as yeast do not contain any proteins homologous to these
receptors. Therefore, we extended our studies of the AF2a region to
ascertain whether the region identified in yeast could function also in
mammalian cells. We were initially surprised to find that the AF2a
identified in the yeast screen functioned analogously in the animal
models; previous studies by Pierrat et al. (22) and Webster
et al. (26) were unable to show activity in mammalian cells.
Webster fused the protein sequence coded by ER exon 4 (aa 254366) to
a GAL4 DBD and did not show any autonomous function. It is possible
that the contrast between our data and previous studies could be
accounted for by differences in reporter constructs or cell lines as we
and others have determined that both of these parameters play a major
role in determining the transcriptional activity of any given AF (11, 13, 27). It is also likely that minor amino acid sequence variations in
the region selected for analysis could account for the variance in the
data obtained by a particular study. Nevertheless, we were able to
demonstrate that aa 282351 were not only important for the
activity of truncated mutants (inactivity of ERN282g as opposed to
activation by 351T), but that this amino acid sequence, when fused to a
GAL4 DBD, could function as an autonomous activation domain. We did
detect some differences in the results regarding transcriptional
activation in yeast and mammalian cells (Figs. 2A
and 4
). Mutants
terminating at aa 324 and 335 demonstrate transcriptional activation
comparable to wtER in yeast, yet are inactive when analyzed in the
HepG2 cell line. The contrast in transcriptional activity between these
two biological systems may be the result of interactions with different
coactivators.
Thus far, no proteins interacting with the N-terminal (AF-1) domain of
the human ER have been identified. The general transcription factor
TAFII30, however, has been shown to associate with the
hinge region of ER in vitro (28), and homologous regions of
related nuclear transcription factors have been implicated in the
binding of nuclear corepressors such as N-CoR (29). Moreover, a
transactivation domain within the glucocorticoid receptor called
2
is located within a homologous region equivalent to the AF2a sequence
isolated in ER (30). Interestingly, a codon 351 mutant (Asp>Tyr)
isolated from a human breast tumor line demonstrated increased
agonistic activity of a fixed-ring tamoxifen analog (31). Thus, this
often overlooked region of the receptor appears to be more important in
transcriptional regulation than previously thought.
The transcriptional activities of AF-1 and AF-2 are greatly influenced
by their cellular and promoter context. Estradiol acts as an ER agonist
regardless of the cellular context, whereas antiestrogens such as
tamoxifen behave as either complete antagonists or partial agonists,
depending on the activity of AF-1 within a given environment (11, 13).
Thus, different ligands demonstrate a differential requirement for AF-1
and AF-2. The recent cloning of proteins that interact with AF-2 (HBD)
of the nuclear receptors has provided insight into the way in which
activation sequences mediate their activities (32, 33, 34). For instance,
it has been shown that in the presence of estradiol, ER can engage the
receptor-interacting protein, mSUG1, whereas tamoxifen, an AF-2
antagonist, disrupts this interaction (35). However, the core AF-2
sequence postulated to mediate the transcriptional activity of the HBD
(36) may not be the only activation sequence within this region. A
recent study demonstrating that SRC-1 can interact with an ER mutant
devoid of the core AF-2 sequence (37) and our own data showing that
GRIP (glucocorticoid receptor interacting protein) can interact with
mutants in which the core AF-2 sequence has been destroyed by point
mutations (our unpublished observations) would indicate that these
receptors may indeed contain many transcriptional activation sequences
and may interact in different ways with receptor coactivators. Thus, it
is likely that nuclear receptor transcriptional activity in a given
cellular environment may ultimately depend on the relative expression
of a multitude of coactivators and corepressors. These findings,
coupled with our current study identifying a previously unrecognized AF
(AF2a) within mammalian cells, could help to explain why compounds such
as raloxifene (14) and GW 5638 (18), which do not permit AF-1 or AF-2
activity, function as full agonists in the bone and cardiovascular
systems. It is possible that the cofactor environment within relevant
tissues permit AF2a to function as a dominant activator. However, until
the target cells and genes in these systems are identified, this
hypothesis cannot be tested.
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MATERIALS AND METHODS
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Enzymes and Chemicals
Restriction and modification enzymes were obtained from Promega
(Madison, WI) Boehringer Mannheim (Indianapolis, IN), or New England
Biolabs Inc. (Beverly, MA). Raloxifene was kindly provided by Dr. Eric
Larsen (Pfizer Inc., Groton, CT). 17ß-Estradiol and 3-aminotriazole
were purchased from Sigma (St. Louis, MO). Oxaliticase was purchased
from Enzogenetics (Corvallis, OR). XL-1-Red cells were purchased from
Stratagene (La Jolla, CA). PCR reagents were purchased from Perkin
Elmer Cetus (Palo Alto, CA) or Promega. Anti-ER antibodies for mobility
shift assays and Western blots were a gift from Dr. Geoffrey Greene
(Ben May Institute, Chicago, IL).
Yeast Plasmids and Strains
Yeast strain BJ5457 (MAT
ura352 trp1 lys2801 leu2
1
his3
200 pep4::HIS3 prb1
1.6R can1 GAL) was used to
express wtER and ER mutants. All screening was performed in yeast
strain YPH500 (MAT
ura352 lys2801 ade2101 trp1-
63
his3-
200 leu2-
1). YPH500 ER was obtained by transforming YPH500
with YEpE22 (21). Reporters pBM2389 (21) and YRpE2 (38) have been
described previously.
Random, Site-Directed, and Oligo-Directed Mutagenesis
Random mutations within ER were generated according to the
manufacturers instructions by transforming YEpE22 into the mutator
E. coli strain XL1-Red. ER mutants 324c, 335c, 343g, and
351t were created by insertion of YEpE22 into m13mp19 followed by
site-directed mutagenesis according to the manufacturers protocol
(Bio-Rad, Hercules, CA). The resulting mutants were then subcloned into
pRST7-ER (20). GAL4-ER fusions were generated using oligo-directed
mutagenesis as previously described (15).
Identification of ER Mutants Using a Phenotypic Screen in
Yeast
Randomly mutated YEpE22 plasmid DNA, along with YRpE2
(ERE-CYC-ß-galactosidase) and the survival plasmid pBM2389, were
introduced into the yeast strain YPH500 by the lithium acetate protocol
(20). Transformants were plated on synthetic defined media lacking
leucine, tryptophan, histidine, or uracil. In addition, 1
mM 3-aminotriazole, 50 µM CuSO4,
and 10-6 M raloxifene (dissolved in ethanol)
were added to the media. Colonies appearing within 5 days were selected
for subsequent analysis. More than 100,000 transformants were
screened.
ß-Galactosidase Assays in Yeast
The transcriptional activities of ER and ER mutants in yeast
were assayed on the ERE-CYC1-ß-galactosidase reporter as previously
described (21). Briefly, mutants were grown overnight in the
appropriate media ± hormone and assayed for ß-galactosidase
activity.
Bandshift Assays
Cell extract containing wtER or ER mutant proteins were isolated
from yeast (BJ5457). Labeled probe (10,000 cpm) was incubated with
yeast extract in a binding buffer consisting of 10 mM
HEPES, pH 7.9, 100 mM KCl, 1 mM dithiothreitol,
0.5 mM EDTA, 2.5 mM MgCl2, 6%
glycerol, and 2% Ficoll. The reactions were allowed to proceed at room
temperature for 10 min. Addition of antibody or competing DNA to the
protein extract occurred before incubation with the labeled probe. The
samples were then subjected to electrophoresis (150 V at room
temperature) in a nondenaturing 5% polyacrylamide gel in 0.5x
Tris-borate-EDTA. Autoradiograms were prepared from dried gels.
Cell Culture and Transient Transfection Assays
HepG2, CV-1, and Hela cells were maintained in modified Eagles
medium (Life Technologies, Grand Island, NY) supplemented with 10% FCS
(Life Technologies). Cells were plated in 24-well plates (coated with
gelatin for transfections of HepG2 cells) 24 h before
transfection. DNA was introduced into the cells using lipofectin (Life
Technologies) as described previously (20).
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ACKNOWLEDGMENTS
|
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The authors would like to thank Maria Huacani, Markus O. Imhof,
and Xiao-Fan Wang for kindly providing plasmids 3X-ERE-tata-Luc,
pBK-cmv-GAL4 (DBD), and 5X-GAL4-tata-Luc, respectively.
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FOOTNOTES
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Address requests for reprints to: Donald P. McDonnell, Department of Pharmacology, Box 3813, Duke University Medical Center, Durham, North Carolina 27710.
This work was supported by NIH Research Grant DK-48807 (to D.P.M.).
Received for publication February 5, 1997.
Accepted for publication March 14, 1997.
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