AF-2-Dependent Potentiation of CCAAT Enhancer Binding Protein ß-Mediated Transcriptional Activation by Glucocorticoid Receptor
Marcin Boruk1,
Joanne G. A. Savory1 and
Robert J. G. Haché
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
 |
ABSTRACT
|
---|
We report glucocorticoid-dependent induction of
transcription from the herpes simplex virus thymidine kinase gene
promoter proximal regulatory region in the absence of glucocorticoid
response elements and independent of the ability of
glucocorticoid receptor (GR) to bind DNA. Examination of the thymidine
kinase promoter localized glucocorticoid responsiveness to a binding
site for CCAAT enhancer-binding proteins (C/EBPs). Further analysis
indicated that GR specifically potentiated the induction of
transcription by C/EBPß, but not C/EBP
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
|
---|
Transcription of the herpes simplex virus thymidine kinase
(HSV-tk) promoter (-109/+51) is regulated by the synergistic
interaction of CCAAT enhancer binding proteins (C/EBPs) and Sp1 (1, 2, 3, 4).
Sp1 is a ubiquitous transcription factor with a zinc-fingered
DNA-binding region that activates transcription of many mammalian genes
but is of particular importance for the transcription of constitutively
expressed structural genes lacking a TATA box (5).
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.
 |
RESULTS
|
---|
The HSV Promoter Is Activated by Glucocorticoids in the Absence of
the Binding of GR to DNA
During the course of experiments examining the mechanism of
transcriptional regulation by GR in Cos7 cells, we observed that the
-109/+51 sequence from the HSV-tk promoter was strongly inducible by
dex in the presence of coexpressed GR (data not shown). This was
unexpected because, although this region of the tk promoter contains
two Sp1 DNA-binding sites and one C/EBP-binding site, it does not
contain a discernible GRE (Fig. 1A
) (3, 4).

View larger version (19K):
[in this window]
[in a new window]
|
Figure 1. Activation of HSV-tk Transcription by
Glucocorticoids
A, Schematic of the HSV-tk promoter proximal (-109 to +51 bp)
regulatory region linked to a CAT reporter gene. This portion of HSV-tk
contains two GC-rich Sp1 DNA binding sites at -105 and -56 and a
CCAAT DNA response element at position -87. B, Cos 7 cells were
cotransfected with the HSV-tk CAT reporter construct and GR or AR
expression plasmids as indicated. Ligand treatments were 0.2
µM dex, 1.0 µM RU 38486, or 0.05
µM DHT in ethanol as indicated. Relative CAT activity is
expressed as fold induction over cells treated with ethanol alone and
is corrected for variations in transfection efficiency. SD
values were calculated from three independent experiments each
performed in duplicate.
|
|
It has been demonstrated previously that N6 methylation of
adenine residues as a result of dam methylation of plasmids
grown in dam + strains of Escherichia
coli can lead to the artefactual creation of cryptic GREs (61).
Therefore, to determine whether dam methylation had resulted
in the creation of a cryptic GRE on our HSV-tk reporter plasmid, we
repeated the transfections with a plasmid prepared in the
dam- dcm- Rb404 E.
coli strain (Fig. 1B
). 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.

View larger version (21K):
[in this window]
[in a new window]
|
Figure 2. The Ligand Binding Domain of GR Is Sufficient for
the Activation HSV- tk Transcription by dex
Cos 7 cells were cotransfected with HSV-tk CAT and expression
constructs for the mutant GRs whose structures and properties are
summarized at the top of the figure. Below, CAT activity
is presented as fold induction of activity in GR expressing cells over
cells transfected with the tk reporter gene alone. For lanes 1, 2, and
4, cells were treated with 0.2 µM dex. Error bars
represent the SD obtained from three independent
experiments performed in duplicate. For lanes 1 and 2, similar effects
were obtained with 33 nM dex (data not shown).
|
|
GR Activates Transcription from the tk CCAAT Element
The tk promoter used in these experiments contains binding sites
for both Sp1 and C/EBP (3). To determine whether the potentiation of tk
transcription by GR was mediated through one of these sequences, we
prepared two CAT constructs with a minimal adenovirus E1B minimal
promoter and four copies of an Sp1-binding site or the C/EBP response
element (Fig. 3
, 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.

View larger version (20K):
[in this window]
[in a new window]
|
Figure 3. GR Potentiates Transcription through a CCAAT
Response Element
Cos7 cells were transfected with a vector expressing
GRL501P and one of four E1B CAT reporter constructs or a
construct with the tk promoter from -109 to +51 with an inactivating
mutation in the C/EBP binding site. The structure of these constructs
is summarized at the top. The fold induction of CAT
activity in response to treatment of the transfected cells with 0.2
µM dex is shown at the bottom. Similar
effects were observed at 33 nM dex.
|
|
Interestingly, when placed adjacent to the E1B promoter, the
promoter-proximal tk-regulatory region was only weakly responsive to
GRL501P and dex, with CAT activity being induced just under
2-fold (lane 4). One possibility suggested by this result was that the
minimal tk promoter was somehow also making an important contribution
to the GR responsiveness. However, recloning the tk sequences into the
E1B promoter also resulted in an increase in spacing of 16 bp between
the DNA response elements in the tk region and the TATA box element.
Thus it is also possible that the decreased response was due to this
change in relative positioning of the tk response elements and the TATA
box, which is equivalent to 1.5 turns of the DNA helix.
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
.
Functional Interaction between GR and C/EBPß Requires the
C-Terminal AF-2 Activity of GR
Our results indicated that the LBD of GR contained a
hormone-dependent ability to potentiate the activation of transcription
by C/EBPß, in the absence of GR binding to DNA. One question raised
by these results was whether C/EBPß could reciprocally potentiate the
activation of transcription of DNA-bound GR in the absence of CCAAT
response elements. To address this question, we tested the ability of
the C/EBPs to potentiate the activation of transcription by a GR LBD
construct fused to the yeast GAL4 DBD. This experiment was performed
using a reporter gene with 5 GAL4-binding sites driving transcription
from the E1B promoter (Fig. 5
).

View larger version (19K):
[in this window]
[in a new window]
|
Figure 5. Interaction of the GR LBD with C/EBPß in
a Mammalian Two-Hybrid Assay Is Dependent on AF-2
Cos 7 cells were cotransfected with a G5E1BCAT reporter plasmid,
GALGRLBD, and GALGRLBD781 fusion protein expression
plasmids and C/EBP expression plasmids as indicated to assess the
ability of C/EBPs to potentiate the activation of transcription by the
LBD of GR. Data are expressed relative to expression of the GAL4 DBD
alone (A) or as fold induction in the presence of GAL-LBD fusion
protein vs. the GAL4 DBD (B). All experiments were
performed in the presence of 0.2 µM dex, which is well
above the concentration necessary to saturate GAL-LBD781.
In panel C, the expression of levels of GAL-LBD and
GAL-LBD781 were compared by Western analysis of whole-cell
extracts probed with GAL4 antibody (Santa Cruz).
|
|
Cotransfection of the C/EBP expression plasmids with pGalO, a plasmid
expressing GAL4 DBD alone, had no effect on E1B expression (Fig. 5A
, lanes 1 and 46). 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 68).
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 1523 (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).

View larger version (70K):
[in this window]
[in a new window]
|
Figure 6. The DBD, but not the LBD, of GR Binds Specifically
to GST-C/EBP and GST-C/EBPß
In vitro translated GR fragments or firefly luciferase
were incubated with GST-C/EBP (A), GST-C/EBPß (B), or GST alone
(C). Specifically bound proteins were resolved on 10% SDS-PAGE gels.
In panel D, 10% of the in vitro translated GRs were
added to the binding assay. All GRs except X616 and N523 were activated
by preincubation with 0.2 µM dex before binding. The GR
peptides contained the following amino acid sequences of GR: GR,
1795; X795, 407795; X781, 407781; X616, 407616; 547C, 547795;
N523, 1523.
|
|
The binding of GR to C/EBPß and
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
57), while little binding was detected in extracts prepared from
control cells lacking GR (lanes 24). 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.

View larger version (38K):
[in this window]
[in a new window]
|
Figure 7. GR Binds Specifically to C/EBPß and C/EBP in
an Immunoprecipitation Assay
A and B, Whole-cell extracts prepared from Sf7 cells with (lanes 57)
or without (lanes 24) stably transfected myc-tagged GR treated for 15
min with 0.2 µM dex (lanes 2 and 5), RU486 (lanes 3 and
6) or 0.4 M KCl (lanes 4 and 7) to transform GR in the
absence of ligand were incubated with in vitro
translated C/EBPß (A) or C/EBP (B). Binding was detected on 15%
SDS-PAGE gels. C, Western blot illustrating the loading of the GRs used
in panel A.
|
|
C/EBP Binding Is a Conserved Property of Steroid/Retinoid
Receptors
Two other nuclear hormone receptors in addition to GR,
estrogen receptor-
(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.

View larger version (62K):
[in this window]
[in a new window]
|
Figure 8. Nuclear Receptors, but Not NF-1, Bind Specifically
to GST-C/EBP and GST-C/EBPß
In vitro translated, 35S-labeled proteins
were incubated with GST-C/EBP (A), GST-C/EBPß (B), or GST alone
(C). Specifically bound proteins were resolved by 10% SDS-PAGE gels.
Panel D shows 10% of the proteins added to the binding assay. All gels
were exposed equally. Before binding, the nuclear receptors were
pretreated with specific ligands as described in Materials and
Methods.
|
|
 |
DISCUSSION
|
---|
Glucocorticoids and C/EBPß converge in the regulation of a large
variety of cellular processes, including responses to inflammation and
stress, and in tissue differentiation, e.g. differentiation
of preadipocytes to mature adipocytes. It is well established that
glucocorticoids and C/EBPß interact cooperatively in the regulation
of the transcription of many of the genes whose induction contributes
to these processes (67, 68, 69). The results presented in this work suggest
that cooperative interactions between C/EBPß and GR in the activation
of specific gene transcription need not be dependent upon the close
proximity of DNA-binding sites for each factor or upon direct
protein-protein interactions between the two factors. Rather, they
suggest that GR can potentiate the transcriptional regulatory potential
of C/EBPß indirectly in a manner that is independent of DNA binding
by GR. Indeed, this effect appeared to be dependent solely upon the
receptor LBD. Moreover, the ability of C/EBPß to potentiate AF-2
dependent transcriptional activation by the DNA-bound GR LBD suggests
that this process may be effective from both GREs and CCAAT-response
elements. Interestingly, however, the transcriptional effects were
specific for C/EBPß, as neither C/EBP
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.

View larger version (43K):
[in this window]
[in a new window]
|
Figure 9. Schematic Presentation of Possible Ways in Which
the GR AF-2 Function Could Stimulate the Activation of Transcription by
C/EBPß
C/EBPß is known to interact with p300/CBP, transcriptional
coactivator molecules that occur within larger transcriptional
coactivator complexes that include molecules such as SRC-1, p/CAF, and
GRIP-1. SRC-1 and potentially other molecules in the complex are also
direct targets of the AF-2 domain of GR. In the first scenario (panel
1), upon ligand binding, GR enters into the coactivator complex that is
associated with C/EBPß and acts to stimulate the activity of this
complex. Possible mechanisms for this effect are discussed in the text.
In a second scenario (panel 2), the GR AF-2 domain interacts with a
factor (X) still unknown, but that functions in the context of the
coactivator complex, or downstream from the complex, to decrease the
efficiency of C/EBPß-mediated transcriptional activation.
|
|
A second possibility (Fig. 9
, 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.
 |
MATERIALS AND METHODS
|
---|
Plasmids
The rat GR eukaryotic expression constructs, p6RGR (89),
p6RGRN525 (64), and p547C (90), have been previously described. Other
constructs were derived from these vectors as described below.
p6RGRL501P was created by site- directed mutagenesis of a p6RGR DNA
fragment. pGaLLBD and pGaLLBD781 were created by PCR
amplification of GR LBD fragments encoding amino acids 540795 and
540781, respectively, into the SalI-XbaI sites
of pGalO (91). C/EBP expression vectors pMSV C/EBP
, 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).
 |
ACKNOWLEDGMENTS
|
---|
We would like to thank Drs. K. Yamamoto, S. Liao, G.
Tomaselli, and W. H. Lee for generously providing us with
plasmid DNAs. The pMSVC/EBP constructs were graciously provided by
Tularik (San Francisco, CA). We are also grateful to our
colleagues, Y. Lefebvre, G. Préfontaine, and C.
Schild-Poulter, for their critical commentary on the manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Robert J. G. Haché, Ottawa Civic Hospital Loeb Research Institute, 1053 Carling Avenue, Ottawa, Ontario, Canada K1Y 4E9. E-mail: rhache{at}lri.ca
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.
 |
REFERENCES
|
---|
-
Osada S, Yamamoto H, Nishihara T, Imagawa M 1996 DNA
binding specificity of the CCAAT/enhancer-binding protein transcription
factor family. J Biol Chem 271:38913896[Abstract/Free Full Text]
-
Seo SJ, Kim HT, Cho G, Rho HM, Jung G 1996 Sp1 and
C/EBP-related factor regulate the transcription of human Cu/Zn SOD
gene. Gene 178:177185[CrossRef][Medline]
-
Graves BJ, Johnson PF, McKnight SL 1986 Homologous
recognition of a promoter domain common to the MSV LTR and the HSV tk.
Cell 44:565576[Medline]
-
McKnight SL, Kingsbury R 1982 Transcriptional control signals
of eukaryotic protein-coding gene. Science 217:316324[Medline]
-
Azizkhan JC, Jensen DE, Pierce AJ, Wade M 1993 Transcription
from TATA-less promoters: dihydrofolate reductase as a model. Crit Rev
Eukaryot Gene Expr 3:229254[Medline]
-
Piccolo S, Marigo V, Girotto D, Volpin D, Bressan GM 1995 Identification of a recognition element for CAAT-enhancer binding
proteins (C/EBPs) in the elastin promoter. Biochim Biophys Acta 1264:4044[Medline]
-
Landschulz WH, Johnson PF, Adashi EY, Graves BJ, McKnight SL 1988 Isolation of a recombinant copy of the gene encoding C/EBP. Genes
Dev 2:786800[Abstract]
-
Landschulz WH, Johnson PF, McKnight SL 1988 The leucine
zipper: a hypothetical structure common to a new class of DNA binding
proteins. Science 240:17591764[Medline]
-
Cao Z, Umek RM, McKnight SL 1991 Regulated expression of
three C/EBP isoforms during adipose conversion of 3T3L1 cells. Genes
Dev 5:15381551[Abstract]
-
Akira S, Isshiki H, Sugita T, Tanabe O, Kinoshita S, Hirano T,
Kishimoto T 1990 A nuclear factor for Il-6 expression (NF-IL6) is a
member of a C/EBP family. EMBO J 9:18971906[Abstract]
-
Chumakov AM, Grillier I, Chumakova E, Chih D, Slater J,
Koeffler HP 1997 Cloning of the novel human myeloid-cell-specific
C/EBP
transcription factor. Mol Cell Biol 17:13751386[Abstract]
-
Descombes P, Chojkier M, Lichtseiner S, Falvey E, Schibler U 1990 LAP, a novel member of the C/EBP gene family, encodes a
liver-enriched transcriptional activator protein. Genes Dev 4:15411551[Abstract]
-
Muller C, Kowenz-Leutz E, Grieser-Ade S, Graf T, Leutz A 1995 NF-M (chicken C/EBPß) induces eosinophilic differentiation and
apoptosis in a hematopoietic progenitor cell line. EMBO J 14:61276135[Abstract]
-
Diehl AM, Johns DC, Yang S, Lin H, Yin M, Matelis LA, Lawrence
JH 1996 Adenovirus-mediated transfer of CCAAT/enhancer-binding
protein-
identifies a dominant antiproliferative role for this
isoform in hepatocytes. J Biol Chem 271:73437350[Abstract/Free Full Text]
-
Trautwein C, Rakemann T, Pietrangelo A, Plumpe J, Montosi G,
Manns MP 1996 C/EBP-ß/LAP controls down-regulation of albumin gene
transcription during liver regeneration. J Biol Chem 271:2226222270[Abstract/Free Full Text]
-
Soriano HE, Bilyeu TA, Juan TS, Zhao W, Darlington GJ 1995 DNA
binding by C/EBP proteins correlates with hepatocyte proliferation. In
Vitro Cell Dev Biol 31:703709
-
Schwarz EJ, Reginato MJ, Shao D, Krakow SL, Lazar MA 1997 Retinoic acid blocks adipogenesis by inhibiting C/EBPß-mediated
transcription. Mol Cell Biol 17:15521561[Abstract]
-
Zhang DE, Hetherington CJ, Meyers S, Rhoades KL, Larson CJ,
Chen HM, Hiebert SW, Tenen DG 1996 CCAAT enhancer-binding protein
(C/EBP) and AML1 (CBF
2) synergistically activate the macrophage
colony-stimulating factor receptor promoter. Mol Cell Biol 16:12311240[Abstract]
-
Screpanti I, Romani L, Musiani P, Modesti A, Fattori E,
Lazzaro D, Sellitto C, Scarpa S, Bellavia D, Lattanzio G, Bistoni F,
Frati L, Cortese R, Gulino A, Ciliberto G, Constantini F, Poli V 1995 Lymphoproliferative disorder and imbalanced T-helper response in
C/EBP ß-deficient mice. EMBO J 14:19321941[Abstract]
-
Mandrup S, Lane MD 1997 Regulating adipogenesis. J Biol
Chem 272:53675370[Free Full Text]
-
Lin FT, Lane MD 1994 CCAAT/enhancer binding protein alpha
is sufficient to initiate the 3T3L1 adipocyte differentiation
program. Proc Natl Acad Sci USA 91:87578761[Abstract]
-
Chen X, Liu W, Ambrosino C, Ruocco MR, Poli V, Romani L,
Quinto I, Barbieri S, Holmes KL, Venuta S, Scala G 1997 Impaired
generation of bone marrow B lymphocytes in mice deficient in C/EBPß.
Blood 90:156164[Abstract/Free Full Text]
-
Chen PL, Riley DJ, Chen Y, Lee WH 1996 Retinoblastoma protein
positively regulates terminal adipocyte differentiation through direct
interaction with C/EBPs. Genes Dev 10:27942804[Abstract]
-
Brun RP, Kim JB, Hu E, Altiok S, Spiegelman BM 1996 Adipocyte
differentiation: a transcriptional regulatory cascade. Curr Opin Cell
Biol 8:826832[CrossRef][Medline]
-
Lee Y-H, Williams SC, Baer M, Sterneck E, Gonzalez FJ, Johnson
PF 1997 The ability of C/EBPß but not C/EBP
to synergize with an
Sp1 protein is specified by the leucine zipper and activation domain.
Mol Cell Biol 12:20382047
-
Sawiris GP, Sydiskis RJ, Bashirelahi N 1994 Hormonal
modulation of herpes simplex virus replication in a mouse neuroblastoma
cell line. J Clin Lab Anal 8:135139[Medline]
-
Beato M 1989 Gene regulation by steroid hormones. Cell 56:335344[Medline]
-
Strahle U, Klock G, Schütz G 1987 A DNA sequence of 15
base pairs is sufficient to mediate both glucocorticoid and
progesterone induction of gene expression. Proc Natl Acad Sci USA 84:78717875[Abstract]
-
Beato M, Herrlich P, Schutz G 1995 Steroid receptors: many
actors in search of a plot. Cell 83:851857[Medline]
-
Laudet V, Hanni C, Coll J, Catzeflis F, Stehelin D 1992 Evolution the nuclear receptor gene superfamily. EMBO J 11:10031013[Abstract]
-
Mangelsdorf DJ, Evans RM 1995b The RXR heterodimers and orphan
receptors. Cell 83:841850
-
Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schütz
G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P, Evans RM 1995a
The nuclear receptor superfamily: the second decade. Cell 83:835839
-
Thummel CS 1995 From embryogenesis to metamorphosis: the
regulation and function of Drosophila nuclear receptor superfamily
members. Cell 83:871877[Medline]
-
Pratt WB, Toft DO 1997 Steroid receptor interactions with heat
shock protein and immunophilin chaperones. Endocr Rev 18:306360[Abstract/Free Full Text]
-
Abraham SE, Lobo S, Yaciuk P, Wang HG, Moran E 1993 p300, and
p300-associated proteins, are components of TATA-binding protein (TBP)
complexes. Oncogene 8:16391647[Medline]
-
Dallas PB, Yaciuk P, Moran E 1997 Characterization of
monoclonal antibodies raised against p300: both p300 and CBP are
present in intracellular TBP complexes. J Virol 71:17261731[Abstract]
-
McEwan IJ, Wright AP, Dahlman-Wright K, Carlstedt-Duke J,
Gustafsson JA 1993 Direct interaction of the tau 1 transactivation
domain of the human glucocorticoid receptor with the basal
transcriptional machinery. Mol Cell Biol 13:399407[Abstract]
-
Baniahmad A, Leng X, Burris TP, Tsai SY, Tsai MJ, OMalley BW 1995 The tau 4 activation domain of the thyroid hormone receptor is
required for release of a putative corepressor(s) necessary for
transcriptional silencing. Mol Cell Biol 15:7686[Abstract]
-
Baniahmad A, Thormeyer D, Renkawitz R 1997 Tau4/tau c/AF-2 of
the thyroid hormone receptor relieves silencing of the retinoic acid
receptor silencer core independent of both tau4 activation function and
full dissociation of corepressors. Mol Cell Biol 17:42594271[Abstract]
-
Barettino D, Vivanco Ruiz MM, Stunnenberg HG 1994 Characterization of the ligand-dependent transactivation domain of
thyroid hormone receptor. EMBO J 13:30393049[Abstract]
-
Durand B, Saunders M, Gaudon C, Roy B, Losson R, Chambon P 1994 Activation function 2 (AF-2) of retinoic acid receptor and 9-cis
retinoic acid receptor: presence of a conserved autonomous constitutive
activating domain and influence of the nature of the response element
on AF-2 activity. EMBO J 13:53705382[Abstract]
-
Leng X, Blanco J, Tsai SY, Ozato K, OMalley BW, Tsai MJ 1995 Mouse retinoid X receptor contains a separable ligand-binding domain
and transactivation domain in its E region. Mol Cell Biol 15:255263[Abstract]
-
Schulman IG, Juguilon H, Evans RM 1996 Activation and
repression by nuclear hormone receptors: hormone modulates an
equilibrium between active and repressive states. Mol Cell Biol 16:38073813[Abstract]
-
Onate SA, Tsai SY, Tsai MJ, OMalley BW 1995 Sequence
and characterization of a coactivator for the steroid hormone receptor
superfamily. Science 270:13541357[Abstract]
-
Kamei Y, Xu L, Heinzel T, Torchia J, Kurokawa R, Gloss B, Lin
SC, Heyman RA, Rose DW, Glass CK, Rosenfeld MG 1996 A CBP integrator
complex mediates transcriptional activation and AP-1 inhibition by
nuclear receptors. Cell 85:403414[Medline]
-
Nordheim A 1994 CREB takes CBP to tango. Nature 370:177178[CrossRef][Medline]
-
Smith CL, Onate SA, Tsai MJ, OMalley BW 1996 CREB binding
protein acts synergistically with steroid receptor coactivator-1 to
enhance steroid receptor-dependent transcription. Proc Natl Acad Sci
USA 93:88848888[Abstract/Free Full Text]
-
Hong H, Kohli K, Trivedi A, Johnson DL, Stallcup MR 1996 GRIP1, a novel mouse protein that serves as a transcriptional
coactivator in yeast for the hormone binding domains of steroid
receptors. Proc Natl Acad Sci USA 93:49484952[Abstract/Free Full Text]
-
Hong H, Kohli K, Garabedian MJ, Stallcup MR 1997 GRIP1, a
transcriptional coactivator for the AF-2 transactivation domain of
steroid, thyroid, retinoid, and vitamin D receptor. Mol Cell Biol 17:27352744[Abstract]
-
Ogryzko VV, Schiltz RL, Russanova V, Howard BH, Nakatani
Y 1996 The transcriptional coactivators p300 and CBP are histone
acetyltransferases. Cell 87:953959[Medline]
-
Spencer TE, Jenster G, Burcin MM, Allis CD, Zhou J, Mizzen CA,
McKenna NJ, Onate SA, Tsai SY, Tsai MJ, OMalley BW 1997 Steroid
receptor coactivator-1 is a histone acetyltransferase. Nature 389:194198[CrossRef][Medline]
-
Chakravarti D, LaMorte VJ, Nelson MC, Nakajima T, Schulman IG,
Juguilon H, Montminy M, Evans RM 1996 Role of CBP/P300 in nuclear
receptor signalling. Nature 383:99103[CrossRef][Medline]
-
Chrivia JC, Kwok RP, Lamb N, Hagiwara M, Goodman MR, Montimy
RH 1993 Phosphorylated CREB binds specifically to the nuclear protein
CBP. Nature 365:855859[CrossRef][Medline]
-
Kwok RPS, Lundblad JR, Chrivia JC, Richards JP, Bachinger
HP, Brennan R, Roberts SGE, Goodman MRGaRH 1994 Nuclear protein CBP is
a coactivator for the transcription factor CREB. Nature 370:223226[CrossRef][Medline]
-
Diamond MI, Miner JN, Yoshinaga SK, Yamamoto KR 1990 c-Jun and
c-Fos levels specify positive or negative glucocorticoid regulation
from a composite GRE. Science 249:12661272[Medline]
-
Heck S, Kulmann M, Gast A, Ponta H, Rahmsdorf HJ, Herrlich P,
Cato AC 1994 A distinct modulating domain in glucocorticoid receptor
monomers in the repression of activity of the transcription factor
AP-1. EMBO J 13:40874095[Abstract]
-
Schüle R, Rangarajan P, Klieweer S, Ransone LJ, Bolado
J, Yang N, Verma IM, Evans RM 1990 Functional antagonism between
oncoprotein c-jun and the glucocorticoid receptor. Cell 62:12171226[Medline]
-
Yang-Yen H-F, Chambard J-C, Sun Y-L, Smeal T, Schmidt TJ,
Drouin J, Karin M 1990 Transcriptional interference between c-Jun and
the glucocorticoid receptor: mutual inhibition of DNA binding due to
direct protein-protein interaction. Cell 62:12051215[Medline]
-
Stocklin E, Wissler M, Gouilleux F, Groner B 1996 Functional
interaction between Stat5 and the glucocorticoid receptor. Nature 383:726728[CrossRef][Medline]
-
Stein B, Yang MX 1995 Repression of the interleukin-6 promoter
by estrogen receptor is mediated by NF-kappa B and C/EBP ß. Mol Cell
Biol 15:49714979[Abstract]
-
Truss M, Bartsch J, Chalepakis G, Beato M 1992 Artificial
steroid hormone response element generated by dam-methylation. Nucleic
Acids Res 20:14831486[Abstract]
-
Lim-Tio S, Keightley M-A, Fuller PJ 1997 Determinants
of specificity of transactivation by the mineralocorticoid of
glucocorticoid receptor. Endocrinology 138:25372543[Abstract/Free Full Text]
-
Schena M, Freedman LP, Yamamoto KR 1989 Mutations in the
glucocorticoid receptor zinc finger region that distinguish
interdigitated DNA binding and transcriptional enhancement activities.
Genes Dev 3:15901601[Abstract]
-
Godowski PJ, Rusconi S, Miesfeld R, Yamamoto KR 1987 Glucocorticoid receptor mutants that are constitutive activators of
transcriptional enhancement. Nature 325:365368[CrossRef][Medline]
-
Strahle U, Schmid W, Schutz G 1988 Synergistic action
of the glucocorticoid receptor with transcription factors. EMBO J 7:33893395[Abstract]
-
Rusconi S, Yamamoto KR 1987 Functional dissection of the
hormone and DNA binding activities of the glucocorticoid receptor. EMBO
J 6:13091315[Abstract]
-
Savoldi G, Fenaroli A, Ferrari F, Rigaud G, Albertini A, Di
Lorenzo D 1997 The glucocorticoid receptor regulates the binding of
C/EPBbeta on the alpha-1-acid glycoprotein promoter in vivo.
DNA Cell Biol 16:14671476[Medline]
-
Renkawitz R, Kaltschmidt C, Leers J, Martin B, Muller M,
Eggert M 1996 Enhancement of nuclear receptor transcriptional
signalling. J Steroid Biochem Mol Biol 56:3945[CrossRef][Medline]
-
Ben-Or S, Okret S 1993 Involvement of a C/EBP-like protein in
the acquisition of responsiveness to glucocorticoid hormones during
chick neural retina development. Mol Cell Biol 13:331340[Abstract]
-
Karin M 1998 New twists in gene regulation by glucocorticoid
receptor: is DNA binding dispensable? Cell 93:487490[Medline]
-
Lefstin JA, Yamamoto KR 1998 Allosteric effects of DNA on
transcriptional regulators. Nature 392:885888[CrossRef][Medline]
-
Glass CK, Rose DW, Rosenfeld MG 1997 Nuclear receptor
coactivators. Curr Opin Cell Biol 9:222232[CrossRef][Medline]
-
Imhof A, Yang XJ, Ogryzko VV, Nakatani Y, Wolffe AP, Ge H 1997 Acetylation of general transcription factors by histone
acetyltransferases. Curr Biol 7:689692[Medline]
-
Korzus E, Torchia J, Rose DW, Xu L, Kurokawa R, McInerney
EM, Mullen TM, Glass CK, Rosenfeld MG 1998 Transcription
factor-specific requirements for coactivators and their
acetyltransferase functions. Science 279:703707[Abstract/Free Full Text]
-
Yang X-J, Ogryzko V, Nishikawa J-i, Howard B, Nakatani Y 1996 A p300/CBP-associated factor that competes with the adenoviral
oncoprotein E1A. Nature 382:319324[CrossRef][Medline]
-
Heery DM, Kalkhoven E, Hoare S, Parker MG 1997 A signature
motif in transcriptional co-activators mediates binding to nuclear
receptors. Nature 387:733736[CrossRef][Medline]
-
Mink S, Haenig B, Klempnauer KH 1997 Interaction and
functional collaboration of p300 and C/EBPß. Mol Cell Biol 17:66096617[Abstract]
-
Voegel JJ, Heine MJS, Zechel C, Chambon P, Gronemeyer H 1996 TIF2, a 160 kDA transcripitonal mediator for the ligand-dependent
activation function AF-2 of nuclear receptors. EMBO J 15:36673675[Abstract]
-
Chen H, Lin RJ, Schiltz RL, Chakravarti D, Nash A, Nagy L,
Privalsky ML, Nakatani Y, Evans RM 1997 Nuclear receptor coactivator
ACTR is a novel histone acetyltransferase and forms a multimeric
activation complex with P/CAF and CBP/p300. Cell 90:569580[Medline]
-
Nishio Y, Isshiki H, Kishimoto T, Akira S 1993 A nuclear
factor for interleukin-6 expression (NF-IL6) and the glucocorticoid
receptor synergistically activate transcription of the rat alpha 1-acid
glycoprotein gene via direct protein-protein interaction. Mol Cell Biol 13:18541862[Abstract]
-
Treisman R, Marais R, Wynne J 1992 Spatial flexibility in
ternary complexes between SRF and its accessory proteins. EMBO J 11:46314640[Abstract]
-
Giffin W, Torrance H, Rodda DJ, Préfontaine GG, Pope L,
Haché RJG 1996 Sequence-specific DNA binding by Ku autoantigen
and its effects on transcription. Nature 380:265268[CrossRef][Medline]
-
Giffin W, Kwast-Welfeld J, Rodda DJ, Préfontaine GG,
Traykova-Andonova M, Zhang Y, Weigel NL, Lefebvre YA, Haché RJ 1997 Sequence-specific DNA binding and transcription factor
phosphorylation by Ku autoantigen/DNA-dependent protein kinase.
Phosphorylation of Ser-527 of the rat glucocorticoid receptor. J
Biol Chem 272:56475658[Abstract/Free Full Text]
-
Chan SK, Jaffe L, Capovilla M, Botas J, Mann RS 1994 The DNA
binding specificity of Ultrabithorax is modulated by cooperative
interactions with extradenticle, another homeoprotein. Cell 78:603615[Medline]
-
Guichet A, Copeland JW, Erdelyi M, Hlousek D, Zavorszky P, Ho
J, Brown S, Percival-Smith A, Krause HM, Ephrussi A 1997 The nuclear
receptor homologue Ftz-F1 and the homeodomain protein Ftz are mutually
dependent cofactors. Nature 385:548552[CrossRef][Medline]
-
Préfontaine GG, Lemieux ME, Giffin W, Schild-Poulter C,
Pope L, LaCasse E, Walker P, Haché RJG 1998 Recruitment of
octamer transcription factors to DNA by glucocorticoid receptor. Mol
Cell Biol 18:34163430[Abstract/Free Full Text]
-
Cole TJ, Blendy JA, Monaghan AP, Krieglstein K, Schmid W,
Aguzzi A, Fantuzzi G, Hummler E, Unsicker K, Schutz G 1995 Targeted
disruption of the glucocorticoid receptor gene blocks adrenergic
chromaffin cell development and severely retards lung maturation. Genes
Dev 9:16081621[Abstract]
-
Reichardt HM, Kaestner KH, Tuckermann J, Kretz O, Wessely O,
Bock R, Gass P, Schmid W, Herrlich P, Angel P, Schutz G 1998 DNA
binding of the glucocorticoid receptor is not essential for survival.
Cell 93:531541[Medline]
-
Pearce D, Yamamoto KR 1993 Mineralocorticoid and
glucocorticoid receptor activities distinguished by nonreceptor factors
at a composite response element. Science 259:11611165[Medline]
-
Picard D, Yamamoto KR 1987 Two signals mediate
hormone-dependent nuclear localization of the glucocorticoid receptor.
EMBO J 6:33333340[Abstract]
-
Sadowski I, Ma J, Trienzenberg S, Ptashne M 1988 GAL4-VP16 is
an unusually potent transcriptional activator. Nature 335:563564[CrossRef][Medline]
-
Chang CS, Kokontis J, Chang CT, Liao SS 1987 Cloning and
sequence analysis of the rat ventral prostate glucocorticoid receptor
cDNA. Nucleic Acids Res 15:9603[Medline]
-
Truss M, Chalepakis G, Slater EP, Mader S, Beato M 1991 Functional interaction of hybrid response elements with wild-type and
mutant steroid hormone receptors. Mol Cell Biol 11:32473258[Medline]
-
Lillie JW, Green MR 1989 Transcription activation by the
adenovirus E1a protein. Nature 338:3944[CrossRef][Medline]
-
Miesfeld R, Rusconi S, Godowski PJ, Maler BA, Okret S,
Wilkstrom A-C, Gustafsson J-A, Yamamoto KR 1986 Genetic complementation
of a glucocorticoid receptor deficiency by expression of cloned
receptor cDNA. Cell 46:389399[Medline]
-
Greene GL, Gilna P, Waterfield M, Baker A, Hort Y, Shine J 1986 Sequence and expression of human estrogen receptor complementary
DNA. Science 231:11501154[Medline]
-
Benbrook D, Lernhardt E, Pfahl M 1988 A new retinoic acid
receptor identified from a hepatocellular carcinoma. Nature 333:669672[CrossRef][Medline]
-
Gander I, Foeckler R, Rogge L, Meisterernst M, Schneider
R, Mertz R, Lottspeich F, Winnacker EL 1988 Purification methods for
the sequence-specific DNA-binding protein nuclear factor I
(NFI)generation of protein sequence information. Biochim Biophys Acta 951:411418[Medline]
-
Towbin H, Staehelin T, Gordon J 1979 Electrophoretic transfer
of proteins from polyacrylamide gels to nitrocellulose sheets:
procedures and some applications. Proc Natl Acad Sci USA 76:43504354[Abstract]