Cooperative Effects of STAT5 (Signal Transducer and Activator of Transcription 5) and C/EBP ß (CCAAT/Enhancer-Binding Protein-ß) on ß-Casein Gene Transcription Are Mediated by the Glucocorticoid Receptor
Shannon L. Wyszomierski and
Jeffrey M. Rosen
Department of Molecular and Cellular Biology Baylor College of
Medicine Houston, Texas 77030-3498
 |
ABSTRACT
|
---|
ß-Casein gene transcription is controlled
primarily by a composite response element (CoRE) that integrates
signaling from the lactogenic hormones, PRL, insulin, and
hydrocortisone, in mammary epithelial cells. This CoRE contains binding
sites for STAT5 (signal transducer and activator of transcription 5)
and C/EBPß (CCAAT/enhancer-binding protein-ß) and several
half-sites for glucocorticoid receptor (GR). To examine how
interactions among these three transcription factors might regulate
ß-casein gene transcription, a COS cell reconstitution system was
employed. Cooperative transactivation was observed when all three
factors were expressed, but unexpectedly was not seen between STAT5 and
C/EBPß in the absence of full-length, transcriptionally active GR.
Cooperativity required the amino-terminal transactivation domain of
C/EBPß, and neither C/EBP
nor C/EBP
was able to substitute for
C/EBPß when cotransfected with STAT5 and GR. Different GR
determinants were needed for transcriptional cooperation between STAT5
and GR as compared with those required for all three transcription
factors. These studies provide some new insights into the mechanisms
responsible for high level, tissue-specific expression conferred by the
ß-casein CoRE.
 |
INTRODUCTION
|
---|
Composite response elements (CoREs) are found in the promoters of
most genes and control spatially and developmentally regulated patterns
of gene transcription (1, 2). CoREs are composed of groups of
transcription factor-binding sites for both positively and negatively
acting transcription factors that integrate signal transduction
pathways. Interestingly, the signal transduction pathways and
downstream transcription factors integrated by CoREs are generally not
individually spatially or developmentally specific. Moreover, the level
of transcription from a CoRE is typically greater than the
transcriptional activity of each transcription factor alone (2).
Although these defining properties, specificity of expression and
enhanced level of transactivation, have been observed repeatedly from
the CoREs in different genes, the mechanisms by which they are
accomplished remain largely undetermined.
The regulation of ß-casein gene transcription is controlled primarily
by a CoRE that integrates signaling from the lactogenic hormones, PRL,
insulin, and hydrocortisone, in mammary epithelial cells (reviewed in
Ref. 3). The individual transcription factors, which bind to and
activate the ß-casein CoRE, have been well characterized in
vitro, and in several cases in vivo analysis has been
performed as well. Signal transducer and activator of transcription
(STAT) 5, glucocorticoid receptor (GR), and CCAATT/enhancer binding
protein-ß (C/EBPß) have been identified as important activators of
transcription (reviewed in Ref. 4 ; schematic representations of the
ß-casein proximal promoter are shown in Refs. 4, 5). The CoRE in
the proximal promoter of the ß-casein gene contains a consensus and a
nonconsensus binding site for STAT5, at least three binding sites for
C/EBP family members, and several half-palindromic binding sites for GR
(half- GREs) (6, 7, 8, 9, 10, 11). These half-GREs are closely interspersed with the
binding sites for the other transcription factors. Although half-GREs
are not the canonical elements known for eliciting GR responses, the
importance of these elements in the ß-casein promoter has been
unequivocally demonstrated by site-directed mutagenesis (7).
Understanding how STATs, GR, and C/EBPs interact with each other and
act in a concerted manner should provide a clearer understanding of how
the ß-casein CoRE conveys high level, mammary-specific gene
expression.
Efforts to understand the mechanism by which transcriptional activity
is enhanced by the ß-casein gene CoRE were initiated by the analysis
of STAT5 and GR interactions using a COS-7 cell reconstitution system.
Direct protein-protein interactions of STAT5 and GR were demonstrated,
resulting in transcriptional synergy at the ß-casein promoter (12).
Two STAT5 proteins, STAT5a and STAT5b, which are encoded by different
genes (13), are both capable of transcriptional synergy with GR (14)
and are associated with GR in the mammary epithelium and HC11
mammary epithelial cells (15).
The C/EBPs are a family of transcription factors that contain an
amino-terminal transactivation domain, which differs among family
members, and a carboxy-terminal basic leucine zipper domain (bZIP)
responsible for dimerization and DNA binding, which is more highly
conserved among family members (16, 17). Multiple C/EBP isoforms can be
generated from the intronless genes, which encode several different
C/EBPs by either differential translation start site utilization (18)
or selective proteolysis (19). For example, from a single C/EBPß
mRNA, at least three transcripts can be translated, two activating
isoforms called LAPs (originally identified as liver-enriched
activating proteins) and one dominant negative isoform called LIP
(originally identified as liver-enriched inhibitory protein) (20).
ß-Casein gene expression is reduced 85% to 100% in mammary
epithelial cells derived from C/EBP ß knockout (KO) mice (21, 22).
Interaction and transcriptional cooperation between C/EBPß and GR
have been studied in transactivation of several genes, including
-1
acid glycoprotein, phosphoenolpyruvate carboxykinase (PEPCK),
and herpes simplex virus thymidine kinase (HSV) (23, 24, 25, 26). Although GR
has been shown to interact with C/EBPß, C/EBP
, and C/EBP
(24, 25), transcriptional cooperation with GR was demonstrated to be
specific for C/EBPß for at least two of these genes (PEPCK and HSV)
(24, 26).
Transcriptional cooperation between C/EBPß and GR had not been
studied previously in ß-casein transactivation. Additionally,
transcriptional cooperation between STAT5 and C/EBP family members had
not been examined in transactivation of any other gene to our
knowledge. Studies were initiated to analyze the potential
cooperative effects between STAT5, C/EBPß, and GR on ß-casein gene
transcription. Transcriptional cooperation of STAT5, C/EBP family
members, and GR on the ß-casein transactivation was shown to be
specific to the LAP C/EBPß isoform. Unexpectedly, STAT5 and C/EBPß
did not exhibit cooperative effects in the absence of GR.
Transcriptional cooperation between the three proteins required
full-length GR in a transcriptionally active state. The determinants
for STAT5 and GR transcriptional cooperativity were also found to be
different from those required for cooperation among all three proteins.
These studies have helped elucidate some of the mechanisms involved in
transcription factor cooperation in ß-casein transactivation and have
provided a better understanding of how the ß-casein CoRE facilitates
interactions and increased transcription not observed with the
individual transcription factors.
 |
RESULTS
|
---|
Cooperative Transactivation Regulates ß-Casein Gene
Expression
COS-1 cells express little or no endogenous STAT5, GR, and
C/EBPß (S. L. Wyszomierski, unpublished observations). They,
therefore, can be used as a versatile reconstitution system to study
the combinatorial effects of these transcription factors on ß-casein
reporter constructs without the complications of variable endogenous
levels of these factors. A PRL receptor expression construct and
reporter construct containing a luciferase gene driven by the
2,300/+490 sequences of the rat ß-casein gene were transiently
cotransfected with different combinations of transcription factor
expression constructs or the corresponding empty vectors. Reporter gene
activity was measured after a desired hormone treatment was
administered for 24 h. As shown previously, transfection of STAT5a
or STAT5b into COS cells followed by PRL treatment leads to a 4- to
5-fold induction of ß-casein promoter activity that is not seen in
the absence of STAT5 (Fig. 1C
, lane 2,
and Ref. 13). Under these conditions, cotransfection of STAT5a and GR
followed by treatment with PRL and hydrocortisone (HC) gave a 55% ±
6% SEM increase in ß-casein promoter activity as
compared with STAT5a alone (Fig. 1A
, lane 5 and Fig. 1C
, lane 8).
Because of the critical role of C/EBPß on ß-casein gene expression
observed in C/EBPß-deficient mammary epithelial cells (21, 22),
studies of the potential cooperative effects between C/EBPß and STAT5
and between C/EBPß and GR at the ß-casein promoter were initiated.
For these studies, an expression construct that expressed only the LAP
isoforms of C/EBPß was used.

View larger version (36K):
[in this window]
[in a new window]
|
Figure 1. Cooperative Regulation of ß-Casein Gene
Transcription by STAT5, GR, and C/EBPß
A, Each transcription factor (50 ng), indicated by a "+", or 50 ng
of the appropriate empty vector as a control, indicated by a "",
was transiently transfected into COS-1 cells in 35- mm wells. Treatment
with either HC alone (black bars) or HC + PRL
(striped bars) was performed for 24 h. A
representative experiment is shown (lanes 18). Each treatment group
was performed in triplicate. Error bars denote the SEM. All
differences reported were statistically significant
(P < 0.05). Fold induction (gray
bars) was calculated vs. the level of
transactivation seen with STAT5a alone without PRL treatment. Lanes
911 show the average fold induction from 40 experiments. Error
bars denote the SEM. B, Increasing amounts of LAP
were added in the presence of 50 ng of STAT5 and GR or empty vector
plasmids. Amounts of LAP expression vectors were as follows: lanes 1,
5, and 11, 5 ng; lanes 2, 7, and 12, 10 ng; lanes 3, 8, and 13, 25 ng;
lanes 4, 9, and 14, 100 ng; data not shown, 200 ng. The experiment was
repeated three times. A representative experiment is shown. Each
treatment group was performed in triplicate. Error bars
denote the SEM. C, Different combinations of transcription
factors were transfected and treated with different combinations of HC
and PRL. Hormone treatments are indicated as follows: HC PRL,
black bars; HC +PRL, white bars; +HC
PRL, dark striped bars, +HC +PRL, light striped
bars. This experiment was repeated nine times. A representative
experiment is shown. Each treatment group was performed in triplicate.
Error bars denote the SEM. All differences
reported were statistically significant (P <
0.05).
|
|
Cotransfection of LAP alone into COS-1 cells resulted in no significant
change in the level of basal transcription from the ß-casein reporter
construct (Fig. 1A
, lane 4). Surprisingly, cotransfection of STAT5a and
LAP also did not lead to a further stimulation of ß- casein
promoter activity over that found with STAT5a alone (Fig. 1A
, lane 6).
When all three transcription factors are cotransfected, however, an
overall increase in the transcriptional activity was observed
reproducibly (Fig. 1A
, lane 8). With LAP present, an increase of 38%
± 6% SEM was seen over the level of transactivation
imparted by STAT5a and GR in the absence of LAP, or 107% ± 10%
SEM compared with STAT5 alone. In terms of fold induction,
reporter gene transcription is increased 4.2-fold by STAT5a alone,
6.5-fold by STAT5a + GR, and 8.5-fold by STAT5a + GR + LAP after
hormone treatment over the uninduced level (Fig. 1A
, lanes 911).
Therefore, the ability of LAP to cooperate with STAT5a was dependent on
the presence of GR. The same response was observed if STAT5b was used
in place of STAT5a (data not shown). Since no difference was seen
between the two highly related STAT5 transcription factors for this
response, all subsequent analyses were performed only with STAT5a.
To determine whether the lack of a cooperative effect between STAT5 and
LAP was due to a limiting amount of LAP expression, titration
experiments were performed. Varying amounts of LAP ranging from 5 ng to
200 ng were cotransfected alone, with STAT5a or with STAT5a + GR. At
all concentrations of LAP tested, GR was necessary for the cooperative
effects with STAT5a (Fig. 1B
, lanes 1114). Cooperativity between
STAT5a and LAP was not seen in the absence of GR at any concentration
of LAP (Fig. 1B
, lanes 69). Additionally, LAP did not affect the
basal level of transcription from the ß-casein promoter at any
concentration (Fig. 1B
, lanes 14).
Even before the specific transcription factors responsible for
conveying these effects were identified, it was known that PRL and
glucocorticoids are both essential for ß-casein gene expression (27, 28). Accordingly, the hormonal dependence of the cooperativity between
STAT5, GR, and LAP was examined. In the absence of PRL, no induction of
the ß-casein promoter was seen, regardless of HC treatment (Fig. 1C
, odd numbered lanes). Consistent with previous observations,
transcriptional cooperation between STAT5a and GR was dependent upon
both HC and PRL (Fig. 1C
, lanes 58). The cooperative transcriptional
effects of STAT5a, GR, and LAP were also dependent on both HC and PRL
(Fig. 1C
, lanes 1316). These results confirm that LAP addition to
this reconstitution system mimics the in vivo hormonal
requirements for ß-casein gene transcription.
The Role of the C/EBPß Transactivation Domain
The bZIP domain of C/EBPß is required for interaction of
C/EBPß with GR (25), but the amino-terminal portions of C/EBPß are
crucial for transcriptional cooperation with GR in PEPCK
transactivation (26). In the COS cell reconstitution system, the
transactivation domain of STAT5 is not required for the transcriptional
cooperation between STAT5 and GR (7, 14). Accordingly, the role of the
transactivation domain of C/EBPß in regulating transcriptional
cooperativity in ß-casein transactivation by these factors was
examined. LIP is a naturally occurring, dominant-negative isoform of
C/EBPß (20). When LIP was cotransfected with STAT5a, an inhibition of
transcription was observed (Fig. 2
, lane
7). Inhibition by LIP also was observed when LIP was cotransfected with
STAT5a and GR (Fig. 2
, lane 10). Therefore, addition of GR does not
circumvent the need for the C/EBPß transactivation domain in
regulating ß-casein gene transcription. Furthermore, LIP inhibited
cooperative transactivation by STAT5, GR, and LAP (Fig. 2
, lane 11).
These data are consistent with the observation that LIP markedly
inhibited ß-casein gene expression in CHOk1 cells (our unpublished
results). CHOk1 cells contain endogenous C/EBPß, STAT5, and GR and
are one of the few nonmammary cell lines that can activate milk protein
gene transcription without the addition of exogenous transcription
factors (our unpublished results and Ref. 29).

View larger version (32K):
[in this window]
[in a new window]
|
Figure 2. LIP Does Not Cooperate with STAT5 and GR
Each transcription factor (50 ng), indicated by a "+", or 50 ng of
the appropriate empty vector as a control, indicated by a "", was
transiently transfected into COS-1 cells in 35- mm wells. All samples
are +HC +PRL. The experiment was repeated three times. A representative
experiment is shown. Each treatment group was performed in triplicate.
Error bars denote the SEM. All differences
reported were statistically significant (P <
0.05).
|
|
Next, to determine whether cooperative transactivation of the
ß-casein promoter fragment was specific for C/EBPß as compared with
other C/EBP family members, cotransfection experiments were performed
with C/EBP
and C/EBP
expression constructs. Neither C/EBP
nor
C/EBP
exhibited cooperative transactivation with STAT5a (Fig. 3A
, lanes 2 and 4) or with STAT5a and GR
(Fig. 3A
, lanes 6 and 8). The same cooperative effect was seen with
C/EBPß as compared with the C/EBPß/LAP- only expression construct
(Fig. 3A
, lane 7). This lack of activity of C/EBP
and C/EBP
on
the ß-casein promoter reporter construct was particularly interesting
given that both C/EBPs were considerably more active than C/EBPß when
their activity was compared using a multimerized C/EBP binding site
[D9-CAT (chloramphenicol acetyltransferase)] reporter
construct in COS-1 cells (data not shown). This finding of C/EBPß
specificity is consistent with previous reports of the selective roles
of different C/EBPs in mammary gland development (see
Discussion).

View larger version (28K):
[in this window]
[in a new window]
|
Figure 3. Transcriptional Cooperation Is Specific for
C/EBPß and Requires Its Amino Terminus
Transfection was performed as described in Fig. 1 . All samples are +HC
+PRL. The experiment in panel A was repeated six times. The experiment
in panel B was repeated three times. Representative experiments are
shown. Each treatment group was performed in triplicate. Error bars
denote the SEM. All differences reported were statistically
significant (P < 0.05).
|
|
To further confirm that the N-terminal transactivation domain of
C/EBPß was required for transcriptional cooperativity, expression
constructs for C/EBP chimeras were obtained (30) containing the
activation domain of C/EBP
fused to the b-ZIP domain of C/EBPß
(C/EBP
ß) or the activation domain of C/EBPß fused to the b-ZIP
domain of C/EBP
(C/EBPß
). Only C/EBPß
elicited a
cooperative effect on ß-casein transactivation with STAT5a and GR
(Fig. 3B
, lane 6). Like C/EBP
, C/EBP
ß did not cooperate with
STAT5a and GR (Fig. 3B
, lane 5), and neither protein cooperated with
STAT5a alone (Fig. 3B
, lanes 13). Taken together, these results
indicate that cooperative transcriptional regulation of ß-casein
promoter activity with STAT5 and GR requires, and is specific to, the
N-terminal transactivation domain of C/EBPß.
Regions of GR Needed for Transcriptional Cooperation
The DNA binding domain (DBD) of GR is required for the
protein-protein interaction with C/EBPß (24), and the transactivation
function, TAF-2, in the ligand binding domain of GR is required for
transcriptional cooperation with C/EBPß (24, 25). Transcriptional
cooperation with STAT5 requires the N-terminal portions of GR (14). A
protein-protein interaction domain in GR for STAT5 interaction has not
been mapped, but is thought to reside in TAF-1 (see Fig. 4
). Given these observations, experiments
were undertaken to determine whether both portions of the GR molecule
would, therefore, be required for cooperative activation of the
ß-casein reporter construct. Expression constructs for N- and
C-terminal truncations of GR (31) were expressed at comparable levels
to the full-length GR in COS-1 cells (Fig. 4
, lane 3 for GR 407795).
The immunoblot shown in Fig. 4
was probed with an anti-GR antibody
recognizing a C-terminal epitope. This antibody (Fig. 4
, lane 2),
therefore, did not detect GR 3556. An antibody to an epitope in the
DBD of GR was used to verify GR 3556 production (data not shown).

View larger version (54K):
[in this window]
[in a new window]
|
Figure 4. Wild-Type and Mutant GRs Employed in Transient
Transfection Assays
Wild-type and mutant GR proteins are represented schematically. The
following abbreviations are used: TAF, transactivation domain/function;
LBD, ligand binding domain. An X indicates a point mutation. Twenty
five micrograms of protein extract were used per lane. Immunoblotting
was performed using the anti-GR (P-20) antibody from Santa Cruz Biotechnology, Inc. Black arrows indicate GR
proteins. Open arrowheads indicate nonspecific
cross-reactive material. The proteins examined are as follows: Lane 1,
wild-type GR in pSTC vector; lane 2, GR 3556; lane 3, GR 407795;
lane 4, GR C482S; lane 5, wild-type GR in pCR3.1 vector, lane 6, GR
108317 ; lane 7, G30IIB; lane 8, nontransfected COS cell extract.
|
|
Consistent with previously published results (14), GR 3556 was
capable of transcriptional cooperativity with STAT5a alone, while GR
407795 was not (Fig. 5A
, lanes 5 and
7). Because GR 3556 is constitutively active, it gave a higher level
of transactivation than wild-type GR when cotransfected with STAT5a
(Fig. 5A
, lane 2 vs. lane 5); however, further
transactivation was not seen upon addition of LAP (Fig. 5A
, lane 6).
Addition of LAP also did not influence the lack of transcriptional
cooperation between STAT5a and GR 407795 (Fig. 5A
, lane 8). These
data indicate that the regions of GR that are necessary for
transcriptional cooperation between STAT5 and GR and between C/EBPß
and GR in pairs are also necessary for transcriptional cooperation
between all three transcription factors on the ß-casein promoter.

View larger version (23K):
[in this window]
[in a new window]
|
Figure 5. Regions of GR Required for Transcriptional
Cooperation
Transfection was performed as described for Fig. 1 . The type of GR,
wild-type (wt) or mutant (mutants are explained in Fig. 4 ) is indicated
below each graph. All samples are +HC +PRL. The
experiments were repeated three times. A representative experiment is
shown for each. Treatment groups were performed in triplicate.
Error bars denote the SEM. All differences
reported were statistically significant (P <
0.05).
|
|
To further examine the importance of the protein-protein interactions
between these transcription factors, a GR mutant, GR C482S, which is
not capable of binding to DNA due to a point mutation in the second
zinc finger of the DBD (Fig. 4
, lane 4), was used. This point mutation
diminished the level of transcriptional cooperativity between STAT5a
and GR (Fig. 5B
, lane 3; in agreement with Refs. 7, 14). Abolishing
DNA binding of GR did not, however, effect transcriptional cooperation
between STAT5a, GR, and LAP (Fig. 5B
, lane 5). Although the
independence of GR transcriptional effects from the DNA binding
activity of GR may be a result of the COS cell overexpression system
(W. Doppler, personal communication), the fact that the transcriptional
cooperativity still occurs emphasizes the importance of the protein-
protein interactions of GR with STAT5 and C/EBPß in regulating
ß-casein gene expression.
The Role of Transactivation by GR
The data thus far suggested several possible mechanisms for the
observed GR- dependent transcriptional cooperation. One possibility was
that GR was playing a structural role. For example, GR might act as a
bridging molecule between STAT5 and C/EBPß, thereby helping to
provide a favorable conformation for cooperative transactivation.
Alternatively, GR could play a transactivational role dependent on the
presence of C/EBPß. To differentiate between these possibilities, the
GR antagonist RU486 and transactivation-deficient GR mutants were
used.
RU486 allows DNA binding of GR but blocks transactivation by
keeping the C-terminal TAF-2 domain of GR in a conformation unable to
interact with the rest of the transcriptional machinery through
coactivators (32). Surprisingly, when STAT5a and GR were cotransfected
and RU486 treatment was performed, the same level of transactivation
was seen as with HC treatment (Fig. 6
, lanes 3 and 4). However, when STAT5a and GR were cotransfected with LAP
and treated with RU486, no additional transactivation occurred (Fig. 6
, lane 6). GR was cotransfected with a mouse mammary tumor virus
(MMTV)-reporter gene to verify that RU486 did not activate consensus
GREs in this cell system. As expected, RU486 did not activate the
MMTV-reporter construct and inhibited HC activation as well (Fig. 6
, gray bars, lanes 710). These data suggest that
transactivation by GR is an important aspect of the transcriptional
cooperativity between STAT5, GR, and C/EBPß.

View larger version (32K):
[in this window]
[in a new window]
|
Figure 6. RU486 Permits Transcriptional Cooperation between
STAT5 and GR but Not STAT5, GR, and C/EBPß
Transfection was performed as described for Fig. 1 . Black
bars indicate ß-casein promoter activity (lanes 16). These
samples are all +PRL. Gray bars indicate MMTV promoter
activity (lanes 710). These samples are all PRL. Treatment with HC
or RU486 is indicated below the graph. H, HC; R, RU486;
HR, HC + RU486. The ß-casein portion of the experiment was repeated
seven times. The MMTV portion of the experiment was repeated twice. A
representative experiment is shown. Each treatment group was performed
in triplicate. Error bars denote the SEM.
All differences reported were statistically significant
(P < 0.05). Note: The small induction seen with
RU486 on MMTV (lane 9) was not statistically significant over the basal
(lane 7).
|
|
To complement these studies using RU486, TAF-1 mutants of GR deficient
in transactivation were obtained. The TAF-1 domain of GR has been shown
not only to be essential for activation of transcription by GR on many
promoters, but is also necessary for repression of AP-1 transcriptional
activity by GR (33). The two GR mutants used are shown schematically in
Fig. 4
. GR 108317
contains a deletion that eliminates the core of
the TAF-1 domain. It is not transcriptionally active and cannot repress
AP-1-dependent transcription. GR30IIB contains three point mutations,
which were identified using a large-scale mutagenesis and screening
strategy in yeast. Mutation of these three amino acids severely
compromises transactivation by GR but does not affect GR repression of
AP-1-dependent transcription (34). The fact that the repressive ability
of GR is maintained suggests that the overall structure of the protein
is not severely altered by the point mutations. Both proteins were
expressed at levels comparable to the wild-type GR in COS-1 cells (Fig. 4
, lanes 6 and 7). In the absence of LAP, GR30IIB was able to cooperate
transcriptionally with STAT5a similar to wild-type GR, while GR
108317
could not (Fig. 7
, lanes 4
and 6). Neither GR mutant was capable of transcriptional cooperativity
with STAT5a and LAP (Fig. 7
, lanes 5 and 7). Thus, the use of the GR
antagonist RU486 and GR mutants containing TAF-1 mutations revealed
that the transcriptional activity of GR is not necessary for
cooperative transactivation between STAT5 and GR. However,
transactivation by GR is an essential component of the transcriptional
cooperativity between STAT5, GR, and C/EBPß at the ß-casein
promoter.

View larger version (28K):
[in this window]
[in a new window]
|
Figure 7. The TAF-1 Domain of GR Is Required for
Transcriptional Cooperation with STAT5 and C/EBPß
Transfection was performed as described for Fig. 3 . All samples are +HC
+PRL. The experiment was repeated twice. A representative experiments
is shown. Each treatment group was performed in triplicate.
Error bars denote the SEM. All
differences reported were statistically significant
(P < 0.05).
|
|
 |
DISCUSSION
|
---|
Despite the fact that STAT and C/EBP sites are found in close
proximity in a number of promoters in addition to the ß-casein
promoter, including the oncostatin M and serine protease inhibitor-3
promoters (35, 36), before these studies, transcriptional cooperativity
between STAT5 and C/EBPß had not been reported. Transcriptional
cooperativity between C/EBPß and GR had also not been studied with
respect to ß-casein transactivation. These analyses revealed that
transcriptional cooperativity between STAT5, GR, and C/EBPß is
dependent on transcriptionally active GR. In addition, this enhancement
of ß-casein transcription was specific for C/EBPß, since
cooperative transactivation was not observed with either C/EBP
or
C/EBP
. The results indicate that the domains of GR necessary for
interaction and transcriptional cooperativity with STAT5 and C/EBPß
individually were also required for cooperativity between all three
proteins. Finally, different determinants are required for
cooperativity between STAT5 and GR alone as compared with STAT5, GR,
and C/EBPß together.
The experiments in this study were performed using a COS-1 cell
reconstitution system. The advantage of this system is its versatility.
Endogenous STAT5, GR, and C/EBPß were not detected by Western
blotting before transfection even when 100 µg of total cellular
protein were analyzed. After transfection, the protein levels of each
transcription factor were easily visualized by Western blotting from as
little as 5 µg of total cellular protein. Additionally, no increase
in reporter gene activity was detected after PRL or hydrocortisone
treatment in the absence of exogenously added transcription factors
(demonstrated in Fig. 1A
, lane 1, and Fig. 2
, lane 1, and in data not
shown). Any effects observed on ß-casein transactivation were
entirely dependent on the transfection of exogenous transcription
factors. This allowed the comparison of combinatorial effects of intact
transcription factors with the analysis of the transcription factors
containing deletions and point mutations, as well as with chimeric
proteins. One limitation of using this reconstitution system is that a
lower level of hormonal induction of ß-casein promoter activity was
observed than has been reported in other cell systems (10, 27, 29). A
second limitation is that the overexpression of these transcription
factors may force interactions not necessarily sufficient at lower
endogenous levels of these same transcription factors. High expression
may help stabilize protein-protein interactions and obviate the need
for weaker protein-DNA interactions (W. Doppler, personal
communication). Although it may not be possible for a reconstitution
system to mimic all aspects of an in vivo process, the
results reported herein correlate well with observations obtained from
studies performed using mammary epithelial cells, mammary gland
extracts, and knockout mice (4).
This finding that C/EBPß transactivation of ß-casein is GR
dependent agrees with previous studies. In a cytotoxic T cell line,
glucocorticoid induction of ß- casein promoter activity was
dependent on the region of the CoRE containing the C/EBP binding sites
(37). Cooperation with STAT5 and GR was specific to C/EBPß. C/EBP
and C/EBP
could not substitute for C/EBPß. This is consistent with
results obtained in mouse models and in experiments using mammary
epithelial cell lines. Four C/EBP binding sites were found in the
ß-casein proximal promoter. Mutation of these binding sites severely
decreased ß-casein promoter activity in stably transfected HC11
mammary epithelial cells. C/EBPß was the predominant protein in
extracts from HC11 cells that bound to the C/EBP sites (6). In mice,
deletion of C/EBPß severely decreased ß-casein gene expression,
while deletion of C/EBP
exhibited no effect on ß-casein gene
expression (22). Although some binding of C/EBP
to the ß-casein
CoRE was detected using extracts from HC11 cells, this interaction was
minor compared with C/EBPß (6). Additionally, C/EBP
levels are
highest in the mammary gland during involution, a stage when ß-casein
expression is down-regulated (38, 39). The ß-casein promoter is one
of only a few promoters on which specificity for a C/EBP family member
has been reported. Transcriptional cooperation with GR is specific for
C/EBPß in transactivation of the PEPCK and HSV promoters as well (24, 26). Synergy with the Sp-1 transcription factor on the CYP2D5 promoter
is also specific for C/EBPß (40). These data suggest that one way of
conferring specificity for individual C/EBP family members is obligate
interaction and cooperation with other proteins at the promoter.
LIP, the dominant negative isoform of C/EBPß, inhibited ß-casein
transactivation both in the presence and absence of LAP. The mechanisms
of the ß-casein gene repression by LIP are particularly interesting
because LIP expression in the mammary gland is high during pregnancy
and is severely decreased during lactation (8). These data strongly
suggest physiological relevance for repression of the ß-casein gene
by LIP. Because LIP has a greater DNA binding affinity than LAP (20),
it is likely that LIP inhibits ß-casein transactivation by binding to
the C/EBP binding sites in the CoRE, preventing LAP from binding.
Elucidation of the mechanism of ß-casein repression by LIP in the
absence of LAP requires further experimentation. Several possibilities
exist. In the CoRE of the rat ß-casein proximal promoter, one of the
C/EBP binding sites overlaps with a nonconsensus STAT5 binding site.
This site alone does not bind STAT5 with high affinity (6, 8) but may
bind STAT5 as a tetramer in cooperation with the consensus STAT5
binding site. Tetramerization of STAT5 on suboptimal DNA binding sites
has been demonstrated for many other promoters (41, 42, 43). Therefore, LIP
binding to the overlapping C/EBP site may inhibit transactivation by
STAT5 by preventing formation of a tetrameric STAT5 complex.
Additionally, the ß-casein proximal promoter contains a Yin Yang-1
(YY1) binding site known to be important for repression of ß-casein
gene transcription (44, 45). LIP interacts directly with YY1 (46) and
YY1 interacts with several histone deacetylases (47, 48). Therefore,
LIP may contribute to the recruitment of histone deacetylases and
active repression of ß-casein transactivation.
One surprising result of the experiments described herein was the
finding that transcriptional cooperation between STAT5 and GR in the
absence of C/EBPß did not require transactivation by GR. RU486-bound
wild-type GR and GR with point mutations in TAF-1 did not eliminate the
increase in transactivation observed when GR was cotransfected with
STAT5 as compared with activation by STAT5 alone. Deletion of amino
acids (a.a.) 108317 of GR did, however, eliminate the cooperative
effect. Although coimmunoprecipitation experiments are required for
confirmation, this further maps the interaction domain between STAT5
and GR within the region previously shown to be essential (a.a. 1407)
(14). These results suggest that cooperative transcription between
STAT5 and GR in this system is the result of a structural effect rather
than a transactivational effect of GR. Binding of STAT5 and GR to their
adjacent elements on this promoter (half-GREs for GR) may strengthen
the STAT5 interaction with the ß-casein promoter, allowing it to
exert increased transcriptional effects. This may either result in or
be a result of prolonged tyrosine phosphorylation of STAT5 (49). GR
also may be mediating chromatin remodeling events, such as those that
have been shown to take place on the MMTV promoter even if GR is bound
to RU486 (50). However, this seems less likely since the reporter genes
in these experiments were transiently introduced into the cells rather
than stably integrated into the chromatin.
There are several nonexclusive, testable models that may explain the
effects of GR on transcriptional cooperativity between STAT5 and
C/EBPß. One model predicts that a component of GR-dependent C/EBPß
activation is a required interaction of C/EBPß with GR to help
relieve an inhibitory conformation of C/EBPß. Experiments using the
D9-CAT reporter gene construct, driven by multimerized C/EBP binding
sites (data not shown), support this hypothesis. Using this reporter
construct, C/EBPß also exhibited very little activity in COS-1 cells
in the absence of GR, but exhibited increased activity when GR was
present. The amino-terminal transactivation domain of C/EBPß contains
two repression domains, which, through an intramolecular interaction,
inhibit transactivation and may decrease the DNA binding of C/EBPß
(51, 52). This repression can be relieved by phosphorylation of
C/EBPß via several kinase-mediated cascades including ras-
activated MAPK cascades (51, 52, 53). This inhibitory conformation can also
be relieved by interaction with other proteins (proposed in Refs. 51, 52). The relief of C/EBPß repression by protein-protein
interactions has been convincingly demonstrated with the myb protein on
the mim-1 promoter. C/EBPß and myb interact (54) and exhibit
transcriptional synergy on the mim-1 promoter (55, 56). In CV-1 cells,
C/EBPß bound to its cis-regulatory element but was
inactive on the mim-1 promoter in the absence of myb. In the presence
of myb, transcriptional synergy was seen (57).
The N terminus of C/EBPß interacts with the E1A region of p300/CBP
(58). It is likely that this interaction requires the open, non
repressed conformation of C/EBPß. In CV-1 cells, p300/CBP exerted
minimal effects on the mim-1 promoter with C/EBPß alone. When
constitutively activated ras was cotransfected, p300/CBP
enhanced transcription by C/EBPß on the mim-1 promoter.
Cotransfection of myb without ras allowed the same
enhancement by p300/CBP to occur, and the transcriptional synergy
previously observed by C/EBPß and myb was greatly enhanced (59).
Therefore, a common theme emerges that may explain some of the
specificity of expression from a CoRE. Interaction of another protein
acting on the CoRE with C/EBPß may relieve the inhibitory
conformation of C/EBPß and allow recruitment of transcriptional
activators such as p300/CBP.
Another model predicts that the transactivation function of GR
contributes a second component to the GR dependence of C/EBPß
activation of ß-casein. It appears unlikely that changing the
C-terminal conformation of GR by binding RU486 and mutating the
N-terminal transactivation domain (TAF-1) would both disrupt the
interaction between GR and C/EBPß. Nevertheless, both methods of
eliminating GR transactivation abolished transcriptional cooperativity
with C/EBPß and STAT5. Additionally, using the D9-CAT reporter, the
GR30IIB mutant activated C/EBPß to a similar extent as wild-type GR
(data not shown). One possibility is that C/EBPß and GR recruit a
coactivator complex together that neither can effectively recruit
alone. Boruk et al. (24) have proposed another mechanism to
explain the transcriptional cooperativity. They theorized that C/EBPß
recruits an activation complex to the HSV promoter after which the
activity of this activation complex is enhanced by TAF-2 of GR. This
enhancement could be independent of GR binding to DNA or interaction
with C/EBPß. It should be noted that this mechanism and the
coactivator mechanism postulated above are not mutually exclusive.
Because C/EBPß and GR cannot activate ß-casein gene transcription
in the absence of STAT5, cooperativity between a STAT5-recruited
activation complex and an activation complex recruited jointly by
C/EBPß and GR seems likely. It has been demonstrated that multiple
coactivator activities are required for transactivation by retinoic
acid receptor, hepatic nuclear factor-1, and NF-
B (60, 61, 62). It is
very likely, therefore, that the recruitment of multiple coactivators
is necessary for high level transactivation of many genes. CoREs may
accomplish this by using multiple transcription factors as a way to
impart specificity of gene expression. Analysis of the coactivators
recruited by STAT5, GR, and C/EBPß to the ß-casein promoter and
their contribution to transactivation is an important area of future
investigation.
There are still some aspects of regulation of the ß-casein CoRE
observed in vivo that cannot be readily explained by these
data. In STAT5a-deficient mice, the level of activated STAT5b is
severely reduced, yet ß-casein expression is only marginally affected
(63). Analysis of ß-casein expression in STAT5a- and STAT5b-deficient
mammary gland transplants has not yet been reported, so it is still not
known whether a small amount of STAT5 is sufficient for transactivation
in vivo. In contrast, transactivation in cell culture
systems is highly dependent on STAT5 (9, 13). Decreasing the level of
STAT5 in the COS-1 reconstitution system severely decreased ß-casein
transactivation even in the presence of GR and C/EBPß (data not
shown). In the reconstitution system, transactivation also is observed
in the absence of C/EBPß, while in C/EBPß-deficient mammary
epithelial cells, ß-casein expression is severely reduced (by
85100%) (21, 22). One possibility is that C/EBPß may play an
additional role in ß-casein transactivation before formation of the
hypothesized activation complexes. C/EBPß was recently found to
interact with the SWI/SNF complex, an ATP-dependent chromatin
remodeling complex (64), and this may be one possible explanation for
the discrepancies between the in vivo and cell culture
observations. Modification of the reconstitution system to examine the
effects of STAT5, C/EBPß, and GR on the ß-casein transactivation
with the reporter construct stably integrated into the chromatin may
provide further information on how these transcription factors act on
the ß-casein CoRE. Additionally, analysis of GR-deficient mammary
epithelial cells may help confirm the importance of GR in
transactivation from the ß- casein CoRE.
In summary, several unique roles for GR at the CoRE located in the
ß-casein proximal promoter have been observed. GR appears to promote
the formation of an activated conformation of C/EBPß as well as
prolong the activated state of STAT5 (49). Additionally, it is likely
that GR, C/EBPß, and STAT5 together recruit an activation complex to
the ß-casein CoRE that cannot be efficiently recruited by any of the
transcription factors individually. Reconciliation of the differences
between the in vivo and cell culture observations and
analysis of the composition and assembly of the proposed activation
complexes are important avenues for future investigation.
 |
MATERIALS AND METHODS
|
---|
Plasmids
For the majority of the studies reported herein, the
2,300/+490 ß-casein promoter subcloned into pGL2 luciferase vector
(Promega Corp., Madison, WI) was used as the reporter
construct. The responsiveness of the 2,300/+490 ß-casein promoter
has been reported previously (27, 65). Two other reporter genes were
used as well: D9-CAT, which contains multiple copies of the C/EBP
binding site from the albumin promoter, which was kindly provided by
Dr. Ueli Schibler (University of Geneva, Geneva, Switzerland) and
MMTVLUC. Both have been previously described (20, 66, 67). PCMVßGAL
was obtained from CLONTECH Laboratories, Inc. (Palo Alto,
CA). The PRL receptor expression vector, pECE-PRL-R-L, which was kindly
provided by Dr. Paul Kelly (INSERM Unité 344 Paris,
France) and the expression vectors for STAT5a and STAT5b,
pRcCMVSTAT5a and pcDNA3STAT5b, have been previously described (49, 68).
Expression vectors for C/EBPß-LAP and C/EBPß-LIP, pSCTLAP
3, and
pSCTLIP, were kindly provided by Dr. Ueli Schibler and have been
previously described (20). For the comparative experiments, we used
expression vectors for C/EBP
, ß, and
, which were potentially
capable of generating multiple isoforms if the alternative downstream
translation start sites were employed, because expression vectors
capable of producing only activating isoforms of C/EBP
and C/EBP
were not readily available. Expression vectors for C/EBP
, C/EBPß,
and C/EBP
in the pSV2sport expression vector as well as expression
vectors for chimeric fusion proteins C/EBP
ß and C/EBP ß
were
kindly provided by Drs. Gerald Elberg and Sophia Tsai (Baylor College
of Medicine) and have been previously described (30). These cDNAs were
subcloned into the pSCT expression vector using restriction enzyme
sites in the multiple cloning sites of both vectors. The original cDNAs
of C/EBP
, C/E 66 Pß, and C/EBP
were kindly provided to Drs.
Elberg and Tsai from Dr. Steven McKnight (University of Texas
Southwestern Medical Center, Dallas, TX). Dr. Rainer Lanz
(Baylor College of Medicine) kindly provided the pSTC and pSCT vectors.
Wild-type GR in the pSTC vector and the following GR mutant constructs,
pSTC GR 3556, pSTC GR 407795 (X-795), and pSTC GR C482S, were
kindly provided by Dr. Rainer Lanz and Dr. Sandro Rusconi (University
of Fribourg, Fribourg, Switzerland) and have been previously described
(69). Wild-type GR in the p6R vector and GR 108317
and GR30IIB in
the same vector were kindly provided by Drs. Jorge Iniguez-Lluhi and
Keith Yamamoto (University of California San Francisco) and have been
previously described (34). Because the Rous sarcoma virus (RSV)
promoter drove these vectors and RSV is C/EBPß responsive, these
cDNAs were subcloned into the pCR3.1 expression vector
(Invitrogen, Carlsbad, CA), which has a CMV promoter like
the vectors used for all the other transcription factors. All plasmids
were purified using QIAGEN DNA maxi-prep kits (QIAGEN,
Valencia, CA).
Cell Culture, Transient Transfection, and Reporter Gene
Assays
DMEM, trypsin-EDTA, donor horse serum, and glutamine were
purchased from JRH Biosciences (Lenexa, KS). FBS was
purchased from Summit Biotechnologies (Fort Collins, CO). Gentamicin,
insulin, and hydrocortisone were purchased from Sigma (St.
Louis, MO). RU486 was obtained from Roussel/UCLAF (Romainville,
France). Ovine PRL (lot AFP10692C) was kindly provided by the National
Hormone and Pituitary program (Bethesda, MD). COS-1 cells were obtained
from the ATCC (Manassas, VA). COS-1 cells were routinely
passaged in DMEM + 10% FBS in the presence of gentamicin. COS-1 cell
transfections were performed 1 day after passaging the cells into the
35-mm wells of six-well tissue culture plates. Transfection was
performed using Superfect Reagent (QIAGEN). In each well,
50 ng of pCMVßgal, 50 ng of pECEPRL-R-L, and 200 ng of -2,300/+490
ß- casein LUC were transiently cotransfected with different
combinations of transcription factor expression constructs or the
corresponding empty vectors as controls. Usually, 50 ng of each
transcription factor were used; 2 µg total DNA and 10 µl of
Superfect were used per 35-mm well. Transfections were performed
according to the manufacturers instructions. During transfection and
thereafter, the cells were maintained in DMEM + 10% charcoal-stripped
horse serum with gentamicin and 5 µg/ml insulin. Twenty-four hours
after transfection, treatment with hydrocortisone (1 µg/ml), RU486
(1 x 10-7 M) and/or ovine PRL
(1 µg/ml) was performed for 24 h as indicated. Luciferase assays
were performed by standard methods on a MLX microtiter plate
luminometer (Dynex Technologies, Chantilly, VA). Luciferin was
purchased from Molecular Probes, Inc. (Eugene, OR) and
used to make substrate containing 1 mM luciferin, 0.1
M Trizma phosphate, 12 mM
MgCl2, and 2.4 mM ATP.
ß-Galactosidase assays were performed by standard protocols (70).
O- Nitrophenyl ß-D-galactopyranoside
was purchased from Sigma. CAT assays were performed using
a CAT enyzme-linked immunosorbent assay (ELISA) kit (Roche Molecular Biochemicals, Indianapolis, IN) according to the
manufacturers instructions.
Statistical Analysis
Univariate ANOVA was used to test for equality of mean relative
light units (RLU)/ßGAL values across treatment groups. The
null hypothesis was that all treatments had the same mean values of
RLU/ßGAL. ANOVA runs were performed using data from triplicate
samples in treatments from a single experiment with necessary
Bonferroni corrections to P values based on the number of
multiple tests. ANOVA runs were also performed using data from all
treatments in all experiments combined. For these analyses, fitted
values of marginal means and their SEs were used
in hypothesis tests for equal means, with necessary Bonferroni
corrections to P values based on the number of multiple
tests. Analysis was performed using the SPSS statistical software
package (SPSS Version 10, SPSS, Inc., Chicago, IL).
Antibodies and Western Blot Analysis
SDS-PAGE and Western blot analysis was performed by standard
protocols that have been previously described (49). STAT5a and GR were
separated on 7.5% running gels and the C/EBPs were separated on 12%
running gels. Affinity purified rabbit polyclonal anti- STAT5a antibody
has been previously described (68). The following anti-GR antibodies
were used: rabbit polyclonal anti-GR (P-20) TransCruz antibody
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at a
1:2,000 dilution and the monoclonal anti-GR antibody, BuGR2
(Affinity BioReagents, Inc. Golden, CO) at a 1:600
dilution. Rabbit polyclonal antibodies anti-C/EBP
(14AA), C/EBPß
(C-19), and C/EBP
(C-22) from Santa Cruz Biotechnology, Inc. were used at a 1:1,000 dilution to detect those
proteins.
 |
ACKNOWLEDGMENTS
|
---|
The authors thank Dr. Leif Peterson for assistance and advice in
statistical analysis of the data presented herein. The authors thank
Drs. Li-yuan Yu-Lee, Rainer Lanz, and Michelle Kallesen for critical
reading of the manuscript and Ms. Alvenia Daniels for secretarial
assistance.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Jeffrey M. Rosen, Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030-3498. E-mail: jrosen{at}bcm tmc.edu.
This work was supported by NIH Grant CA-16303.
Received for publication September 15, 2000.
Revision received November 6, 2000.
Accepted for publication November 9, 2000.
 |
REFERENCES
|
---|
-
Jiang J, Levine M 1993 Binding affinities and cooperative
interactions with bHLH activators delimit threshold responses to the
dorsal gradient morphogen. Cell 72:741752[Medline]
-
Kel OV, Romaschenko AG, Kel AE, Wingender E, Kolchanov NA 1995 A compilation of composite regulatory elements affecting gene
transcription in vertebrates. Nucleic Acids Res 23:40974103[Abstract]
-
Rosen JM, Zahnow C, Kazansky A, Raught B 1998 Composite
response elements mediate hormonal and developmental regulation of milk
protein gene expression. Biochem Soc Symp 63:101113[Medline]
-
Rosen JM, Wyszomierski SL, Hadsell D 1999 Regulation of milk
protein gene expression. Annu Rev Nutr 19:407436[CrossRef][Medline]
-
Lechner J, Welte T, Doppler W 1997 Mechanism of interaction
between the glucocorticoid receptor and Stat5: role of DNA-binding.
Immunobiology 198:112123[Medline]
-
Doppler W, Welte T, Philipp S 1995 CCAAT/enhancer-binding
protein isoforms ß and
are expressed in mammary epithelial cells
and bind to multiple sites in the ß-casein gene promoter. J Biol
Chem 270:1796217969[Abstract/Free Full Text]
-
Lechner J, Welte T, Tomasi JK, Bruno P, Cairns C, Gustafsson
J, Doppler W 1997 Promoter-dependent synergy between glucocorticoid
receptor and Stat5 in the activation of ß-casein gene transcription.
J Biol Chem 272:2095420960[Abstract/Free Full Text]
-
Raught B, Liao WS, Rosen JM 1995 Developmentally and
hormonally regulated CCAAT/enhancer-binding protein isoforms influence
ß-casein gene expression. Mol Endocrinol 9:12231232[Abstract]
-
Schmitt-Ney M, Doppler W, Ball RK, Groner B 1991 ß-Casein
gene promoter activity is regulated by the hormone-mediated relief of
transcriptional repression and a mammary gland-specific nuclear factor.
Mol Cell Biol 11:37453755[Medline]
-
Wakao H, Gouilleux F, Groner B 1994 Mammary gland factor (MGF)
is a novel member of the cytokine regulated transcription factor gene
family and confers the prolactin response [published erratum appears
in EMBO J 1995 Feb 15;14(4):8545]. EMBO J 13:21822191[Abstract]
-
Welte T, Philipp S, Cairns C, Gustafsson JA, Doppler W 1993 Glucocorticoid receptor binding sites in the promoter region of milk
protein genes. J Steroid Biochem Mol Biol 47:7581[CrossRef][Medline]
-
Stocklin E, Wissler M, Gouilleux F, Groner B 1996 Functional
interactions between Stat5 and the glucocorticoid receptor. Nature 383:726728[CrossRef][Medline]
-
Liu X, Robinson GW, Gouilleux F, Groner B, Hennighausen L 1995 Cloning and expression of Stat5 and an additional homologue (Stat5b)
involved in prolactin signal transduction in mouse mammary tissue. Proc
Natl Acad Sci USA 92:88318835[Abstract]
-
Stoecklin E, Wissler M, Moriggl R, Groner B 1997 Specific DNA
binding of Stat5, but not of glucocorticoid receptor, is required for
their functional cooperation in the regulation of gene transcription.
Mol Cell Biol 17:67086716[Abstract]
-
Cella N, Groner B, Hynes NE 1998 Characterization of Stat5a
and Stat5b homodimers and heterodimers and their association with the
glucocortiocoid receptor in mammary cells. Mol Cell Biol 18:17831792[Abstract/Free Full Text]
-
Cao Z, Umek RM, McKnight SL 1991 Regulated expression of three
C/EBP isoforms during adipose conversion of 3T3L1 cells. Genes Dev 5:15381552[Abstract]
-
Williams SC, Cantwell CA, Johnson PF 1991 A family of
C/EBP-related proteins capable of forming covalently linked leucine
zipper dimers in vitro. Genes Dev 5:15531567[Abstract]
-
Calkhoven CF, Muller C, Leutz A 2000 Translational control of
C/EBP
and C/EBPß isoform expression. Genes Dev 14:19201932[Abstract/Free Full Text]
-
Welm AL, Timchenko NA, Darlington GJ 1999 C/EBP
regulates
generation of C/EBPß isoforms through activation of specific
proteolytic cleavage. Mol Cell Biol 19:16951704[Abstract/Free Full Text]
-
Descombes P, Schibler U 1991 A liver-enriched transcriptional
activator protein, LAP, and a transcriptional inhibitory protein, LIP,
are translated from the same mRNA. Cell 67:569579[Medline]
-
Robinson GW, Johnson PF, Hennighausen L, Sterneck E 1998 The
C/EBPß transcription factor regulates epithelial cell proliferation
and differentiation in the mammary gland. Genes Dev 12:19071916[Abstract/Free Full Text]
-
Seagroves TN, Krnacik S, Raught B, Gay J, Burgess-Beusse B,
Darlington GJ, Rosen JM 1998 C/EBPß, but not C/EBP
, is essential
for ductal morphogenesis, lobuloalveolar proliferation, and functional
differentiation in the mouse mammary gland. Genes Dev 12:19171928[Abstract/Free Full Text]
-
Alam T, An MR, Mifflin RC, Hsieh CC, Ge X, Papaconstantinou J 1993 Trans-activation of the
1-acid glycoprotein gene acute phase
responsive element by multiple isoforms of C/EBP and glucocorticoid
receptor. J Biol Chem 268:1568115688[Abstract/Free Full Text]
-
Boruk M, Savory JG, Hache RJ 1998 AF-2-dependent potentiation
of CCAAT enhancer binding protein ß- mediated transcriptional
activation by glucocorticoid receptor. Mol Endocrinol 12:17491763[Abstract/Free Full Text]
-
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
1-acid
glycoprotein gene via direct protein-protein interaction. Mol Cell Biol 13:18541862[Abstract]
-
Yamada K, Duong DT, Scott DK, Wang JC, Granner DK 1999 CCAAT/enhancer-binding protein ß is an accessory factor for the
glucocorticoid response from the cAMP response element in the rat
phosphoenolpyruvate carboxykinase gene promoter. J Biol Chem 274:58805887[Abstract/Free Full Text]
-
Doppler W, Groner B, Ball RK 1989 Prolactin and glucocorticoid
hormones synergistically induce expression of transfected rat
ß-casein gene promoter constructs in a mammary epithelial cell line.
Proc Natl Acad Sci USA 86:104108[Abstract]
-
Topper YJ, Freeman CS 1980 Multiple hormone interactions in
the developmental biology of the mammary gland. Physiol Rev 60:10491106[Free Full Text]
-
Lesueur L, Edery M, Ali S, Paly J, Kelly PA, Djiane J 1991 Comparison of long and short forms of the prolactin receptor on
prolactin-induced milk protein gene transcription. Proc Natl Acad Sci
USA 88:824828[Abstract]
-
Elberg G, Gimble JM, Tsai SY 2000 Modulation of the murine
peroxisome proliferator-activated receptor
2 promoter activity by
CCAAT/enhancer-binding proteins. J Biol Chem 275:2781527822[Abstract/Free Full Text]
-
Godowski PJ, Rusconi S, Miesfeld R, Yamamoto KR 1987 Glucocorticoid receptor mutants that are constitutive activators of
transcriptional enhancement [published erratum appears in Nature 1987
Mar 511;326(6108):105]. Nature 325:365368[CrossRef][Medline]
-
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 receptors. Mol Cell Biol 17:27352744[Abstract]
-
Pearce D, Yamamoto KR 1993 Mineralocorticoid and
glucocorticoid receptor activities distinguished by nonreceptor factors
at a composite response element. Science 259:11611165[Medline]
-
Iniguez-Lluhi JA, Lou DY, Yamamoto KR 1997 Three amino acid
substitutions selectively disrupt the activation but not the repression
function of the glucocorticoid receptor N terminus. J Biol Chem 272:41494156[Abstract/Free Full Text]
-
Kordula T, Travis J 1996 The role of Stat and C/EBP
transcription factors in the synergistic activation of rat serine
protease inhibitor-3 gene by interleukin-6 and dexamethasone. Biochem J 313:10191027[Medline]
-
Ma Y, Streiff RJ, Liu J, Spence MJ, Vestal RE 1999 Cloning and
characterization of human oncostatin M promoter. Nucleic Acids Res 27:46494657[Abstract/Free Full Text]
-
Chida D, Wakao H, Yoshimura A, Miyajima A 1998 Transcriptional
regulation of the ß-casein gene by cytokines: cross-talk between
STAT5 and other signaling molecules. Mol Endocrinol 12:17921806[Abstract/Free Full Text]
-
Gigliotti AP, DeWille JW 1998 Lactation status influences
expression of CCAAT/enhancer binding protein isoform mRNA in the mouse
mammary gland. J Cell Physiol 174:232239[CrossRef][Medline]
-
Gigliotti AP, DeWille JW 1999 Local signals induce
CCAAT/enhancer binding protein-
(C/EBP-delta) and C/EBP-ß mRNA
expression in the involuting mouse mammary gland. Breast Cancer Res
Treat 58:5763[CrossRef][Medline]
-
Lee YH, 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 17:20382047[Abstract]
-
Yoshimura A, Ichihara M, Kinjyo I, Moriyama M, Copeland NG,
Gilbert DJ, Jenkins NA, Hara T, Miyajima A 1996 Mouse oncostatin M: an
immediate early gene induced by multiple cytokines through the
JAK-STAT5 pathway. EMBO J 15:10551063[Abstract]
-
Meyer WK, Reichenbach P, Schindler U, Soldaini E, Nabholz M 1997 Interaction of STAT5 dimers on two low affinity binding sites
mediates interleukin 2 (IL-2) stimulation of IL-2 receptor
gene
transcription. J Biol Chem 272:3182131828[Abstract/Free Full Text]
-
John S, Vinkemeier U, Soldaini E, Darnell Jr JE, Leonard WJ 1999 The significance of tetramerization in promoter recruitment by
Stat5. Mol Cell Biol 19:19101918[Abstract/Free Full Text]
-
Meier VS, Groner B 1994 The nuclear factor YY1 participates in
repression of the ß-casein gene promoter in mammary epithelial cells
and is counteracted by mammary gland factor during lactogenic hormone
induction. Mol Cell Biol 14:128137[Abstract]
-
Raught B, Khursheed B, Kazansky A, Rosen J 1994 YY1 represses
ß-casein gene expression by preventing the formation of a
lactation-associated complex. Mol Cell Biol 14:17521763[Abstract]
-
Bauknecht T, See RH, Shi Y 1996 A novel C/EBP ß-YY1 complex
controls the cell type-specific activity of the human papillomavirus
type 18 upstream regulatory region. J Virol 70:76957705[Abstract]
-
Yang WM, Inouye C, Zeng Y, Bearss D, Seto E 1996 Transcriptional repression by YY1 is mediated by interaction with a
mammalian homolog of the yeast global regulator RPD3. Proc Natl
Acad Sci USA 93:1284512850[Abstract/Free Full Text]
-
Yang WM, Yao YL, Sun JM, Davie JR, Seto E 1997 Isolation and
characterization of cDNAs corresponding to an additional member of the
human histone deacetylase gene family. J Biol Chem 272:2800128007[Abstract/Free Full Text]
-
Wyszomierski SL, Yeh J, Rosen JM 1999 Glucocorticoid
receptor/signal transducer and activator of transcription 5 (STAT5)
interactions enhance STAT5 activation by prolonging STAT5 DNA binding
and tyrosine phosphorylation. Mol Endocrinol 13:330343[Abstract/Free Full Text]
-
Fryer CJ, Kinyamu HK, Rogatsky I, Garabedian MJ, Archer TK 2000 Selective activation of the glucocorticoid receptor by steroid
antagonists in human breast cancer and osteosarcoma cells. J Biol
Chem 275:1777117777[Abstract/Free Full Text]
-
Kowenz-Leutz E, Twamley G, Ansieau S, Leutz A 1994 Novel
mechanism of C/EBP ß (NF-M) transcriptional control: activation
through derepression. Genes Dev 8:27812791[Abstract]
-
Williams SC, Baer M, Dillner AJ, Johnson PF 1995 CRP2 (C/EBP
ß) contains a bipartite regulatory domain that controls
transcriptional activation, DNA binding and cell specificity. EMBO J 14:31703183[Abstract]
-
Nakajima T, Kinoshita S, Sasagawa T, Sasaki K, Naruto M,
Kishimoto T, Akira S 1993 Phosphorylation at threonine-235 by a
ras-dependent mitogen-activated protein kinase cascade is essential for
transcription factor NF-IL6. Proc Natl Acad Sci USA 90:22072211[Abstract]
-
Mink S, Kerber U, Klempnauer KH 1996 Interaction of C/EBPß
and v-Myb is required for synergistic activation of the mim-1 gene. Mol
Cell Biol 16:13161325[Abstract]
-
Burk O, Mink S, Ringwald M, Klempnauer KH 1993 Synergistic activation of the chicken mim-1 gene by v-myb and C/EBP
transcription factors. EMBO J 12:20272038[Abstract]
-
Ness SA, Kowenz-Leutz E, Casini T, Graf T, Leutz A 1993 Myb and NF-M: combinatorial activators of myeloid genes in heterologous
cell types. Genes Dev 7:749759[Abstract]
-
Oelgeschlager M, Krieg J, Luscher-Firzlaff JM, Luscher B 1995 Casein kinase II phosphorylation site mutations in c-Myb affect DNA
binding and transcriptional cooperativity with NF-M. Mol Cell Biol 15:59665974[Abstract]
-
Mink S, Haenig B, Klempnauer KH 1997 Interaction and
functional collaboration of p300 and C/EBPß. Mol Cell Biol 17:66096617[Abstract]
-
Oelgeschlager M, Janknecht R, Krieg J, Schreek S, Luscher B 1996 Interaction of the co-activator CBP with Myb proteins: effects on
Myb-specific transactivation and on the cooperativity with NF-M. EMBO J 15:27712780[Abstract]
-
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]
-
Sheppard KA, Rose DW, Haque ZK, Kurokawa R, McInerney E,
Westin S, Thanos D, Rosenfeld MG, Glass CK, Collins T 1999 Transcriptional activation by NF-
B requires multiple coactivators.
Mol Cell Biol 19:63676378[Abstract/Free Full Text]
-
Soutoglou E, Papafotiou G, Katrakili N, Talianidis I 2000 Transcriptional activation by hepatocyte nuclear factor-1 requires
synergism between multiple coactivator proteins. J Biol Chem 275:1251512520[Abstract/Free Full Text]
-
Liu X, Robinson GW, Wagner KU, Garrett L, Wynshaw-Boris A,
Hennighausen L 1997 Stat5a is mandatory for adult mammary gland
development and lactogenesis. Genes Dev 11:179186[Abstract]
-
Kowenz-Leutz E, Leutz A 1999 A C/EBP ß isoform recruits the
SWI/SNF complex to activate myeloid genes. Mol Cell 4:735743[Medline]
-
Lee KF, DeMayo FJ, Atiee SH, Rosen JM 1988 Tissue-specific
expression of the rat ß-casein gene in transgenic mice. Nucleic Acids
Res 16:10271041[Abstract]
-
Grimm SL, Nordeen SK 1999 A composite enhancer element
directing tissue-specific expression of mouse mammary tumor virus
requires both ubiquitous and tissue- restricted factors. J Biol
Chem 274:1279012796[Abstract/Free Full Text]
-
Nordeen SK 1988 Luciferase reporter gene vectors for analysis
of promoters and enhancers. Biotechniques 6:454458[Medline]
-
Kazansky AV, Raught B, Lindsey SM, Wang YF, Rosen JM 1995 Regulation of mammary gland factor/Stat5a during mammary gland
development. Mol Endocrinol 9:15981609[Abstract]
-
Rusconi S, Severne Y, Georgiev O, Galli I, Wieland S 1990 A
novel expression assay to study transcriptional activators. Gene 89:211221[CrossRef][Medline]
-
Sambrook J, Fritsch EF, Maniatis T 1989 Molecular
Cloning, A Laboratory Manual. Cold Spring Harbor Press, Cold Spring
Harbor, NY