From the
We have previously reported that the transcriptional activation
function AF-1, located in the A/B region of the human estrogen
receptor, exhibits cell-type and promoter context specificity in both
animal cells and yeast. To further characterize AF-1, we have
constructed a number of deletion mutants spanning the A/B region in the
context of either the whole human estrogen receptor or the A/B region
linked to the GAL4 DNA binding domain, and tested their transcriptional
activity in chicken embryo fibroblasts and in yeast cells, two cell
types in which AF-1 efficiently activates transcription on its own.
Additionally, we utilized HeLa cells in which AF-1 is poorly active but
can synergize with the transcriptional activation function AF-2 located
in the hormone binding domain. We show that in animal cells the
``independent'' activity of AF-1 is embodied in a rather
hydrophobic proline-rich 99-amino acid activating domain (amino acids
51-149), whereas amino acids 51-93 and 102-149 can
independently synergize with AF-2. Interestingly, in yeast, three
discrete activating domains (amino acids 1-62, 80-113, and
118-149) are almost as active on their own as the whole A/B
region, indicating that multiple activating domains can operate
independently in yeast. Our study also demonstrates that, within the
context of the whole human estrogen receptor, the same AF-1 activating
domains are ``induced'' by either estradiol or
4-hydroxytamoxifen.
In eukaryotes, sequence-specific DNA binding proteins control
initiation of transcription and play a major role in tissue-specific
and temporal regulation of gene expression (for reviews, see Mitchell
and Tjian (1989), Carey (1991), Ham et al. (1992), Hori and
Carey (1994), and Tjian and Maniatis (1994)). Functional analyses have
shown that transactivators contain separable and interchangeable
domains responsible for DNA binding and for transcriptional activation.
Little is known concerning the nature of the activating domains (for
reviews, see Mitchell and Tjian (1989), Ptashne and Gann (1990), Carey
(1991), Hahn (1993), and Tjian and Maniatis (1994)). No strong primary
sequence similarity has been found between the activating regions of
different transcription factors. However, some activating domains are
characterized by their high content in acidic amino acids such as the
yeast transactivators GAL4 or GCN4, the human glucocorticoid receptor,
or the herpes simplex virus activator VP16 (Gill and Ptashne, 1987; Ma
and Patshne, 1987; Hope et al., 1988; Hollenberg and Evans,
1988; Triezenberg et al., 1988; Sadowski et al.,
1988; Gill et al., 1990; Cress and Triezenberg, 1991), which
may be arranged to form amphipathic
The estrogen
receptor (ER)
In the present
study, the AF-1 transcriptional activation function present in the hER
A/B region has been functionally dissected in animal cells and in
yeast. We have identified a hydrophobic, proline-rich, 99-amino
acid-long region, which is responsible for AF-1 activity in CEF cells
and for synergistic activation with AF-2 in HeLa cells. We also show
that shorter segments of the A/B region are sufficient for efficient
transcriptional activation in yeast. Finally, we provide further
evidence that estradiol (E2) and 4-hydroxytamoxifen have similar
effects on AF-1 activity within the context of the whole ER.
The
oligonucleotide 5`-GGCCGCAAGGTCGGAGGACTGTCCTCCGAAGCC-3`
3`-CGTTCCAGCCTCCTGACAGGAGGCTTCGGAGCT-5` containing a 17M GAL4 binding
site was cloned into the NotI- XhoI sites of pY0,
resulting in pY017M. The BglII fragments containing AB-GAL and
mutant AB-GAL sequences isolated from the pSG5b vectors (see above)
were cloned into the BglII site of pY017M. pYERE1/HEG0 has
been described (Tora et al., 1989b), and the HEG0 mutant
derivatives were cloned into pYERE1 as described for HEG0 (Tora et
al., 1989b).
In animal
cells, transcriptional activation by AB-GAL, HEG0 (wild-type hER, see
Tora et al. (1989b)) and their mutant derivatives was
investigated by transfection of the corresponding expression vectors
together with specific reporter plasmids. The amount of expression
vector used was such that the reporter plasmid was in excess relative
to the transactivator, so that the chloramphenicol acetyltransferase
(CAT) activity of the deletants compared with that of the parental
activator was not dependent on the amount of expression vector (see
Tora et al. (1989a); similar relative levels of expression
were obtained by transfection of 20, 100, or 500 ng of expression
vectors, data not shown). For CAT activity measurement, cell extracts
were standardized for transfection efficiency using the
In yeast, AB-GAL, HEG0, and their respective
deletion, mutants were expressed from PY017M (see ``Materials and
Methods'') and PYERE1 (Metzger et al., 1988) vectors as
appropriate, and transcriptional activation was obtained by
determination of the
The transcriptional activity obtained in the presence of E2 or
OHT was also determined in yeast using the most informative mutants on
the estrogen-responsive chimeric GAL1 promoter. In the presence of 1
µ
M OHT, HEG0 was 80% as active as in the presence of 10
n
M E2, in agreement with previous results (Metzger et
al., 1992). Mutants containing large deletions in the A/B region
of hER, such as HE 303, HE 311, HE 315/304, HE 311/388, and HE 303/384,
which were active in the presence of E2, were also highly active in the
presence of OHT. Reciprocally, mutants poorly active in the presence of
E2, ( e.g. HE 307 and HE 356) were also poorly active in the
presence of OHT.
These results demonstrate that, in both animal and
yeast cells, the hER A/B segments, which are responsible for activation
of transcription in the presence of estradiol, are also those
responsible for activation in the presence of 4-hydroxytamoxifen.
The estrogen receptor contains two activation functions,
which activate transcription in a constitutive manner (AF-1) or in a
hormone-dependent manner (AF-2) when isolated from the rest of the ER
sequences. Previous studies have shown that both transcriptional
activation functions act in a promoter context and cell-type specific
manner (see Introduction) and that AF-1 is located within amino acids
1-180 (region A/B) of the human ER. In particular, the A/B region
is transcriptionnally active on its own in CEFs (Kumar et al.,
1987; Webster et al., 1988b; Tora et al., 1989a;
Berry et al., 1990), as well as in yeast (White et
al., 1988; Berry et al., 1990; Metzger et al.,
1992).
Using systematic deletional mutagenesis, we have now
localized the amino acid sequences within the A/B region responsible
for AF-1 activity in CEF cells. A/B region deletion mutants were
created using both the chimeric activator AB-GAL and HEG0, in order to
test AF-1 activity in the absence and in the presence of the hormone
binding domain (containing AF-2), respectively. We show that the
amino-terminal 149 amino acids contain all of the sequences required
for transcriptional activation by the A/B region. Thus, AF-1 is encoded
within the first exon of the ER gene (amino acids 1-150; see
Ponglikitmongkol et al. (1988)). Amino acids 151-180,
which are not involved in AF-1 activity, are encoded in the same exon
as the first zinc finger of ER DBD. Furthermore, deletion of amino
acids 1-50 has little effect on transactivation using the present
responsive promoters. A mutant that contains only amino acids
51-149 of the A/B region was as active as HEG0, whereas deletions
within amino acids 51-149 caused a drastic reduction in
transactivation, showing that the whole of this region is required for
the activity of AF-1 on its own (Fig. 5).
We and others have shown that steroid
receptors can activate transcription when expressed in yeast cells (see
Introduction). We have also shown that a hER mutant, which contains a
deletion of the HBD, can activate transcription constitutively and as
efficiently as the wild-type receptor (White et al., 1988;
Berry et al., 1990; Metzger et al., 1992). In fact,
as it is the case for a minimal promoter in CEF cells, AF-2 contributes
very little to transcription activation in yeast from a chimeric
GAL1-LacZ reporter construct. Accordingly, we found here that the A/B
region appended to the GAL4 DBD efficiently activates transcription in
yeast. To further characterize the AF-1 function in this simple
eukaryotic system, we have tested deletion mutants of the A/B region
linked to the GAL4 DBD or to CDEF regions of hER. We have identified
three small regions that can activate transcription efficiently on
their own: amino acids 1-62, 80-113, and 118-149 are
80, 60, and 70% as active as the whole A/B region, respectively,
indicating that multiple activating domains can operate independently
in yeast. Two of these regions are comprised within the region
(51-149) required for transcriptional activity in CEF cells
(Fig. 5). However, the 62 amino-terminal amino acids, which
contribute very little, if at all, to the activity of AF-1 in CEF cells
are highly active in yeast, which raises the possibility that this
region may be involved in some cell-specific and/or promoter-specific
transcriptional activation in animal cells.
None of the sequences
that are required for AF-1 activity in animal or in yeast cells are
significantly similar to those that have been characterized as
activating domains in other transactivators, if one excepts
similarities in amino acid composition. Examination of the amino acid
composition of the core of the activating domain in CEF cells (amino
acids 51-149) reveals significantly high proline (16% versus 5.8% for the complete protein) and tyrosine (8% versus 3.8%) content, and low levels of isoleucine (0% versus 3.1%), aspartic acid (0% versus 4%), lysine (0%
versus 5%) and cysteine (0% versus 2%). Proline-rich
regions have been identified in the transcriptional activation domains
of several transcription factors ( e.g. CTF/NF1, progesterone
receptor, Oct2, AP2) (Mermod et al., 1989; Gerster et
al., 1990; Williams and Tjian, 1991; Meyer et al., 1992).
A comparison of the amino acid composition of the 99-amino acid core of
AF-1 with such proline-rich activation regions did not reveal any
primary sequence similarity. The small amino acid stretches of the A/B
region ( i.e. 1-62, 80-113 and 118-149),
which are highly active in yeast, are also characterized by a high
proline content (10, 18, and 19%, respectively). One phosphorylation
site (Ser-118) involved in the transcriptional activity of the A/B
region has been identified in hER expressed in COS-1 cells and HeLa
cells (Ali et al., 1993b; Le Goff et al., 1994).
However, Ser-118 is not phosphorylated in yeast, and mutation of this
amino acid to an alanine does not significantly affect the
transcriptional activity of A/B region in yeast.
We have previously shown that the agonistic activity of
OHT can be ascribed to AF-1 (Berry et al., 1990; Metzger
et al., 1992). Using A/B deletants in CEF cells and in yeast,
we further show here that the mechanisms underlying the induction of
AF-1 activity are most probably the same in the presence of E2 or OHT,
since the same sequences of the A/B region are required in the two
instances.
We and others have proposed that different classes of
activators may interact with the basic transcriptional machinery
through TIFs (Tasset et al., 1990; Martin et al.,
1990; Goodrich et al., 1993; Hoey et al., 1993; Gill
et al., 1994). In this respect, the observation that the
minimal hER AF-1 domain, which is efficient in animal cells, contains
two activating domains that can independently activate transcription in
yeast suggests that some higher eukaryotic activating domains were
formed by the combination of primordial activating modules. The
presence of the corresponding primordial TIFs in yeast may account for
the observation that many higher eukaryotic transactivators are
functional in yeast (see Introduction for references). These primordial
TIFs may not be expressed in all cell types of higher eukaryotes, which
instead would contain new TIFs recognizing activators generated through
multiple combinations of a limiting number of primordial activating
modules. In the course of evolution this may have led to a
combinatorial generation of the numerous cell-specific transcriptional
activators, which are required for the complex spatio-temporal control
of gene expression in higher eukaryotes.
Transcriptional activities of the activators in CEF and
yeast were determined as described in Figs. 2 and 4, respectively, in
the presence of either E2 (10 n
M) or OHT (1 µ
M)
as indicated. The -fold induction (ratio between transcriptional
activation obtained in the presence and absence of ligand) is given in
brackets.
We thank D. Tasset for the gift of AB-GAL and J. White
and Y. Lutz for the gift of 2GV3 and 3GV2 antibodies. We also thank Dr.
A. Wakeling and ICI (UK) for provinding 4-hydroxytamoxifen and S.
Johnston for the gift of the yeast strain SC30. We also thank the cell
culture group for providing cells, F. Ruffenach and A. Staub for
oligonucleotide synthesis, the secretarial staff for typing the
manuscript, C. Werlé, S. Metz, B. Boulay, and J. M. Lafontaine
for preparing the figures, and J. Clifford for critical reading of the
manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-helices or
sheets
(Giniger and Ptashne, 1987; van Hoy et al., 1993; Leuther
et al., 1993). Other activating domains, rich in proline or
glutamine residues or in hydroxylated amino acids, have also been
described (Mermod et al., 1989; Courey and Tjian, 1988; Theill
et al., 1989). However, the importance of these amino acids is
not yet clear (see Mitchell and Tjian (1989) and Seipel et al. (1994)). Interestingly, the basic mechanisms controlling
initiation of transcription appear to be conserved throughout
eukaryotes. Several subunits of RNA polymerase II(B) are highly
conserved from yeast to man (Sentenac and Sawadogo, 1990; Young, 1991),
and the yeast TATA-binding factor protein can to some extent
functionally replace the corresponding HeLa cell factor (Buratowski
et al., 1988; Cavallini et al., 1988). Moreover,
yeast transcriptional activators can function in animal and plant cells
(Kakidani and Ptashne, 1988; Webster et al., 1988a; Fisher
et al., 1988; Ma et al., 1988), and many mammalian
transcriptional activators have been shown to activate transcription in
yeast (see Metzger et al. (1988), Schena and Yamamoto (1988),
Heery et al. (1993), and references therein).
(
)
belongs to a superfamily of
ligand-inducible transregulators, which includes receptors for steroid
and thyroid hormones, vitamin D3, retinoic acid, and peroxisome
proliferator-activated receptors (Evans, 1988; Green and Chambon, 1988;
Beato, 1989; Gronemeyer, 1991; Laudet et al., 1992; Leid
et al., 1992; Kastner et al. 1994; Mangelsdorf et
al., 1994). Cloning of ER cDNAs from different species led to the
definition of six regions (see Fig. 1 A, A-F)
exhibiting different degrees of amino acid sequence conservation (Krust
et al., 1986). This division was subsequently extended to all
members of the nuclear receptor superfamily. Molecular genetic analyses
of ER have identified separable domains responsible for DNA binding,
hormone binding, and transactivation (see Fig. 1 A).
Region C, which is highly conserved across species, corresponds to the
DNA binding domain (DBD). It is responsible for specific binding to
estrogen response elements (EREs) of target genes (for Refs., see
Gronemeyer (1991)). The carboxyl-terminal region E, which is also well
conserved between species, contains the hormone binding domain (HBD)
and a hormone-dependent transcription activation function (AF-2,
previously called TAF-2) (Webster et al., 1988b; Bocquel
et al., 1989; Lees et al., 1989; Tora et
al., 1989a; Berry et al., 1990; Tasset et al.,
1990; Danielian et al., 1992; Durand et al., 1994).
Figure 1:
Localization of the AF-1 activating
domain using the chimeric AB-GAL activator in CEF cells. A,
schematic representation of hER and the reporter gene 17M/ERE-G.CAT.
The regions containing the AFs, DBD, and HBD of hER are indicated. The
numbers refer to amino acid positions for hER and to nucleotides for
the reporter gene. The +1 arrow represents the
transcriptional start site. B, transcriptional activity of the
AB-GAL deletion mutant series. The amino acids of the A/B region are
represented in gray. The deletion is indicated by a
line, and the deleted amino acids are indicated. Amino acids
1-147 of GAL4 are represented by an open box.
The induction of CAT activity obtained with AB-GAL relative to the
parental pSG5 vector is taken as 100; the corresponding -fold induction
is given in parentheses. Transcriptional activities of
deletants are normalized to that of AB-GAL. Each value is an average
(±10%) of at least four independent experiments. C, a
Western blot analysis of transfected COS-1. Each lane contains
10 µg of cell extract: lane 1, transfection with
pSG5 expression vector; lanes 2-12,
transfections with the indicated expression vector. The extracts were
immunoprobed with 2GV3 and 3GV2 antibodies. Note that in this
particular transfection series, 306-GAL and 313-GAL were expressed at a
lower level than the other AB-GAL derivatives. However, this decreased
expression was not seen in other transfection series. The position of
the molecular mass standards are indicated in kDa. D, a
representative CAT assay. CEF cells were transfected with the reporter
plasmid 17M/ERE-G.CAT together with the expression vector as indicated.
Extracts were assayed for CAT activity after normalization for
-galactosidase activity (see ``Materials and
Methods'').
Another transcriptional activation function (AF-1, previously called
TAF-1) was characterized in the ER amino-terminal A/B region and was
shown to function in a hormone independent manner when isolated from
the HBD (Bocquel et al., 1989; Lees et al., 1989;
Tora et al., 1989a; Tasset et al., 1990). This ER
amino-terminal region, which is less conserved between species (Krust
et al., 1986), corresponds to a region that exhibits little or
no conservation among the nuclear receptor superfamily (see Segraves
(1991) for references). A number of nuclear receptor genes encode
isoforms that differ exclusively in their amino-terminal amino acid
sequences (for reviews, see Gronemeyer (1991), Leid et al. (1992), and Chambon (1994)). Interestingly, the two chicken and
human progesterone receptor isoforms A and B have been shown to
differentially activate transcription of target genes (Tora et
al., 1988; Kastner et al., 1990), and we have previously
shown that AF-1 of the human ER (hER) exhibits cell-type and promoter
context specificity. In HeLa cells, AF-1 activates transcription very
poorly on its own, but it can synergize with AF-2 to activate
transcription from some promoters. In contrast, in chicken embryo
fibroblasts (CEF), AF-1 can efficiently activate transcription on its
own (Kumar et al., 1987; Tora et al., 1989a; Berry
et al., 1990). In the yeast Saccharomyces cerevisiae,
hER transactivates from reporter genes containing the GAL1 promoter
into which an ERE has been inserted (Metzger et al., 1988).
Interestingly, deletion of the HBD results in a constitutive ER mutant
that is almost as active as hER (White et al., 1988), whereas
little or no transcriptional activity remains when the A/B region is
deleted (Berry et al., 1990; Metzger et al., 1992).
However, when tested on different promoters, an A/B region-deleted hER
is more active than a region E-deleted hER, indicating that the
activities of AF-1 and AF-2 are also promoter context dependent in
yeast (Metzger et al., 1992; Pham et al., 1992). We
have also shown that the partial agonistic activity of the
anti-estrogen 4-hydroxytamoxifen (OHT) can be ascribed to the activity
of AF-1, whereas AF-2 activity is inhibited by OHT (Berry et
al., 1990; Metzger et al., 1992). Comparative functional
analyses of hER AF-1 and AF-2 and of the acidic activation functions of
GAL4 and VP16, have shown that their synergistic and transcriptional
interference (``squelching'') properties are different and
suggested that AF-1 and AF-2 may interact with different intermediary
factors (TIF) (mediators or coactivators) also required for mediating
the activity of acidic activators, whereas acidic activators may
require an additional mediator (Tora et al. (1989a) and Tasset
et al. (1990) and references therein).
Recombinants for Animal Cell Studies
HEG0 has
been described previously (Tora et al., 1989b). The AB-GAL
expression vector (a gift of D. Tasset) was constructed as follows. The
EcoRI- KpnI fragment encoding amino acids 1-184
of hER was excised from HE28 (Green and Chambon, 1987) and inserted 5`
of the GAL4 DBD into the EcoRI- HindIII sites of pG4M
poly(I) (Webster et al., 1989) using a
KpnI- HindIII adaptor; the stop codon between the
HindIII site and the ATG of the GAL4 DBD was removed by
site-directed mutagenesis leading to the presence of the extra amino
acids GTGTSL between the A/B region and the GAL4 DBD. The
EcoRI site located 5` to the AB-GAL coding sequences was
blunt-ended, and a BglII linker was cloned in, resulting in
pSG5bAB-GAL. The deletions were constructed by site-directed
mutagenesis of single-stranded pSG1HEG0 or pSG5bAB-GAL using synthetic
oligonucleotides (36-mer) complementary to 15 nucleotides located 5`
and 3` to the deletion and containing the sequence 5`-CTCGAG-3`
corresponding to a XhoI restriction site in the middle. All
constructs were verified by sequencing. The reporter plasmids
ERE-TATA-CAT and 17M/ERE-G.CAT have been described (Tora et
al., 1989a).
Recombinants for Yeast Cell Studies
pY0 was
constructed by exchanging the XmaI- XhoI restriction
fragment from pYERE1 (Metzger et al., 1988) containing the ERE
sequence with a linker 5`-CCGGGTTTGCGGCCGCATC-3`
3`-CAAACGCCGGCGTAGAGCT-5` containing a NotI site.
Cell Transfection and CAT Assays
COS-1, CEF, and
HeLa cell transfections were performed using the standard calcium
phosphate co-precipitation technique (Tora et al., 1989a,
Berry et al., 1990). COS-1 cells were transfected with 5
µg of expression vector and 10 µg of carrier Bluescribe
M13+ DNA (BSM+, Stratagene). CEF cells were transfected with
either 500 ng of 17M/ERE-G.CAT or 2 µg of ERE-TATA-CAT as reporter
genes, together with either 500 ng of AB-GAL, HEG0, or mutant
derivatives as activator genes. CEF cell transfections included 1
µg of the reference plasmid pCH110 (Pharmacia Biotech Inc.) and
Bluescribe M13+ DNA as carrier DNA to make a total of 15 µg of
DNA. HeLa cells were transfected with 2 µg of ERE-TATA-CAT reporter
gene, together with 1 µg of HEG0 or A/B region deletants as
activator genes, 2 µg of pCH110 and Bluescribe M13+ DNA up to
15 µg DNA. Ethanol (as a control), E2 (10 n
M) or OHT (100
n
M) prepared in ethanol were added 1 h post-transfection,
where appropriate. CEF and HeLa cell extracts and CAT assays were
performed as described (Berry et al., 1990).
Yeast Transformation and
Yeast strain TGY14.1 was transformed as described (Metzger
et al., 1988), and yeast strain SC30 (MATa, gal4-Galactosidase
Assay
,
ura3-52, leu 2-3, 112, his3
, ade
)
was transformed by electroporation (Becker and Guarente, 1991).
Transformants were grown in selective medium, ligands were added as
appropriate, and
-galactosidase activity determined as described
previously (Metzger et al., 1988; Tora et al.,
1989b).
Immunoblots
COS-1 cells were extracted (100
µl/plate) in high salt buffer containing 400 m
M KCl, 20
m
M Tris-HCl, pH 7.5, 1 m
M EDTA, 1 m
M
dithiothreitol, 10% glycerol (v/v), 1 m
M phenylmethylsulfonyl
fluoride, protease inhibitor mixture (2.5 µg/ml of leupeptin,
pepstatin, chymostatin, antipain, and aprotinin) by three cycles of
freeze (-70 °C)/thaw (+4 °C) and centrifugation at
10,000 g for 15 min at 4 °C. Yeast extracts were
prepared in the same buffer, and Western blotting was performed as
described previously (Metzger et al., 1988), using the
monoclonal antibody F3 directed against the F region of the hER (Ali
et al., 1993a) or 2GV3 and 3GV2 directed against the GAL4 DBD
(White et al., 1992).
Experimental Design
To determine which amino
acid sequences of the hER A/B region are responsible for
transcriptional activation in animal cells and yeast, we have created
deletion mutants spanning the A/B region appended to either the
amino-terminal 147 amino acids of the yeast transactivator GAL4
(containing the DNA binding domain, the dimerization domain, and the
nuclear localization signal (Silver et al., 1988; Carey et
al., 1989)) or regions CDEF of hER (see Figs. 1-4). To
minimize variations in expression levels of the mutant proteins (which
may be related to the amino acid composition of the amino-terminal
extremity of the protein (Bachmair et al., 1986)), the
amino-terminal-most two amino acids of hER were maintained in all of
the mutants. A restriction site was inserted at the position of the
deletion in order to facilitate the screening of the recombinants, thus
resulting in the insertion of two amino acids (Leu-Glu) in each mutant.
The mutations were verified by DNA sequencing on double-stranded
plasmid DNA, and the size and expression levels of the proteins were
controlled by Western blotting analysis of transfected COS-1 or yeast
cell extracts, using monoclonal antibodies directed against the GAL4
DNA binding domain (2GV3 and 3GV2; see White et al. (1992)) or
a monoclonal antibody directed against region F of hER (F3 antibody)
(Ali et al., 1993a). All of the mutants used in this study
were expressed at levels similar to that of the parental activator (see
Figs. 1 C and 2 C, and data not shown).
-galactosidase activity obtained from the vector pCH110
(Pharmacia). A representative CAT assay is shown in
Fig. 1D.
-galactosidase activity generated from
chimeric reporter genes containing a single GAL4 response element (17M)
or an ERE (see Figs. 3 A and 4 A). The AB-GAL chimera
and its derivatives were assayed in the yeast strain SC30 (kind gift of
S. Johnston) containing a deleted GAL4 gene in order to avoid problems
due to endogenous yeast GAL4 protein, whereas HEG0 and its derivatives
were tested in the previously used TGY14.1 strain (Metzger et
al., 1988). hER AF-1 Includes Amino Acids 51-149 in CEFs-In CEF cells,
GAL4 (1-147) was inactive on a CAT reporter construct placed
under the control of a chimeric promoter composed of a globin promoter
region containing a single 17M GAL4 binding site (17M/ERE-G.CAT; see
Fig. 1A). In contrast, the chimeric AB-GAL construct
activated transcription
5-fold (Fig. 1, B and
D, compare lanes 3 and 9 with
lanes 1 and 8), as did GAL-ER(AB), which was
used in previous studies (Tora et al., 1989a) (Fig. 1,
B and D, compare lanes 2 and
3). Thus, AF-1 of hER activates transcription when tethered
either amino- or carboxyl-terminally to the GAL4 DNA binding domain. To
determine which amino acid stretches are involved in transcriptional
activation by AF-1, a series of deletion mutants was generated from
AB-GAL (Fig. 1 B). Removal of amino acids 3-50 did
not affect transcriptional activation (344-GAL: 110%), and deletion to
amino acid 61 (343-GAL) maintained most of the transcriptional
activation capacity. Further deletions within the A/B region resulted
in a progressive decrease in transcriptional activation, and deletion
to amino acid 101 (302-GAL) resulted in less than 10% of the
transcriptional activation capacity of AB-GAL being retained. Thus
amino acids 51-184 are sufficient for efficient transcriptional
activation, and amino acids 62-101 appear to be particularly
important. Deletion from the carboxyl-terminal end showed that removal
of the last 35 amino acids of the A/B region (as well as the six amino
acids located between region A/B and the GAL4 DBD) had relatively
little effect on transcriptional activation (411-GAL, 80% and 384-GAL,
75%). Note that deletion of the same carboxyl-terminal amino acids from
the A/B region of HE15F2 (an hER mutant lacking the HBD
(
282-552), see Fig. 2 B) had no effect on
transcriptional activation using this reporter plasmid (data not
shown), indicating that the A/B activating domain may be exposed in a
slightly different manner in the chimeric construct (see also below in
the case of HEG0). Larger deletions resulted in reduced activity; the
transcriptional activity of 306-GAL (
64-184) was only 10%
that of AB-GAL, and 313-GAL (
35-184) was totally inactive.
Taken together, these results indicate that in CEF cells, amino acids
1-50 and amino acids 150-184 are dispensable for
transcriptional activation by AF-1. Accordingly, deletion mutant
384/344-GAL (
3-50/
150-184) retains most, but not
all, of the wild-type activity, indicating that amino acids
51-149 are crucial for transcriptional activation in CEF cells
and that the flanking amino acids contribute to some extent to full
activity, perhaps by allowing proper exposure of the activating domain.
Figure 2:
Localization of the AF-1 activating domain
using human ER (HEG0) in CEF and HeLa cells. A, schematic
representation of the reporter gene. The numbers refer to nucleotides,
and the arrow refers to the transcription start site.
B, transcriptional activity of HEG0 deletion mutants. The A/B
region and deletions are represented as in Fig. 1. The induction of CAT
activity obtained with HEG0 relative to the parental pSG1 vector in the
presence of E2 is taken as 100 (the corresponding -fold induction is
given in parentheses). The average values (±10%) from
at least four independent experiments are given. C, a
representative Western blot analysis of transfected COS-1 cells. Each
lane contains 10 µg of cell extract: lane 1, transfection with 1 µg of pSG1 vector; lanes 2-13, transfection with 1 µg of the indicated
expression vector. The extracts were immunoprobed with the F3
monoclonal antibody. The arrowhead points to a nonspecific
interaction. The position of the molecular mass standards are indicated
in kDa.
To investigate whether AF-1 activity could be influenced by the
presence of the ER hormone binding domain, a similar deletional
analysis was performed with mutants derived from HEG0. The mutants were
tested in CEF cells on the reporter plasmid ERE-TATA-CAT, on which AF-1
is highly active and AF-2 is inactive (see Fig. 2) (Tora et
al., 1989a; Berry et al., 1990). Mutants containing
deletions of amino acids 3-50 (HE 344) or 150-178 (HE 384)
were as active as HEG0 and, furthermore, were active only in the
presence of E2 (data not shown). The activity of mutants containing
larger deletions was decreased depending on the size of the deletion,
and deletion of amino acids 3-101 (HE 302) abolished
transcriptional activity, indicating that amino acids 51-101 are
crucial for transcriptional activation by HEG0 from this promoter. When
deleting into the A/B region from the carboxyl-terminal end, removal of
amino acids 94-178 (HE 314) almost totally abolished
transcriptional activation, indicating that amino acids 94-149
are also required for transcriptional activation. Accordingly, deletion
mutant HE 344/384, which retains amino acids 51-149 only, was as
active as HEG0, while deletion of additional 11 amino acids from the
amino terminus or nine residues from the carboxyl terminus of this
region greatly decreased transcriptional activity (HE 384/343 and HE
344/368; 40% activity of HEG0). Removal of amino acid stretches within
this region also resulted in a significant decrease in transcriptional
activity (see HE 354, HE 359, HE 353, and HE 363,
Fig. 2B). Thus, we conclude that hER amino acids
51-149 include all of the sequences involved in AF-1
transcriptional activity in CEF cells.
Characterization of the hER A/B Sequences Required for
Synergism with AF-2 in HeLa Cells
Previous studies have shown
that both AF-1 and AF-2 are inactive on their own on a minimal promoter
containing only an ERE and a TATA box (ERE-TATA-CAT) in HeLa cells and
that synergism between AF-1 and AF-2 is required for HEG0 to activate
transcription from this promoter (Tora et al., 1989a; see also
Fig. 2
). The activity of HEG0-derived constructs deleted in the
A/B region was tested on this promoter to investigate whether the
sequences required for AF-1 activity in CEF cells are the same as those
necessary for synergism with AF-2 in HeLa cells. We found that amino
acids 51-149 were required for synergism with AF-2 (Fig.
2 B). However, in contrast to what was seen in CEF cells for
the activity of AF-1 on its own, the decrease in synergistic activity
was less dramatic, such that deletion of amino acids 3-101 (HE
302) gave 40% activity in HeLa cells, while transcriptional activation
was abolished in CEF cells (Fig. 2 B). Similarly,
although deletion of amino acids 141-178 (HE 368) had no effect
on AF-1 activity in CEF or in HeLa cells, deleting amino acids
94-178 (HE 314) resulted in little activation by AF-1 on its own
in CEFs, but 45% of the synergistic activity was retained in HeLa
cells. Further amino- and carboxyl-terminal deletions caused a drastic
fall in transcriptional activation in HeLa cells (deleting from the
amino terminus to amino acid 117 (HE 303) or from the carboxyl terminus
to amino acid 85 (HE 310) resulted in very little transactivation).
Note, however, that deletion of amino acids 91-118 (HE 353)
resulted in a 50% loss of transcriptional activity in CEF cells but did
not affect the synergistic AF-1 activity in HeLa cells. We have
previously shown that there is ligand-inducible phosphorylation of
serine 118 in HeLa, COS-1, and CEF cells. We have also shown that in
HeLa and COS-1, but not in CEF cells, mutation of Ser-118 to an alanine
residue reduces transactivation, whereas mutation to a glutamic acid
residue increases transactivation (Ali et al. (1993b) and data
not shown). Deletion of amino acids 91-118 leads to the
replacement of Ser-118 by a glutamic acid (see ``Experimental
Design'' and ``Materials and Methods''). This suggests
that, in HeLa cells but not in CEF cells, the presence of a glutamic
acid residue at this position may compensate for a loss of
transcriptional activity due to deletion of amino acids 91-118.
In any event, the above results indicate that deletants that have only
a weak AF-1 in CEF cells can synergize with AF-2 in HeLa cells and that
amino acids 51-93 and 102-149 are involved in the
synergistic activity.
Analysis of AF-1 Activity in Yeast
We have
previously shown that in yeast an hER truncated for the HBD (HE15 or
HE15F2, 282-595 and
282-552, respectively, see
Fig. 2B) activates transcription constitutively from
chimeric GAL-1 gene-derived promoters containing an ERE (White et
al., 1988; Berry et al., 1990; Metzger et al.,
1992). In order to demonstrate that the A/B region can activate
transcription in yeast when linked to a heterologous DBD, we tested the
above described AB-GAL chimera on a reporter gene in which
-galactosidase expression is driven by a GAL-1 gene-derived
promoter containing a single GAL4 binding site (17M; see 17M-GAL1-LacZ
in Fig. 3 A). AB-GAL efficiently activated transcription
from this promoter, whereas GAL1-147 was inactive (data not
shown, and see Fig. 3 B). The stimulation by AB-GAL was
clearly mediated through the 17M GAL4 binding site, since its deletion
in the reporter gene rendered the promoter nonresponsive (data not
shown). Thus, AF-1 is transcriptionally active in yeast independent of
any other hER domain. To determine whether the same amino acid
51-149 region, which is responsible for AF-1 activity in CEF and
HeLa cells, is also involved in transactivation in yeast, A/B region
deletions were investigated using AB-GAL-derived constructs
(Fig. 3 B). Interestingly, mutants bearing extensive
deletions from the amino terminus showed little loss in AF-1 activity,
while they were totally inactive in CEF and HeLa cells (303-GAL). Only
deletions beyond amino acid 117 caused a drastic decrease in
transactivation, showing that amino acids 118-184 are sufficient
for full activation by the A/B region in yeast. Strikingly, when
deleting from the carboxyl terminus, this region could be deleted with
no loss in activity relative to AB-GAL, and amino acids 1-84
appeared to be sufficient for full transactivation. Thus sequences
located between amino acids 1-84 and 118-184 could
independently activate transcription in yeast. This conclusion was
further supported by the results obtained with mutants 384/303-GAL
(
3-117/
150-184), 368/303-GAL
(
3-117/
141-184), and 306/346-GAL
(
3-28/
64-184), which retained 100, 30, and 45% of
the AB-GAL activity, respectively. We conclude that the hER A/B region
contains at least two discrete, apparently redundant activation
functions (amino acids 29-63 and 118-149), which can
activate transcription in yeast.
Figure 3:
Localization of the AF-1-activating domain
using the chimeric AB-GAL activator in yeast. A, schematic
representation of the reporter gene. 17M corresponds to the GAL4
binding site. The numbers refer to nucleotides (+1 corresponds to
the first nucleotide of the relevant coding sequence), the triangle and the arrow refer to the TATA box and RNA start site,
respectively. B, activators and their corresponding
-galactosidase activity (
-gal) obtained from 17
M-GAL1-LacZ (activators are represented as in Fig. 1). The induction of
-galactosidase activity obtained with AB-GAL relative to the
parental vector pY017M is taken as 100% (the corresponding -fold
induction is given in parentheses). The transcriptional
activity of the deletants is normalized to that of AB-GAL. Each value
is an average (±20%) of at least three independent
experiments.
The transcriptional activity of the
A/B region was also investigated in yeast using deletion mutants of the
complete receptor (HEG0) and the ERE1-GAL1-LacZ reporter gene
(Fig. 4, A and B). Deletion of amino acids
3-79 (HE 304) or 85-178 (HE 310) resulted in mutants having
a transcriptional activity comparable with that of HEG0
(Fig. 4 B). Larger deletions progressively decreased the
transcriptional activity of the mutants, and deletion of amino acids
3-139 (HE 307) or 12-178 (HE 356) resulted in near complete
loss of transactivation. Internal deletions of up to 66 amino acids (HE
354; see Fig. 4 B) did not decrease the transcriptional
activity of the mutant, and some internal deletion mutants were even
more active than HEG0. Further functional dissection of the A/B region
showed that it contains several discrete stretches of amino acids that
result in levels of activation greater than 60% relative to HEG0 (Fig.
4 B, see for example mutants HE 311/388 (containing amino acids
18-62), HE 315/304 (containing amino acids 80-113), and HE
303/384 (containing amino acids 118-149)). Interestingly, these
mutants and even some mutants with smaller deletions ( e.g. HE
354, HE 303, and HE 310) did not activate transcription in CEF or HeLa
cells (compare with Fig. 2) (data not shown).
Figure 4:
Localization of the AF-1 activating domain
using human ER (HEG0) in yeast. A, schematic representation of
the reporter gene. The symbols are the same as in Fig. 3 A. ERE
corresponds to the ER response element. B, activators and
their corresponding -galactosidase activity (
-gal).
The
-galactosidase activity obtained with HEG0 in the presence of
E2 relative-pYERE1 is taken as 100 (the corresponding -fold induction
is given in parentheses). The transcriptional activity of the
deletants is normalized to HEG0. Each value is an average (±20%)
of at least three independent experiments.
4-Hydroxytamoxifen and Estradiol Play a Similar Role in
AF-1 Activity
We have previously ascribed the agonistic activity
of the OHT observed in CEF cells and yeast to AF-1 (Berry et al. 1990; Metzger et al., 1992). The above hER mutants were
used to investigate whether the same amino acid sequences are involved
in transcriptional activation induced by E2 and OHT. The
transcriptional activity of HEG0 and of some key mutants was determined
in CEF cells in the presence of 10 n
M E2 or 100 n
M OHT using the ERE-TATA-CAT reporter gene (Fig. 2 A). In
agreement with previous results (Berry et al., 1990), in the
presence of OHT, HEG0 was about 30% as active as in the presence of E2
(). Deletions of amino acids from HEG0 that did not affect
the E2-induced transcriptional activity, also did not affect
transcriptional activity obtained with OHT (HE 344, HE 384, and HE
344/384 were as active as HEG0 in the presence of OHT, ).
On the other hand, deletions that reduced activation in the presence of
E2 (HE 302, HE 314, and HE 354) had a similar effect on the presence of
OHT.
Figure 5:
Localization of the activating domains in
hER AF-1. The hER A/B region is represented by a line, and the
numbers refer to the amino acid positions. The amino acids
responsible for AF-1 transcriptional activity in animal and yeast cells
are boxed; those responsible for synergism with AF-2 in HeLa
cells are dotted.
We have previously
demonstrated that AF-1, on its own, is unable to activate transcription
in HeLa cells from any promoter (Kumar et al., 1987; Tora
et al., 1989a; Berry et al., 1990) and that AF-2,
which activates transcription very weakly from minimal promoters,
requires the synergistic effect of AF-1 to activate transcription
efficiently from such promoters in HeLa cells (Tora et al.,
1989a). We show here that deletion mutants of AF-1, which are only
weakly active on their own, can nevertheless synergize with AF-2 to
transactive in HeLa cells. Our data suggest that amino acids
51-93 and 102-149 are sufficient for synergism between AF-1
and AF-2 (Fig. 5).
(
)
Table: Comparison of transcriptional activity of
HEG0 and A/B deletants in the presence of E2 or OHT in CEF and yeast
cells
-estradiol; CAT, chloramphenicol acetyltransferase.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.