The High Mobility Group Protein 1 Enhances Binding of the Estrogen Receptor DNA Binding Domain to the Estrogen Response Element
Lorene E. Romine,
Jennifer R. Wood,
LuAnne A. Lamia,
Paul Prendergast,
Dean P. Edwards and
Ann M. Nardulli
Department of Molecular and Integrative Physiology (L.E.R., J.R.W.,
L.A.L., A.M.N.) University of Illinois Urbana, Illinois
61801
Department of Pathology (P.P., D.P.E.) University
of Colorado Health Sciences Center Denver, Colorado 80262
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ABSTRACT
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We have examined the ability of the high-mobility
group protein 1 (HMG1) to alter binding of the estrogen receptor
DNA-binding domain (DBD) to the estrogen response element (ERE). HMG1
dramatically enhanced binding of purified, bacterially expressed DBD to
the consensus vitellogenin A2 ERE in a dose-dependent manner. The
ability of HMG1 to stabilize the DBD-ERE complex resulted in part from
a decrease in the dissociation rate of the DBD from the ERE. Antibody
supershift experiments demonstrated that HMG1 was also capable of
forming a ternary complex with the ERE-bound DBD in the presence of
HMG1-specific antibody. HMG1 did not substantially affect DBD-ERE
contacts as assessed by methylation interference assays, nor did it
alter the ability of the DBD to induce distortion in ERE-containing DNA
fragments. Because HMG1 dramatically enhanced estrogen receptor DBD
binding to the ERE, and the DBD is the most highly conserved region
among the nuclear receptor superfamily members, HMG1 may function to
enhance binding of other nuclear receptors to their respective response
elements and act in concert with coactivator proteins to regulate
expression of hormone-responsive genes.
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INTRODUCTION
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Steroid hormones play critical roles in development and
maintenance of reproductive tissues. Upon binding to the estrogen
receptor (ER), estrogenic ligands induce changes in receptor
conformation that in turn promote binding of the ER dimer to an
estrogen response element (ERE). Because the ER-ERE interaction
initiates changes in target gene transcription that result in new
protein synthesis, this interaction provides a crucial link in the
chain of events that are required for estrogen responsiveness.
It has recently become apparent that association of receptors with
activators may be involved in modulating hormone-responsive gene
transcription. A number of candidate coactivator proteins have been
identified, including SRC1, ERAP 140 and 160, RIP140 and 160, TIF 1,
TIF 2, TAFII30, and CBP/p300 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10). It is thought that these and
other proteins (11, 12) may act in concert with ER to activate
transcription. Corepressors, which inhibit steroid hormone
receptor-induced transcription, have also been identified (13, 14).
Thus, overall transcription of steroid hormone-responsive genes may be
subject to the additive effects of coactivators and corepressors.
The high-mobility group protein 1 (HMG1) is a ubiquitous intracellular
protein that binds with low affinity in a sequence-independent manner
to single- and double-stranded linear DNA, supercoiled DNA, bent or
kinked DNA, and DNA structures such as four-way junctions or cruciform
DNA (15, 16, 17, 18). HMG1 has been conserved evolutionarily in protozoan (19),
yeast (20), plant (21), vertebrate (22), and mammalian (16, 23) cells.
This high degree of conservation among such diverse organisms may
indicate that HMG1 plays an important role in cell function. In fact, a
number of functions have been attributed to HMG1, including DNA bending
(24, 25) and loop formation (18), assembly of nucleoprotein structures
(26), decondensation of chromatin (27), and transcription activation
(28, 29) and repression (30, 31, 32).
In vitro assays have demonstrated that HMG1 enhances binding
of the ER and the progesterone receptor (PR) to DNA (33, 34). However,
the receptor regions required for this HMG1-enhanced DNA binding have
not been delineated. ER and PR are both members of the nuclear receptor
superfamily, which have common structural and functional domains. The
most highly conserved region among these nuclear receptor superfamily
members is the DNA-binding domain (DBD). It seemed possible that HMG1
might be capable of interacting with nuclear receptor DBDs to enhance
receptor binding. To test this hypothesis, we have examined the ability
of purified HMG1 to enhance binding of purified ER DBD to
ERE-containing DNA fragments. The Xenopus laevis DBD used in
these studies encompasses amino acids 171281, which includes two zinc
fingers and an acidic domain from the hinge region (35), and retains
many of the characteristics of the intact ER. The purified DBD is a
monomer in solution, binds effectively and with great specificity to
consensus and imperfect ERE sequences, and enhances transcription of an
estrogen-responsive reporter plasmid in transient transfection assays
(36, 37). In addition, because the DBD is the most highly conserved
region of steroid hormone receptors, examination of the interaction of
the DBD with target DNA may provide clues as to how steroid hormone
receptors exert their effects.
We find that purified HMG1 dramatically enhances binding of the DBD to
the ERE. This enhanced binding is in part due to the decreased
dissociation rate of the DBD-ERE complex in the presence of HMG1.
However, neither the mobility of the DBD-ERE complex nor the DBD
contacts with DNA are substantially different in the presence or
absence of HMG1. These findings suggest that HMG1 may function to
enhance binding of other nuclear receptors to their respective response
elements and thereby influence expression of hormone-responsive
genes.
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RESULTS
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The DBD Binds to the ERE as a Homodimer
Binding of bacterially expressed, purified ER DBD to
ERE-containing DNA fragments was first examined in the absence of HMG1.
Increasing concentrations (065 ng) of purified DBD were incubated
with 427-bp ERE-containing 32P-labeled DNA fragments and
then fractionated on a nondenaturing acrylamide gel. A single DBD-DNA
complex (Fig. 1
,
) was observed over a
range of DBD concentrations. When 10 ng DBD were added to the binding
reaction, a faint gel-shifted band was observed. Increasing the amount
of DBD in the binding reaction proportionately increased the amount of
DBD-DNA complex formed. When 65 ng DBD were added to the binding
reaction, which constitutes more than a 250-fold excess of DBD over ERE
half-sites, nearly all of the 32P-labeled DNA fragments
were present in the DBD-ERE complex. Two slower migrating bands, which
were sometimes present in individual probe preparations, were also
observed but were unaffected by DBD addition. The presence of a single
gel-shifted band with identical mobility at both low and high DBD
concentrations suggested that the DBD bound either as a monomer or as a
dimer at all concentrations tested.

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Figure 1. The DBD Forms a Single Complex with
ERE-Containing DNA Fragments
Increasing amounts of purified DBD (065 ng) were incubated with 427
bp 32P-labeled DNA fragments, each containing a single
consensus ERE. The DBD-DNA mixtures were fractionated on a
nondenaturing acrylamide gel, which was then dried and subjected to
autoradiography. The DBD-ERE complex is indicated ( ).
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To determine whether the DBD was interacting with the consensus ERE as
a monomer or as a dimer, deoxyribonuclease I (DNase I) footprinting was
carried out using a 281-bp ERE-containing DNA fragment that had been
labeled with 32P on the coding strand. As seen in Fig. 2
, both ERE half-sites were equally
protected when the DBD was added to the binding reaction over a wide
range of DBD concentrations. Regardless of whether the DBD
concentration was low or high, the 5'- and 3'-half-sites were equally
protected. These findings in combination with the gel mobility shift
assays, in which only one DBD-ERE complex was observed, demonstrate
that the DBD bound only as a homodimer to the ERE and are in agreement
with previous gel shift assays using a different, much smaller DNA
fragment (37) and with crystal structure studies of the human ER DBD
(38, 39). Interestingly, a hypersensitive site was produced at the 3'-
but not at the 5'-end of the ERE and may reflect changes in DNA
structure (40). DNase I footprinting of the opposite strand also
displayed equal protection of both ERE half-sites (data not shown).

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Figure 2. The DBD Binds as a Dimer to the ERE
The coding strand of a 281-bp DNA fragment was radioactively labeled,
combined with increasing amounts of purified DBD, and subjected to
DNase I digestion for 2.5 min at room temperature. The cleaved DNA
fragments were fractionated on an 8% denaturing acrylamide gel. The
gel was dried and subjected to autoradiography. The position of the ERE
sequence and a hypersensitive site (*) are indicated to the
right of the figure.
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HMG1 Enhances DBD Binding to the ERE
To determine whether HMG1 enhanced ER DBD binding to DNA, a
constant amount of purified ER DBD and 32P-labeled
ERE-containing DNA fragments was incubated with increasing amounts of
HMG1. When these protein-DNA mixtures were fractionated on an
acrylamide gel, the DBD-ERE complex was barely visible with 8 ng
purified DBD in the absence of HMG1 (Fig. 3
, 0 ng HMG1). However, addition of
purified HMG1 to the binding reaction enhanced the protein-DNA complex
formation in a dose-dependent manner. Addition of as little as 10 ng
HMG1, which constituted a 1.6-fold excess of DBD monomer over HMG1,
enhanced protein-DNA complex formation. However, the exact DBD/HMG1
stoichiometry required for optimal DBD binding is difficult to
determine since activity of the purified HMG1 varied somewhat with
individual preparations. As seen with the purified DBD alone (Fig. 1
),
a single protein-DNA complex, which comigrated with the DBD-ERE
complex, was formed when 1060 ng HMG1 were included in the binding
reactions. The enhanced binding observed in the presence of HMG1 was
not due to stabilization of the DBD by increased protein concentrations
since addition of increasing concentrations of either ovalbumin or BSA
failed to elicit a dose-dependent increase in DBD binding (data not
shown). However, to ensure that protein concentrations present in the
binding reactions did not influence DBD-DNA complex formation,
ovalbumin was added to each of the samples to maintain constant total
protein levels while varying the amount of HMG1.

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Figure 3. HMG1 Increases Protein-DNA Complex Formation
Increasing amounts of HMG1 (060 ng) were incubated with 8 ng purified
ER DBD and 32P-labeled ERE-containing DNA fragments.
Ovalbumin was included to maintain protein levels at 2.8 µg. Binding
reactions were incubated as described in Materials and
Methods and then fractionated on a nondenaturing polyacrylamide
gel. The gel was dried and subjected to autoradiography. The
protein-DNA complex is indicated ( ).
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Although HMG1 significantly enhanced the formation of a protein-DNA
complex that comigrated with the DBD-DNA complex, it was not clear
whether HMG1 was part of this complex. HMG1, which has a molecular mass
of 28,000 Da and could feasibly bind to the 32P-labeled DNA
fragments, is nearly identical in size to a DBD dimer formed by two
13,700-Da monomers. To determine which proteins were present in the
protein-DNA complexes, antibodies made against the ER DBD and HMG1 were
included in the binding reactions. P1A3, a monoclonal antibody made
against the purified Xenopus laevis ER DBD (41),
supershifted the protein-DNA complexes in the absence (Fig. 4
, lane 2) and in the presence (lane 4)
of HMG1, indicating that the DBD was present in both complexes. When
the HMG1-specific monoclonal antibody 854E10 was included in the
binding reaction, a small portion of the protein-DNA complex was
supershifted (lane 5). Higher concentrations of the HMG1 antibody
failed to supershift more of the DBD-ERE complex (data not shown). The
supershifting of a portion of DBD-ERE complexes suggests that HMG1 was
weakly associated with the DBD-ERE complex in solution and that
antibody binding stabilized the ternary HMG1-DBD-ERE complex
sufficiently so that it could withstand the 3-h electrophoresis period.
The HMG1-specific antibody 854E10 has previously demonstrated the
ability to supershift a portion of PR-HMG1-DNA complexes (42).
HMG1-DBD-ERE complexes that migrated more slowly than the DBD-ERE
complex were not observed in the absence of the HMG1 antibody (lane 3).
As an additional control, HMG1 was incubated with ERE-containing DNA
fragments in the absence of the DBD, and no supershifted complex was
observed (data not shown). Thus, these data suggested that the HMG1 was
weakly associated with the DBD-DNA complex.

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Figure 4. Antibodies to DBD and HMG1 Supershift the DBD-DNA
Complex
Purified DBD was incubated with 32P-labeled ERE-containing
DNA fragments in the absence (lanes 1 and 2) or in the presence of HMG1
(lanes 35). DBD-specific ( DBD) or HMG1-specific ( HMG1)
monoclonal antibodies were included in the binding reactions as
indicated. Ovalbumin was added to lanes without antibody to maintain
total protein concentration. The DBD-DNA mixtures were fractionated on
a nondenaturing acrylamide gel. The gel was dried and subjected to
autoradiography.
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HMG1 Does Not Alter the DBD Footprint
The DNA fragments used in the gel shift assays were large (427
bp), and the possibility existed that HMG1 might modify the interaction
of the DBD with the ERE. To more precisely map the DBD-ERE boundaries,
methylation interference experiments were carried out. This procedure
utilizes the alkylating agent dimethyl sulfate to chemically methylate
the N7 position of guanine residues. Guanine methylation,
in turn, inhibits protein binding and identifies individual nucleotides
that are required for protein-DNA interactions.
Purified DBD was incubated with methylated 32P-labeled DNA
fragments in the absence and in the presence of HMG1. Protein-DNA
complexes and free DNA were resolved on a nondenaturing gel,
electroeluted, cleaved with piperidine, fractionated on a denaturing
acrylamide gel, and visualized by autoradiography. As seen in Fig. 5
, only the ERE was involved in the
DBD-DNA interaction regardless of whether HMG1 was present. The three
guanine residues in the two ERE half-sites
(GGTCAcagTGACC) were important for DBD binding
(lanes 2 and 3). Fragments that were methylated at these crucial
guanine residues were predominantly present in the free probe (lanes 1
and 4). In contrast, methylation of the guanine residue in the 3-bp
spacer between the two ERE half-sites disrupted DBD-ERE complex
formation somewhat, but was clearly less important than the guanine
residues within the ERE half-sites. These findings indicate that HMG1
does not significantly alter the DBD-ERE contacts.

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Figure 5. Methylation Interference Demonstrates That Only the
ERE Is Involved in the DBD-DNA Interaction in the Absence and in the
Presence of HMG1
Methylated 281-bp ERE-containing DNA fragments, which had been labeled
on the coding strand, were incubated with 100 ng purified DBD (lanes 1 and 2) or 50 ng purified DBD
plus 250 ng purified HMG1 (lanes 3 and 4) and fractionated on a
nondenaturing acrylamide gel. The complexed (C) and free (F) DNA from
each lane were electroeluted, precipitated, cleaved with piperidine,
and fractionated on a sequencing gel. The gel was dried and subjected
to autoradiography. The sequence and position of the ERE are
indicated.
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HMG1 Stabilizes DBD-ERE Binding
HMG1 could enhance DBD-ERE complex formation by increasing the
association rate, decreasing the dissociation rate, or a combination of
these two events. To determine whether HMG1 influenced the association
rate of the DBD with the ERE, purified DBD was incubated with
32P-labeled ERE-containing DNA fragments in the absence and
in the presence of HMG1 for 0.510 min at 4 C and then loaded onto a
running acrylamide gel. The DBD-ERE association occurred very rapidly
in the absence and in the presence of HMG1 (Fig. 6
). Despite the fact that the reactions
were carried out at 4 C in the presence of 15% glycerol to slow the
association, maximal binding was achieved at the earliest timepoint
measured (0.5 min). Because the DBD-ERE association was so rapid, we
were unable to determine whether HMG1 affected the DBD-ERE association
rate. It is clear, however, that the DBD associates very rapidly with
the ERE in the absence and in the presence of HMG1 and that maximal
binding is achieved by 0.5 min.

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Figure 6. DBD Association with the ERE Is Rapid in the
Absence and in the Presence of HMG1
A, Schematic representation of experimental protocol. B,
32P-labeled ERE-containing DNA fragments were combined with
purified DBD alone or with DBD plus 75 ng HMG1 and incubated at 4 C.
Aliquots were removed 0.5, 1, 2, 5, and 10 min after DBD addition and
loaded directly onto a running acrylamide gel. The gel was dried and
subjected to autoradiography.
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To determine whether HMG1 influenced the dissociation
rate of the DBD from the ERE, purified DBD was incubated with
ERE-containing DNA fragments in the absence and in the presence of
HMG1. An excess of unlabeled ERE (300 ng) was added to the binding
reaction after a 10-min incubation and 20-µl aliquots were removed at
specified times and loaded directly onto a running acrylamide gel. In
the absence of HMG1, the DBD dissociated very rapidly from the ERE, so
that 2 min after addition of the unlabeled ERE the DBD-ERE complex was
nearly undetectable (Fig. 7
). Addition of
75 ng HMG1 consistently slowed dissociation of the DBD from the ERE
when the unlabeled ERE oligo was present, so that DBD-ERE complexes
were still detectable 30 min after addition of the unlabeled ERE. In
the absence of unlabeled ERE oligo, the DBD-DNA complex remained stable
for the 30-min incubation period (data not shown). Thus, HMG1
helped to stabilize the DBD-ERE interaction by decreasing the
dissociation rate of the DBD from the ERE. Specific DBD/HMG1 ratios may
be needed to stabilize the DBD-ERE interaction since 50 ng HMG1 failed
to stabilize and 100 ng did not further stabilize DBD-ERE complex
formation (data not shown). However, due to batch-to-batch variations
in HMG1 activity, the DBD/HMG1 ratio required for DBD stabilization
must be empirically determined for each HMG1 preparation.

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Figure 7. HMG1 Decreases the Rate of DBD-ERE Dissociation
A, Schematic representation of experimental protocol. B,
32P-labeled ERE-containing DNA fragments were combined with
purified DBD alone or with DBD plus 75 ng HMG1 and incubated for 10 min
at 4 C. A 20-µl aliquot was removed and loaded directly onto a
running acrylamide gel, and 300 ng of a 30-bp oligo containing the ERE
were immediately added to the incubation. Aliquots (20 µl) were
removed 0.2530 min after addition of the ERE oligo and loaded onto a
running acrylamide gel. The gel was dried and subjected to
autoradiography. C, Results from six (DBD alone) or seven (DBD and
HMG1) independent experiments were combined, and values are presented
as the mean ± SE. Several error bars
are quite small and are not visible.
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HMG1 Does Not Affect the Ability of the DBD to Distort DNA
We have previously demonstrated that the DBD induces distortion in
ERE-containing DNA fragments (43, 44, 45). Since HMG1 recognizes bent DNA
structures and induces DNA bending (46, 47), it seemed plausible that
HMG1 might affect the ability of the DBD to distort DNA. Therefore,
circular permutation analysis was carried out using a series of DNA
fragments, each of which contained a consensus ERE at varying
positions. Either 25 ng DBD alone or 8 ng DBD plus 70 ng HMG1 were
incubated with 32P-labeled ERE-containing DNA fragments. As
evidenced by the differential migration of the DBD-DNA complexes, the
DBD induced distortion in ERE-containing DNA fragments in the absence
and in the presence of HMG1 (Fig. 8
).
DNA-bending standards (48) were used to determine that the degree of
distortion induced by DBD binding was 33o in the presence
and in the absence of HMG1. These studies are in good agreement with
earlier studies examining DBD-induced distortion in DNA fragments (43)
and demonstrate that HMG1 does not alter the degree of distortion
induced by DBD binding.

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Figure 8. HMG1 Does Not Increase the Magnitude of the
DBD-Induced DNA Bend
32P-labeled DNA fragments (AE) were prepared by digesting
the circular permutation vector ERE BendI with restriction
endonucleases so that the ERE was located either at the end of the
fragment (A and E), in the middle of the fragment (C), or at an
intermediate position (B and D). These fragments were incubated with 25
ng DBD or 8 ng DBD and 70 ng HMG1. The DBD-DNA mixtures were
fractionated on a nondenaturing acrylamide gel. The gel was dried and
subjected to autoradiography. DNA fragments containing 36° and 54°
intrinsic bending sequences were also included on the gel.
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DISCUSSION
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A number of studies have reported that purified steroid hormone
receptors bind poorly to their recognition sequences when compared with
receptors associated with other cellular proteins. The fact that
addition of crude cellular or purified proteins restores the ability of
the purified receptors to bind to DNA (12, 34, 49) suggests that
nuclear receptors do not function in isolation, but that they require
the participation of other cellular proteins to efficiently bind to
DNA. One protein that has demonstrated the ability to enhance binding
of ER and PR to their respective response elements is HMG1 (33, 34, 42). In this work we demonstrated that incremental addition of HMG1
induced a dose-dependent increase in binding of the ER DBD to the
ERE.
Three guanine residues were particularly important for DBD-ERE binding
in the presence and in the absence of HMG1. Crystal structure studies
of the human ER DBD have demonstrated that these guanines
(GGTCAcagTGACC) form hydrogen bonds with lysine
and arginine residues in the DBD recognition helix (38). Since the zinc
finger regions of the human and Xenopus laevis ER DBDs are
completely conserved, except for a single serine residue at position
184 in the first finger of the Xenopus DBD, one would
anticipate that protein-DNA contacts formed when the Xenopus
and human ER DBDs bind to the ERE would be very similar. Our
methylation interference experiments suggest that the crystal structure
for the human ER can be used as a template to predict the position of
the amino acids in the Xenopus ER DBD. Although the three
highly conserved guanine residues present in the ERE half-sites were
extremely important in the DBD-ERE interaction, the guanine residue in
the 3-bp spacer between the two ERE half-sites was significantly
less important. This might be expected since the spacer
region varies significantly with individual ERE sequences.
Interestingly, HMG1 enhanced binding of the DBD to ERE-containing DNA
fragments but did not alter the mobility of the protein-DNA complexes.
These findings are reminiscent of studies carried out with PR and the
HOXD9 homeodomain protein, in which HMG1 enhanced binding but did not
alter the mobility of the protein-DNA complex in gel mobility shift
experiments (34, 42, 50). HMG1 association with HOXD9-DNA complex was
detected by methods that did not require the extended time periods
required for gel mobility shift assays (50). Likewise, a ternary
PR-DNA-HMG1 complex was detected in antibody supershift experiments
when an HMG1-specific antibody was present (42). Taken together, these
studies imply that HMG1 may assist in transcription factor binding and
participate in formation of a ternary complex, but that it is weakly
associated with the protein-DNA complex. This view is supported by our
antibody supershift experiments in which an HMG1-specific antibody
supershifted a portion of the DBD-DNA complex. Since no supershifted
complex was observed in the absence of DBD, this supershifted complex
must represent a ternary DBD-HMG1-DNA complex that was stabilized by
antibody binding. In the absence of antibody, the ternary complex is
most likely unstable during the prolonged electrophoretic process. The
ability of an antibody to stabilize this ternary complex is similar to
the stabilization of the ER-ERE complex by ER-specific antibody
(51).
Travers et al. (52) have hypothesized that HMG1 may serve as
a DNA "chaperone" to enhance conformational changes in DNA and
facilitate the assembly of nucleoprotein complexes. In this capacity
HMG1 could interact directly with DNA to enhance DBD-ERE interaction by
binding and distorting the ERE into a more thermodynamically favorable
conformation so that subsequent binding by the DBD would be
facilitated. In support of this idea, one group has reported that HMG1
enhances binding of the full-length ER to the ERE only when HMG1 is
present before addition of the receptor (33). Although our data
demonstrate that HMG1 has a profound effect on the ability of the DBD
to bind to the ERE when HMG1 is the last component added to the binding
reaction, we did not find that the order of addition substantially
affected the ability of HMG1 to enhance DBD binding. These findings do
not necessarily rule out the possibility that HMG1 assists DBD binding
by inducing conformational changes in DNA structure. In fact, we have
previously demonstrated that local DNA structure does influence DBD
binding to the ERE. When the ERE is bent in a direction that opposes
the ER-induced DNA bend (45), the DBD-ERE complex is less stable than
when the ERE is bent in the same direction as the ER-induced DNA bend
(53). Thus, it is possible that HMG1 may enhance binding of the ER DBD
and the full-length ER to the ERE by prebending the DNA.
Like HMG1, the DBD binds and induces distortion in DNA structure (24, 26, 44, 45). Since the 33° DBD-induced distortion angle was not
altered by the presence of HMG1, our data support the idea that the
DBD, but not HMG1, is strongly associated with the ERE-containing DNA
fragments. Our findings with the ER DBD are also consistent with
previous studies in which HMG1 enhanced binding of the intact PR to
DNA, but did not alter the magnitude of the PR-induced DNA bending
(42).
HMG1 stimulates transcription activation (28, 29), transcription
repression (30), decondensation of chromatin (27), and nucleosome
assembly (26). A common feature of these processes is the assembly of
higher order nucleoprotein complexes. Since endogenous
hormone-responsive genes contain multiple transcription factor-binding
sites that are separated by varying distances, HMG1 may play a role in
formation of the higher order nucleoprotein complexes by promoting DNA
flexibility and transcription factor binding. This hypothesis is
consistent with a recent study by Verrier et al. (33), which
demonstrated that although HMG1 alone was unable to enhance ER-mediated
transcription activation, HMG1 in combination with TAFII30 activates
transcription. Thus, HMG1 may be necessary, but not sufficient, for
transcription activation and may require the participation of other
transcription factors to bring about its effects.
Like histone H1, HMG1 binds to linker DNA located between nucleosomes
and is thought to play a role in organizing chromatin structure by
positioning nucleosomes or looping DNA (22, 31). Although nucleosome
repositioning has not been reported for estrogen-responsive genes,
it is a critical step in the regulation of glucocorticoid-responsive
genes (54, 55). Even if repositioning of nucleosomes is not required
for transcription activation of estrogen-responsive genes, HMG1-induced
DNA looping could help provide the chromatin structure required for
transactivation of genes. DNA looping, which could be assisted by HMG1,
has been documented in the estrogen-responsive PRL (56) and
vitellogenin (57) genes. Thus, the ability of HMG1 to bend DNA,
combined with the ability of the receptor to recognize specific DNA
sequences, could work in concert to provide the geometry and sequence
specificity required for transcription of hormone-responsive genes.
We have demonstrated that the only ER region needed for HMG1-enhanced
binding to the ERE is the DBD. It is interesting to note that the only
region of the HOXD9 homeodomain protein required for HMG1-enhanced
binding is the DBD (50). Given the high degree of conservation in DBD
amino acid sequence among the steroid receptor superfamily members, one
might predict that the binding of other steroid hormone receptors would
also be enhanced by the presence of HMG1 and that HMG1 may be involved
in regulating transcription of a number of hormone-responsive
genes.
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MATERIALS AND METHODS
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Isolation of Purified Proteins
HMG1 was purified from calf thymus as previously described (34).
The Xenopus laevis ER DBD was expressed in BL21(DE3) plys S
cells (58) and purified using BioRex 70 (Bio-Rad Laboratories,
Hercules, CA) and phosphocellulose chromatography as described
previously (37). Purified proteins were stored at -70 C and thawed on
ice before use.
Plasmids and Preparation of 32P-Labeled
DNA Fragments
The construction of the circular permutation vectors ERE Bend I
(43) and ERE Bend III were previously described (59).
For gel mobility shift assays, ERE Bend I (43) was digested with
EcoRI, HindIII, EcoRV,
NheI, or BamHI to produce 427-bp DNA fragments
containing a consensus ERE at the 3'-end, at an intermediate
3'-position, in the middle, at an intermediate 5'-position, or at the
5'-end of the DNA fragment, respectively. The DNA fragments were
end-labeled with
[32P]ATP as previously described (37, 43). DNA-bending standards, kindly provided by A. Landy (48), were
digested and labeled as described (43).
For DNase I and methylation interference footprinting, the circular
permutation plasmid B3consERE (59) was digested with EcoRV
and HindIII to produce 281-bp ERE-containing DNA fragments,
which were fractionated on a 5% acrylamide gel, cut out of the gel,
and electroeluted. The purified end-labeled DNA fragments were filled
in with
[32P]dATP and
[32P]dGTP using
Klenow DNA polymerase (GIBCO-BRL, Gaithersburg, MD). The32P-labeled probes were separated from unincorporated
nucleotides using a G-25 Quick Spin Column (Boehringer Mannheim,
Indianapolis, IN) according to the manufacturers instructions.
Gel Mobility Shift Assays
Purified DBD was combined with 500010,000 cpm32P-labeled ERE-containing DNA fragments and 50 ng poly(dI-dC)
in binding reaction buffer (15 mM Tris, pH 7.9, 0.2
mM EDTA, 80 mM KCl, 4 mM
dithiothreitol, and 10% glycerol) to a final volume of 20 µl.
Varying amounts of ovalbumin were included in each binding reaction to
maintain total protein levels at 2.8 µg. HMG1 was added to the
binding reaction last. Protein-DNA mixtures were incubated for 10 min
on ice, followed by 5 min at room temperature, and then fractionated on
a low ionic strength, nondenaturing polyacrylamide gel (60). For
antibody supershift experiments, the monoclonal antibody P1A3, which
was made against the purified Xenopus laevis ER DBD (41), or
monoclonal antibody 854E10 (42), which was made against purified calf
thymus HMG1, was added to the incubation just before addition of the
HMG1. For association and dissociation rate determinations, glycerol
concentrations were increased to 15% and all reactions were maintained
at 4 C. To determine the association rate, 120-µl samples were
incubated at 4 C and 20-µl aliquots were removed and loaded onto a
running gel 0.5 to 10 min after DBD addition. To determine the
dissociation rate, samples were maintained at 4 C for 10 min. After a
20-µl aliquot was removed and loaded onto a running gel (time 0), 300
ng of a 30-bp annealed oligo containing the ERE was immediately added.
Twenty-microliter aliquots were removed and loaded onto a running gel
0.2530 min after addition of the ERE oligo. All gels were run at 4 C
with buffer recirculation, dried, and subjected to autoradiography. For
circular permutation experiments, DNA bending standards (48) were
included on gels to determine the magnitude of the DBD-induced DNA bend
as previously described (43). Relative mobilities of DNA bending
standards, free DNA, and protein-DNA complexes were determined using a
Molecular Dynamics PhosphorImager and Image Quant software (Molecular
Dynamics, Sunnyvale, CA).
DNase I Footprinting
DNase I footprinting was carried out essentially as described
(41). Briefly, EcoRV/HindIII-digested,
end-labeled DNA fragments (30,000 cpm) containing the consensus ERE
were combined with 01 µg of purified DBD in binding reaction buffer
with 1.25 mM MgCl2 and 0.5 mM
CaCl2. Ovalbumin was included so that the total protein
concentration in each sample was maintained at 1 µg. The binding
reaction was incubated for 15 min at room temperature. 0.5 U of RQ1
ribonuclease-Free DNase I (Promega, Madison, WI) was added to each
sample and incubated at room temperature for 2.5 min. DNase I digestion
was terminated with stop solution (200 mM NaCl, 1% SDS,
and 30 mM EDTA). The DNA was extracted with
phenol-chloroform, precipitated, washed twice with 70% ethanol, dried,
and resuspended in 8 µl of loading buffer (95% formamide, 20
mM EDTA, 0.05% bromophenol blue, and 0.05% xylene
cyanol). Samples were incubated at 90 C for 1.5 min before loading onto
a denaturing 8% polyacrylamide gel. The gel was electrophoresed,
dried, and exposed to x-ray film with an intensifying screen for 1216
h at -70 C.
Methylation Interference
Methylation interference assays were carried out essentially as
described (41). Briefly, the
EcoRV/HindIII-digested, end-labeled DNA fragments
(2,000,000 cpm) were methylated for 5 min with 0.5% dimethyl sulfate
in a reaction buffer containing 50 mM sodium cacodylate, pH
8.0, and 1 mM EDTA (211 µl final volume). The reaction
was terminated with 50 µl stop buffer (1.5 M sodium
acetate, pH 7.0, 1 M ß-mercaptoethanol, and 100 µg/ml
tRNA) and chilled ethanol, precipitated twice, and resuspended in TE.
Approximately 500,000 cpm methylated probe was combined with binding
reaction buffer, 125 ng poly(dI-dC), and either 100 ng purified DBD or
50 ng purified DBD plus 250 ng purified HMG1. The 50-µl reaction
mixture was fractionated on an 8% polyacrylamide nondenaturing gel as
described above. The wet gels were exposed to film for 26 h to detect
free probe and protein-DNA complexes. Free and complexed DNA were
excised from the gel, isolated by electroelution, and precipitated. The
modified DNA was cleaved for 30 min with 10% piperidine at 90 C. After
evaporation of the piperidine solution, the DNA was resuspended in 30
µl water, lyophilized, resuspended in 20 µl water, and lyophilized.
The DNA fragments were resuspended in loading buffer, incubated at 90 C
for 1.5 min, and electrophoresed on an 8% denaturing gel 3 h at
constant power (30 watts). The gel was dried and visualized by
autoradiography.
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. Arthur Landy for DNA bending standards and Dr.
Robin Dodson for helpful suggestions on the preparation of this
manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Ann M. Nardulli, Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, 524 Burrill Hall, 407 South Goodwin Avenue, Urbana, Illinois 61801.
This work was supported by NIH Grant R29 HD-31299 (to A.M.N.) and USPHS
Grant CA-46938 (to D.P.E.). J.R.W. was supported by NIH Reproductive
Biology Training Grant PHS 2T32 HD-072819.
Received for publication October 24, 1997.
Revision received January 30, 1998.
Accepted for publication February 2, 1998.
 |
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