High-Mobility Group (HMG) Protein HMG-1 and TATA-Binding Protein-Associated Factor TAFII30 Affect Estrogen Receptor-Mediated Transcriptional Activation
Carmel S. Verrier,
Nady Roodi,
Cindy J. Yee,
L. Renee Bailey,
Roy A. Jensen,
Michael Bustin and
Fritz F. Parl
Department of Pathology (C.S.V., N.R., C.Y., L.R.B., R.A.J.,
F.F.P.) Vanderbilt University Nashville, Tennessee
37232
Laboratory of Molecular Carcinogenesis (M.B.)
National Cancer Institute Bethesda, Maryland 20892
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ABSTRACT
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The estrogen receptor (ER) belongs to a family of
ligand-inducible nuclear receptors that exert their effects by binding
to cis-acting DNA elements in the regulatory region of
target genes. The detailed mechanisms by which ER interacts with the
estrogen response element (ERE) and affects transcription still remain
to be elucidated. To study the ER-ERE interaction and transcription
initiation, we employed purified recombinant ER expressed in both the
baculovirus-Sf9 and his-tagged bacterial systems. The effect of
high-mobility group (HMG) protein HMG-1 and purified recombinant
TATA-binding protein-associated factor TAFII30
on ER-ERE binding and transcription initiation were assessed by
electrophoretic mobility shift assay and in vitro
transcription from an ERE-containing template
(pERE2LovTATA), respectively. We find that
purified, recombinant ER fails to bind to ERE in spite of high
ligand-binding activity and electrophoretic and immunological
properties identical to ER in MCF-7 breast cancer cells. HMG-1
interacts with ER and promotes ER-ERE binding in a concentration- and
time-dependent manner. The effectiveness of HMG-1 to stimulate ER-ERE
binding in the electrophoretic mobility shift assay depends on the
sequence flanking the ERE consensus as well as the position of the
latter in the oligonucleotide. We find that
TAFII30 has no effect on ER-ERE binding either
alone or in combination with ER and HMG-1. Although HMG-1 promotes
ER-ERE binding, it fails to stimulate transcription initiation either
in the presence or absence of hormone. In contrast,
TAFII30, while not affecting ER-ERE binding,
stimulates transcription initiation 20-fold in the presence of HMG-1.
These results indicate that HMG-1 and TAFII30
act in sequence, the former acting to promote ER-ERE binding followed
by the latter to stimulate transcription initiation.
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INTRODUCTION
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The estrogen receptor (ER) belongs to a family of ligand-inducible
nuclear receptors that exert their effects by binding to
cis-acting DNA elements (hormone response elements) in the
regulatory region of target genes (1, 2). The detailed mechanisms by
which ER interacts with the estrogen response element (ERE) and affects
transcription remains to be elucidated. The ER is commonly divided into
regions A-F, with each region carrying out a specific function. The
N-terminal regions A and B comprise a ligand-independent
transactivation function, AF-1 (3). Region C is the cysteine-rich
DNA-binding domain whereas region D harbors nuclear localization
signals. The C-terminal region E contains the hormone-binding domain as
well as a ligand-dependent transcription activation domain, AF-2 (4, 5). Functionally, it is believed that the estrogen-dependent activation
of gene transcription occurs in a series of steps. In the first step,
estrogen binds to the hormone-binding domain and induces the formation
of stable ER homodimers (6, 7). In the second step, the
hormone-activated ER dimer interacts with the ERE, characterized as a
13-bp palindrome with 5-bp stems separated by a 3-bp spacer and a
consensus sequence of GGTCAnnnTGACC (8, 9, 10). In the final step, it is
assumed, in analogy with the progesterone receptor, that the ER-ERE
complex promotes the recruitment of general transcription factors
and/or stabilizes their interaction with the promoter of
estrogen-responsive genes such that high levels of transcription can
ensue (11, 12, 13).
There is increasing evidence that the ER-ERE interaction is
influenced by other proteins. For example, recombinant human ER
purified from either HeLa or yeast cells fails to bind ERE (14, 15).
Addition of yeast extract to purified ER restored formation of the
ER-ERE complex. Mukherjee and Chambon (14) identified the yeast factor
and characterized it as a 45-kDa single-stranded DNA-binding protein,
termed ER DNA-binding stimulatory factor. Since then, various other
proteins of sizes ranging from 30 to 160 kDa have been reported to
associate with ER (16, 17, 18, 19, 20, 21, 22, 23). Some of these interacting proteins were
identified by expressing the hormone-binding domain of ER fused to
glutathione-S-transferase (GST-AF2). In the presence of
10-8 M 17ß-estradiol, several proteins from
ZR-75 human breast cancer cells with molecular masses of approximately
160, 100, and 50 kDa were retained by GST-AF2 preloaded on
glutathione-coupled beads (16). Using similar techniques, another group
identified 160- and 140-kDa proteins (17). More recently, the cloned
human TAFII30, which complexes with TATA-binding protein
(TBP), has also been shown to interact directly with ER (18). Neither
estradiol nor estrogen antagonists influenced this binding.
Although the number of ER-associated proteins is growing, their
interaction and precise role in DNA binding or transcriptional
activation remains to be defined. Recently, it was reported that
binding of purified progesterone receptor to its response element is
enhanced by HMG-1 (24, 25). HMG-1 is a 28-kDa-member of the
high-mobility group (HMG) family of nonhistone chromosomal proteins
that is involved in diverse aspects of eukaryotic gene expression,
including determination of nucleosome structure and stability, as well
as transcription and/or replication (26, 27). Higher eukaryotes contain
three families of HMG proteins, the HMG-1/-2 family, the HMG-14/-17
family, and the HMG-I/-Y family, each of which contains distinct
sequence motifs (28). HMG-1 is an abundant, highly conserved protein
present in all vertebrate nuclei that has been shown to nonspecifically
bind and bend different DNA structures as well as facilitate the
binding of transcription factors to template DNA (28, 29). Two groups
conclusively proved the ability of HMG-1 to induce curvature in
double-stranded DNA by testing its effect on ligase-dependent
cyclization of short linear DNA fragments. Covalently closed circles
were formed only in the presence of HMG-1, indicating that HMG-1 is
capable of introducing bends into the linear duplex (30, 31). Because
the two groups used different DNA fragments, their results also
indicate that the effect of HMG-1 is independent of DNA sequence.
In addition, HMG-1 and the related HMG-2 have been shown to facilitate
the DNA-binding of general and specific transcription factors, such as
TFIID-TFIIA, MLTF, HOX, and octamer transcription factor 2 (Oct2)
(32, 33, 34, 35). Thus, HMG-1 should be considered as an "architectural
element" (29, 32), which bends DNA and facilitates binding of
DNA-binding proteins to their target. Based on this interaction, it has
been proposed that HMG-1 may function as a general class II
transcription factor by stimulating the formation of transcription
initiation complexes of RNA polymerase II and III (36, 37, 38). Conversely,
HMG-1 has been proposed to inhibit formation of the preinitiation
complex by interacting with TBP, leading to an inhibition of RNA
polymerase II transcription (39).
In light of these findings, we decided to determine whether HMG-1 and
TAFII30 could facilitate ER to ERE binding and whether that
promotion of binding is associated with any changes in the level of
estrogen-dependent transcription. We find that recombinant, purified
human ER binds to its cognate response element only in the presence of
HMG-1. HMG-1 and TAFII30 act in sequence, the former acting
to promote ER-ERE binding followed by the latter to stimulate
transcription initiation.
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RESULTS
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ER-ERE Binding Requires Additional Factors
To assess binding requirements of ER to its specific DNA
element, we overexpressed ER in baculovirus. Western blot analysis of
the baculovirus-generated ER demonstrated that the overexpressed
protein had electrophoretic and immunological properties identical to
those of ER expressed in the ER-positive breast cancer cell line, MCF-7
(Fig. 1A
). Moreover, the ER overexpression increased
with time, reaching a peak 48 h post infection. When tested for
ERE binding by electrophoretic mobility shift assay (EMSA), the
overexpressed ER failed to show binding with ERE (Fig. 1B
), even though
hormone-binding assays revealed high estradiol binding (Fig. 1C
). ER
overexpressed in bacteria (Fig. 1
, D and E) similarly failed to bind
with ERE (results not shown). This failure of recombinant ER to bind
ERE, therefore, suggested that additional proteins may be necessary to
promote formation of the complex.

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Figure 1. Recombinant ER Does Not Bind ERE
A, ER in MCF-7 (lane 1) and Sf9-hER (lanes 24) cells was analyzed by
Western blot using anti-ER monoclonal antibody, D547; B, EMSA; and C,
hormone-binding assay with results expressed in femtomoles per mg.
Sf9-hER cells, analyzed at 24, 48, and 72 h post infection,
contain increasing amounts of immunochemically detectable ER with some
degradation products at the later time points. An ER-ERE complex formed
with MCF-7 whole-cell extract is indicated by the arrow.
While the hormone binding increases in parallel with the overexpressed
ER, no corresponding ER-ERE complexes are observed with Sf9-hER cells
at all three time points. Identical results were obtained with
recombinant ER expressed in bacteria as His-tagged protein. D, Purified
His-tagged ER (lane 2) was run on an SDS-polyacrylamide gel and stained
with Coomassie blue (lane 1, mol wt markers) as well as analyzed by
Western blot (E).
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HMG-1 Promotes ER-ERE Binding and Interacts with ER
To determine whether HMG-1 can promote ER-ERE binding, EMSAs were
carried out. Under the conditions of our assays, when purified HMG-1
was incubated alone with ERE, no detectable complex was formed.
However, in the presence of HMG-1, a strong ER-ERE complex was formed,
which comigrated with the MCF-7 control (Fig. 2A
). In
competition experiments, addition of increasing concentrations of cold
ERE led to a progressive decrease in the intensity of the ER-ERE
complex (Fig. 2B
). To determine whether the complex contained ER, we
carried out an antibody supershift analysis. Incubation with the
monoclonal anti-ER antibody, H222, further retarded band migration,
indicating the presence of ER in the complex (Fig. 2C
). On the other
hand, using a rabbit antiserum to HMG-1, we were not able to
demonstrate the presence of HMG-1 in the ER-ERE complex. EMSAs carried
out with histone H1 failed to show a promoting effect on ER-ERE
binding, ruling out the possibility of a nonspecific effect of nuclear
proteins on ER-ERE complex formation.

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Figure 2. EMSA of ER-ERE Interactions in the Presence of
HMG-1
A, ER complexes were incubated for 30 min at 4 C with
32P-labeled ERE in the presence or absence of purified
HMG-1 protein. Human ER-ERE complexes formed (denoted by the
arrow) were resolved on a 4.5% polyacrylamide gel.
MCF-7 whole-cell extracts were used as a positive control. HMG-1
appears to promote ER-ERE binding. This binding can be competed off
with cold ERE. B, Competition experiment demonstrating that the ER-ERE
complex promoted by HMG-1 can be suppressed by addition of cold ERE. C,
The human ER monoclonal antibody, H222, was first incubated with ER,
and the complex was further incubated for 30 min at 4 C with
[32P]ERE in the presence of HMG-1. The supershifted band
demonstrates the presence of ER in the complex formed.
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To test whether HMG-1 can interact with ER, we performed
coimmunoprecipitation experiments. When recombinant ER was mixed with
purified HMG-1, it could be coimmunoprecipitated with affinity-purified
antibody specific for HMG-1 coupled to protein A-Sepharose (Fig. 3
). This coprecipitation was dependent on the presence
of both the HMG-1 protein and the antibody against HMG-1, confirming
the specificity of the precipitation reaction. When recombinant
TAFII30 or BSA as a control were substituted for ER, no
coimmunoprecipitation occurred. These results suggest that the HMG1-ER
interaction is a property of the native proteins.

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Figure 3. Coimmunoprecipitation Assays
Using an affinity-purified antibody to HMG-1, the latter was
precipitated in the presence of either purified ER or purified
TAFII30. Unbound proteins were collected in the
supernatant. Bound proteins were washed three times, then eluted off
the Protein A-Sepharose by boiling in SDS-sample buffer. Proteins were
transferred onto nitrocellulose membrane, which was probed with: A, the
ER antibody, H222; or B, the TAFII30 antibody, 2F4; or C,
the affinity-purified antibody to HMG-1. Whereas ER can be
coprecipitated with HMG-1, TAFII30 cannot.
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HMG-1-Induced ER-ERE Binding Is Dependent Upon Oligonucleotide
Length and Position of the ERE within the Oligonucleotide
Having identified HMG-1 as a factor capable of promoting ER-ERE
binding, we next sought to elucidate the mechanism by which HMG-1
promotes formation of the complex. To accomplish that task, we took
into consideration that HMG-1 is a DNA-bending protein. We reasoned
that the time at which HMG-1 is added to the reaction mixture might
determine whether or not an ER-ERE complex is formed. Thus, HMG-1 was
incubated with ERE before or after addition of the overexpressed
receptor. As can be seen in Fig. 4
, to promote ER-ERE
binding, HMG-1 must be incubated with the DNA probe before addition of
ER. When the order is reversed, HMG-1 no longer facilitates ER-ERE
complex formation. In addition, the promotion of ER-ERE binding brought
about by HMG-1 is concentration-dependent (Fig. 4
). Moreover, if DNA
bending by HMG-1 is a critical factor for ER-ERE complex formation, the
length of the oligonucleotide as well as the position of the
palindromic ERE within the synthetic oligo deserve consideration. Two
25-mers containing the ERE consensus sequence, either at the end or in
the middle of the oligo, were employed instead of the usual 35-mer. The
binding of ER to these two 25-mers differed significantly when tested
in the presence of HMG-1. Figure 5A
, lane 1, shows
binding obtained with the usual 35-mer used in previous EMSAs. With the
25-mers, when the ERE consensus is placed at the end of the oligo, a
significant reduction in ER-ERE interaction was observed. In contrast,
when the ERE is centrally placed within the oligo, ER-ERE binding is
restored to levels higher than that observed with the asymmetric 25-mer
but still lower than that obtained with the 35-mer. The effect of HMG-1
was also dependent on the sequence flanking the ERE consensus. When the
ERE consensus was flanked symmetrically by GCs to create another
symmetric 25-mer, ER-ERE binding mediated by HMG-1 was reduced as
compared with the symmetric 25-mer flanked by mostly ATs. Yet, the
symmetry of this GC-rich 25-mer ERE allowed it to bind ER at a much
higher level than did the asymmetric 25-mer (Fig. 5A
, compare lane 2
vs. 4). Competition experiments showed that increasing
amounts of cold 25-mer suppressed ER-ERE complex formation in a
dose-dependent manner, whereas equimolar concentrations of cold 15-mer
containing the ERE without flanking sequences reduced the ER-ERE
formation much less (Fig. 5B
). Thus, the effectiveness of HMG-1 to
stimulate ER-ERE binding in the EMSA depends on oligonucleotide length
and position of the ERE within the nucleotide.

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Figure 4. EMSA of ER-ERE in the Presence of HMG-1: Promotion
of ER to ERE Binding by HMG-1 is Dependent Upon Time of Addition and
Concentration of HMG-1
Lane 1, MCF-7 whole-cell extract was incubated with
[32P]ERE for 30 min at 4 C. ER-ERE complex
(arrow) was resolved on a 4.5% nondenaturing
polyacrylamide gel. Lanes 210 represent the incubation of
[32P]ERE with increasing concentrations (10, 50, 200 ng,
respectively) of HMG-1 in the absence (lanes 24) or presence (lanes
510) of ER extracted from Sf9-hER. To promote ER-ERE binding, HMG-1
must be incubated with the probe before the addition of ER (lanes
810). When ER is incubated with HMG-1 before the addition of labeled
probe, ER-ERE binding is no longer facilitated by HMG-1, regardless of
concentration (compare lanes 57 vs. lanes 810).
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Figure 5. EMSA of ER-ERE in the Presence of HMG-1: Length of
Oligonucleotide, Position of ERE, and Bases Flanking the ERE Affect
HMG-1-Mediated ER-ERE Binding
ER complexes were incubated for 30 min at 4 C with equimolar amounts of
each of the respective labeled probes indicated above in the presence
of HMG-1. HMG-1-mediated ER-ERE complexes were resolved on 4.5%
polyacrylamide gel. A, When a 35-mer ERE is used, strong ER-ERE
complexes are formed when HMG-1 is present (lane 1). Reducing the
length of the ERE to a 25-mer causes a significant reduction in ER-ERE
complex formation (lanes 24). This reduction in complex formation is
further reduced when the ERE is either positioned at the end of the
oligo (lane 2) or flanked by GCs (lane 4). B, When competition
experiments are carried out with 50- or 100-fold excess of either the
symmetrical, AT-rich 25- or the 15-mer ERE, it can be seen that the
25-mer is a much better competitor than the 15-mer ERE.
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HMG-1 and TAFII30 Are Important
Components in ER-Mediated Transcription Initiation
To determine whether the ER-ERE binding promoted by HMG-1
has any functional significance, we carried out in vitro
transcription assays using purified ER, HMG-1, and TAFII30.
TAFII30 was bacterially expressed and purified by means of
a nickel-nitrilotriacetic acid (Ni-NTA) column. In contrast to HMG-1,
TAFII30 appears to have no effect on ER-ERE interaction
(Fig. 6
). By itself, TAFII30 neither
promotes ER-ERE binding nor does it further induce the HMG-1-mediated
ER-ERE binding. In light of these findings, we assessed the functional
role, if any, that HMG-1 and TAFII30 might have on
transcription initiation. The results of the in vitro
transcription are presented in Fig. 7
. When the plasmid
pLovTATA, which is devoid of any ERE, is used, only basal level or no
transcription can be obtained with ER alone or in the presence of HMG-1
or TAFII30 or both (lanes 13). Similarly, when the
ERE-containing plasmid, pERE2LovTATA, was used,
transcription remained basal in the presence of ER alone or in
combination with HMG-1 or TAFII30 (lanes 4, 5, and 7).
However, transcription was enhanced 20-fold by addition of
TAFII30 to ER and HMG-1 (lane 6). Addition of estradiol
further increased transcription (lanes 911). The hormone induction
was approximately 5-fold for ER alone, ER plus HMG-1, and ER plus HMG-1
and TAFII30.

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Figure 6. EMSA of ER-ERE Binding in the Presence of
TAFII30
TAFII30 was expressed in E. coli as a
histidine-tagged protein for one-step purification onto a Ni-NTA
column. By EMSA, ER-ERE complexes formed were resolved on a 4.5%
nondenaturing polyacrylamide gel. Lane 1 is the ER-ERE complex formed
using MCF-7 cell extracts, a positive control. TAFII30
alone neither complexes with ERE (lane 2) nor promotes ER-ERE binding
(lane 3). The HMG-1-mediated ER-ERE complex formation is not further
induced by the presence of TAFII30 (compare lane 4
vs.5).
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Figure 7. In Vitro Transcription Assay
The control template, pLovTATA, and test template,
pERE2LovTATA, were incubated with either ER, ER and HMG-1,
ER and TAFII30, or ER and HMG-1 and TAFII30.
The transcription reactions were initiated by addition of 5 U of
Drosophila embryo nuclear extract and incubated at 30 C
for 1 h. Transcripts were recovered by ethanol precipitation and
analyzed by electrophoresis on a 6% acrylamide, 8 M urea
sequencing gel. As an internal control, the plasmid pMLcas190, which
contains the AdML promoter linked to a G-free cassette of 180 bp, was
used. With the control template, pLovTATA, only basal or undetectable
levels of transcription are obtained (lanes 13). With the test
template, pERE2LovTATA, HMG-1 has no effect on
transcription in the absence or presence of hormone (lanes 45
vs. 89). However, when TAFII30 is added to
ER and HMG-1, a 20-fold increase in transcription is seen (lane 6), and
this increase is further enhanced by addition of hormone (lane 11).
Interestingly, while TAFII30 appears to be needed in
ER-dependent transcription initiation, it failed to initiate
ER-dependent transcription in the absence of HMG-1 (lane 7). Ethanol as
a vehicle has no significant effect on transcription (lane 5
vs. 10).
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DISCUSSION
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The experiments show that overexpressed ER, although it displays
high hormone binding and is electrophoretically and immunologically
identical to MCF-7 ER, does not by itself bind ERE. This observation
suggested that other factors may be necessary to promote ER-ERE
binding. We find that the HMG chromatin protein, HMG-1, enhances
binding of ER to ERE in a dose-dependent fashion in mobility shift
assays. In addition, coimmunoprecipitation experiments show direct
interaction of HMG-1 with ER in the absence of DNA. To study in greater
detail the mechanism by which HMG-1 promotes ER-ERE binding, we felt it
was necessary to obtain purer ER fractions. Although the baculovirus
expression system allows high level expression of recombinant ER, we as
well as others (40) encountered several problems when an attempt was
made to purify the receptor. First, high levels of degradation products
are obtained post infection from the baculovirus system; second,
additional breakdown products are generated when conventional column
chromatography techniques are carried out. Therefore, we opted for
expression in the bacterial system that not only offers better control
over expression via alterations in growth conditions but also allows ER
to be obtained by one-step purification onto the Ni-NTA column (see
Materials and Methods). Even though expression of ER in the
bacterial system yields much lower levels of full-length ER as had been
reported by others (41), we have successfully expressed the receptor in
bacteria with less degradation products being generated. EMSAs in which
the bacterially expressed ER was used further support the findings
obtained with baculovirus ER.
HMG-1 has previously been shown to enhance the sequence-specific DNA
binding of the progesterone receptor (24, 25). However, the binding of
the retinoic acid receptor
to its cognate responsive element is not
enhanced by HMG-1 (34), indicating that the effect of HMG-1 on DNA
binding is not generalized for all nuclear receptors. HMG-1 also
enhances the sequence-specific DNA binding of HOX proteins,
developmentally active transcription factors (34). Addition of HMG-1 to
the HOX-DNA binding reaction did not result in the formation of slower
migrating complexes in EMSA, indicating that a DNA-HMG1-HOX ternary
complex was not formed or dissociated very rapidly, similar to the lack
of a ternary ERE-HMG1-ER complex in the present study. In any case,
coimmunoprecipitation experiments demonstrated that HMG-1 formed
protein-protein contacts with HOX proteins in the absence of DNA (34),
again similar to the HMG1-ER interaction in this study. HMG-1 and the
closely related HMG-2 were also shown to increase the sequence-specific
DNA binding of Oct proteins (35). Interestingly, HMG-2 protein,
although not present in the Oct-DNA complex detected by EMSA, forms
protein-protein contacts with Oct, as demonstrated by
coimmunoprecipitation. Thus, HMG1/2 is capable of enhancing the
sequence-specific DNA binding of several unrelated transcriptional
activators and of establishing protein-protein contacts with these same
activators in the absence of DNA.
It has been suggested that HMG1-like proteins may exert a DNA
chaperone action by binding transiently to DNA, bending it into a
thermodynamically unfavorable conformation and then exchanging with the
protein that must eventually form a stable complex with its DNA target
(42). This scenario is indeed attractive also in the context of
ER-mediated transcription. HMG-1 enhanced binding of ER to ERE in a
time-dependent fashion, i.e. the promotion of binding was
apparent only when HMG-1 was incubated with the probe before the
addition of ER. However, the DNA chaperone model does not
necessarily predict any form of direct protein-protein interaction as
demonstrated for HOX, Oct, and ER. To account for the protein-protein
interaction, Zappavigna et al. (34) favor an alternative
interpretation, although not mutually exclusive with the one described
above. They propose that the physical contact between HMG-1 and its
protein partner directs both to adjacent or overlapping DNA segments,
generating a complex that is endowed with both geometric and sequence
specificity.
Using purified ER, we sought to determine how the length as well as the
position of the ERE within the oligonucleotide might affect the
HMG-1-mediated ER-ERE binding. We found that when the ERE was
positioned at the very end of the oligonucleotide, a significant
reduction in binding was noted. Flanking sequences around the ERE were
also important factors contributing to the ER-ERE complex formation
induced by HMG-1. ERE flanked by ATs bound ER at a higher yield than
did the ERE flanked by GCs. While HMG-1 has no consensus DNA-binding
site, it shows a preference for binding AT-rich sequences (38). In
terms of length, we have also noted a significant reduction in binding
when a 25-mer ERE was used in place of the usual 35-mer. We believe
that this reduction in binding is a result of decreasing the DNA site
HMG-1 needs for efficient interaction with sequences flanking the ERE.
The footprint size of HMG-1 was determined to be 14 [plusm] 3 bp
(28). Therefore, it is conceivable that as the number of bases flanking
the ERE is reduced, HMG-1-mediated ER-ERE binding becomes affected as
well. In the case of PR, the effect of HMG-1 on DNA binding was
assessed in relation to the position of the progesterone response
element within a 142-bp oligonucleotide (25). In EMSA, Prendergast
et al. (25) found a subtle difference in band migration that
was dependent upon the position of the progesterone response element
and reflected the degree of DNA flexure induced by HMG-1. In this
study, no difference in migration was noted between the 35-mer and
25-mer, because both are much smaller that the 142-bp PRE.
Although HMG-1 plays a clearly defined role in DNA binding, its
function in transcriptional activation remains uncertain (36, 37, 38, 39). For
this reason, we decided to determine its interaction with a bona fide
ER-specific transcriptional activator, TAFII30 (18). By
EMSA, we find that TAFII30 alone does not promote ER-ERE
binding and does not have an effect on HMG-1-mediated ER-ERE binding.
However, for transcription to be initiated from an ERE-containing
template, TAFII30 must be present. Thus, although HMG-1
promotes ER-ERE binding, it fails to stimulate transcription initiation
either in the presence or absence of hormone. This is consistent with
the notion that although DNA bending may be involved in transcriptional
regulatory mechanisms, it is not sufficient, by itself, for
transcription (43, 44). Therefore, HMG-1 might fall into the growing
class of transcription factors that act by bending DNA to facilitate
assembly of higher order nucleoprotein complexes (45, 46, 47, 48, 49). In this
context, HMG-1 may be providing the structural framework necessary for
other transcription factors to interact and function. In fact, as seen
by in vitro transcription assay, TAFII30, while
not affecting ER-ERE binding, stimulates transcription initiation when
in the presence of HMG-1. We observed a 20-fold induction of
transcription initiation, even in the absence of hormone, when ER,
TAFII30, and HMG-1 are incubated with the test template,
pERE2LovTATA. In the presence of hormone, an additional
effect on transcription initiation was apparent. We believe that the
induction of transcription initiation in the absence of hormone is due
to the lack of repressors in our in vitro system. A
repressor protein, SSN6, that specifically interacts with the
N-terminal AF1 of the ER was identified in yeast (50). Estradiol
promotes dissociation of SSN6 allowing interaction of AF1 and AF2 with
the transcription apparatus. However, a mammalian counterpart of yeast
SSN6 has not been identified. Other repressors have been identified for
the thyroid hormone and retinoic acid receptors as SMRT (silencing
mediator for retinoid and thyroid receptors) and N-Cor (nuclear
receptor corepressor) that have the ability to silence thyroid hormone
receptor- and retinoic acid receptor-dependent gene expression
(51, 52, 53). Upon addition of ligand, SMRT and N-Cor dissociate from the
receptor, thereby permitting ligand-induced transcriptional
activation.
In summary, HMG-1 enhances the binding of ER to ERE. The ER thus joins
the progesterone receptor and the HOX and Oct proteins as a group of
transcription factors whose sequence-specific DNA binding is promoted
by HMG-1. These findings are in agreement with the proposed role of
HMG-1 as an "architectural element" (29, 32) that bends DNA and
facilitates the stable binding of DNA binding proteins to their target.
Once formed, the stable ER-ERE complex enhances the recruitment of
specific transcription factors, such as TAFII30, that can
assume the role of a bridging protein between ER, TBP, and other
components of the transcriptional machinery that are essential for
ER-induced transcription initiation. In this process, HMG-1 and
TAFII30 appear to act in sequence, the former acting to
promote ER-ERE binding followed by the latter to stimulate
transcription initiation. Extensive work will be required to define the
role of additional proteins in this process and to gain a complete
understanding of ER-mediated gene transcription.
 |
MATERIALS AND METHODS
|
---|
Baculovirus Expression of ER
The insect cell line Spodoptera frugiperda (Sf9) was
obtained from American Type Culture Collection (Rockville, MD) and
grown at 27 C in EXCELL 400 (JR Scientific; Lenexa, KS) medium.
Autographa californica nuclear polyhedrosis virus (AcNPV)
was purchased from Invitrogen (San Diego, CA). Sf9 cells were infected
with a multiplicity of infection of
10 plaque-forming units/cell
for protein expression studies and 0.11.0 plaque forming units/cell
for virus stock production. The baculovirus transfer vector pVL1392 was
purchased from Invitrogen. The plasmid pSG5 HEGO containing the human
ER cDNA was a generous gift from Dr. Pierre Chambon (Illkirch, France).
Insertion of the ER cDNA into pVL1392 was accomplished by digesting
pSG5 HEGO with EcoRI. This fragment was then cloned into
pVL1392, which had been similarly digested and purified by isolation on
NA45 membranes (Schleicher & Schuell, Keene, NH). This fragment was
oriented by digestion with SmaI and designated pVL1392-hER.
Recombinant baculovirus was produced by cotransfecting 2 x
106 Sf9 cells with AcNPV DNA (1 µg) and pVL1392-hER (2
µg) using the calcium phosphate transfection. The resulting culture
supernatants were harvested after 4 days and screened for homologous
recombination by visual inspection of plaques, which were confirmed by
dot-blot hybridization using the respective 32P-labeled,
nick-translated cDNA probes. Purified recombinant baculovirus was
obtained after three rounds of plaque purification and designated
Ac-hER. Sf9 cells (9 x 106) infected with Ac-hER were
harvested at 24, 48, and 72 h post infection by centrifugation,
and lysed in 500 µl of buffer A (20 mM HEPES, pH 7.5, 0.1
mM EDTA, 40 mM KCl, 20% glycerol) containing 5
mM dithiothreitol (DTT), 1 mM
phenylmethylsulfonyl fluoride, 2.5 µg/ml aprotinin, 2.5 µg/ml
pepstatin, and 2.5 µg/ml leupeptin. The lysates were clarified by
centrifugation at 14,000 x g for 10 min and stored
frozen at -70 C.
Purification of ER from Baculovirus Extracts
Approximately 35 x 106 cells were homogenized
in 3.2 ml of ice-cold extraction buffer (0.4 M KCl, 10
mM Tris, pH 7.9, 1 mM EDTA, 5 mM
DTT, 10% glycerol) containing protease inhibitors as listed above. The
resulting homogenate was centrifuged for 3 min at 4 C in an Eppendorf
microfuge at 14,000 rpm. The salt concentration of the supernatant (3
ml) was reduced to 40 mM KCl with extraction buffer
containing no salt. The sample was loaded onto a Heparin-Sepharose
column that had been preequilibrated with extraction buffer containing
40 mM KCl. The column was washed with the same buffer used
for equilibration and step-eluted with extraction buffer containing
200, 400, and 800 mM KCl, respectively. A 10-µl aliquot
of the 400 and 800 mM fractions containing ER was used for
protein determination by standard methods to adjust the protein
concentration to 1.0 mg/ml using incubation buffer (10 mM
Tris, pH 7.9, 1 mM EDTA, 10 mM
monothioglycerol, 10% glycerol) containing protease inhibitors before
carrying out EMSAs.
Bacterial Expression and Purification of His-tagged ER and
TAFII30
The ER cDNA was amplified using the sense primer,
5'-CGGGATCCATGACCATGACCCTCCACACCAAAGC-3' and the antisense primer,
5'-GGGGTACCCGTGTGGGAGCCAGGGAGCT-3'. TAFII30 cDNA was
amplified from a cDNA stock of the normal mammary cell line, HBL-100.
Primers for amplification of TAFII30 were the sense primer,
5'-CGGGATCCAGCTGCAGCGGCT CC-3' and the antisense primer,
5'-GGGGTACCTACATTTAGGTTGGGTGGCTCAG GTG-3'. Both sets of primers were
designed to contain BamHI and KpnI sites,
respectively, at the 5'-ends. The amplification reaction was carried
out in 100 µl volume containing 10 mM Tris-HCl, pH 8.3,
50 mM KCl, 1.5 mM MgCl2, 200
mM each of the four deoxyribonucleotides, native
Pfu DNA Polymerase (2.5 U; Stratagene, La Jolla, CA), and
each oligonucleotide at either 50 ng/ml or 150 ng/ml. Amplification
conditions for ER cDNA consisted of a denaturing step at 97 C,
annealing at 67 C, and extension at 72 C for a total of 20 cycles. For
TAFII30, conditions consisted of a denaturing step at 95 C,
annealing at 64 C, and extension at 72 C for a total of 30 cycles. The
amplified cDNAs were purified using the QIAEX gel purification kit
(QIAGEN, Chatsworth, CA), digested with BamHI and
KpnI, and repurified by the same method. Ligation of each
cDNA into the similarly digested vector pQE-30 (QIAGEN), which encodes
an N-terminal hexahistidine tag, followed. Each ligated vector/insert
was used to transform M15 Escherichia coli strain (QIAGEN)
as described by the manufacturer. A picked colony harboring the correct
size insert (as judged by restriction digest and DNA sequencing) was
used to express the ER or TAFII30 protein. Expression
conditions consisted of growing the cells in Luria-Broth (supplemented
with kanamycin at 25 mg/ml and Ampicillin at 100 mg/ml) at 30 C until
an A600 reading of 0.6 was reached, induction with 0.5
mM isopropyl-ß-D-thiogalactopyranoside, and
collection of cells 1.5 h after induction. The cells were lysed by
sonication and freeze/thaw cycles. Tagged ER or TAFII30 was
purified using the Ni-NTA resin as specified by the manufacturer
(QIAGEN).
HMG-1
Human HMG-1 was purified as previously reported (54, 55).
Histone H1
Histone H1 was purified by 5% perchloric acid extraction
followed by ion exchange chromatography on Amberlite IRC-50 columns
(ICN Pharmaceuticals, Costa Mesa, CA) (56).
ER Immunoblotting
Whole-cell lysates from either Sf9 or E. coli were
separated by SDS-PAGE and transferred to Immobilon-P membranes
(Millipore; Bedford, MA) in 25 mM Tris, 192 mM
glycine, 0.025% SDS, and 15% methanol for 2 h at 200 mA.
Nonspecific binding was blocked with nonfat dry milk, and the blots
were incubated with rat anti-human ER monoclonal antibodies D547 or
H222 (Abbott Laboratories, North Chicago, IL). The filters were
washed four times with Tris-buffered saline/0.05% Tween-20 and bound
antibody was detected with enhanced chemiluminescence (Amersham;
Arlington Heights, IL).
Hormone-Binding Assay
The ER content of whole-cell extracts from the baculovirus or
bacterial system was determined using dextran-coated charcoal to absorb
free hormone in a six-point binding assay of
17ß-[3H]estradiol. Scatchard analysis was performed to
calculate binding capacity and affinity.
Oligonucleotides
All double-stranded oligonucleotides used in EMSAs were
purchased from Integrated DNA Technologies (Coralville, IA). EREs used
were: 1) 35-mer ERE of the Xenopus vitellogenin A2 gene
5'-GTCCAAAGTCAGGTCACAGTGACCTGATCAAAGTT-3'; 2)
two 25-mers from the vitellogenin A2 gene differing in the position of
the ERE, 5'-AGGTCACAGTGACCTGATCAAAGTT-3' and
5'-AAGTCAGGTCACAGTGACCTGA TCA-3'; 3) a
consensus ERE flanked by GCs,
5'-GGCCCCGGTCACAGTGACCGG CCCC-3'. The oligos
were dissolved in TE buffer (10 mM Tris, 1 mM
EDTA, pH 8.0), and equimolar amounts of each strand were annealed by
heating to 95 C and cooling to room temperature over a period of
1.5 h. Double-stranded oligos were end-labeled using
[
-32P]ATP and T4 polynucleotide kinase. Free isotope
was removed by passing the labeled, double-stranded oligos through
Chroma spin-10 columns (Clontech; Palo Alto, CA).
EMSA
The assay was performed essentially as previously described
(57). HMG-1 (1 ng) was first incubated with 0.30.5 ng of the
32P-labeled double-stranded ERE oligomer for 15 min at 4 C.
Aliquots of partially purified ER (10 µg/ml) and 1.0 µg
poly(deoxyinosinic)·poly(deoxycytidylic)acid were then added to the
reaction mixture for a final volume of 20 µl. After a 15-min
incubation at 4 C, the protein-ERE complexes were separated by
electrophoresis through 4.5% acrylamide (38:2, acrylamide:bis) gels
using 1x TBE buffer (10 mM Tris, 10 mM boric
acid, 0.02 mM EDTA, pH 8.0). Gels were vacuum-dried and
autoradiographed. The ER-containing human breast cancer cell line MCF-7
was used as a positive control for ERE binding. For antibody shift
experiments, ER was preincubated with the monoclonal antibody H222
before the assay.
Coimmunoprecipitation Assays
For coimmunoprecipitation experiments, 25 µl preswollen
protein A insolubilized on Sepharose CL-4B (Sigma, St. Louis, MO) were
washed twice with 500 µl binding buffer (100 mM NaCl, 20
mM Tris, pH 7.0, 10% glycerol, 1% Triton-X). Between
washes, the protein A-Sepharose was recovered by centrifugation for 2
min at 3500 rpm. To the washed beads, affinity-purified anti-HMG-1 (1
µg), purified HMG-1 (100 ng), and 100 ng purified bacterial ER or
TAFII30 were added. The reaction volume was brought up to
400 µl using binding buffer containing DTT and the protease
inhibitors phenylmethylsulfonylfluoride, aprotinin, and leupeptin. The
reactions were incubated overnight at 4 C on a rotating wheel.
Immunoprecipitates were collected by centrifugation, washed three times
with 500 µl binding buffer, and recovered by boiling the precipitate
for 5 min in SDS-sample buffer. The supernatant and pooled washes were
each concentrated using Centricon-10 concentrators (Amicon, Beverly,
MA). Supernatant, wash, and precipitate fractions were run on an
SDS-PAGE gel. Proteins were transferred onto nitrocellulose membranes,
and Western blot analysis was carried out with the ER-antibody, H222
(Abbott Laboratories) or TAFII30 antibody, 2F4 (kindly
provided by Dr. Pierre Chambon). The same membranes were stripped in
100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM
Tris/HCl, pH 7.0, at 50 C and reprobed with the affinity-purified
anti-HMG-1.
In Vitro Transcription Assays
Assays were performed using the test template,
pERE2LovTATA, and the control template, pLovTATA, kindly
provided by Drs. Ming-Jer Tsai and Bert OMalley (Baylor University,
Houston, TX). Both templates are ultimately derived from
pML(C2AT)19, a plasmid containing a 377-bp
G-free cassette linked to the TATA box region of the adenovirus-2
major late promoter (58). Accurate initiation 30 bp downstream from the
TATA box is expected to generate a 360-nucleotide transcript devoid of
G residues. As an internal control, the plasmid pMLcas190, which
contains the AdML promoter linked to a G-free cassette of 180 bp (58),
was used. Typical reactions contained the following components in a 30
µl volume: 1) 7.5 mM HEPES, pH 7.6, 60 mM
potassium glutamate, 3.75 mM MgCl2, 0.03
mM EDTA, 1.5 mM DTT, 3% glycerol, and 0.5
mM each ATP, CTP, GTP, 20 µM UTP, and 15
µCi of [
-32P]UTP; 2) 500700 ng ERE-test or control
template and internal control template; 3) ER-containing extract at 100
ng/ml and, when indicated, ER was preincubated with 10-8
M estradiol dissolved in ethanol; 4) 1 ng HMG-1; 5) 10 ng
TAFII30; and 6) 10 U of ribonuclease T1. Reactions were
initiated by adding 5 U of Drosophila embryo nuclear extract
(Promega; Madison, WI). After a 60-min incubation at 30 C, the
transcription reactions were terminated by treatment with 170 µl of
stop solution [20 mM Tris-HCl, pH 7.5, 10 mM
EDTA, 0.5% SDS] containing 200 µg/ml yeast tRNA, and 400 µg/ml
proteinase K. After addition of 200 µl of 7 M urea (in 10
mM Tris-HCl, 1 mM EDTA, pH 8.0), transcripts
were recovered by ethanol precipitation and analyzed by electrophoresis
on 6% acrylamide (38:2, acrylamide-bis), 8 M urea
sequencing gels.
 |
ACKNOWLEDGMENTS
|
---|
We thank P. Chambon for HEGO ER cDNA and
anti-TAFII30 antibody, M. J. Tsai and B. W. OMalley for
pLovTATA and PERE2LovTATA, and Abbott Laboratories for
anti-ER antibodies. We also thank Martha Bass for excellent technical
help.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Fritz F. Parl, M.D., Ph.D., 4918 The Vanderbilt Clinic, 22nd Ave South at Pierce, Nashville, Tennessee 37232.
This research was supported by Public Health Service Grant HD-07043 (to
C.S.V.) and US Army Breast Cancer Training Grant DAMD-17-94-J4024 (to
C.J.Y. and L.R.B.).
Received for publication August 15, 1996.
Accepted for publication April 17, 1997.
 |
REFERENCES
|
---|
-
Beato M, Herrlich P, Schutz G 1995 Steroid hormone
receptors: many actors in search of a plot. Cell 83:851857[Medline]
-
Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G,
Umesono K, Blumberg B, Kastner P, Mark M, Chambon P, Evans RM 1995 The
nuclear receptor superfamily: the second decade. Cell 83:835839[Medline]
-
Tora L, White J, Brou C, Tasset D, Webster N, Scheer E,
Chambon P 1989 The human estrogen receptor has two independent
nonacidic transcriptional activation functions. Cell 59:477487[Medline]
-
Kumar V, Green S, Staub A, Chambon P 1986 Localisation of the
oestradiol-binding and putative DNA-binding domains of the human
oestrogen receptor. EMBO J 5:22312236[Abstract]
-
Webster NJG, Green S, Jin JR, Chambon P 1988 The
hormone-binding domains of the estrogen and glucocorticoid receptors
contain an inducible transcription activation function. Cell 54:199207[Medline]
-
Linstedt AD, West NB, Brenner RM 1986 Analysis of
monomeric-dimeric states of the estrogen receptor with monoclonal
antiestrophilins. J Steroid Biochem 24:677686[CrossRef][Medline]
-
Kumar V, Chambon P 1988 The estrogen receptor binds tightly
to its responsive element as a ligand-induced homodimer. Cell 55:145156[Medline]
-
Klein-Hitpass L, Schorpp M, Wagner U, Ryffel GU 1986 An
estrogen-responsive element derived from the 5' flanking region of the
Xenopus vitellogenin A2 gene functions in transfected human cells. Cell 46:10531061[Medline]
-
Klock G, Strahle U, Schutz G 1987 Oestrogen and
glucocorticoid responsive elements are closely related but distinct.
Nature 329:734736[CrossRef][Medline]
-
Seiler-Tuyns A, Walker P, Martinez E, Merillat AM, Givel F,
Wahli W 1986 Identification of estrogen-responsive DNA sequences by
transient expression experiments in a human breast cancer cell line.
Nucleic Acids Res 14:87558770[Abstract]
-
Ptashne M 1988 How eukaryotic transcriptional activators work.
Nature 335:683689[CrossRef][Medline]
-
Ptashne M, Gann AAF 1990 Activators and targets. Nature 346:329331[CrossRef][Medline]
-
Bagchi MK, Tsai MJ, OMalley BW, Tsai SY 1992 Analysis of the
mechanism of steroid hormone receptor-dependent gene activation in
cell-free systems. Endocr Rev 13:525535[Medline]
-
Mukherjee R, Chambon PA 1990 Single-stranded DNA-binding
promotes the binding of the purified oestrogen receptor to its
responsive element. Nucleic Acids Res 18:57135716[Abstract]
-
Nardulli AM, Geoffrey LG, Shapiro DJ 1993 Human estrogen
receptor bound to an estrogen response element bends DNA. Mol
Endocrinol 7:331340[Abstract]
-
Cavailles V, Dauvois S, Danielian PS, Parker MG 1994 Interaction of proteins with transcriptionally active estrogen
receptors. Proc Natl Acad Sci USA 91:2000910013
-
Halachmi S, Marden E, Martin G, Mackay H, Abbondanza C, Brown
M 1994 Estrogen receptor-associated proteins: possible mediators of
hormone-induced transcription. Science 264:14551458[Medline]
-
Jacq X, Brou C, Lutz Y, Davidson I, Chambon P, Tora L 1994 Human TAFII30 is present in a distinct TFIID complex and is
required for transcriptional activation by the estrogen receptor. Cell 79:107117[Medline]
-
Landel CC, Kushner PJ, Greene GL 1994 The interaction of human
estrogen receptor with DNA is modulated by receptor-associated
proteins. Mol Endocrinol 8:14071419[Abstract]
-
Cavailles V, Dauvois S, LHorset F, Lopez G, Hoare S, Kushner
PJ, Parker MG 1995 Nuclear factor RIP140 modulates transcriptional
activation by the estrogen receptor. EMBO J 14:37413751[Abstract]
-
Le Dourain B, Zechel C, Garnier JM, Lutz YTL, Pierrat B, Heery
D, Gronemeyer H, Chambon P, Losson R 1995 The N-terminal part of TIF1,
a putative mediator of the ligand-dependent activation function (AF-2)
of nuclear receptors, is fused to B-raf in the oncogenic protein T18.
EMBO J 14:20202033[Abstract]
-
Onate SA, Tsai SY, Tsai MJ, OMalley BW 1995 Sequence and
characterization of a coactivator for the steroid hormone receptor
superfamily. Science 270:13541357[Abstract]
-
vom Baur E, Zechel C, Heery D, Heine MJS, Garnier JM, Vivat V,
Le Douarin B, Gronemeyer H, Chambon P, Losson R 1996 Differential
ligand-dependent interactions between the AF-2 activating domain of
nuclear receptors and the putative transcriptional intermediary factors
mSUG1 and TIF1. EMBO J 15:110124[Abstract]
-
Onate SA, Prendergast P, Wagner JP, Nissen M, Reeves R,
Pettijohn DE, Edwards DP 1994 The DNA-bending protein HMG-1 enhances
progesterone receptor binding to its target DNA sequence. Mol Cell Biol 14:33763391[Abstract]
-
Prendergast P, Pan Z, Edwards DP 1996 Progesterone
receptor-induced bending of its target DNA: distinct effects of the A
and B receptor forms. Mol Endocrinol 10:393407[Abstract]
-
Johns EW 1982 The HMG Chromosomal Proteins. Academic Press,
London
-
Bustin M, Lehn DA, Landsman D 1990 Structural features of the
HMG chromosomal proteins and their genes. Biochim Biophys Acta 1049:231243[Medline]
-
Landsman D, Bustin M 1993 A signature for the HMG-1 box DNA
binding proteins. Bioessays 15:539546[Medline]
-
Grosschedl R, Giese K, Pagel J 1994 HMG domain proteins:
architectural elements in the assembly of nucleoprotein structures.
Trends Genet 10:94100[CrossRef][Medline]
-
Pil PM, Chow CS, Lippard SJ 1993 High-mobility-group protein
mediates DNA bending as determined by ring closures. Proc Natl Acad Sci
USA 90:94659469[Abstract]
-
Paull TT, Haykinson MJ, Johnson RC 1993 The non-specific
DNA-binding and -bending proteins HMG1 and HMG2 promote the assembly of
complex nucleoprotein structures. Genes Dev 7:15211534[Abstract]
-
Shykind BM, Kim J, Sharp PA 1995 Activation of the TFIID-TFIIA
complex with HMG-2. Genes Dev 9:13541365[Abstract]
-
Watt F, Molloy PL 1988 High mobility group proteins 1 and 2
stimulate binding of a specific transcription factor to the adenovirus
major late promoter. Nucleic Acids Res 16:14711486[Abstract]
-
Zappavigna V, Falciola L, Citterich MH, Mavilio F, Bianchi ME 1996 HMG1 interacts with HOX proteins and enhances their DNA binding
and transcriptional activation. EMBO J 15:49814991[Abstract]
-
Zwilling S, Konig H, Wirth T 1995 High mobility group protein
2 functionally interacts with the POU domain of octamer transcription
factors. EMBO J 14:11981208[Abstract]
-
Tremethick DJ, Molloy PL 1988 Effects of high mobility group
proteins 1 and 2 on initiation and elongation of specific transcription
by RNA polymerase II in vitro. Nucleic Acids Res 16:1110711123[Abstract]
-
Waga S, Mizuno S, Yoshida M 1990 Chromosomal protein HMG1
removes the transcriptional block caused by the cruciform in
supercoiled DNA. J Biol Chem 265:1942419428[Abstract/Free Full Text]
-
Singh J, Dixon GH 1990 High mobility group proteins 1 and 2
functions as general class II transcription factors. Biochemistry 29:62956302[Medline]
-
Ge H, Roeder RG 1994 The high mobility group protein HMG1 can
reversibly inhibit class II gene transcription by interaction with the
TATA-binding protein. J Biol Chem 269:1713617140[Abstract/Free Full Text]
-
Seielstad DA, Carlson KE, Katzenellenbogen JA, Kushner PJ,
Greene GL 1995 Molecular characterization by mass spectrometry of the
human estrogen receptor ligand-binding domain expressed in E.
coli. Mol Endocrinol 9:647658[Abstract]
-
Wittliff JL, Wenz LL, Dong J, Nawaz Z, Butt TR 1990 Expression
and characterization of an active human estrogen receptor as a
ubiquitin fusion protein from Escherichia coli. J Biol
Chem 265:2201622022[Abstract/Free Full Text]
-
Ner SS, Travers AA, Churchill MEA 1994 Harnessing the writhe:
a role for DNA chaperones. Trends Biochem Sci 19:185187[CrossRef][Medline]
-
Kim J, Klooster S, Shapiro DJ 1995 Intrinsically bent DNA in a
eukaryotic transcription factor recognition sequence potentiates
transcription activation. J Biol Chem 270:12821288[Abstract/Free Full Text]
-
Nardulli AM, Grobner C, Cotter D 1995 Estrogen receptor
induced DNA bending: orientation of the bend and replacement of
estrogen response element with an intrinsic DNA binding sequence. Mol
Endocrinol 9:10641076[Abstract]
-
Giese K, Cox J, Grosschedl R 1992 The HMG domain of lymphoid
enhancer factor 1 bends DNA and facilitates assembly of functional
nucleoprotein structures. Cell 69:185195[Medline]
-
Thanos D, Maniatis T 1992 The high mobility group protein HMG
I(Y) is required for NF-kB-dependent virus induction of the human
IFN-beta gene. Cell 71:777789[Medline]
-
Natesan S, Gilman MZ 1993 DNA bending and
orientation-dependent function of YY1 in the c-fos promoter. Genes Dev 7:24972509[Abstract]
-
Pontiggia A, Rimini R, Harley VR, Goodfellow PN, Lovell-Badge
R, Bianchi ME 1994 Sex-reversing mutations affect the architecture of
SRY-DNA complexes. EMBO J 13:61156124[Abstract]
-
Wolffe AP 1994 Architectural transcription factors. Science 264:11001101[Medline]
-
McDonnell DP, Vegeto E, OMalley BW 1992 Identification of a
negative regulatory function for steroid receptors. Proc Natl Acad Sci
USA 89:1056310567[Abstract]
-
Chen JD, Evans RM 1995 A transcriptional co-repressor that
interacts with nuclear hormone receptors. Nature 377:454457[CrossRef][Medline]
-
Horlein AJ, Naar AM, Heinzel T, Torchia J, Gloss B, Kurokawa
R, Ryan A, Kamei Y, Soderstrom M, Glass CK, Rosenfeld MG 1995 Ligand-independent repression by the thyroid hormone receptor mediated
by a nuclear receptor co-repressor. Nature 377:397403[CrossRef][Medline]
-
Kurokawa R, Soderstrom M, Horlein A, Halachmi S, Brown M,
Rosenfeld MG, Glass CK 1995 Polarity-specific activities of retinoic
acid receptors determined by a co-repressor. Nature 377:451457[CrossRef][Medline]
-
Romani M, Rodman TC, Vidali G, Bustin M 1979 Serological
analysis of species specificity in the high mobility group chromsomal
proteins. J Biol Chem 254:29182922[Abstract]
-
Hamada H, Bustin M 1985 Chromosomal proteins HMG1 and 2
distinguish between S1 sensitive sites in supercoiled DNA. Biochemistry 24:14281433[Medline]
-
Bustin M 1973 Arrangement of histones in chromatin.
Nature New Biol 245:207209[Medline]
-
Foster BD, Cavener DR, Parl FF 1991 Binding analysis of
the estrogen receptor to its specific DNA target site in human
breast cancer. Cancer Res 51:34053410[Abstract]
-
Sawadago M, Roeder RG 1985 Factors involved in
specific transcription by human RNA polymerase II: analysis by
a rapid and quantitative in vitro assay. Proc Natl Acad Sci
USA 82:43944398[Abstract]