Heterodimerization between the Glucocorticoid Receptor and the Unrelated DNA-Binding Protein, Xenopus Glucocorticoid Receptor Accessory Factor
Brian Morin,
Glenna R. Woodcock,
LaNita A. Nichols and
Lené J. Holland
Department of Physiology University of Missouri-Columbia School
of Medicine Columbia, Missouri 65212
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ABSTRACT
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The adrenal steroid hormones, glucocorticoids,
control many physiological responses to trauma, including elevated
synthesis of fibrinogen, a major blood-clotting protein. Glucocorticoid
regulation of the
-fibrinogen subunit gene in Xenopus
laevis is mediated by a binding site for Xenopus
glucocorticoid receptor accessory factor (XGRAF) and a contiguous
glucocorticoid response element (GRE) half-site. Here, we characterize
the protein:DNA complex formed by a cooperative interaction between
XGRAF, GR, and the DNA. We demonstrate that the complex contains XGRAF
by competition in a gel shift assay. The presence of GR is established
by two criteria: 1) size dependence of the XGRAF:GR:DNA complex on the
size of the GR component and 2) interference with complex formation by
GR antibody. Cooperative binding of XGRAF and GR to the DNA was
quantitated, showing that GR favors binding to XGRAF:DNA compared with
free DNA by a factor of 30. The cooperative interaction between XGRAF
and GR can occur on nicked DNA but is disrupted when 1 bp is inserted
between the XGRAF binding site and half-GRE. Significantly, this loss
of physical association in vitro correlates with loss of
XGRAF amplification of GR activity in transiently transfected primary
Xenopus hepatocytes. The simplest explanation for
cooperativity between XGRAF and GR is formation of a DNA-bound
heterodimer of these two proteins. This mechanism represents a new mode
of transcriptional regulation in which GR and a nonreceptor protein
form a heterodimer, with both partners contacting their specific DNA
sites simultaneously.
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INTRODUCTION
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Steroid hormones exert their myriad physiological effects by
binding to specific intracellular proteins that selectively regulate
gene transcription in target tissues (1). These hormone receptors
belong to the large nuclear receptor superfamily, which is divided into
two general classes based on DNA binding specificity (2, 3, 4). The first
includes the receptors for glucocorticoid, mineralocorticoid, androgen,
and progestin steroids (recognizing 5'-TGTTCT-3'). The second class
encompasses the receptors for estrogenic steroids and nonsteroids such
as retinoids, as well as many orphan receptors with no known ligand
(recognizing 5'-TGACCT- 3'). In the classical
mechanism of action, the steroid receptors bind as homodimers to
inverted repeats of their recognition sequences, such as the consensus
glucocorticoid response element (GRE), 5'-GGTACAnnnTGTTCT
-3' (see Table 1
A)
(5).
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Table 1. Nucleotide Sequences of DNA Fragments from the
Upstream Region of the Xenopus -Fibrinogen Gene Used in
Gel Shift and Transfection Assays
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Consistent with their large number and diversity of functions, the
nuclear receptors operate by a variety of mechanisms in addition to
homodimerization. For instance, members in the second class of
receptors frequently heterodimerize on paired DNA sites (2, 3), whereas
this mechanism has been described in only a few instances between
members of the first class (6, 7, 8). Also, an association between
receptors of the two different classes has been observed (9). Finally,
interaction of the nuclear receptors with transcription factors in
completely different protein families occurs (10). For example, the
estrogen receptor can enhance binding of the promoter-specific
transcription factor Sp1 to DNA (11) and GR can be tethered to the DNA
by signal transducer and activator of transcription-5 (Stat5) (12) but,
in both of these cases, a specific DNA binding site for the steroid
receptor is not required.
A different steroid receptor mechanism controls transcription of
the gene coding for the
-subunit of fibrinogen in the liver of the
frog Xenopus laevis (13, 14, 15). The transcriptional regulatory
region of the
-fibrinogen subunit gene has a binding site for a
monomer of GR (TGTTCC) at positions 168 to 163 upstream of the
transcription start site. The immediately adjacent sequence GAGTTAA at
175 to 169 (see Table 1
A) binds Xenopus glucocorticoid
receptor accessory factor (XGRAF) (13, 14, 15). The XGRAF binding site
occupies the position of an upstream half-GRE in a full consensus GRE
(5), but the DNA-binding domain of GR does not bind to this sequence
(13). The recognition sequence for XGRAF does not correspond to the
binding sites of nuclear receptors or other transcription factors,
suggesting that XGRAF is a novel protein (13, 14). The segment of DNA
containing the GR and XGRAF binding sites is sufficient to confer
glucocorticoid induction of transcription (15). XGRAF alone does not
mediate hormone responsiveness, but it increases fold induction by GR
(15). The ability of XGRAF to bind to DNA and amplify hormonal
induction defines it as an accessory factor (16). Here, we describe a
new mechanism of GR action in which GR and XGRAF form a heterodimer on
the DNA.
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RESULTS
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Demonstration of the Presence of XGRAF and GR in the XGRAF:GR:DNA
Complex
We have shown previously in gel mobility shift assays that GR and
XGRAF bind individually to their respective recognition sites on a
fragment of the
-gene upstream DNA encompassing positions 189 to
157 (13, 14, 15). When both proteins are present, a protein:DNA complex
forms that is larger than either GR:DNA or XGRAF:DNA (15). The
formation of this complex implies that XGRAF and GR bind to the
-gene DNA simultaneously and that both proteins make contact with
the DNA (15). Here we use gel shift assays that incorporate
competition, size variation, and antibody displacement to confirm the
identity of the protein components in the XGRAF:GR:DNA complex.
Figure 1
A shows the migration
position of the
(189 to 157) DNA bound to XGRAF (lane 1). This
complex was eliminated by competition with nonradioactive DNA
containing a binding site for XGRAF but not for GR (lane 2). Consistent
with our previous work (13), binding of native GR in the nuclear
extract to
-gene DNA was not seen. Because endogenous GR in a crude
nuclear extract is generally not readily detectable in gel shift
assays, experiments are usually carried out with GR synthesized from
cDNA (17, 18, 19). Here, we used a truncated form of rat GR synthesized in
Escherichia coli, designated GR(L) (see Materials and
Methods). GR(L) bound to
(189 to 157) DNA primarily as a
monomer (lane 3, lower band), with a small amount of dimer
present (lane 3, upper band). The upper band in
lane 3 was deduced to be GR dimer by comparison to GR bound to a probe
with a strong consensus GRE (lane 4), where binding as a dimer is
expected to be the predominant form (13, 20). When XGRAF and GR(L) were
both present, a new larger band, XGRAF:GR(L):DNA, also formed (lane 5).
The complex was distinctly different from GR homodimer (compare lane 5
to lanes 3 and 4). Excess competitor DNA containing the XGRAF binding
site completely eliminated both XGRAF:DNA and XGRAF:GR(L):DNA, without
interfering with GR binding to the DNA (lane 6). Thus, XGRAF is
required for formation of the XGRAF:GR(L):DNA complex. This result,
together with our previous demonstration that XGRAF:GR(L):DNA
does not form on a probe with a mutated XGRAF binding site (15),
confirms that XGRAF is an integral part of the complex.

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Figure 1. Characterization of the XGRAF:GR:DNA Complex Using
Gel Mobility Shift Assays
The reactions were carried out as described in Materials and
Methods with either wild-type (WT) DNA or consensus GRE (C)
probe (see Table 1 A) and, as indicated, 56 µg
Xenopus liver nuclear extract and the nonradioactive
competitor 1 DNA (Table 1 A) at 100-fold molar excess over probe. A, WT
DNA and C DNA were at 0.2 ng and GR(L) was at 20 ng. B, WT DNA was at
0.2 ng and C DNA at 0.01 ng. GR(S) was used at 5 ng (lane 4) or 10 ng
(lanes 3, 5, and 6).
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The presence of GR in the XGRAF:GR:DNA complex was established using
the same experimental scheme with a smaller form of the rat GR, GR(S)
(see Materials and Methods). When both XGRAF and GR(S) were
present, XGRAF:GR(S):DNA formed (Fig. 1
B, lane 5). This complex was
shown to contain XGRAF by competition (lane 6). XGRAF:GR(S):DNA
migrated slightly above XGRAF:DNA and, therefore, was in a distinctly
different position from XGRAF:GR(L):DNA (compare Fig. 1B
, lane 5, to
Fig. 1
A, lane 5). The size difference of the complexes is directly
attributable to the two different sizes of GR used.
GR was confirmed to be required for assembly of XGRAF:GR(S):DNA by
including a GR-specific antibody in the gel shift reactions. The
antibody primarily blocked formation of GR(S):DNA (Fig. 2
, compare lane 4 to lane 3), although a
small amount of material was detectable as a large supershifted band on
a darker image (lane 4'). When the DNA was incubated with both XGRAF
and GR(S), the GR-specific antibody caused the GR(S):DNA and the
XGRAF:GR(S):DNA bands to disappear, but did not interfere with
XGRAF:DNA formation (compare lane 6 to lane 5). The largest band in
lanes 6 and 6' is identified as GR(S):antibody:DNA since it comigrated
with the supershifted GR(S):DNA in lane 4'. The nuclear extract
apparently stabilized the supershifted complex. GR-specific antibody
interference with XGRAF:GR(S):DNA assembly provides additional support
for the presence of GR in the complex.

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Figure 2. Supershift or Immunodepletion of Protein-DNA
Complexes Containing GR
The gel shift reactions were carried out as described in
Materials and Methods with WT DNA probe at 0.2 ng, GR
antibody (Ab) at 1 µg, and GR(S) at 20 ng. Lanes 4'-6' are a darker
image of lanes 46.
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Quantitation of Cooperative Binding of XGRAF and GR to
-Fibrinogen Gene DNA
The existence of the XGRAF:GR:DNA complex raised the possibility
that GR and XGRAF bind cooperatively to the
-gene DNA. To test this
hypothesis, we adapted an approach described by La Baer and Yamamoto
(21) to calculate a relative value for the equilibrium association
constants for formation of XGRAF:GR:DNA and GR:DNA. The equilibrium
association constants are derived from the following reaction
scheme:

The equations for the equilibrium association constants
KD and KB are:
Equation 1a:
KD=[XGRAF:GR:DNA]/[XGRAF:DNA][GR]
Equation 1b:
KB=[GR:DNA]/[DNA][GR]
The ability of GR to bind to the XGRAF:DNA complex as
compared with free DNA is expressed as the ratio of
KD to KB:
A value of 1.0 for
KD/KB signifies that GR has
equal affinity for XGRAF:DNA and free DNA, indicating that binding is
not cooperative. Values greater than 1.0 denote that GR binds
preferentially to XGRAF:DNA. Using the
KD/KB ratio avoids
uncertainties with regard to absolute concentrations of the reactants
since a relative number is calculated (21). Thus, our modification of
this powerful, yet simple, approach allows quantitative analysis of
heterodimer formation using crude nuclear extract. The system can also
be thought of in terms of the preference of XGRAF to bind GR:DNA
compared with free DNA, and it is worthy of note that, upon
rearrangement of terms, the ratio
KC/KA is identical to the
ratio KD/KB.
To determine KD/KB, the
amount of radioactive wild-type
DNA in XGRAF:GR:DNA, XGRAF:DNA,
GR:DNA, and free DNA in each lane of a gel shift assay was quantitated
by phosphorimaging and substituted into Equation 2. Using GR(L),
KD/KB was determined in six
independent experiments (with 46 individual lanes per experiment) to
be 30 ± 4 (mean ± SEM) (e.g. Fig. 3
A, lanes 25). Using GR(S), a similar
value of 29 ± 3 in eight experiments (with 38 individual lanes
per experiment) was obtained (B. Morin and L. J. Holland,
unpublished data). These values are substantially greater than 1.0,
indicating that GR and XGRAF bind cooperatively to their adjacent
binding sites on the DNA. In contrast, binding of GR(L) alone to
(189 to 157) DNA yielded (when the above reaction scheme was
rewritten for binding of two molecules of GR) a
KD/KB value of 1.9 ±
0.3 (Fig. 3
B), demonstrating minimal GR:GR cooperativity as predicted
by the lack of a full GRE.

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Figure 3. Gel Shift Assay for Quantitation of Cooperative
Binding of XGRAF and GR to DNA
A, The gel shift reactions contained 4 ng GR(L) and 0.2 ng C DNA
(lane 1) or 20 ng GR(L) and 0.2 ng WT DNA (lanes 25).
Xenopus liver nuclear extract was used at 8, 7, 6, and 5
µg (lanes 25). B, WT DNA was at 0.2 ng and GR(L) was at 100 ng
(lanes 13).
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Demonstration of Cooperativity by Competition between Labeled
GR:DNA and Unlabeled Free DNA
We have used a competition gel shift assay previously to
quantitate the preference of XGRAF to bind wild-type
-gene DNA
compared with mutated competitors (14). Here this assay was adapted to
examine the preference of XGRAF to bind radioactively labeled GR:DNA
instead of unlabeled competitor free DNA. In this assay, a large amount
of GR was used to form a substantial amount of the GR:DNA complex (Fig. 4
). Under these conditions XGRAF:DNA was
not visible because the XGRAF:GR:DNA complex was formed
preferentially. A nonradioactive competitor DNA, which contains
the XGRAF binding site but no GR binding site, was added to the
reactions. Since this competitor can only form the XGRAF:DNA complex,
this method computes the affinity of XGRAF for the radioactive GR:DNA
complex compared with its affinity for nonradioactive free DNA. The
amount of radioactivity in the XGRAF:GR:DNA complex over a range of
competitor concentrations was quantitated by phosphorimaging (lanes
29). A Scatchard analysis of the data, described in Materials
and Methods, revealed that a 25-fold excess of competitor over
GR:DNA was required to reduce the concentration of XGRAF:GR:DNA by
50%. The 25-fold preference of XGRAF to bind GR:DNA compared with free
DNA determined by this approach is in excellent agreement with the
30-fold value calculated by the more direct method in the previous
section.

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Figure 4. Demonstration of Cooperative Binding of XGRAF and
GR to DNA by Competition
The gel shift reactions contained 7 µg Xenopus liver
nuclear extract, 50 ng GR(L), 0.05 ng WT probe, and competitor 2 (see
Table 1 A) at the indicated fold excess over GR:DNA.
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Interaction between XGRAF and GR on a Nicked DNA Probe
Cooperativity could be due to a protein-induced
conformational change in the DNA that increases the binding affinity of
the second protein (22). To examine this possibility, in the gel shift
assay we used a DNA probe with a nick in one strand between the GR and
XGRAF binding sites. The nick should disrupt transmission of an
alteration in the DNA structure. For these experiments, we used a probe
of 42 nucleotides from positions 189 to 148. Figure 5
shows that this probe has similar
binding characteristics for GR(L) (lane 1), XGRAF (lane 2), and the
XGRAF:GR(L):DNA complex (lane 3) as the 33 mer
(189 to 157) DNA
(see Figs. 1
A, 3A, and 4). When this probe had a nick in the sense
strand between the XGRAF and GR binding sites, binding of GR(L) (lane
5), XGRAF (lane 6), and XGRAF:GR(L) (lane 7) also occurred. For both
probes, the presence of XGRAF in the XGRAF:DNA and XGRAF:GR(L):DNA
bands was confirmed by including an unlabeled competitor DNA that has a
binding site for XGRAF but not for GR (lanes 4 and 8, respectively).
XGRAF bound equally well to the intact or nicked probes. However, GR
binding was substantially impaired by the nick, lowering the absolute
amount of XGRAF:GR(L):DNA that formed on the nicked probe relative to
the intact probe. Nonetheless, the
KD/KB ratios, calculated
from three separate reactions, showed that cooperativity was similar
for the intact and nicked 42-mer probes. These values were comparable
to that calculated for the 33-mer probe. Since a nick in the DNA failed
to reduce cooperativity, it is unlikely that a conformational change in
the DNA accounts for the preferential simultaneous binding of XGRAF and
GR to the DNA.

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Figure 5. Analysis of the Effect of a Nick in the DNA between
the XGRAF and GR Binding Sites on Cooperative Binding
The gel shift reactions contained, as indicated, 6 µg
Xenopus liver nuclear extract and either 2 ng (lanes 1,
3, and 4) or 20 ng (lanes 5, 7, and 8) GR(L). The 42-mer intact probe
(lanes 14) or 42-mer nicked probe (lanes 58) was present at 0.2 ng
and competitor 1 (see Table 1 A) at 20 ng. See Materials and
Methods for a description of the DNA probes.
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Disruption of Cooperativity by Separation of the XGRAF and GR
Binding Sites
A second possibility to account for the cooperativity is a
direct protein-protein interaction that stabilizes XGRAF:GR binding to
the DNA (22). To determine the importance of protein-protein contacts,
the contiguity of the XGRAF and GR binding sites was disrupted. Gel
shift experiments were carried out with a DNA probe containing 1 bp
inserted between the XGRAF binding site and GRE half-site (Table 1
A).
This DNA was able to bind GR or XGRAF singly (Fig. 6
, lanes 1 and 2, respectively) in a
manner similar to the wild-type probe (lanes 4 and 5, respectively).
However, the XGRAF:GR:DNA band observed using wild-type DNA (lane 6)
was undetectable with the DNA containing the 1-bp insertion (lane 3).
While XGRAF and GR may still bind to this DNA simultaneously, without
the cooperative interaction the amount of the trimeric complex would be
reduced by 30-fold and would be indiscernible. Since the immediate
adjacency of the sites is required for cooperativity, we conclude that
a direct interaction between XGRAF and GR occurs.

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Figure 6. Analysis of the Effect of Separation of the XGRAF
and GR Binding Sites on Cooperative Binding to DNA
Gel shift reactions contained, as indicated, 6 µg
Xenopus liver nuclear extract, 20 ng GR(S), and 0.2 ng
either WT or +1 bp probe (see Table 1 A).
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Correlation between Loss of Cooperativity in Vitro and
Loss of Function in Vivo
The effect of separation of the binding sites on function in
vivo was analyzed by transient transfection. DNA vectors
containing the
-gene regulatory region were introduced into
Xenopus primary hepatocytes (15). Hormonal induction of
transcription by the XGRAF and GR binding sites was shown using the
GRU(
-104) construct (Fig. 7
).
Consistent with our previous demonstrations that XGRAF enhances GR
function (13, 14, 15), mutation of the XGRAF binding site in the construct
GRUmutX(
-104) reduced glucocorticoid responsiveness. A 1-bp
separation of the two binding sites in the GRU+1bp(
-104) construct
also decreased hormonal activation in comparison to the GRU(
-104)
control. The level of induction was equivalent whether the XGRAF
binding site was completely inactivated by mutation or was moved away
from the GR binding site. Thus, when the binding sites are
noncontiguous, the loss of cooperative binding in the gel shift assay
correlates with the reduced glucocorticoid responsiveness in intact
cells. This observation strongly supports the hypothesis that
interaction between XGRAF and GR, and not simply binding of the two
proteins to the DNA, is crucial for XGRAF to exert its stimulatory
effect on GR.
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DISCUSSION
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We have shown here that the preferential simultaneous
binding of GR and XGRAF to their respective sites on the DNA involves a
cooperative interaction between the two proteins. Quantitation of the
cooperativity established that GR favored binding to XGRAF:DNA compared
with free DNA by a factor of 30, as determined by direct comparison of
equilibrium binding constants. This value was corroborated by a
competition binding analysis. One explanation for cooperative
binding is that protein-induced changes in DNA conformation increase
individual binding affinity (Fig. 8
).
This interpretation is unlikely since a nick in the DNA between the
binding sites, which is expected to disrupt transmission of changes in
DNA structure (22), did not interfere with cooperative binding (Fig. 5
). An alternative explanation for cooperativity is that XGRAF and GR
physically contact one another when bound to the DNA (Fig. 8
). Support
for this model was obtained when separation of the two binding sites,
which would reposition potential interaction surfaces of the proteins,
eliminated cooperativity (Fig. 6
).

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Figure 8. Models of Formation and Disruption of the XGRAF:GR
Heterodimer
XGRAF is represented by the white triangle, GR by the
gray oval, and DNA by the black
bars. The 1-bp insertion is denoted by the white
box.
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While GR classically binds to DNA as a homodimer, monomeric GR
has been shown to function with other transcription factors (23, 24).
Therefore, the XGRAF:GR:DNA complex could contain either one or two
molecules of GR. We have shown previously that XGRAF binds to DNA in
the major groove (14), occluding the position where a second molecule
of GR normally binds to DNA as a homodimer (25). Thus, if the complex
contains two molecules of GR, one GR monomer must be tethered rather
than contacting the DNA directly (13). To distinguish between one or
two molecules of GR, we examined the size of the XGRAF:GR:DNA complex.
Although the native gels of the gel shift assay cannot be used for
absolute mass determination, XGRAF behaves as a protein somewhat larger
than GR(L), since XGRAF:DNA migrated more slowly than GR(L):DNA (Fig. 1
A, compare lane 1 to lane 3). Furthermore, the XGRAF:GR:DNA
complex migrated slightly above the GR(L) dimer (Fig. 1
A, compare lane
5 with lanes 3 and 4), indicating that the complex most likely contains
one molecule each of XGRAF and GR(L). If the complex contained two
molecules of GR in addition to XGRAF, its apparent size would be larger
than three molecules of GR(L) bound to the DNA. However, we have
determined that GR(L):GR(L):GR(L):DNA migrates much more slowly than
XGRAF:GR(L):DNA (L. J. Holland, unpublished data).
Taken together, the in vitro binding results are most
consistent with a model of heterodimerization between GR and XGRAF.
This mechanism, in which GR and an unrelated partner protein both
contact the DNA simultaneously, has not been described previously. The
region of GR that interacts with XGRAF was delineated using truncated
forms of GR. Both GR(L) and GR(S), which have only amino acids 407525
in common, interact cooperatively with XGRAF. Thus, the surface of GR
responsible for heterodimerization with XGRAF must be contained within
these amino acids. This region also contains domains for DNA binding
(25), GR homodimerization (25), and physical or functional interaction
with other transcription factors (26, 27). Furthermore, this region is
conserved across the nuclear receptor superfamily (2), suggesting that
XGRAF could heterodimerize with other members of the family.
The cooperative binding between XGRAF and the small GR fragments
in vitro parallels XGRAF enhancement of native GR function
in vivo. Therefore, it is likely that the transcriptional
activation of the
-fibrinogen gene by full-length GR in intact cells
involves a cooperative interaction with XGRAF. Since the in
vitro binding studies were carried out with mammalian GR,
heterodimerization with XGRAF is not restricted to the amphibian
receptor. In addition, we have evidence for a protein with similar
DNA-binding specificity to XGRAF in a human liver-derived cell line
(K. D. Fohey and L. J. Holland, unpublished data). Thus, this
mechanism is potentially applicable to activation of other
glucocorticoid-regulated genes in a wide variety of animals, including
humans.
We have shown that glucocorticoid-induced gene transcription can
be mediated through a heterodimer of GR and an unrelated
DNA-binding protein, in which both proteins bind to their specific
sites on the DNA. Heterodimerization between some related nuclear
receptors is common (2, 3). However, this is the first demonstration,
to our knowledge, of any member of the large nuclear receptor
superfamily forming a DNA-bound heterodimer (as opposed to a tethered
ternary complex) with a protein that has a completely different
DNA-binding specificity. This mechanism provides a new explanation for
how the DNA can dictate the assembly of specific transcriptional
regulatory complexes on different genes, to achieve diverse patterns of
gene activation in response to a hormone signal.
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MATERIALS AND METHODS
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Gel Mobility Shift Assays and Phosphorimager Analysis
The 33-mer oligonucleotide probes used in Figs. 1
, 2
, 3
, 4
, and 6
are described in Table 1
A. Oligonucleotide probes were purified and
radioactively labeled at the 5'- end as described (13). The 42-mer
probes used in Fig. 5
had the following sequence on the sense strand:
-189CTCCAGACAGAAAAGAGTTAATGTTCCCTCTTATacTaACTA-148.
The XGRAF binding site is in bold and the GRE half-site is
underlined. The lower case letters indicate
mutations that inactivate a second GR binding site at positions 156
to 151 (13, 15). The nicked 42-mer probe contained a single-strand
break between nucleotides 169 and 168. This probe was assembled by
annealing two independently synthesized (Genosys, The Woodlands, TX)
21-mer sense strands, 189 to 169 and 168 to 148, to an intact
antisense 42-mer from 148 to 189. A 42-mer instead of 33-mer probe
was used to ensure a stable double-stranded structure. Both the intact
and nicked 42-mer probes were radioactively labeled at the 5'-end as
described above. We confirmed by denaturing gel electrophoresis (28)
that the nicked probe consisted of labeled 21-mer and 42-mer strands
and that the nick did not become ligated during incubation for the gel
shift assay (L. J. Holland, unpublished data).
Bacterially synthesized, truncated forms of rat GR were kindly
provided by the laboratory of Dr. Keith Yamamoto. T7EX525, here
designated GR(L), comprises amino acids 106318 adjoined to amino
acids 407525, which includes the DNA-binding domain (29). T7X556,
here designated GR(S), consists of amino acids 407556 (17, 30). It is
necessary to use truncated forms of GR because full-length GR is too
large for optimal electrophoretic separation and quantitation of all
the protein-DNA complexes. The GR antibody BuGR 2 (Affinity BioReagents, Inc., Golden, CO) recognizes amino acids
410416 (31). Xenopus liver nuclear extract served as the
source of XGRAF (15). The binding reactions and gel electrophoresis
conditions were as described previously (15).
Quantitation of products in the gel shift assays was performed with a
phosphorimager and ImageQuant 3.3 software (Molecular Dynamics, Inc., Sunnyvale, CA). In each lane the amount of radioactivity
in XGRAF:GR:DNA, XGRAF:DNA, GR:DNA, and free DNA was determined. An
individual background for each lane was determined and subtracted from
the values for the protein-DNA complexes. As an example, normalized
data for Fig. 3A
are shown in Table 2
.
Quantitation of Preferential Binding by Competition
Assay
The competition gel shift assays (14) were carried out under
conditions where XGRAF was visible only in XGRAF:GR:DNA. Unlabeled
competitor DNA containing an XGRAF binding site but no GR binding site
was added in increasing amounts. Radioactivity was quantitated by
phosphorimaging, and the data were analyzed with a Scatchard plot (14)
generated by the following equation:
The amount of XGRAF bound to the competitor DNA, designated
[DNA·XGRAF], was computed as the fraction lost from
XGRAF:GR:DNA. [DNA]t, representing total
competitor DNA, was expressed as fold excess over GR:DNA, since the
ability of XGRAF to bind either GR:DNA or the competitor was being
compared. The quantity of GR:DNA in each reaction was calculated as the
fraction of radioactivity in the GR:DNA complex compared with
radioactivity in the entire lane. The term C50
represents the fold excess of competitor required to displace 50% of
XGRAF from the XGRAF:GR:DNA. The y-intercept was used to obtain a value
for C50. This value was normalized to the
C50 value obtained in a parallel experiment for
competition of XGRAF from wild-type DNA in the absence of GR.
Transfection of Primary Hepatocytes and Assay of Hormonal
Induction
The transfection vectors containing Xenopus
-fibrinogen gene regulatory DNA were constructed with either
wild-type or mutated
(187 to 157) (see Table 1
B) linked to
(104 to +41) (15). The vectors were transiently transfected into
Xenopus primary hepatocytes following published methods (15)
except cells were plated at 4 x 105 per
well in 24-well plates with 1.6 ml of medium, with a final composition
as described (32). Experiments were conducted in accordance with the
NIH Guide for the Care and Use of Laboratory Animals. As described in
detail previously (15), hormonal induction is expressed as a percentage
of the control using the following equation: Fold Induction (% of
control) = [(Fold induction of test construct 1)/(Fold
induction of control construct 1)] x 100. The value 1 was
subtracted from each fold induction to account for the baseline,
representing no hormone response. The induction for each construct is
expressed as the percentage ± SEM of that
observed for the GRU(
-104) control in four separate experiments.
Statistical analyses of raw data for fold hormonal induction in each
independent transfection were done with the Student- Newman-Keuls
test (33).
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ACKNOWLEDGMENTS
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We thank the laboratory of K. Yamamoto for generously providing
the purified GR fragments and M. Hannink, M. Martin, A. McClellan, and
R. Woodward for helpful comments on the manuscript.
 |
FOOTNOTES
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Address requests for reprints to: Lené J. Holland, Department of Physiology, University of Missouri-Columbia School of Medicine, Columbia, Missouri 65212.
This work was supported by the American Heart Association
(Grants-in-Aid 9708034A and 0051320Z).
Received for publication September 22, 2000.
Revision received November 28, 2000.
Accepted for publication November 29, 2000.
 |
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