From the Department of Physiology, University of Missouri School of
Medicine, Columbia, Missouri 65212
In addition to the glucocorticoid receptor,
DNA-binding proteins called accessory factors play a role in hormone
activation of many glucocorticoid-responsive genes. Hormonal regulation
of the
-fibrinogen subunit gene from the frog Xenopus
laevis requires a novel DNA sequence that binds a liver nuclear
protein called Xenopus glucocorticoid receptor accessory
factor (XGRAF). Here we demonstrate that the recognition site for XGRAF
encompasses GAGTTAA at positions
175 to
169 relative to the start
site of transcription. This sequence is not closely related to the
binding sites for known transcription factors. The two guanosines make close contact with XGRAF, as shown by the methylation interference assay. Single-point mutagenesis of every nucleotide in the 9-base pair
region from positions
177 to
169 showed an excellent correlation between ability to bind XGRAF in vitro and ability to
amplify hormone-induced transcription from DNA transfected into
Xenopus primary hepatocytes. Conversely, XGRAF had little
or no effect on basal transcription of the
-fibrinogen gene. Maximal
hormonal induction also requires three half-glucocorticoid response
elements (half-GREs) homologous to the downstream half of the consensus GRE. Interestingly, the XGRAF-binding site is immediately adjacent to
the most important half-GRE. This close proximity suggests a new
mechanism for activation of a gene lacking a conventional full GRE.
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INTRODUCTION |
Steroid hormones, which include glucocorticoids and
mineralocorticoids from the adrenal cortex and estrogens, progestins, and androgens from the gonads, regulate a vast array of physiological processes that are essential for development, differentiation, growth,
metabolism, homeostasis, behavior, and reproduction in vertebrate
organisms. In the classical model of steroid hormone action (1), the
steroid ligands bind to specific intracellular protein receptors in
target cells. The hormone-receptor complexes interact with particular
short nucleotide sequences in the chromosomal DNA and modulate
transcription of nearby genes. This model, however, cannot account
fully for the complex tissue-specific and gene-specific actions of
hormones. Transcriptional induction by steroids is influenced by many
factors, such as local chromatin structure (2), stages of the cell
cycle (3), cellular morphology or differentiation state (4, 5),
specific hormone ligand (6), and physiological state (7). Differential
hormone responsiveness depends in part on the availability of other
transcriptional regulatory proteins including coactivators and
corepressors, which do not themselves bind to DNA (8), and accessory
factors, which are DNA-binding proteins (9). For
glucocorticoid-regulated genes, several accessory factors have been
identified (2, 10-17), but the mechanisms by which these proteins
potentiate hormonal activation of transcription are not known.
To understand the role of accessory DNA-binding proteins in determining
responsiveness to a steroid hormone signal, we are investigating
glucocorticoid induction of fibrinogen gene expression in the liver.
Fibrinogen is the precursor of fibrin, the major structural protein of
a blood clot, and its synthesis is regulated by adrenal steroids both
in basal homeostasis and following physiological stresses such as
infections, inflammation, surgery, burns, etc. (18, 19). Using primary
liver cells from the frog Xenopus laevis as a model system
(20), we have demonstrated that glucocorticoids stimulate transcription
of the three separate genes coding for the fibrinogen subunits, termed
A
, B
, and
(21).
Identification of the specific DNA sequences that mediate steroid
regulation of the Xenopus fibrinogen genes was accomplished by linking the 5'-flanking DNA of these genes to the firefly luciferase reporter gene and transfecting the DNA into primary Xenopus
hepatocytes. Hormonal activation of the B
gene occurs through a
single element (22) with a close match to the consensus glucocorticoid
response element (GRE),1
GGTACAnnnTGTTCT (23). Full stimulation of the
gene, on the other
hand, requires three closely spaced weak binding sites for the
glucocorticoid receptor (GR) between nucleotides
168 and
135
relative to the start site of transcription (24). These sites have
homology only to the downstream portion of the consensus GRE and
therefore are referred to as half-GREs. In addition, steroid responsiveness is affected by bases within the sequence AAGAGTTAA at
positions
177 to
169, immediately adjacent to the 5'-most half-GRE
(24). This tract is unrelated to the conventional GRE and does not
match other known transcription factor-binding sites. A protein present
in Xenopus liver nuclei binds to this DNA sequence in
vitro. Experiments with DNA containing blocks of mutations within
and around bases
177 to
169 showed that the DNA-protein interaction
correlates with hormonal activation of transcription. Thus, we named
the nuclear protein Xenopus glucocorticoid receptor accessory factor (XGRAF). Compared with other known GR accessory factors, the location of the XGRAF-binding site is unusual since it
replaces the sequence that would normally constitute the upstream half
of a GRE.
In this work, we describe the detailed mutational analysis of the 9-bp
region to which XGRAF binds. This investigation located the 5'- and
3'-boundaries of the recognition sequence and revealed which
nucleotides are most critical for DNA binding in vitro and for glucocorticoid-stimulated transcription of transfected DNA in
Xenopus primary hepatocytes.
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EXPERIMENTAL PROCEDURES |
Introduction of Point Mutations into Reporter Gene
Constructs--
All possible single-point mutations in the potential
XGRAF-binding site in the
gene upstream region were obtained by the following general strategy. 1) The
gene sequence from positions
187 to +41 was synthesized by the polymerase chain reaction (PCR) using a downstream primer with wild-type sequence and an upstream primer consisting of a mixed population of oligonucleotides with mutations in the XGRAF-binding region from positions
177 to
169. 2)
The PCR products were inserted into a luciferase reporter vector, and
the DNA was cloned by transformation into bacteria. 3) The nucleotide
sequence of the
DNA in individual clones was determined to
ascertain which nucleotide(s) of the XGRAF region had been mutated.
The upstream primer,
5'-GGGGTACCAGACAGAAAAGAGTTAATGTTCCCTCTTATGTTC-3',
was synthesized (Genemed Biotechnologies, Inc.) in a single reaction,
with the reservoir for each nucleotide in the potential binding site
(underlined) deliberately contaminated to a concentration of 2.5% with
each of the three nondesignated bases. PCR products from this primer
yielded 15 of the 27 possible single-point mutants. A second population
of mixed primers was synthesized (Genosys Biotechnologies), with
contamination at the desired positions to optimal levels based on the
formula described by Derbyshire et al. (25), yielding
several additional mutants. The few remaining mutants were synthesized
in PCRs with specific individual primers.
The PCR amplification was carried out with the pLL
187 construct
(24) as the template DNA (which contains
gene DNA from positions
187 to +41), the upstream primers described above, a downstream
primer within the vector, and Pfu polymerase (Stratagene) following the protocol from the manufacturer. The PCR products were
digested upstream with KpnI and adjacent to position +41 downstream with HindIII, purified through 2%
low-melting-temperature agarose gels (26), and cloned into
KpnI- and HindIII-digested pLuc-Link 2.0 (27).
Transformation into Escherichia coli DH5
was as described
(24). The nucleotide sequences of the
DNA and of the junctions with
vector DNA were confirmed for each mutant. Plasmid DNA was purified
over anion-exchange resin and, for transfection, over a cesium chloride
gradient (24).
Gel Shift Assays--
Both the probe and the competitors for the
gel shift assays contained the
sequence extending from positions
187 to
115 and were generated by PCR using Pfu
polymerase. In addition, the molecules included 19 bases of vector
sequence upstream of position
187 and a MfeI restriction
enzyme site downstream of position
115. Wild-type pLL
187 was
used as the template for making the probe, and the point mutation
constructs described above were the templates for the competitors. The
PCR products were purified through 2% low-melting-temperature agarose
gels (26), and the probe was 5'-end-labeled (28).
Nuclear extracts were made from X. laevis primary
hepatocytes after 4 days in culture exactly as described (24), except that the concentration of HEPES-KOH in buffer C+ was 20 mM. The gel shift assays were carried out as described (24)
in a final volume of 15 µl with 0.5 ng of radioactive probe (7000-51,000 cpm) and either no specific competitor or a 0.5-500-fold molar excess of specific competitor. Native 5% polyacrylamide gels
(24) were run at 350 V for 6-7 h at 4 °C, dried at 80 °C for
2 h, exposed to XAR film (Eastman Kodak Co.) at
80 °C with one Lightning Plus intensifying screen (Dupont), and exposed to a
PhosphorImager screen (Molecular Dynamics, Inc.). The data from the
phosphorimaging scan were analyzed with ImageQuant 3.3 software (Molecular Dynamics, Inc.).
Data Analysis for Gel Shift Assays--
The ability of XGRAF to
bind
DNA with mutations in the putative XGRAF-binding region was
determined by competition gel shift assays, which contained a constant
amount of radioactively labeled wild-type
DNA and either no
competitor or various concentrations of DNA with a single-point
mutation. An example of the gel shift assay with mutant
A
171 as the competitor is shown in Fig.
1A. Radioactivity in the
shifted DNA·XGRAF complex in each lane was quantitated from the
phosphorimaging scan. The total amount of XGRAF in the complex with
radioactive DNA in the absence of any competitor (Fig. 1A,
lane 2) was defined as 1.0, and the amount of XGRAF in the
complex with radioactive DNA in the presence of a 100-fold molar excess
of wild-type competitor was defined as zero (Fig. 1A,
lane 1). The quantity of XGRAF remaining in the complex with
radioactive DNA in the presence of mutated competitor (Fig.
1A, lanes 3-17) was expressed as a fraction of 1.0. The difference between the total and the fraction still bound to
the radioactive probe equaled the fraction of XGRAF bound to the
competitor DNA.

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Fig. 1.
Quantitation of binding of XGRAF to DNA
with a single-point mutation at position 171. A, gel shift
assay. The binding of XGRAF in Xenopus liver nuclear extract
to a DNA fragment spanning positions 187 to 115 of the
-fibrinogen subunit gene upstream region was analyzed. The reactions
contained the wild-type (WT) DNA sequence as the radioactive
probe and the following competitors: lane 1, 100-fold molar
excess of wild-type DNA; lane 2, no competitor; lanes
3-17, three replicates (labeled sets A, B,
and C) of DNA from positions 187 to 115 with the
single-point mutation (Mut) A 171 at 50-, 100-, 200-, 300-, and 500-fold molar excesses. B, Scatchard
analysis. The data from A were plotted according to Equation 3 (see "Experimental Procedures"). Bound XGRAF on the
x axis corresponds to [DNA·XGRAF], and Bound
XGRAF/Competitor DNA on the y axis corresponds to
[DNA·XGRAF]/[DNA]t in Equation 3. Bound XGRAF is
expressed as a fraction of total XGRAF, and competitor DNA is expressed
as the -fold excess over the radioactive probe.
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The equilibrium binding equation describes the interaction between
XGRAF and DNA (Equation 1),
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(Eq. 1)
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where [XGRAF] indicates free XGRAF protein concentration,
[DNA] represents free competitor DNA concentration, and
[DNA·XGRAF] represents concentration of XGRAF complexed with
competitor DNA. Since competitor DNA is in excess over XGRAF, the
concentration of total competitor DNA, [DNA]t, can be
substituted for the concentration of free competitor DNA. Also,
[XGRAF] can be expressed as [XGRAF]t minus [DNA·XGRAF],
and the binding equation can be rearranged to the Scatchard equation
(Ref. 29) (Equation 2),
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(Eq. 2)
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where f is the ratio of bound XGRAF to total XGRAF,
[DNA·XGRAF]/[XGRAF]t. As explained above,
[XGRAF]t has been defined as 1.0. Since we are not
calculating an absolute value for the equilibrium constant,
Kd is substituted with the term
C50, which represents the -fold excess of
competitor required to displace 50% of XGRAF from the probe DNA, and
Equation 2 is simplified to Equation 3.
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(Eq. 3)
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[DNA·XGRAF] is expressed as a fraction of
[XGRAF]t. [DNA]t is expressed in units of -fold
excess of unlabeled competitor DNA over radioactively labeled DNA. In
Fig. 1B, the data from Fig. 1A were plotted
according to Equation 3, and C50 was calculated
as the reciprocal of the y intercept. For each of the 27 single-point mutations, C50 was determined in
this way in three independent experiments. Values for [DNA·XGRAF]
below 0.1 or above 0.9 were not included in the Scatchard plots.
Essentially the same results were obtained when
C50 was calculated as the negative reciprocal of
the slope.
Methylation Interference Footprinting--
DNA probes containing
gene upstream sequence from positions
232 to
6,
32P-labeled on the sense strand, and from positions
232
to
115, 32P-labeled on the antisense strand, were
produced by PCR with 0.1 µM primers and Taq
enzyme (24). The PCR products were purified through a native 6%
polyacrylamide gel (26) followed by organic extraction. The end-labeled
DNA fragments (~1 × 106 cpm/pmol) were partially
methylated at the guanine moieties by incubation for 2-3 min with
dimethyl sulfate without carrier (30); in some cases underwent organic
extraction; were precipitated twice with ethanol; and were dissolved in
10 mM Tris-HCl and 0.1 mM EDTA (pH 8.0). For
preparative binding, the reaction volumes described above for the gel
shift assay were scaled up 15-30-fold, with ~300,000 cpm of DNA
probe, and electrophoresis was carried out at 250 V for 2.5-3.5 h at
4 °C. Wet gels were exposed to XAR film at 4 °C overnight;
portions of the gel containing bound and unbound DNA were excised; and
DNA was eluted by shaking overnight once or twice at 37 °C in 0.2 M NaCl, 10 mM Tris-HCl (pH 7.5), and 0.1 mM EDTA. The eluted DNA was purified over a NACS column (Life Technologies, Inc.) according to the manufacturer's
instructions, and 8-16 µg of carrier yeast RNA was added. After
ethanol precipitation, piperidine cleavage was performed (31), and
piperidine was removed (24). The entire recovered bound DNA fraction
and an approximately equal amount of radioactivity of the unbound DNA
were analyzed by gel electrophoresis as described previously for
methylation protection footprinting (24). The gels were exposed to XAR
film at
80 °C with one Lightning Plus intensifying screen.
Transfection and Assays for Reporter Gene
Activity--
Isolation of hepatocytes from three adult female
X. laevis frogs and transfection were as described
previously (24), except that 10 µg of control plasmid pCMV-
gal
(32) was used, and electroporation was conducted at a setting of 130 V. The cells were divided equally and incubated with or without
10
7 M dexamethasone and 10
9
M triiodothyronine in 12-well Primaria plates (Falcon) with
1 × 106 cells in 4 ml of medium. After 44-48 h, cell
extracts were prepared, and the activities of
-galactosidase (using
the Galactolight method) and of luciferase were assayed as described
(24).
Data Analysis for Transfection Assays--
Luciferase values
were normalized to the
-galactosidase activity in each transfection.
-Fold hormonal induction was calculated by dividing the amount of
luciferase activity/
-galactosidase activity in hormone-treated cells
by the amount of luciferase activity/
-galactosidase activity in
untreated cells. The data for each mutant are expressed relative to
wild-type DNA as follows. XGRAF activity with wild-type DNA is defined
as FWT
FD, where FWT represents the -fold increase in
transcription in response to hormone treatment for the wild-type
construct pLL
187 and FD represents the
-fold increase in transcription in response to hormone treatment for
the mutation D construct, which has multiple mutations in the XGRAF
site. Mutation D (
175ACT
173) abolished the
XGRAF-binding site, so the remaining -fold response is due solely to
the half-GREs (24). For each single-point mutant, the percent of
wild-type XGRAF activity was calculated as ((FM
FD)/(FWT
FD)) × 100, where FM is
the -fold increase in transcription in response to hormone treatment
for each mutant. In a total of eight experiments, the average
FWT was 5.3 ± 0.6 (mean ± S.E.), and the
average FD was 2.2 ± 0.2 (mean ± S.E.). Each mutant was tested in three independent experiments.
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RESULTS |
Identification of Specific Guanosines Involved in XGRAF
Binding--
Previously, we mapped the DNA elements between 177 and
135 bp upstream of the transcription start site that are important for
glucocorticoid regulation of the Xenopus
-fibrinogen
subunit gene (24). Three sites designated half-GRE1 (positions
168 to
163), half-GRE2 (positions
156 to
151), and half-GRE3 (positions
140 to
135) in Fig. 2C
have a close match to the downstream half of the consensus GRE, bind to
the DNA-binding domain of GR in vitro, and contribute to
steroid-induced transcription in primary hepatocytes. Half-GRE1 is the
most critical of the three GREs for hormonal induction. In addition,
the tract from positions
177 to
169 is necessary for full hormone
responsiveness even though it does not match the consensus GRE. The
nuclear protein XGRAF binds to this region of the DNA (Fig.
2C).

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Fig. 2.
Identification of guanosines in the
-fibrinogen subunit gene upstream regulatory region that contact
XGRAF. A, sense strand methylation interference. The assay
used DNA from positions 232 to 6 synthesized by PCR with an
end-labeled sense strand primer. Lane G+A, DNA sequencing
ladder; lane B, protein-bound DNA; lane F, free
DNA. Numbers indicate genomic sequence positions. Arrowheads
mark the bands with reduced intensity in the protein-bound DNA fraction
compared with free DNA (lane B versus lane F). B,
antisense strand methylation interference. The conditions were the same
as described in A, except that the probe was DNA from
positions 232 to 115, 32P-labeled on the antisense
strand. C, the nucleotide sequence of the upstream
region from positions 187 to 130. Arrowheads at
positions 184, 175, and 173 indicate those guanosines at which
methylation inhibited binding of XGRAF. The squares above
the sequence indicate 10-bp intervals. All the elements that contribute
to glucocorticoid responsiveness of the gene are indicated below
the sequence: the XGRAF-binding region between positions 177 and
169 and the three half-GREs (positions 168 to 163, 156 to
151, and 140 to 135).
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To identify bases that have direct contacts with XGRAF, we used the
methylation interference assay, which disrupts protein binding by
methylation of critical guanines within a recognition site. A
radioactively labeled DNA fragment including nucleotides
177 to
169
of the
gene upstream region was partially methylated in
vitro. The DNA was incubated with Xenopus liver nuclear
extract, and both protein-bound DNA and free DNA were isolated by
preparative native gel electrophoresis. The DNA was cleaved at all
methylated guanosines, and the two populations of DNA were compared for
relative abundance of fragments ending at particular positions (Fig.
2). When the sense strand was radioactively labeled (sequence shown in
Fig. 2C), the two DNA fragments ending at positions
175
and
173 were significantly reduced in intensity in the protein-bound DNA fraction (Fig. 2A, lane B) as compared with
the free DNA fraction (Fig. 2A, lane F),
indicating that methylation of these guanines interfered with binding
of a protein in the nuclear extract. Thus, bases G
175 and
G
173 are important contact points for XGRAF. The
intensity of the fragment ending at G
184 was also
reduced. This nucleotide is not considered to be part of the core
XGRAF-binding site since the intervening bases from positions
178 to
181 are not required for XGRAF binding or function (24). When the
antisense strand was radioactively labeled, no differences in
intensities of bands were observed (Fig. 2B, compare lanes F and B). Although no guanosines are
present on the antisense strand within the putative XGRAF-binding site
between positions
177 and
169, this experiment confirmed that the
binding site does not extend upstream to position
182 or downstream
to position
164, where the nearest guanosines are located.
Effect of Point Mutations on XGRAF Binding Ability--
The
relative contribution of each nucleotide to the specific interaction
between XGRAF and the
DNA was assessed by saturation mutagenesis of
the site from positions
177 to
169, generating all 27 single-point
mutants in the 9-bp sequence. The effect of the mutations on DNA
binding in vitro was analyzed by the gel shift assay, using
the mutated DNA sequences as competitors for binding of XGRAF to
radioactively labeled wild-type DNA, as described under "Experimental
Procedures." Binding ability is expressed as
C50, the -fold excess of competitor required to
displace half of XGRAF from the wild-type probe. For each mutant, the
C50 value was calculated in three independent
experiments, and the results are presented as the mean ± S.E. of
the three determinations (Fig. 3 and
Table I). The wild-type DNA had a
C50 value of 1.7-fold excess.

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Fig. 3.
Relative ability of single-point mutants of
the DNA from positions 177 to 169 to bind XGRAF. Using the
gel shift assay and Scatchard analysis described under "Experimental
Procedures" and exemplified in Fig. 1, the ability of each mutant to
bind to XGRAF was quantitated. The wild-type (WT)
nucleotides at positions 177 to 169 and their corresponding
single-point mutations (mut) are indicated along the y
axis. C50 on the x axis
represents the -fold excess of mutant DNA required as competitor to
displace half of XGRAF from the radioactively labeled wild-type DNA
probe. The data are shown as the mean ± S.E. of values determined
in three independent experiments. For each mutant, three independently
produced preparations of competitor DNA and at least two different
batches of nuclear extract were used (except for mutant
A 172, for which the data were derived from two
experiments with two different preparations of competitor and one
nuclear extract).
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Table I
Relative ability of the 27 single-point mutants at positions 177 to
169 of the gene to bind XGRAF, to enhance glucocorticoid
induction of transcription, and to regulate basal transcription
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At positions
177,
176, and
174, all of the mutants bound strongly
to XGRAF, with low C50 values of 1.7-3.8-fold
excess. Therefore, these positions are not critical determinants for
binding specificity since any nucleotide can be substituted without
significantly affecting the interaction with XGRAF. Similarly, changing
G
175 to either C or T was not deleterious to binding.
However, when this position was mutated to A, the binding ability
was reduced, with a C50 of 36-fold excess. It is
interesting that the introduction of A at position
175 creates a
stretch of six adenosines, which may interfere with binding due to
structural alterations rather than the specific nucleotide substitution
(33).
In contrast, nearly all changes in the bases from positions
173 to
169 substantially impaired XGRAF binding ability (Fig. 3 and Table
I), with C50 values from 20-fold excess for
mutant C
171 to 323-fold excess for mutant
C
173. The only exception was mutant C
169,
which retained relatively strong XGRAF binding
(C50 = 6.3-fold excess).
Effect of Point Mutations on Glucocorticoid Responsiveness--
We
also examined the effects of the point mutations in the XGRAF-binding
site on glucocorticoid induction of transcription. The mutated
gene
upstream region was inserted into a luciferase reporter vector, and the
constructs were transfected into primary Xenopus
hepatocytes. Transfected cells were divided for plus or minus
glucocorticoid treatment for 2 days, and lysates were analyzed for
luciferase activity. The total -fold increase in luciferase levels in
hormone-treated cells reflects the role of not only the XGRAF site, but
also the three half-GREs in the upstream regulatory region of the
gene (Fig. 2C). As described under "Experimental Procedures," the effect of the mutations only on the XGRAF
contribution to the hormonal stimulation was assessed by comparing each
single-point mutant with wild-type DNA, representing 100% induction,
and with a triple mutant in the XGRAF-binding site, which eliminated
XGRAF binding and therefore represented 0% activity of XGRAF in the induction. The triple mutant, which changed
175GAG
173 to
175ACT
173, has been shown previously to
reduce glucocorticoid responsiveness through effects on the
XGRAF-binding site rather than the GRE (24). Retention of at least 60%
of XGRAF activity was considered normal function, whereas activity
below 47% was defined as impaired.
As shown in Fig. 4 and Table I, full
XGRAF activity ranging from 79 to 135% of the value obtained with
wild-type DNA was achieved for all single-nucleotide substitutions at
positions
177,
176, and
174. Hence, these positions are not
essential for conferring XGRAF function on the
gene. At position
175, XGRAF activity was reduced to 33% when the site was mutated to A, but 85 and 107% of wild-type function were attained with the T and
C substitutions, respectively. Thus, the only functionally deleterious
mutation from positions
177 to
174 is the G to A transition at
position
175.

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Fig. 4.
Relative ability of single-point mutants of
the DNA from positions 177 to 169 to enhance glucocorticoid
induction of transcription. Transfection of Xenopus
primary hepatocytes with constructs containing mutations in the
XGRAF-binding region of the DNA and maintenance of the cells with
or without hormone treatment are described under "Experimental
Procedures." The wild-type (WT) nucleotides at positions
177 to 169 and their corresponding single-point mutations
(mut) are indicated along the y axis. The data
are reported as XGRAF activity, which is defined as the portion of the
hormonal induction attributable to XGRAF (rather than GR) for each
mutant compared with that of wild-type DNA (see "Experimental
Procedures"). The values are the mean ± S.E. of three
independent experiments. The value obtained in each experiment was the
average of usually triplicate, and in a few cases duplicate,
measurements.
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Conversely, almost all mutations at positions
173 to
169
significantly decreased XGRAF activity to between 11 and 46% of function with wild-type DNA (Fig. 4 and Table I). The most dramatic departure from this general pattern is the G to C transversion at
position
173, which improved the glucocorticoid induction, apparently
to 453% of normal XGRAF activity. The other two exceptions are mutants
G
172 and C
171, which allowed 60 and 71% of
wild-type activity, respectively.
Effect of Point Mutations on Basal Transcription--
The level of
transcription of the single-point mutants in the absence of hormone
treatment ranged from 48 to 142% of that of wild-type DNA (Table I).
Each data point for basal expression was obtained from an independent
transfection event, whereas changes due to hormone treatment were
assessed on a single cell population that was divided after the
transfection.
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DISCUSSION |
Correlation between XGRAF Binding to DNA in Vitro and Stimulation
of Transcription--
In Table I, the ability of DNA with single-point
mutations at positions
177 to
169 to bind to XGRAF in the gel shift
assay is classified as strong (+) if half-maximal competition was
achieved with <7-fold molar excess of mutated competitor over
wild-type probe or as weak (
) if 20-fold or greater excess competitor
was needed. Similarly, in Table I, the ability of DNA with each
single-point mutation to enhance glucocorticoid-induced transcription
in transfected primary hepatocytes is labeled as high (+) if at least
60% of wild-type XGRAF activity was retained or low (
) if 46% or
less of wild-type XGRAF activity was observed. With only four
exceptions, which will be discussed below, high activity in the
transfection assay correlated with strong binding ability in the gel
shift assay, whereas low functional activity corresponded with weak binding of XGRAF to the mutated DNA in vitro. This
correlation can be seen clearly in a plot of XGRAF activity as a
function of C50 (Fig.
5). The excellent agreement between
binding and function firmly supports the conclusion that XGRAF, defined
by its interaction with DNA in vitro, is the same protein
that plays a role in hormonal stimulation of gene transcription
in vivo.

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Fig. 5.
Correlation between the ability of the
single-point mutants in region 177 to 169 to amplify
hormone-induced transcription and to bind XGRAF. The portion of
the glucocorticoid response attributable to XGRAF, expressed as XGRAF
activity with mutant DNA as a percentage of that with wild-type DNA, is
plotted as a function of the ability of the DNA to bind XGRAF in
vitro, expressed as C50. See
"Experimental Procedures" and "Discussion" for details.
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The most striking discrepancy between physical association with XGRAF
and ability to amplify glucocorticoid responsiveness was seen with
mutant C
173, for which the hormonal induction was much
higher than for any other construct, while ability to bind XGRAF was
the weakest (Table I). This mutant was not included in Fig. 5 because
its functional activity was much greater than that of the other
mutants. Our method of computation attributed the stimulation of
transcription to XGRAF, but we believe that the effect in this case was
actually due to GR. The C
173 mutation generated the
following sequence:
177AAGACTnnnTGTTCC
163,
with two matches to the upstream half of the consensus GRE
(GGTACAnnnTGTTCT) in addition to the five out of six matches to the
downstream half. We have shown previously that the C at position
173
is essential for GR to interact with this site as a dimer and for
hormonal stimulation greater than 10-fold (24). Therefore, the strong glucocorticoid response seen with the single-point mutation to C
173 could be explained by strong GR binding that
eliminated the role of XGRAF in the induction. A comparable effect was
not expected with any of the other nucleotides because even when all
the bases except
173 were changed to match the consensus GRE, no
increase in GR dimer binding or hormonal induction was observed
(24).
Two other mutants, G
172 and C
171, were also
classified in Table I as having weak binding while retaining the
capacity to stimulate transcription. The levels of XGRAF
transcriptional activity were, however, moderate at 60 and 71%,
respectively, the lowest values that were still considered positive.
The binding abilities (C50 values of 24- and
20-fold excess), although defined as weak, were intermediate between
the strongest and weakest. In Fig. 5, the data for these mutants are
represented by the two points in the center of the plot, which lie
close to the line and therefore show good correlation between binding
and function.
Mutant C
169 had a striking disjunction between strength
of binding to DNA and ability to amplify glucocorticoid action. XGRAF bound quite well to the C
169 mutant since only a 6.3-fold
molar excess of this DNA was required to displace half of XGRAF from
the wild-type probe in the gel shift assay (Table I). Nonetheless,
hormonal stimulation was very poor, with only 25% of wild-type XGRAF
activity. The distinction between the C
169 mutant and all
other constructs is evident from the anomalous position of the
C
169 data in the lower left portion of Fig. 5. These
results cannot be explained by changes in the interaction of GR with
the DNA since the mutation lies within the 3-bp region between the two half-sites of the GRE, which is not critical for GR binding and function (23). Although the C
169 mutant binds fairly
tightly to XGRAF in vitro, we hypothesize that it is
incapable of conferring a functionally important conformational change
on the protein that would occur upon binding to the natural recognition
sequence.
Another important question is whether XGRAF had general effects on
transcription independent of its amplification of GR action. The level
of basal transcription for each of the single-point mutants is shown in
Table I. Basal transcription was inherently more variable than -fold
hormonal induction in the transfection assay because each data point
was derived from an independently transfected sample. When
transcriptional activity in the absence of hormone treatment was
plotted versus binding ability (Fig. 6), only a slight correlation was
observed. Therefore, we conclude that XGRAF may have a small effect on
general transcription of the
subunit gene, but that the major
function of XGRAF is to enhance glucocorticoid induction in response to
GR. This specificity is in contrast to many other glucocorticoid
receptor accessory factors (such as nuclear factor-1; activator
protein-1; cAMP response element-binding protein; hepatocyte nuclear
factor-1, -3, and -4; and chicken ovalbumin upstream promoter
transcription factor), which also stimulate basal transcription (2,
10-17).

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Fig. 6.
Correlation between the ability of the
single-point mutants in region 177 to 169 to stimulate basal
transcription and to bind XGRAF. Basal transcription, the
luciferase activity/ -galactosidase activity in untreated cells
transfected with mutant DNA expressed as a percentage of that with
wild-type DNA, is plotted as a function of the ability of the DNA to
bind XGRAF in vitro, expressed as C50. See
"Experimental Procedures" and "Discussion" for details.
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Consensus Sequence for the XGRAF Recognition Site--
Based on
the physical and functional data presented here, a consensus sequence
can be derived for the XGRAF-binding site that reflects the most
favorable nucleotide at each position. Bases
177 and
176 are no
longer considered part of the recognition sequence since no
substitutions at these positions affected XGRAF binding or activity.
For nucleotides
175 to
169, the consensus sequence is BNGTTAA
(B = C, G, or T; N = A, C, G, or T). Even with this more well
defined site, we found no striking matches to recognition sequences in
the transcription factor site data base TRANSFAC 3.2 (34) using the
TESS searching program (36).2
Thus, XGRAF appears to have a novel sequence specificity for binding to
DNA.
Models for Interaction of XGRAF and GR with Contiguous or
Overlapping Binding Sites--
Previously, we presented four possible
models for the interaction of XGRAF and GR with DNA at closely
juxtaposed sites (24). Model 1 depicted simultaneous binding of XGRAF
to its site at nucleotides
175 to
169 and GR binding as a dimer at
nucleotides
177 to
163, which would constitute a full-length GRE.
To bind these sites concurrently, XGRAF must contact the DNA in the
minor groove since GR is known to occupy the major groove (35).
However, the methylation interference experiment (Fig. 2) established
that XGRAF also binds in the major groove since modification of the N-7
positions of guanines, which are accessible only in the major groove,
interfered with binding. Therefore, Model 1 is not a likely mechanism
for interaction of GR and XGRAF with their respective sites.
Model 2 proposed an interaction between XGRAF and a monomer of GR.
Model 3 depicted a trimeric complex consisting of one molecule of XGRAF
and a dimer of GR, with GR contacting only the downstream half of the
GRE. Both of these scenarios are possible but must take into account
that the XGRAF- and GR-binding sites are directly contiguous. In Model
4, binding of XGRAF and GR was sequential rather than simultaneous,
which would obviate problems of steric hindrance for two protein
molecules binding to abutted recognition sites. Experiments are in
progress to distinguish between these mechanisms.
Classically, the presence of a receptor defined a tissue as being a
target for a steroid hormone, and the presence of a high affinity
receptor-binding site on the DNA was a prerequisite for a responsive
gene. It is becoming increasingly clear that steroid hormone action is
dramatically influenced by many other aspects of the local cellular
environment and the structure of the gene regulatory region. Accessory
DNA-binding proteins such as XGRAF can play as important a role as the
receptor in determining the extent of hormonal induction. The fact that
diverse cellular responses rely on unique combinations of accessory
DNA-binding proteins, coactivators, corepressors, and other factors
makes it possible for multiple control mechanisms to regulate genes
differentially in the same cell in response to a single hormonal
stimulus.
We gratefully acknowledge insightful
discussions with Drs. Mark Milanick and Mark Hannink and helpful
comments on the manuscript from Brian Morin.