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Departments of Medicine (R.J.G.H.) and Biochemistry (M.B., J.G.A.S, R.J.G.H.) University of Ottawa Ottawa Civic Hospital Loeb Research Institute Ottawa, Ontario, Canada K1Y 4E9
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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or 
, and that full
induction could be obtained by the ligand-binding domain (LBD) of GR
alone. C/EBPß, but not C/EBP or , reciprocally potentiated
transcriptional activation by DNA-bound GR LBD. However, C/EBPß was
unable to increase activation by a GR LBD with a short C-terminal
truncation, indicating that the functional interaction between the two
factors was dependent upon the GR AF-2. Surprisingly, despite the
specificity in functional effects, all three C/EBPs bound
indistinguishably to GR in GST pull-down and immunoprecipitation
assays. Indeed, several nuclear receptors, including the estrogen
(ER), progesterone, retinoic acid (RAR), and androgen receptors,
displayed a similar potential to bind C/EBPs. Previous reports have
demonstrated that ER and RARs repress transcriptional activation by
C/EBPß in ways that were dependent on their related AF-2 functions.
Therefore, the GR AF-2 may encode functional features that distinguish
the transcriptional regulatory potential of GR from that of ER and RAR.
Finally, C/EBP binding mapped to the GR DNA-binding domain, which was
not required for functional interaction with C/EBPß. Thus, the
potentiation of C/EBPß-mediated transcription by GR would appear to
require the presence of an intermediary factor. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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By contrast, the C/EBPs are a subfamily of the tissue-restricted bZip
transcription factors, which regulate transcription through CCAAT DNA
sequence motifs (6, 7, 8, 9). There are several C/EBPs genes and many
different isoforms of the C/EBP proteins (1, 10, 11, 12, 13). The bZip domains
of most of the C/EBPs are highly conserved (8). However, the remainder
of the proteins vary considerably between individual family members.
C/EBPs have been shown to play determining roles in the differentiation
(14) and function of hepatocytes (15, 16), adipogenesis (17, 18, 19, 20, 21, 22, 23, 24), and
the functional regulation and homeostatic control of lymphoid (19, 22)
and hematopoietic cells (19). Interestingly, C/EBPß (also known as
NF-IL6, Il-6DBP, LAP, AGP/EBP, CRP2, and NF-M), but not C/EBP, has
been shown to specifically interact with Sp1 in a manner that allows it
to regulate transcription from the rat CYP2D5 P450 gene
(25).
HSV replication occurs more efficiently in cells treated with the synthetic glucocorticoid dexamethasone (dex) (26). However, glucocorticoids have not previously been reported to directly induce or otherwise influence transcription of the viral tk gene. Glucocorticoids mediate transcriptional regulation through an intracellular nuclear hormone receptor that binds as a homodimer with high affinity to specific glucocorticoid-responsive DNA sequences [glucocorticoid-responsive elements (GREs) (27, 28)]. The promoter-proximal regulatory region of the tk gene does not contain a sequence resembling GRE (4).
GR, like all nuclear receptors, is a modular protein with a central DNA- binding domain flanked by carboxy- and amino-terminal transcriptional regulatory functions (29, 30, 31, 32, 33). In the absence of hormone, glucocorticoid receptor (GR) normally occurs in the cytoplasm in a high molecular weight complex with heat shock proteins and immunophilins (34). Steroid binding induces a conformational change in the receptor ligand-binding domain (LBD), which promotes dissociation of the GR-heat shock protein complex and allows translocation of the free receptor to the nucleus (34).
The activation of transcription by nuclear receptors is accomplished through interactions with transcriptional coregulatory proteins that promote the modification of chromatin structure and that interact with the basal transcriptional machinery (35, 36, 37). While the N-terminal activation functions appear to be unique to each receptor, the AF-2 activation functions at the C terminus of GR and other nuclear receptors (38, 39, 40, 41, 42, 43) interact in an overlapping manner with a series of transcriptional coactivator protein complexes that include proteins such as SRC-1 (44), CBP (45, 46, 47), and GRIP-1 (48, 49) and have histone acetylase activity (50, 51). Interestingly, many other transcription factors also appear to interact with the same coactivator complexes. This creates the potential for competition by nuclear receptors and other transcription factors for a limited pool of coactivator molecules. For example, it is now clear that GR competes with other nuclear hormone receptors and transcription factors such as CREB and AP-1 for CBP-containing coactivator complexes (45, 52, 53, 54). However, the differential interaction of transcription factors with common coactivators also may explain elements of the transcriptional synergism observed between nuclear receptors and other sequence-specific transcription factors on complex promoters.
Not all effects of GR on transcription result from the direct binding of receptor homodimers to canonical GREs. A number of transcriptional effects resulting from direct protein-protein interaction of GR with other sequence-specific transcription factors have been described. For example, direct interaction between GR and AP-1 has been demonstrated to be required to direct transcription from composite response elements that bind both factors together (55). Transcription is enhanced or repressed by this complex, depending on the specific c-fos family member in the jun/fos AP-1 heterodimer (55, 56, 57, 58). Recently, it also has been demonstrated that GR can act essentially as a coactivator to potentiate the activation of transcription from PRL-responsive promoters in the absence of a GRE by binding to DNA-bound Stat5 (59).
In the present study we have determined that glucocorticoids activate
transcription from the HSV-tk proximal promoter despite the absence of
a GRE. The results of our analysis suggest that this effect is mediated
through a functional interaction between the AF-2 of GR and C/EBPß.
These results contrast with the recent demonstrations that the AF-2
activities of retinoic acid receptor and estrogen receptor- (ER)
can act to repress C/EBPß-mediated transcription (17, 60). Therefore,
our results indicate one way in which the GR AF-2 may be functionally
distinct from the AF-2s of other steroid/retinoid receptors.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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) (3, 4).
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). Dex treatment of cells cotransfected with
the tkCAT reporter and a rat GR expression plasmid resulted in a 4-fold
induction of chloramphenicol acetyltransferase (CAT) activity (lane 1).
Thus, the glucocorticoid responsiveness of the tk promoter was not due
to dam methylation. The activation of transcription was
dependent upon GR and hormone agonist, as no induction was detected in
the absence of cotransfected receptor expression plasmid (lane 4) or
when GR-expressing cells were treated with the glucocorticoid
antagonist RU486 (lane 2). Further, androgen receptor (AR) was unable
to substitute for GR (lane 3). Indeed, dihydrotestosterone (DHT)
treatment of cells expressing AR reproducibly led to a 2-fold
repression of CAT activity. Finally, to confirm that the observed
effect was not due to treatment of the cells with high levels of dex,
we repeated the experiment at 33 nM dex, the optimal
concentration for the use of this glucocorticoid in the Cos7 parental
line CV1 (62), with similar results (data not shown).
To confirm that this effect was mediated in the absence of GR binding
to DNA and to begin to localize the determinants on GR required for
this effect, we examined the transcriptional response of the tk
promoter to three additional GR constructs (Fig. 2). First, expression of full-length GR
with an L501P mutation in the DNA-binding domain (DBD) that abrogates
sequence-specific DNA binding (63) actually led to a slightly stronger
response, with the induction of CAT activity from the tk promoter
increased from 4- to 6-fold (lanes 1 and 2). However, expression of the
N-terminal 525 amino acids of GR had no effect on reporter gene
activity (lane 3). By contrast, N525 constitutively activates
transcription from a GRE (64). Finally, expression of a GR fragment
N-terminally truncated at amino acid 547 at the border of the LBD was
as efficient in activating tk transcription as WT GR (lane 4). Thus,
the ligand-binding domain of GR appeared to be sufficient for full
induction of tk expression.
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, top). As a
control, a similar construct was prepared with four copies of an
octamer motif, which does not occur in the tk promoter. In addition, we
also recloned the -109/-29 HSV-tk promoter-proximal regulatory region
in front of the E1B promoter to determine whether similar effects could
be detected with a heterologous promoter. Transfections were then
performed to determine the response of these constructs to the
activation of GRL501P by dex (Fig. 3, bottom).
In GRL501P-transfected cells, dex treatment failed to
activate transcription appreciably from the SP1-responsive promoter or
control construct with four octamer motifs inserted adjacent to the E1B
sequence (lanes 1 and 2). By contrast, the CCAAT/E1B construct was
hormone responsive, with dex treatment inducing CAT activity 4-fold
(lane 3). This suggested that GR specifically targeted factors acting
through the C/EBP-binding site in the tk promoter.
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Finally, to determine whether the entire effect of GR on tk transcription was mediated through the C/EBP-binding site in the tk promoter, we determined the response of a -109 to +51 tk reporter gene in which site-directed mutagenesis had been used to convert the C/EBP-binding site to a nonfunctional sequence that has previously been described (65). Dex treatment of cells cotransfected with GRL501P and the HSV-tk C/EBPmut reporter plasmid was completely unable to induce reporter gene activity. Therefore, the C/EBP binding site in the tk promoter was both required for and sufficient for the induction of transcription by GR in Cos7 cells.
GR Potentiates the Activation of Transcription by C/EBPß, but Not
by C/EBP or
The results obtained in the experiment shown in Fig. 3
suggested that GR had the ability to potentiate the activation of
transcription by one or more isoforms of C/EBP. To evaluate the
selectivity of dex induction of transcription through the CCAAT
element, we examined the effect of coexpressing GRL501P and
three C/EBP proteins, C/EBP, ß, and , on the induction of
transcription of the CCAAT/E1B CAT reporter gene (Fig. 4). In the absence of dex, expression of
C/EBP, ß, and each resulted in a 5- to 8-fold induction of CAT
activity. The same result was obtained in the absence of cotransfected
GR, and no induction was observed on the parent E1B reporter construct
lacking the CCAAT response elements (data not shown). Treatment of
cells cotransfected to express C/EBP or and GRL501P
with dex had no significant additional effect on transcription (Fig. 4A, lanes 2 and 4). However, when C/EBPß was coexpressed with
GRL501P, dex treatment led to a strikingly further
induction of transcription (lane 3). Reexpression of the data as fold
induction by dex (Fig. 4B) highlights that GRL501P induced
CAT activity 4-fold above the level induced by C/EBPß, but had only a
minimal effect on the transcription induced by C/EBP and
C/EBP.
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).
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, lanes 1 and 4–6). Expression of the GAL-LBD construct in the presence
of dex induced CAT activity approximately 8-fold above the level
obtained with GalO (lane 2 Fig. 5 B, lane 1). Coexpression of C/EBPß
with GAL-LBD increased the induction of transcription in response to
dex treatment a further 4-fold (Fig. 5B, lane 3). By contrast,
coexpression of C/EBP or resulted in no significant additional
transcriptional activation above the level induced by GAL-LBD alone
(lanes 2 and 4). Therefore, while the potentiation of transcription
again appeared to be a specific property of the GR LBD and C/EBPß,
which partner was tethered to DNA appeared to be unimportant.
The inability of RU486-treated GR to potentiate the activation of tk
transcription (Fig. 1) suggested that the GR-C/EBPß interaction could
be linked to AF-2 function of GR, which is unresponsive to RU486.
Deletion of 14 amino acids from the C-terminal end of GR inactivates
the AF-2 function, with a decrease in ligand-binding affinity that can
be compensated for by treatment with pharmacological concentrations of
hormone (66). To determine whether the AF-2 function of the GR LBD was
required for C/EBPß to potentiate GAL-LBD-mediated E1B transcription,
we repeated our experiment with GAL-LBD781 (Fig. 5). As
expected, GAL-LBD781 was ineffective in activating E1B
transcription (Fig. 5A, lane 3), and no additional activity was
observed upon coexpression of C/EBP, or (Fig. 5B, lanes 6–8).
However, in this instance, coexpression of C/EBPß also failed to
increase reporter gene transcription. Western blotting, shown in Fig. 5C, demonstrated that GAL-LBD and GAL-LBD781 were expressed
at similar levels. Thus, our results indicate that functional
interaction between the GR LBD and C/EBPß was dependent on the
integrity of the GR AF-2.
The Potentiation of C/EBPß-Mediated Transcriptional Activation
Occurs Independent of Binding to GR
To investigate whether the functional interactions observed
between the GR LBD and C/EBPß might correlate with protein-protein
interactions between the two factors, we tested the ability of in
vitro translated GR peptides to bind to C/EBP and C/EBPß
expressed as GST fusion proteins. The results of this experiment are
displayed in Fig. 6. Contrary to
expectations, full-length, dex-treated GR bound strongly to both
C/EBPs, not just C/EBPß (lanes 1 and 8). Deletion of the N terminus
of GR up to the DBD (X795) had no effect on binding (lanes 2 and 9),
nor did deletion of AF-2 (X781, lanes 3 and 10). Similarly, both C/EBPs
were bound by a GR peptide containing amino acids 1–523 (lanes 6 and
13), a fragment of GR that was unable to potentiate C/EBP activity in
transfection experiments (Fig. 2). By contrast, the LBD of GR (547C),
which was sufficient for the potentiation of C/EBPß activity in
transfection experiments, failed to bind either C/EBP (lanes 5 and 12).
Finally, a GR peptide containing only the DBD (X616) retained full
C/EBP-binding activity in this assay (lanes 4 and 11).
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was investigated further by
incubating in vitro translated C/EBPs with GR
immunoprecipitated from fibroblasts expressing a stably transfected WT
GR with an N-terminal C-myc antibody tag (Fig. 7). To investigate the hormonal
requirements for binding, GRs were prepared by salt extraction from
cells treated with dex or RU486 and from untreated cells. In this
experiment, both in vitro translated C/EBPß (Fig. 7 A) and
C/EBP (Fig. 7 B) were coimmunoprecipitated to approximately the same
extent from all three extracts prepared from GR-positive cells (lanes
5–7), while little binding was detected in extracts prepared from
control cells lacking GR (lanes 2–4). In further contrast to the
transcription results, C/EBP binding was the same for RU486-treated GRs
and GRs transformed by heat and salt, as it was for dex-treated
receptors.
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(ER) and retinoic acid receptor-
(RAR),
have been reported to interact functionally with C/EBPß in an
AF-2-dependent manner (17, 60). However, in contrast to the inductive
effects of GR, both ER and RAR were observed to repress
C/EBPß-mediated transcription. Further, peptides including the DBD of
ER have previously been shown to bind to C/EBPß in
vitro (60). As GR bound C/EBPß in an apparently similar manner,
we wondered whether C/EBP binding was a conserved property of
steroid/retinoid receptors. Our results, displayed in Fig. 8, indicate that ER, AR, and retinoic
acid receptor ß (RARß) also bound to both C/EBP and ß in a GST
pull-down assay. The same result was also obtained with RAR (data
not shown). By contrast, an unrelated transcription factor, nuclear
factor 1, and firefly luciferase did not interact with the
C/EBPs. Thus, it appears that the ability to bind C/EBPs is a property
of several nuclear hormone receptors and may be important for the
repression of C/EBPß-activated transcription by ER, RAR, and
potentially AR, but is dispensable for the potentiation of
C/EBPß-activated transcription by GR. However, the functional
significance of this potential for direct binding between
steroid/retinoid receptors and the three C/EBP isoforms remains to be
completely elucidated.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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nor C/EBP interacted
productively with GR in our experiments. To date, three main mechanisms for the regulation of gene transcription by glucocorticoids have been established: direct activation through GREs; direct repression through negative GREs; and transcriptional interference resulting from the direct interaction of GR with other sequence-specific transcription factors (29, 70, 71). Our results suggest a fourth mechanism, transcriptional cooperativity mediated indirectly through an interaction between GR and the transcriptional machinery downstream from the binding of C/EBPß to DNA.
The activation of transcription by GR and C/EBPß, like most transcription factors, is mediated through interactions with transcriptional coactivator molecules, proteins with histone acetyltransferase activity that do not bind DNA themselves, but function as bridging molecules between sequence-specific transcription factors and the basal transcriptional machinery (72, 73). Recently, it has become apparent that many of these coactivator molecules exist in larger coregulatory complexes that include several different coactivator molecules (74). For example, p300/CBP occurs in complexes with SRC-1, p/CAF, GRIP-1, and potentially other factors (75).
The activation functions of many sequence-specific transcription
factors bind directly to a variety of sites on individual coactivators
(76). Recently, it has been demonstrated that nuclear receptors and
other transcription factors exhibit different requirements for
coactivators within a coactivator complex, and it has been suggested
that coactivator complexes exist in multiple alternative configurations
(74). Thus, liganded nuclear receptors, including GR, RAR, and ER,
interact with p/CAF and SRC-1, while C/EBPß and CREB interact with
p300/CBP (45, 49, 52, 74, 77, 78).
Two schemes to explain how the functional interaction between GR and
C/EBPß observed in our experiments might take place in the absence of
DNA binding by GR are presented in Fig. 9. As the reciprocal potentiation of
transcriptional activation of GR and C/EBPß in our experiments was
mediated indirectly, and both factors interact differently with
coactivators that occur in the same complex, it is plausible that the
functional interaction between GR and C/EBPß occurred at the level of
the coactivator complex (Fig. 9, panel 1). In this scenario, in
response to dex treatment, liganded GR interacting at a second site on
the C/EBPß-coactivator complex would enhance the ability of the
coactivators to activate transcription. The effect on transcriptional
activation could be mediated allosterically or by inducing changes in
the composition of the complex. Certainly, the feasibility for the
formation of such a complex has been established (74, 79). It should
also be noted that this model also suggests a mechanism for
transcriptional synergism when both GR and C/EBPß are bound to DNA.
In this instance, the binding of GR and C/EBPß to DNA might be
expected to further stabilize the recruitment of the larger regulatory
complex.
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, panel 2) would be that GR interacts with
a molecule [X] that acts negatively downstream from the
C/EBPß-coactivator complex to decrease transcriptional activation. In
this instance, GR would remove or titrate a block on the communication
of the C/EBPß-coactivator complex with basal transcription factors.
This possibility seems less likely as there are presently no potential
candidates for this activity. Interestingly, one potential compromise
between these two possibilities is that the binding of GR to the
coactivator complex could relieve a repressive activity that occurs
directly within the coactivator complex.
Our results clearly dissociated the binding of GR to C/EBPß from the
potentiation of C/EBPß-mediated transcription. Indeed, the minimum GR
fragment required for the potentiation of transcription activated by
C/EBPß was the only GR fragment tested in binding assays that failed
to bind C/EBPß. Further GR also bound to C/EBP and C/EBP, but
had no effect on the activation of transcription by these factors under
our experimental conditions. These results clearly sever the previously
proposed linkage between GR-C/EBPß binding and the potentiation of
C/EBPß-mediated transcription. However, it remains possible that
GR-C/EBP binding will prove to be biologically relevant in other cell
types or in response to additional signaling molecules not included in
the present study. Alternatively, it is also possible that the binding
observed here for C/EBP, ß, and , and reported previously for
C/EBPß, does not reflect a productive association between these
factors in the cell.
In the present study, we observed that GR-C/EBPß binding in
vitro requires the GR DBD, while a previous study
demonstrated that binding required the bZIP DBD of C/EBPß (80). As
this is the conserved region of the C/EBPs, it would seem probable that
the binding of GR to C/EBP and would also be to the bZIP domain.
Thus, a third possibility would be that the binding of the C/EBPs to
DNA could interfere with the protein-protein interaction with GR. The
DBDs of some factors, including GR, can simultaneously accommodate
protein-protein and protein-DNA interactions (59, 80, 81, 82, 83, 84, 85). By contrast,
we have recently demonstrated that a direct interaction between the GR
DBD and the POU DNA binding domain of transcription factors
Oct-1 and Oct-2 was dissociated by the binding of GR to a GRE (86). For
GR and the octamer factors, the protein-protein interaction is
nonetheless productive, as it serves to promote the binding of the
octamer factors to response elements adjacent to DNA-binding sites for
GR. Thus determining how GR-C/EBP binding responds to the presentation
of GREs and CCAAT elements may suggest how this interaction might occur
productively in the cell.
While GR potentiates the ability of C/EBPß to activate transcription,
there are reports that ER and RAR act to repress the activation of
transcription by C/EBPß (17, 60). In our study, we also observed that
AR repressed the activation of transcription by C/EBPß and that
RARß, ER, and AR bound C/EBPß similarly to GR. For ER and
RAR, repression also required AF-2 (17, 60), which would appear to
suggest a difference in the function of the AF-2 of GR and that of
ER and RAR. For example, it is possible that the differences in
effect reflect differences in the association of GR and ER-RAR with
a common coactivator complex.
For ER, however, the repression of transcription induced by C/EBPß
also was dependent upon the receptor DBD (60). Indeed, we note that,
upon deletion of the DBD, ER reverted from a repressor of
C/EBPß-induced transcription to an activator similar to GR. Thus a
second possibility is that the difference in the effect of ER and GR
on C/EBPß may be explained by differences in the way ER and GR
bind to C/EBPß. A resolution of the molecular basis for the
differences in the interaction of GR, ER, and RAR with C/EBPß will
require a direct comparative study of their individual effects.
GR is required for viability, as mice lacking a GR gene die shortly
after birth from a defect that results in the lack of production of
surfactant proteins in the lung (87). The recent demonstration, that
mutant GRs compromised for DNA-binding and DNA-dependent dimerization
are viable (88), highlights that many important functional activities
of GR are mediated in the absence of direct contact of the receptor
with DNA. The most intensively investigated DNA-independent effects of
GR have been in the interference with the activities of NF
B and AP1.
Our results suggest that potentiation of the transactivation potential
of C/EBPß may be another important way in which GR may exert
physiological effects in the absence of DNA binding. Functional
interaction between GR and C/EBPß is most obvious in their effects on
inflammation and in the differentiation of preadipocytes. It will be
interesting to determine to what degree the effects of glucocorticoids
on these processes are dependent on the interaction between GR and
C/EBPß reported here.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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, ß, and
have been previously described (9). The rat AR was expressed from pSV40
AR (92). The HSV-tk CAT reporter vector was essentially that previously
described containing HSV-tk sequences -109/+51 (93). Adenovirus E1B
reporter constructs were prepared by cloning into the XbaI
site at -45 adjacent to the minimal E1B promoter of pG5E1BCAT
(94). Four copies of C/EBP (5'-CTA GGA GTG TCA TTG GCG
AGG-3') binding sites, octamer motifs (5'-AGGAGC TTG CTT ATG CAA
ATA AGG TG-3'), and Sp1 (5'-CTA GCG ACC CCG CCC AGC
GTG-3') binding sites were cloned into pG5E1BCAT to generate
p4Sp1G5E1BCAT, p4C/EBPG5E1BCAT, and p4OctG5E1BCAT reporter
plasmids. PCR amplification was used to clone the -109/-29
promoter-proximal regulatory region of HSV-tk adjacent to the E1B
promoter at -45 in pG5E1BCAT to generate pG5tkE1BCAT. Mutagenesis of
the C/EBPß response element in the tk promoter was performed by
changing the sequence of the CCAAT element at -88 to -90
-GAGTCGGACA-80 (65), by performing PCR amplification with a mutated
oligo and recloning the amplified product into the
BamHI/RsrII sites of original HSV-tkCAT reporter
gene. All constructs were verified by DNA sequencing. In all
experiments pRSV ß-gal was cotransfected to monitor transfection
efficiency. C/EBPß and C/EBP plasmids for in vitro
translation were created by isolating EcoRI/BamHI
ß and fragments from pMSVC/EBP ß and and recloning into
pGEM-7Z (Promega, Madison, WI). The plasmids used for in vitro translations, GRWT (pRDN93) (95), X795, X781, X616, and 547C (66), have all been described previously. N523 was generated by digesting T7N556 (66) with PstI. The AR (92), ER (96), RARß (97), and nuclear factor 1 (98) vectors have been described previously. pSP6Luciferase was from Promega. The pTL-MTG GR expression vector has been described previously (86).
In initial experiments, and for all plasmids not used as reporters in
transient transfections, DNA was prepared from E.
coli-DH5. Reporter plasmids were prepared from E.
coli Rb404 (strain) to preclude the presence of cryptic GRE
resulting from bacterial methylation (61).
Transient Transfection Analyses
Cos 7 cells were maintained in DMEM containing 10% FBS at 37 C.
Sixteen hours before transfection, 2 x 105 cells were
seeded onto 35-mm plates. Transfections were performed using
Lipofectamine (Life Technologies, Gaithersburg, MD; 5 µl per 35-mm
plate). Each transfection was performed using 0.3 µg CAT reporter
plasmid, 0.3 µg ß-galactosidase reporter, and, as indicated, 0.6
µg steroid receptor expression plasmids and 0.3 µg C/EBP expression
plasmids. Sixteen hours posttransfection, the medium was replaced with
DMEM-10% FBS supplemented with steroidal ligands or ethanol alone as
described in individual experiments. Dex (Steraloids, Wilton,
NH) was added to 0.2 µM, RU38486 (RU486) was
added to 1.0 µM, and DHT (Steraloids) was added
to 0.05 µM. In selected transfections the lower
concentration of 33 nM dex was used with similar results.
Cells were then allowed to grow for an additional 48 h.
ß-Galactosidase (used to normalize results for variations in transfection efficiency) and CAT assays were performed essentially as previously described (86). Conversion of acetylated chloramphenicol was quantified using phosphorimager analysis (Bio-Rad, Richmond, CA). CAT activity was corrected for ß-activity. Each data point represents the average of a minimum of three independent experiments each performed in duplicate. All error bars represent the SEM.
GR-C/EBP Binding Assays
GST fusion proteins were prepared and purified on glutathione
Sepharose essentially as described (23). 35S-labeled
proteins were produced using the Coupled Transcription-Translation TNT
Reticulocyte Lysate System (Promega). Steroid binding to in
vitro translated receptors was done by adding 1 µM
all-trans-retinoic acid to RAR, DHT to AR,
diethlystilbestrol (DES) to ER, and dex to GR for 2 h at 4 C. For
GST-binding assays, 35S-labeled proteins were incubated
with 0.5 µg immobilized GST-fusion protein in 200 µl binding buffer
[(15 mM HEPES, pH 7.9, 60 mM KCl, 12%
glycerol, 1 mM EDTA, 1 mM dithiothreitol, 0.1
mM phenylmethylsulfonyl fluoride] for 90 min at 4 C and
washed three times. The proteins retained on the affinity matrix were
eluted in SDS sample buffer, resolved by SDS-PAGE, and visualized by
autoradiography. A fraction representing 10% of the in
vitro translated proteins added to the binding assay was loaded on
identical gels and exposed together with the gels containing the bound
fractions.
For immunoprecipitation assays, myc-tagged GR was immunoprecipitated from cellular extracts of Sf-7 murine fibroblasts stably transfected with a vector expressing WT GR with an N-terminal myc tag (86). The protein A-Sepharose beads complex was preblocked in 150 µl binding buffer and 5 µl rabbit reticulocyte lysate at 4 C for 2 min. Samples were then centrifuged at 4000 rpm (4 C), after which the precipitate was resuspended in another 150 µl binding buffer. Equivalent amounts (as determined by phosphoimage analysis) of the desired in vitro translated C/EBP isoform were added and allowed to bind to the affinity-purified GR for 2 h at 4 C. Samples were washed three times with 500 µl binding buffer. After the washes, samples were resuspended in 20 µl SDS sample buffer, boiled for 5 min, and run on SDS-PAGE. The gel was then dried and bands were quantified by phosphorimager analysis. Binding to immunoprecipitates from MTG GR-containing extracts was compared with that of the parental Sf-7 cells, which do not express MTG GR. MTG GR loading in each experiment was confirmed by Western immunoblotting.
Western blotting for GR was done as previously described (99). After SDS-PAGE, protein samples were electroblotted from the SDS-PAGE gel to a polyvinylidene fluoride membrane. The primary antibody used was anti-myc antibody, 9E10 (1:2000 dilution). Detection of 9E10 signal was done by enhanced chemiluminescence (ECL, Amersham, Arlington Heights, IL) using horseradish peroxidase-conjugated sheep antimouse antibody (1:50,000 dilution) (Amersham), as the secondary antibody. Expression levels for the pGALO constructs were verified by Western blotting with the an anti-GAL4 antibody (Santa Cruz Biotechnology, Santa Cruz, CA).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by operating funds from the Medical Research Council of Canada (to R.J.G.H.). R.J.G.H. is a Scholar of the Medical Research Council of Canada and the Cancer Research Society, Inc.
1 M. Boruk and J. Savory contributed equally to this work and should be
considered co-first authors. 
Received for publication December 31, 1997. Revision received July 1, 1998. Accepted for publication July 23, 1998.
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