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J. Biol. Chem., Vol. 276, Issue 43, 39885-39891, October 26, 2001
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,¶, and
**
From the Department of Molecular Physiology and
Biophysics, § Department of Biological Sciences, Vanderbilt
University School of Medicine and Department of Veterans Affairs
Medical Center, Nashville, Tennessee 37232
Received for publication, June 11, 2001, and in revised form, August 6, 2001
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Glucocorticoid induction of the
phosphoenolpyruvate carboxykinase (PEPCK) gene requires a
glucocorticoid response unit (GRU) comprised of two non-consensus
glucocorticoid receptor (GR) binding sites, GR1 and GR2, and at least
three accessory factor elements (gAF1-3). DNA-binding accessory
proteins are commonly required for the regulation of genes whose
products play an important role in metabolism, development, and a
variety of defense responses, but little is known about why they are
necessary. Quantitative, real time homogenous assays of cooperative
protein-DNA interactions in complex media (e.g. nuclear
extracts) have not previously been reported. Here we perform
quantitative, real time equilibrium and stopped-flow fluorescence
anisotropy measurements of protein-DNA interactions in nuclear
extracts to demonstrate that GR binds to the GR1-GR2 elements poorly as
compared with a palindromic or consensus glucocorticoid response
element (GRE). Inclusion of either the gAF1 or gAF2 element with
GR1-GR2, however, creates a high affinity binding environment for GR.
GR can undergo multiple rounds of binding and dissociation to the
palindromic GRE in less than 100 ms at nanomolar concentrations. The
dissociation rate of GR is differentially slowed by the gAF1 or gAF2
elements that bind two functionally distinct accessory factors,
COUP-TF/HNF4 and HNF3, respectively.
The balance between glucose production and disposal must be
tightly regulated to ensure glucose homeostasis. Many genes, whose products are control points in metabolism, are regulated through multicomponent hormone response units in which DNA-binding accessory proteins modulate hormone receptor function (1-3). Similar
arrangements are found in genes whose products play an important role
in development and in a variety of host defense responses (1-7).
Little is known, however, about how accessory factors function
in these hormone response units.
Glucocorticoid induction of the
PEPCK1 gene, which encodes
a key enzyme involved in gluconeogenesis, is accomplished through a
glucocorticoid response unit (GRU) comprised of two non-consensus glucocorticoid receptor binding sites (GR1, GR2) and at least three
accessory factor elements (gAF1-3) that bind HNF4/COUP-TF, HNF3 Here we perform quantitative, real time equilibrium and stopped-flow
fluorescence anisotropy measurements of protein-DNA interactions in
nuclear extracts to demonstrate that the multicomponent complexes that
regulate PEPCK gene transcription form and dissociate with kinetics
appropriate for the rapid changes in the transcription of this gene
mediated by hormones (15). Further, two functionally distinct accessory
factor elements, which bind HNF3 Synthesis, Labeling, and Annealing of Fluorescently Labeled
Oligonucleotides--
Oligonucleotides were made by an Expedite 8909 oligonucleotide synthesizer (Perspective Biosystems, Framingham, MA).
The sequences of all oligonucleotides used in this study are shown in
Table I. A 5'-amino group on a 6-carbon
atom linker arm (Glen Research Catalog No. 10-1916-02) was added to the
oligonucleotides labeled with a fluorescent dye. Rhodamine X (Molecular
Probes Catalog No. X-491) and 5- and 6-carboxyfluorescein (Molecular
Probes Catalog No. C-1311) were attached to the linker, purified, and
annealed as previously described (16). This method ensures that
only complete oligonucleotides are labeled. No single-stranded
fluorescently labeled oligonucleotides were detected by non-denaturing
gel electrophoresis after annealing. Double-stranded oligonucleotides
used for competition have the same sequence and were prepared in the
same way as the corresponding fluorescently labeled oligonucleotides,
but the fluorescent dye was not included in the reaction.
Nuclear Extract Preparation--
H4IIE hepatoma cells were
cultured as previously described (17). Cells were treated with 500 nM dexamethasone for 45 min and collected, and nuclear
extracts were prepared as described previously (8, 18), aliquoted, and
stored at Steady State Anisotropy Measurements--
A SPEX (Edison, NJ)
1681 fluorolog spectrofluorimeter was used to make fluorescence
anisotropy measurements of the GRE-containing oligonucleotides in the
presence or absence of GR from the nuclear extracts (for more
background on fluorescence anisotropy see Ref. 19). Anisotropy was
calculated as previously described (20). The assay is homogenous,
without distinct aqueous and solid-support phases, as in filter binding
and electrophoretic mobility shift assays. No separation of free and
complex-bound DNA is required; thus the equilibrium conditions
necessary to study cooperative binding are easily maintained (21-23).
A 10 nM concentration of rhodamine-labeled oligonucleotide
provides a strong fluorescent signal. Samples were excited with
vertically polarized light (Oriel dichroic sheet polarizers) with a
wavelength of 585 nm. Emission at 620 nm was detected through two photo
detectors, assembled in T format, which allows simultaneous
determination of the horizontal and vertical components of emitted
light. At these excitation/emission wavelengths, the contribution of
background fluorescence from the buffer or from protein scattering is
negligible. For each fluorescence anisotropy determination, 60-100
consecutive 0.5-s measurements were made and averaged. Using this
technique, 95% confidence intervals of individual measurements are
typically less than 0.0001 anisotropy unit; thus even small changes in
anisotropy can be measured.
The binding buffer used contained 20 mM Tris, pH 7.6, 100 mM NaCl, 250 µM ZnSO4, 6%
glycerol, 6.25 mM EDTA, 10 mg/ml bovine serum albumin, and
500 nM dexamethasone and was prepared fresh for each
experiment. The concentrations of zinc and dexamethasone used provide
for optimal binding of GR (24). There is no difference in the binding
capacity of GR if the extract is preincubated at 25 °C for 30 min or
kept on ice for the duration of the experiment, presumably because of
the presence of dexamethasone in both cell culture and binding buffer
(25). Nonspecific binding was determined by two complementary methods;
first, by competition with unlabeled GRE and secondly by binding of
nuclear extract to a labeled oligonucleotide with point mutations in
the GRE and without necessary flanking sequences for GR binding (26,
27). These methods produced identical results. The nonspecific binding
experiment was repeated three times with an S.E. < 0.001 anisotropy
unit. Linear regression through these data (Sigma Plot 5.0) was used to
generate the line for nonspecific binding shown in Fig. 1b.
Specific binding was determined by subtracting nonspecific binding from
total binding. Only specific binding is shown in the remaining
steady-state experiments (Figs. 1c and 2, a-d).
Titrations of nuclear extract up to 20 µg are shown. There is no
increase in specific binding with further additions. The nonspecific
oligonucleotide used in Fig. 1c contains the gAF2 element to
demonstrate that competition with gAF2 itself does not eliminate GR
binding. Other nonspecific oligonucleotides produced similar results.
For antibody experiments, nuclear extracts were preincubated with the
indicated antibodies on ice for 30 min (anti-GR was a gift from
K. Yamamoto; anti-mineralocorticoid receptor was obtained from
Santa Cruz Biotechnology). Curves were generated using non-linear
regression analysis (Sigma Plot 5.0). In graphs in which multiple
curves are presented (Fig. 2, a, b, and
c), the results represent the mean of at least three
experiments. The standard error at each data point between experiments
was typically less than 0.001 anisotropy unit.
Pre-steady-state Stopped-flow Anisotropy Measurements--
A
pre-steady-state stopped-flow analysis allows complex assembly and
disassembly to be observed in real time. This set of binding
experiments uses a fluorescence depolarization assay similar to that
employed in the previous experiments (Figs. 1 and 2), except that
fluorescence excitation utilized a Coherent Inova 310 argon-ion laser
(Santa Clara, CA) directed into the sample cell. Excitation was
vertically polarized at 488 nm. This wavelength is near the excitation
maximum of the fluorescent dye fluorescein; thus the same
oligonucleotides used in Figs. 1 and 2 were conjugated with fluorescein
for detection (Fig. 1a). Fluorescent emission was observed
simultaneously in the vertical and horizontally polarized planes
through Oriel 20 nm band pass filters centered at 520 nm and
Glan-Thompson polarizers. A Biologic SFM4 stopped-flow unit (Molecular
Kinetics, Pullman, MA) was used with a 50-µl FC15 fluorescence cuvette and hard-stop shutter. More details about this experimental setup have been reported (20). All buffers were extensively degassed
before use. Data for all experiments were collected with a fraction of
data channels devoted to internal controls. Data were collected for 20 ms per channel for 8192 total channels. Dilute solutions of labeled
oligonucleotide and buffer, or oligonucleotide and nuclear extract,
were rapidly mixed (1:1) to determine free oligonucleotide anisotropy
and association rates, respectively. The dead time is ~15 ms for flow
rates of 2 ml/s. To measure dissociation rates, complexes were
preformed on ice for 15 min, allowed to equilibrate to room
temperature, and mixed 1:1 with a 50× excess of unlabeled palindromic
GRE. The ratio of fluorescently labeled oligonucleotide and nuclear
extract used corresponds to a saturating level with regard to GR
binding detected in steady-state experiments. 16-25 kinetic
measurements, or shots, were summed to obtain adequate signal to noise
ratios. All shots are precisely synchronized with regard to mixing by a
sync-out pulse from the SFM4 control unit.
Differences in intrinsic depolarization between rhodamine X and
fluorescein account for the differences in the free oligonucleotide anisotropy values seen for the same oligonucleotides (0.184 for the
palindromic GRE as observed by rhodamine emission and 0.048 as observed
by fluorescein emission). The equilibrium and kinetic properties of the
protein-DNA complexes quantitated in this study were found to be
independent of the specific fluorophore used for detection.
High Affinity Binding of GR from Nuclear Extracts as Measured by
Changes in Fluorescence Anisotropy--
The dynamics of assembly of
the GRU were studied using fluorescence anisotropy, an assay based on
the principle that DNA in solution undergoes rotational diffusion that
varies with molecular weight. The rotational motion of DNA is slowed
when proteins are bound and can be detected through changes in the
fluorescence anisotropy of the labeled oligonucleotides, which include
various segments of the PEPCK gene promoter represented in Fig.
1a (21-23) (for more
background on fluorescence anisotropy see Ref. 19). H4IIE hepatoma
cell nuclear extracts provided the GR and accessory factors in
appropriate physiologic concentration. This protein preparation is
particularly useful for studying the function of GR, which is not
easily purified and is unstable in solution in the absence of
appropriate stabilizing factors (28, 29). Induction and repression of
PEPCK gene transcription are rapid events (15, 30); thus the most
important advantage of this assay is that dynamic binding events can be
observed. No separation of free and complex-bound DNA is required; thus
the equilibrium conditions necessary to study cooperative binding can
be maintained.
The requirements for GR binding to a simple response element in
vitro have been well characterized (26, 31). We used this knowledge to demonstrate that GR in a nuclear extract can specifically bind to a palindromic GRE contained within a double-stranded
oligonucleotide 29 base pairs in length (29-mer) labeled at the 5' end
with the fluorescent dye rhodamine X (filled circles, Fig.
1b). Addition of GR-containing nuclear extract shows the
expected saturation (open triangles, Fig. 1b),
and nonspecific binding increases linearly (dashed line,
Fig. 1b).
Competition with an unlabeled palindromic GRE eliminates nearly all
specific binding (Fig. 1, b and c, bar
3). Addition of a nonspecific oligonucleotide that does not bind
GR has little effect (Fig. 1c, bar 4). A
competitor containing a consensus GRE competes for this binding as well
as the palindromic GRE (data not shown); thus competition is dependent
on affinity of the competitor for GR and is specific. The binding seen
in Fig. 1b can be recapitulated using partially purified GR
(data not shown). We quantified the GR content in the nuclear extract
preparations using this partially purified GR as a standard. There are
~0.16 pmol of GR/µg of nuclear extract, providing an approximate
Kd of binding to the palindromic GRE of 5 nM. Binding of purified GR to this palindromic GRE has a
Kd of 7.5 nM (data not shown); thus GR
from the nuclear extracts closely recapitulates the activity of the purified protein. The observed binding of GR from the nuclear extract
is not restricted to the palindromic GRE. A consensus GRE with flanking
sequences that maximize affinity for GR (26, 32) binds GR with a
Kd of 1.6 nM GR (Fig. 1c,
bars 5 and 6, and Fig.
2a, filled
circles). Preincubation of the nuclear extract with an antibody
raised against the DNA binding domain of GR significantly reduces the
ability of GR to associate with the consensus GRE, whereas an antibody
raised against the mineralocorticoid receptor does not inhibit binding
(Fig. 1c, bars 8 and 9). The observed
binding of GR to the palindromic and consensus GRE elements correlates
with the measured affinity of GR for these sites (32, 33).
Accessory Factor Elements Restore Affinity of GR for Non-consensus
PEPCK GR1 and GR2 Elements--
GR binds with very low affinity to the
PEPCK GR1 and GR2 elements (13). To test whether the accessory factors
enhance binding of GR for the low affinity GR1 and GR2 elements, we
constructed a double-stranded rhodamine X-labeled oligonucleotide that
spans the gAF2, GR1, and GR2 elements from the PEPCK gene promoter
(Fig. 1a). The presence of the gAF2 element restores the
affinity of the GR-accessory factor complex binding to this promoter
segment to a level comparable with that exhibited by GR for the
consensus GRE (compare the filled circles in Fig. 2,
a and b). This binding is completely
dependent on association of the accessory factor with the gAF2 element,
as a block mutation in gAF2, which prevents accessory factor binding
and reduces the glucocorticoid effect (10), leaves only GR1 level
affinity (compare open triangles in Fig. 2, a and
b).
The gAF2-GR1-GR2 construct (a 76-mer) has a higher free anisotropy
value than the palindromic GRE (a 29-mer); thus we predict that binding
of a large protein complex would be needed to produce the observed
anisotropy changes seen in Fig. 2b (19). If the association
of accessory factors with the gAF2 element does indeed restore affinity
of GR for this complex, the GR should represent most of the bound mass
on the gAF2-GR1-GR2 promoter segment. This prediction is based on the
presumption that the complex is comprised of HNF3 (40 kDa) and four
monomers of GR (each 90 kDa), as shown schematically in Fig.
1a. Preincubation of the nuclear extract with the anti-GR
antibody prevents ~70% of the mass assembled to this complex from
forming, based on the anisotropy changes seen (Fig. 2d,
bar 3). Thus, the complex formed in vitro
recapitulates the complex that is required for functional induction of
PEPCK gene transcription. The anti-GR antibody reduces the effective concentration of GR able to participate in this complex. The remaining mass on this promoter segment may indicate that this antibody is not
100% effective but likely represents the remaining mass of the
gAF2-bound accessory factor, which binds to its response element with
high affinity in the absence of GR (data not shown).
HNF3 interacts with GR in vitro (14). Furthermore, the
helical spacing between the gAF2 element and the GR1 element is
critical for the glucocorticoid response (14), suggesting that a
physical interaction between HNF3 and GR may be responsible for the
facilitated binding seen here. In support of this hypothesis, we show
that although GR represents the majority of the protein mass assembled to this complex (Fig. 2d, bar 3), there is
significantly less competitor-induced dissociation of GR from this
complex than from the palindromic GRE (compare Fig. 2d,
bars 2 and 4, with Fig. 1c, bars
2 and 3), indicating that this complex is quite stable with regard to GR binding. Whereas the gAF2 element can bind both HNF3
and C/EBP in vitro (10), the facilitated binding of GR seen
here requires HNF3, as the TTR HNF3 element can substitute for gAF2,
whereas a gAF2 element with point mutations that only permit C/EBP
binding, cannot (data not shown). This correlates precisely with the
ability of these proteins to function as accessory factors in
vivo (10).
A complete set of accessory factors is required for induction of PEPCK
gene transcription by glucocorticoids (1, 13, 14). By contrast, when
the GR1 element is replaced with a palindromic GRE, the gAF2 element is
still required for the glucocorticoid response, but the gAF1 and gAF3
elements are not (13). This observation suggests that two functionally
distinct classes of accessory factors are required for the PEPCK gene
glucocorticoid response. The gAF1 element binds both HNF4 and COUP-TF,
whereas the gAF3 element binds only COUP-TF (Fig. 1a) (8,
9). There is no functional consequence of replacing gAF3 with gAF1 and
vice versa (14). We took advantage of this observation to create a GRU
segment that contains the GR1 and GR2 elements but where the associated
accessory factor element is gAF1 (Fig. 1a). This allowed us
to generate an oligonucleotide of the same length and free anisotropy
value of gAF2-GR1-GR2. As with the gAF2 element, the association of
gAF1 also restores affinity of the GR-accessory factor complex to the
PEPCK gene promoter to a level comparable with the binding of GR to the
consensus GRE (compare filled circles in Fig. 2,
c and a). Again, GR contributes the majority of
the mass in this protein-DNA complex (Fig. 2d, bar
7). Whereas GR binding to the GRU is facilitated by either class
of accessory factor element, the GR1-GR2-gAF1 complex is less stable
with regard to GR binding than is gAF2-GR1-GR2, as competition with an
unlabeled consensus GRE oligonucleotide effectively eliminates GR
binding from the former (compare Fig. 2d, bars 4 and 8). A block mutation that prevents HNF4 and COUP-TF
binding is much less effective in recruiting GR to GR1-GR2 (open
triangles, Fig. 2c, and Ref. 8). Although it is not
known if HNF4 or COUP-TF can interact with GR, the set of experiments
shown in Fig. 2 suggests that the factors bound to the gAF1 element
facilitate binding of GR by a mechanism different from that employed by
HNF3 at gAF2.
Dissociation of GR from the PEPCK Gene GRU Is Modulated by
Accessory Factors--
The increases in anisotropy seen by binding of
GR to the consensus GRE and PEPCK gene GRU segments occur too rapidly
to be measured by steady-state techniques (Figs. 1 and 2).
Pre-steady-state stopped-flow analysis provides the additional
advantages of rapid mixing and detection to these experiments, so that
complex assembly and disassembly can be observed in real time. For the
palindromic GRE, binding is effectively complete in less than 100 ms
(Fig. 3a). A slower second
phase of association is observed but is of undetermined significance
(Fig. 3a). Competition with an unlabeled palindromic GRE
gives a rapid decrease in anisotropy, again with most dissociation
complete in less than 100 ms (Fig. 3b). The association-dissociation rates measured here represent multiple rounds
of binding and dissociation of GR to the palindromic GRE before
equilibrium is reached (34).
The experiments shown in Fig. 2 demonstrate that association of the
PEPCK accessory factor elements facilitates GR binding to the GRU.
Using fluorescein-labeled oligonucleotides that span GR1-GR2, and
including either gAF2 or gAF1 (Fig. 1a), we sought to
determine the association rates of the protein-DNA complexes. The
observed formation of an enhanceosome-like structure by either gAF2 or
gAF1 occurs rapidly (Fig. 3, c and e). Both GRU
segments form complexes with kinetics of binding similar to that
observed when GR binds to the palindromic GRE (Fig. 3a).
Whereas all of the complexes formed equally rapidly within the
detection limits of this system, dissociation of GR is dramatically slowed by the presence of the adjacent accessory factor elements. Indeed, there is substantially less dissociation of GR from the complex
bound to gAF2-GR1-GR2 (Fig. 3d). These results are
qualitatively similar to the steady-state composition of this complex,
where there is very little GR dissociation (Fig. 2d,
bar 4), despite GR contributing ~70% of the mass of this
complex (Fig. 2d, bar 3). The physical
association of GR with HNF3 may account for the stability of this
complex (14). By contrast, GR readily dissociates from the GR1-GR2-gAF1
complex (Fig. 2d, bar 8), albeit about 10 times
more slowly than dissociation of GR from the palindromic GRE (compare
b and f in Fig. 3).
Binding to an oligonucleotide containing only the gAF2 element is also
complete within 100 ms (data not shown); thus it may be that prior
association of the accessory factors creates a high affinity binding
environment to the GR1-GR2 elements that, as a complex, functions as
well as the palindromic GRE. Indeed, in this study we show that high
affinity binding of GR to the PEPCK gene promoter is strictly dependent
on associated accessory factor elements. It was not possible to test
the individual roles of HNF3, HNF4, and COUP-TF as accessory factors in
these biophysical experiments (as described for GR in Fig. 2) because
antibodies that prevent DNA binding for these factors are not currently
available. However, in support of this model, we have shown by the
chromatin immunoprecipitation assay that the accessory factors
described here are constitutively bound to the PEPCK gene promoter
in vivo, creating a high affinity binding environment for
GR, which only binds to the promoter in the presence of glucocorticoids
(35).2 Additionally, we show
that GR binding to a GRE is intrinsically rapid but can be altered by
association with accessory factors.
We speculate that even small changes in the dynamics of the structures
involved in gene regulation could have a large impact on the
physiological end points of the processes controlled by these gene
products, such as glucose production. Simultaneous changes in the
regulation of multiple genes whose products are involved in the same
pathway have a large combined impact on glucose production (36). For
instance, overexpression of an HNF3 variant that reduces the responses
of the PEPCK and glucose-6-phosphatase genes to glucocorticoids by
~50% nearly abolishes glucocorticoid-induced glucose production in
hepatoma cells. Although GR binding to a GRE is dynamic (Fig. 3 and
Ref. 37), and such binding is stabilized by accessory factors, these
complexes still form and dissociate within a few seconds (Fig. 3). We
propose that dynamic structures such as the PEPCK gene GRU provide for
the adaptable regulation of metabolic genes that is seen in
vivo.
The maintenance of glucose homeostasis in the face of the gradient of
nutrient availability imposed by the extremes of the fasting and fed
states is created by changes in the rates of glucose production and
utilization. These adaptive responses are achieved by the products of a
concert of genes that require accessory factors for hormonal regulation
of their expression. Complex hormone response units, in which receptor
binding and dissociation are modulated by accessory factors, provide
variable rates of gene transcription and could be a mechanism by which
a gradient of glucose production is generated. Gradients of gene
products necessary for processes such as development, differentiation,
and host defense responses may also be achieved by this mechanism.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
,
and COUP-TF, respectively (Fig. 1a) (1, 8-10). HNF3 and
HNF4 are expressed in a tissue-specific manner that correlates with the
ability of glucocorticoids to induce this gene (11, 12). The GR1 and
GR2 elements correspond to the consensus GR binding element at only 7 of 12 and 6 of 12 nucleotides, respectively, and bind GR with very low
affinity relative to the consensus (glucocorticoid response element
(GRE)) (13). The GR1 and GR2 elements, alone or in combination, are not
able to confer glucocorticoid responsiveness to a heterologous
promoter-reporter construct (13). A mutation of any one of the
accessory elements results in a 50-60% reduction of
glucocorticoid-stimulated PEPCK gene transcription in H4IIE rat
hepatoma cells (1, 13, 14). Any combination of two mutations of
gAF1/gAF3 or gAF2 abolishes the response (1, 14). This series of
observations suggests that the accessory factors required for the
glucocorticoid response may facilitate GR binding to the GR1 and GR2 elements.
and HNF4, differentially promote
the stability of GR in the GRU complex. These results suggest that the
magnitude of gene regulation in response to glucocorticoids may be
achieved by modulation of GR binding by accessory factors.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
Oligonucleotides used, sense strand 5' to 3' (X represents 5'-amino
modifier)
70 °C. Aliquots were only used one time to avoid the
effects of freeze-thawing on binding. These nuclear extracts have
abundant amounts of GR and the necessary accessory factors.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (17K):
[in a new window]
Fig. 1.
High affinity binding of GR from nuclear
extracts as determined by steady state anisotropy. a,
schematic diagram of the PEPCK gene GRU and accessory factors required
for induction of gene transcription by glucocorticoids. Bars
represent fluorescently labeled double-stranded oligonucleotides that
contain sequences spanning the indicated regions of the GRU.
b, the steady-state anisotropy for 10 nM
rhodamine X-labeled palindromic GRE increases when titrated with H4IIE
nuclear extract (filled circles). The corresponding
unlabeled palindromic GRE oligonucleotide was used in 20× excess for
competition of specific binding (Spec. Comp.). Nonspecific
binding (dashed line) was determined as described under
"Experimental Procedures." Specific binding (open
triangles) was determined by subtracting nonspecific binding from
total binding. Data points represent the mean ± S.E. of at least
three experiments. c, free oligonucleotide anisotropy values
of the palindromic and consensus GRE oligonucleotides are each 0.184 as
determined by rhodamine emission (bars 1 and 5).
This value is set at 0, and changes in anisotropy are shown. After a
titration experiment to reach saturated binding (bar 2),
specific binding is competed efficiently with a 20× excess of an
unlabeled palindromic GRE (bar 3). A nonspecific
oligonucleotide produces only a small decrease in anisotropy (bar
4). A rhodamine-labeled consensus GRE exhibits specific binding
similar to the palindromic GRE (bars 5 and 6).
Preincubation of nuclear extracts with an anti-GR antibody prevents
specific binding, whereas an antibody raised against the
mineralocorticoid receptor is without effect (bars 8 and
9). Results represent the mean ± S.E. of at least
three experiments.
View larger version (18K):
[in a new window]
Fig. 2.
Accessory factor elements restore affinity of
GR for non-consensus PEPCK GR1 and GR2 elements as determined by
steady-state anisotropy. a, specific binding to a
consensus GRE (filled circles) is compared with the PEPCK
GR1 element (open triangles). Kd* is
expressed in units of µg of nuclear extract. This corresponds to an
approximate Kd of 1.6 nM GR for the
consensus GRE and 48 nM GR for the PEPCK GR1 element or a
30× affinity difference. b, specific binding to a 76-base
pair, rhodamine-labeled segment of the PEPCK gene GRU containing the
gAF2 accessory factor element and the GR1 and GR2 GR-binding elements
(filled circles). A corresponding segment with a block
mutation that prevents accessory factor binding to gAF2 does not
promote complex formation (open triangles). c, a
76-base pair segment of the GRU spanning GR1-GR2 and gAF1 also forms a
complex with high affinity (filled circles). A block
mutation in gAF1 that prevents accessory factor binding markedly
reduces complex formation (open triangles). d,
the gAF2-GR1-GR2 complex is more stable with regard to GR binding than
GR1-GR2-gAF1. Most of the mass associated with the gAF2-GR1-GR2
fragment can be prevented by preincubating the nuclear extracts with an
anti-GR antibody (bar 3); however, competition with a 50×
excess consensus GRE can eliminate only approximately one-third of
specific binding (bar 4). By contrast, competition of
binding of GR from GR1-GR2-gAF1 is efficient (bar 8),
indicating that these complexes have a different capacity to retain
bound GR. Preincubation of nuclear extract with an anti-GR antibody
prevents most of this complex from forming (bar 7).
View larger version (46K):
[in a new window]
Fig. 3.
Time course of complex assembly and GR
dissociation as determined by pre-steady-state stopped-flow
analysis. Left panels, association rates of nuclear
extract binding to palindromic GRE only (a), gAF2-GR1-GR2
(c), and GR1-GR2-gAF1 (e). Right
panels, competitor-induced dissociation of GR from palindromic GRE
(b), gAF2-GR1-GR2 (d), and GR1-GR2-gAF1
(f). Although all complexes form at similar rates (compare
a, c, and e), there is considerable
difference in the ability of GR to dissociate from the complexes.
Dissociation of GR is rapid from the palindromic GRE (b),
poor from gAF2-GR1-GR2 (d), and efficient but slower from
GR1-GR2-gAF1 (f).
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ACKNOWLEDGEMENTS |
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We thank Dr. Keith Yamamoto (University of California, San Francisco) for providing the anti-GR antibody, Drs. Lee Limbird, Mary Waltner-Law, Utpal Banik, and Jen-Chywan Wang for helpful advice, and Deborah Caplenor Brown for helping to prepare this manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants DK20593 (Vanderbilt Diabetes Research and Training Center), DK35107, GM 07347 (Vanderbilt Medical Scientist Training Program), and GM 55056 and the Veterans Affairs Research Service.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Present address: Molecular Probes Inc., Biosciences, Eugene, OR 97402.
** To whom correspondence should be addressed: Dept. of Molecular Physiology and Biophysics, 707 Light Hall, Vanderbilt University School of Medicine, Nashville, TN 37232. Tel.: 615-322-7004; Fax: 615-322-7236; E-mail: daryl.granner@mcmail.vanderbilt.edu.
Published, JBC Papers in Press, August 22, 2001, DOI 10.1074/jbc.M105370200
2 D. Duong and D. K. Granner, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: PEPCK, phosphoenolpyruvate carboxykinase; GRU, glucocorticoid response unit; GR, glucocorticoid receptor; GRE, glucocorticoid response element; COUP-TF, chicken ovalbumin upstream promoter transcription factor; HNF, hepatocyte nuclear factor.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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