Cooperative Mechanism of Transcriptional Activation by a Cyclic
AMP-response Element Modulator
Mutant Containing a Motif for
Constitutive Binding to CREB-binding Protein*
Daniel M.
Fass
,
Johanna C.
Craig
,
Soren
Impey, and
Richard H.
Goodman§
From the Vollum Institute, Oregon Health Sciences University,
Portland, Oregon 97201
Received for publication, September 10, 2000, and in revised form, November 21, 2000
 |
ABSTRACT |
Cyclic AMP-response element modulator
(CREM
) is a transcription factor that is highly related to
cAMP-response element-binding protein (CREB) but represses cAMP-induced
gene expression from simple artificial promoters containing a
cAMP-response element (CRE). CREM
lacks two glutamine-rich Q regions
that, in CREB, are thought to be necessary for transcriptional
activation. Nevertheless, protein kinase A stimulation induces
CREM
to activate the complex native promoter in the
phosphoenolpyruvate carboxykinase (PEPCK) gene. To study this
phenomenon in the absence of protein kinase A stimulation, we
introduced a mutation into CREM
to allow constitutive binding to the
coactivator CREB-binding protein. This mutant, CREM
DIEDML, constitutively activated the PEPCK
promoter. By engineering the leucine zipper regions of
CREM
DIEDML and CREBDIEDML to direct their
patterns of dimerization, we found that only CREM
DIEDML homodimers fully activated the PEPCK promoter. By using a series of
deletion and block mutants of the PEPCK promoter, we found that
activation by CREM
DIEDML depended on the CRE and two
CCAAT/enhancer-binding protein (C/EBP) sites. A dominant negative
inhibitor of C/EBP, A-C/EBP, suppressed activation by
CREM
DIEDML. Furthermore, a GAL4-C/EBP
fusion protein
and CREM
DIEDML cooperatively activated a promoter
containing three GAL4 sites and the PEPCK CRE. Thus, we propose that
the C/EBP sites in the PEPCK promoter allow CREM
to activate
transcription despite its lack of Q regions.
 |
INTRODUCTION |
The cyclic AMP-response element modulator
(CREM)1 gene is alternatively
spliced to generate multiple transcription factors that mediate the
regulation of transcription by cAMP (reviewed in Ref. 1). These CREM
factors form homodimers and heterodimers with the related protein CREB
that bind specifically to CREs (TGACGTCA) via their conserved
C-terminal basic leucine zipper (bZIP) domains (2). Three domains
encoded by the CREM and CREB genes contribute to transcriptional
activation: the kinase-inducible domain (KID) and two glutamine-rich
regions designated as Q-1 and Q-2 (3). Phosphorylation of a serine
residue within the KID by PKA allows CREB and CREM to bind to the
coactivator CREB-binding protein (CBP) (4, 5). The Q regions are
thought to interact with components of the basal transcriptional
machinery (6). In CREB, the deletion of the Q-2 region prevents
transcriptional activation (7). Moreover, CREM isoforms that lack Q
regions have been shown to act as repressors of cAMP-induced
transcription (2). These data have lead to the hypothesis that Q
regions are required for CREB-mediated transcriptional activation.
The CREM
isoform contains a KID but lacks both Q regions (2).
CREM
represses transcription induced by PKA-activated CREB at a
minimal promoter containing the somatostatin CRE (2). This repression
may be mediated by CREB-CREM
heterodimers, which are only weak
activators, or by CREM
homodimers that cannot activate transcription
at the somatostatin CRE (8). These data form the bulk of the evidence
for the hypothesis that CREM
is a repressor of cAMP-induced
transcription. However, tests of this hypothesis at native promoters
have produced mixed results. For example, CREM
has been shown to be
a repressor at the native c-fos and insulin promoters (9, 10)
but activates transcription from the PEPCK promoter (11).
The PEPCK gene has a complex promoter that contains multiple elements
to allow regulation by a variety of hormone-triggered signaling
pathways and tissue-specific factors (reviewed in Ref. 12). In
addition, cAMP induces transcription via multiple PEPCK promoter
elements (13). Several transcription factors have been proposed to be
involved in the cAMP response: 1) CREB and CREM proteins, which can
bind to a nonconsensus CRE (TTACGTCA) ~85 base pairs upstream of the
PEPCK transcription start site; 2) CCAAT/enhancer-binding proteins
(C/EBPs), which can bind to the CRE and three upstream sites;
and 3) c-Jun, which can bind within the region encompassing the
C/EBP sites. All of the above factors are necessary for the full cAMP
response at the PEPCK promoter, suggesting that cooperativity among the
factors is the crucial aspect of the activation mechanism.
In this paper, we examined the mechanism of activation of PEPCK
transcription by CREM
. The most direct way to do this was to study
the activity of CREM
in isolation from other effectors of PKA at the
PEPCK promoter. Thus, we developed a constitutively active
PKA-independent mutant of CREM
, CREM
DIEDML, that
contains a six-amino acid substitution within the KID (RRPSYR to
DIEDML). In the related CREB protein, this substitution has been shown to confer constitutive binding to CBP, resulting in constitutive transcriptional activity (14). We tested whether the CREM
mutant could activate transcription from the PEPCK promoter as a homodimer or
whether heterodimerization with another bZIP factor is required. Also,
we used a fluorescence polarization equilibrium binding assay to
demonstrate that CREM
binds with high affinity to the PEPCK CRE.
Finally, we used the CREM
mutant to ask whether its transcriptional
activity depended on other factors that bind to the PEPCK promoter. Our
findings led us to propose that the recruitment of CBP to CREM
homodimers can result in the activation of complex promoters containing
binding sites for C/EBP.
 |
EXPERIMENTAL PROCEDURES |
Cell Culture and Transfections--
Hep G2 cells were grown as
described previously (11) except that heat-inactivated fetal bovine
serum was used in most experiments. Cells were seeded onto five 10-cm
plates (for experiments shown in Figs. 1, 2A, 4, 5, and
6C) or five 24-well plates (for experiments shown in Figs.
2B, 3, and 6A) per T75 flask. Transfections were performed with calcium phosphate (Life Technologies, Inc.), TransFast (Promega) (Fig. 2B only), or FuGENE 6 (Roche Molecular
Biochemicals) (Figs. 3 and 6A only) according to the
manufacturer's instructions. All transfection methods produced
equivalent results. Cells were harvested after incubation for 48 h. In the experiment shown in Fig. 2B, cells were
serum-starved for the final 16 h of incubation before harvesting.
Reporter gene activities were determined as described previously (14).
Relative luciferase (LUC) or chloramphenicol acetyltransferase (CAT)
activities were determined by normalization to
-galactosidase
activity (values are given as the mean ± S.D. or the mean ± S.E. in Fig. 3). All experiments were repeated at least three times.
Plasmids--
The reporter genes
490PEPCK-LUC, fos-LUC,
and
109G3-CAT have been described previously (15-17). Block mutants
of
490PEPCK-LUC were made with the QuikChange site-directed
mutagenesis kit (Stratagene) using mutations described previously (13).
Mouse CREM
(2) was cloned into the expression vector pRcRSV
(Invitrogen). RSV-CREB and the DIEDML mutation have been
described previously (14). The ZIP3 and ZIP4 mutations were made in
CREM
DIEDML and CREBDIEDML with the
QuikChange kit. In CREB ZIP3, lysine replaces glutamate 319, and
glutamate replaces lysine 333. In CREB ZIP4, glutamate replaces
arginine 314, and lysine replaces glutamate 328. In CREM
ZIP3,
lysine replaces glutamate 207, and glutamate replaces lysine 221. In
CREM
ZIP4, glutamate replaces arginine 202, and lysine replaces
glutamate 216. GAL4-C/EBP
is G
2 as described previously (17).
A-C/EBP is described (see Ref. 18). RSV-
-GAL was used for
normalization for transfection efficiency.
Fluorescence Polarization Measurements--
Fluorescence
polarization measurements were made as described previously (14) with
the following exceptions. The 5'-fluoresceinated oligonucleotide
corresponding to the top strand of the PEPCK CRE (5'-CCTTACGTCAGCCCCCTGACGTAAGG-3') was purchased from Life Technologies, Inc. The CRE is in boldface. The binding reactions in 1 ml of solution contained 1 nM
fluoresceinated oligonucleotide in 25 mM Tris-HCl, pH 7.6, 50 mM NaCl, 0.5 mM EDTA, 5% glycerol, 6 µg
of bovine serum albumin, and 10 mM MgCl2.
C/EBP
protein was a generous gift of Dr. Peter Johnson (Frederick
Cancer Research and Development Center, Frederick, MD).
Glutathione S-Transferase Pull-down Assay--
The binding of
CBP in HeLa nuclear extracts to glutathione
S-transferase-CREM
DIEDML-(1-171) was assayed
as described previously (19), using CBP C-20 antibody (Santa Cruz Biotechnology).
 |
RESULTS |
Cardinaux et al. (14) recently developed a
constitutively active CREB mutant (CREBDIEDML). This mutant
(Fig. 1A) was made by
replacing six residues (RRPSYR, which includes S133)
from the KID domain of CREB with six residues (DIEDML) from the sterol
response element-binding protein, a transcription factor that binds CBP
in the absence of phosphorylation. Whereas wild-type CREB must be
phosphorylated at Ser133 to bind to CBP, the
substitution of the DIEDML motif conferred constitutive
phosphorylation-independent binding. CREBDIEDML activated transcription from CRE-containing reporter genes under basal conditions in a variety of cell types. Thus, the activity of
CREBDIEDML could be studied in isolation from other
activities stimulated by PKA. We sought to develop a similar mutant of
CREM
. Because the relevant regions of CREB and CREM
are
identical, we substituted the residues DIEDML for RRPSYR in CREM
to
generate the mutant CREM
DIEDML. We tested the activity
of this mutant with a reporter gene driven by 490 base pairs of the
PEPCK promoter upstream from the transcription start site (
490
PEPCK). It has been demonstrated that the PEPCK promoter can be
activated by wild-type CREM
in the presence of cotransfected PKA
catalytic subunit in the Hep G2 human liver-derived cell line (11).
Fig. 1B shows that, in the absence of cotransfected PKA,
CREM
DIEDML produced an ~4-5-fold activation of the
PEPCK promoter, whereas wild-type CREM
produced only marginal
activation. We also performed a glutathione S-transferase
pull-down assay to confirm that CREM
DIEDML binds CBP
(data not shown). Thus, CREM
DIEDML constitutively
activates PEPCK transcription by recruiting CBP.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 1.
Constitutively active CREM
mutant, CREM DIEDML,
activates the PEPCK promoter under basal conditions in Hep G2
cells. A, comparison of part of the CBP interaction
domains in CREB and CREM and in the mutants CREBDIEDML
and CREM DIEDML. B, activation of PEPCK-LUC
reporter gene by CREM and CREM DIEDML. Hep G2 cells
were transfected with 5 µg of PEPCK-LUC and 6 µg of CREM or
CREM DIEDML.
|
|
We next tested the efficacy and specificity of
CREM
DIEDML. To measure efficacy, we compared the ability
of CREBDIEDML and CREM
DIEDML to activate the
PEPCK promoter. Fig. 2A shows
that CREM
DIEMDL produced a slightly greater activation
than CREBDIEDML at all doses. Thus, despite lacking the Q
regions, CREM
appears to be a better activator than CREB in the
context of the PEPCK promoter. To test specificity, we measured the
activity of CREM
DIEDML at the c-fos promoter. Fig.
2B shows that CREM
DIEDML had little effect on
c-fos reporter gene transcription, whereas CREBDIEDML produced ~9-fold activation. Thus, it appears that
CREM
DIEDML induces transcription only from promoters at
which wild-type CREM
can produce activation. These data suggest that
CREM
DIEDML can be an effective and specific reagent for
testing the mechanism of transcriptional activation by CREM
.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 2.
Comparison of the activities of
CREM DIEDML and
CREBDIEDML at the PEPCK (A) and
c-fos (B) promoters. Hep G2 cells were
transfected with 5 µg of PEPCK-LUC (A) or 80 ng of fos-LUC
(B) and 160 ng of CREB, CREBDIEDML, CREM , or
CREM DIEDML (B), or as indicated
(A).
|
|
In principle, the differences in the activity of
CREM
DIEDML and CREBDIEDML at the PEPCK
promoter could be due to differences in their ability to bind the PEPCK
CRE. Electrophoretic mobility shift assays have demonstrated that
CREM
, CREB, and C/EBP
can all bind to the PEPCK CRE (11, 15), but
the quantitative accuracy of these assays may be compromised by the
separation of bound and free transcription factors during gel
electrophoresis. Thus, we used a fluorescence polarization assay (20)
to determine the affinities of these interactions under equilibrium
conditions in solution. Fig. 3 shows that
CREM
, CREB, and C/EBP
all bind to the PEPCK CRE with
Kd = ~1 nM. Thus, all three
transcription factors have the ability to interact with the PEPCK CRE
with similarly high affinity.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 3.
Binding of CREM ,
CREB, and C/EBP to the PEPCK CRE.
Representative examples of the changes in fluorescence polarization in
milliPolarization (mP) units (20) due to the binding
of CREM ( ), CREB ( ), and C/EBP ( ) are shown. In
these experiments, Kd values were 1.2 nM
(CREM ), 1 nM (CREB), and 1.5 nM
(C/EBP ).
|
|
Although it has been presumed that the transfection of
CREM
DIEDML into Hep G2 cells results in the formation of
stable homodimers, it is possible that CREM
DIEDML
heterodimerizes with endogenous CREB. To test the ability of
CREM
DIEDML homodimers and heterodimers to activate the
PEPCK promoter, we made two leucine zipper mutants of
CREM
DIEDML and CREBDIEDML termed ZIP3 and
ZIP4. ZIP3 and ZIP4 contain mutations that are designed to cause them
to preferentially associate as heterodimers (8). The mechanism of
heterodimerization between ZIP3 and ZIP4 mutants is explained in Fig.
4A, which shows these
mutations in the context of the crystal structure of the CREB
bZIP·CRE complex (21). As indicated, the substitution of the
charged residues in the e and g positions of the leucine zipper results in electrostatic repulsion except in the context of the ZIP3/ZIP4 heterodimer. Thus, by cotransfecting the ZIP3 and ZIP4 mutants, we can ensure the formation of the intended dimers. Fig. 4B shows that only the CREM
DIEDML
homodimer
(CREM
DIEDMLZIP3/CREM
DIEDMLZIP4) can
activate the PEPCK promoter to the same degree as
CREM
DIEDML containing a wild-type leucine zipper.
CREM
DIEDML/CREBDIEDML heterodimers
(CREM
DIEDMLZIP3/CREBDIEDMLZIP4 or
CREM
DIEDMLZIP4/CREBDIEDMLZIP3) produced
only marginal activation. Thus, CREM
can activate the PEPCK promoter
as a homodimer. Therefore, the requirement of Q regions for
CREM-mediated transcriptional activation is highly dependent on
promoter context.

View larger version (48K):
[in this window]
[in a new window]
|
Fig. 4.
CREM DIEDML activates the
PEPCK promoter as a homodimer. A, ZIP3 and ZIP4 mutants
of the CREB bZIP. Mutations are depicted in the context of the crystal
structure of the CREB bZIP dimer bound to the somatostatin CRE (21).
The left panel shows the wild-type dimer. The ZIP3 and ZIP4
mutations should result in charge repulsions in ZIP3/ZIP3 and ZIP4/ZIP4
homodimers (middle two panels) and in heterodimers of ZIP3
or ZIP4 with endogenous CREB family proteins. In contrast, the
favorable electrostatic interactions of the wild-type zippers should be
present in the ZIP3/ZIP4 heterodimer (right panel).
Analogous mutations were made in the highly related CREM bZIP.
B, activation of the PEPCK promoter by
CREBDIEDML and CREM DIEDML ZIP3 and ZIP4
mutants. In this panel, CREM and CREB refer to
CREM DIEDML and CREBDIEDML. Hep G2 cells were
transfected with 100 ng of PEPCK-LUC reporter, 100 ng of
CREBDIEDML or CREM DIEDML, and 50 ng each of
ZIP3 or ZIP4 mutant.
|
|
One possible explanation for the context specificity of the function of
CREM
is that it can only activate transcription in cooperation with
other factors. The
490 PEPCK promoter contains three groups of
cis-regulatory elements (Fig.
5A) (reviewed in Ref. 12). To
determine which factors cooperate with CREM
to produce activation,
we tested the ability of CREM
DIEDML to activate a series
of PEPCK promoter deletion mutants. Fig. 5B shows that deleting group 3 elements had no effect on reporter gene expression levels produced by CREM
DIEDML. In contrast, deleting
both groups 3 and 2 greatly diminished activation by
CREM
DIEDML. Moreover, CREM
DIEDML did not
activate a PEPCK promoter mutant lacking all regulatory elements
upstream of the CRE. Thus, CREM
DIEDML must cooperate
with transcription factors that bind to the group 2 region to produce
full activation of the PEPCK promoter.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 5.
PEPCK promoter regulatory group 2 is required
for activation by CREM DIEDML.
A, a schematic of three groups of regulatory elements in the
PEPCK promoter (reviewed in Ref. 22). B, activation
of PEPCK promoter deletion mutants by CREM DIEDML.
Mutants were made by deleting group 3 (PEPCK groups 2 + 1),
groups 3 and 2 (PEPCK group 1), or groups 3 and 2 and site
P1 (PEPCK CRE). Hep G2 cells were transfected with 5 µg of
PEPCK reporter wild-type or deletion mutants and 6 µg of
CREM DIEDML. IRE, insulin regulatory element;
GRE, glucocorticoid regulatory element; TRE,
thyroid hormone regulatory element.
|
|
Five cis-regulatory elements have been identified in the
group 2 region of the PEPCK promoter (Fig. 5A) (reviewed in
Ref. 12), three of which (P3I, P3II, and P4) are required for full activation by cAMP (13). To determine which of these elements binds factors that cooperate with CREM
DIEDML, we used
block mutants that were previously characterized (13). The block
mutations prevent the binding of rat liver nuclear proteins and greatly diminish the response of the promoter to the catalytic subunit of PKA.
Fig. 6 shows the effect of the block
mutations on the ability of CREM
DIEDML to activate the
PEPCK promoter. Block mutation of the P3I site partially reduced
activation by CREM
DIEDML. The activation was further
reduced when both the P3I and P4 sites were mutated. In contrast, block
mutations in the P2 and P3II sites had little effect on the activation
by CREM
DIEDML (data not shown). Thus, CREM
activates
transcription in cooperation with factors that bind to the P3I and P4
sites of the PEPCK promoter.

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 6.
Two sites in regulatory group 2 of the PEPCK
promoter are required for activation by
CREM DIEDML. Cells were
transfected with 5 µg of wild-type or mutant reporter plasmids and 6 µg of CREM DIEDML. Fold activation values were
determined by normalizing the relative luciferase activities in the
presence of CREM DIEDML to control activity of the
wild-type PEPCK promoter.
|
|
The only transcription factors known to bind the P3I and P4 sites in
the PEPCK promoter are members of the C/EBP family (22). To test
whether C/EBP proteins are required for activation of the PEPCK
promoter by CREM
DIEDML, we used a dominant negative inhibitor of C/EBP, A-C/EBP (18). Fig.
7A shows that A-C/EBP suppressed the stimulatory effect of CREM
DIEDML. Thus,
C/EBP proteins appear to be required for the activation of the PEPCK promoter by CREM
. Of the various C/EBP proteins, C/EBP
has been reported to be most critical for activation by cAMP (23). To test
whether C/EBP
could cooperate with CREM
DIEDML, we
used a reporter gene driven by three binding sites for the yeast
transcription factor GAL4 and a partial PEPCK promoter containing only
the CRE (Fig. 7B) (termed
109G3 in Ref. 17). Fig.
7C shows that
109G3 was activated to a greater degree by
the combination of both Gal4-C/EBP
and CREM
DIEDML
than by each factor alone. These data suggest that the factor that
cooperates with CREM
to allow activation of the PEPCK promoter is
C/EBP
.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 7.
CREM DIEDML cooperates with
C/EBP to activate the PEPCK promoter.
A, the dominant negative C/EBP mutant (A-C/EBP)
inhibits activation of the PEPCK promoter by CREM DIEDML.
Hep G2 cells were transfected with 75 ng of PEPCK-LUC reporter plasmid,
75 ng of CREM DIEDML, and 375 ng of A-C/EBP.
B, schematic of the reporter gene (-109G3-CAT) used to test
for cooperativity between GAL4-C/EBP and CREM DIEDML.
C, CREM DIEDML and GAL4-C/EBP cooperatively
activate the reporter gene shown in B. Hep G2 cells were
transfected with 7 µg of -109G3-CAT reporter plasmid, 5 µg of
CREM DIEDML, and 10 ng of GAL4-C/EBP .
|
|
 |
DISCUSSION |
In this study, we examined the mechanism by which CREM
activates PEPCK transcription. To do this, we developed a
constitutively active mutant of CREM
, CREM
DIEDML.
CREM
DIEDML probably functions by constitutive binding to
CBP as does the related mutant, CREBDIEDML (14). The
phosphorylation-independent activity of these mutants allowed us to
study their activity in isolation from other effectors of PKA at the
PEPCK promoter (e.g. AP1) (24, 25). In principle, the
activation of PEPCK transcription by CREM
DIEDML (Fig. 1) could have been produced by CREM
DIEDML homodimers or by
heterodimers of CREM
DIEDML with endogenous
unphosphorylated CREB. In the latter case, the unphosphorylated CREB
molecule could provide the Q regions capable of contacting the basal
transcriptional machinery, which has been proposed to be a requirement
for CREB-mediated transcriptional activation. We tested these two
possibilities by engineering the leucine zipper regions of
CREM
DIEDML and CREBDIEDML to direct their
pattern of dimerization. Also, we tested whether activation of PEPCK
transcription by CREM
DIEDML depended on cooperative interactions with other factors that bind the PEPCK promoter.
Our finding that CREM
homodimer activates the PEPCK promoter (Fig.
4C) shows that Q regions are not always necessary for transcriptional activation. Indeed, Q regions appear to be detrimental to the activation at the PEPCK promoter. CREBDIEDML is
slightly less active than CREM
DIEDML (Fig.
2A), and CREM
DIEDML/CREBDIEDML heterodimers produce only marginal activation (Fig. 4C). In
contrast, the importance of these regions at some promoters is
indicated by the observation that deletion of the Q-2 region eliminates activation of a somatostatin CRE reporter gene by CREB (7). Moreover,
we found that the c-fos promoter could not be activated by
CREM
DIEDML. Thus, the function of the Q regions appears
to be highly dependent on promoter context.
CREM
DIEDML only marginally activated transcription from
a promoter containing just the PEPCK CRE (Fig. 5) or a PEPCK promoter with mutations in two C/EBP sites (Fig. 6). Thus, the recruitment of
CBP by CREM
is not sufficient for activation of the PEPCK promoter.
Rather, it appears that CREM
must cooperate with C/EBP
to achieve
full activation (Fig. 7). Perhaps C/EBP
also interacts with CBP,
either directly or through an intermediate protein. This interaction
might serve to stabilize the binding of CBP with CREM
at the PEPCK
promoter as has been proposed for C/EBP
and Myb at the Mim-1
promoter (26). Further work will be required to elucidate the mechanism
of cooperation between CREM
and C/EBP
at the PEPCK promoter.
Our data with CREM
DIEDML led us to propose the model
shown in Fig. 8 for the mechanism of
activation of the PEPCK promoter. In this model, a CREM
homodimer
binds to the PEPCK CRE. Direct contacts between this homodimer and the
basal transcriptional machinery are not required. Phosphorylation of
CREM
leads to the recruitment of CBP. In addition, the binding of
two C/EBP
dimers at the P3I and P4 sites appears to be required for
transcriptional activation. C/EBP
may help to further stabilize the
interaction of CBP with CREM
. The model implies that the
characterization of CREM
as a transcriptional repressor may pertain
only to certain promoters. At some promoters, cooperative interactions
with factors such as C/EBP
may allow CREM
to function as a
transcriptional activator.

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 8.
Model of cooperative activation of the PEPCK
promoter by CREM and
C/EBP . This model does not depict the
histone acetyltransferase function of CBP (e.g. Ref. 28),
which is also believed to contribute to gene activation.
GTFs, general transcription factors.
|
|
One prediction that could be made based on our data with the PEPCK
promoter is that CREM
may be able to activate any promoter containing CRE and C/EBP
binding sites. However, the c-fos promoter contains a C/EBP binding site (27), and it is not activated by
CREM
DIEDML (Fig. 2B). The spacing of the
C/EBP binding sites and the CREs differ in the c-fos and PEPCK
promoters, and this difference may be a critical determinant of whether
CREM
can serve as an activator. Certainly cooperative activation by
CREM
and C/EBP
is very much dependent on promoter context. It is
possible, however, that CREM
and C/EBP
may cooperatively activate
other genes.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Richard Hanson and William
Roesler for providing plasmids. We also thank Drs. Peter Johnson and
James Lundblad for the generous gifts of C/EBP
and CREM
proteins.
 |
FOOTNOTES |
*
This work was supported by grants from the National
Institutes of Health.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
These authors contributed equally to this work.
§
To whom correspondence should be addressed: Vollum Inst., Oregon
Health Sciences University, 3181 S. W. Sam Jackson Park Rd., Portland, OR 97201. Tel.: 503-494-5078; Fax: 503-494-4353; E-mail: goodmanr@ohsu.edu.
Published, JBC Papers in Press, November 22, 2000, DOI 10.1074/jbc.M008274200
 |
ABBREVIATIONS |
The abbreviations used are:
CREM, cyclic
AMP-response element modulator;
CREB, cAMP-response element-binding
protein;
CRE, cAMP-response element;
bZIP, basic leucine zipper;
KID, kinase-inducible domain;
PKA, protein kinase A;
CBP, CREB-binding
protein;
PEPCK, phosphoenolpyruvate carboxykinase;
C/EBP, CCAAT/enhancer-binding protein;
CAT, chloramphenicol acetyltransferase;
LUC, luciferase.
 |
REFERENCES |
1.
|
Sassone-Corsi, P.
(1995)
Annu. Rev. Cell Dev. Biol.
11,
355-377[CrossRef][Medline]
[Order article via Infotrieve]
|
2.
|
Foulkes, N. S.,
Borrelli, E.,
and Sassone-Corsi, P.
(1991)
Cell
64,
739-749[Medline]
[Order article via Infotrieve]
|
3.
|
Laoide, B. M.,
Foulkes, N. S.,
Schlotter, F.,
and Sassone-Corsi, P.
(1993)
EMBO J.
12,
1179-1191[Abstract]
|
4.
|
Chrivia, J. C.,
Kwok, R. P. S.,
Lamb, N.,
Hagiwara, M.,
Montminy, M. R.,
and Goodman, R. H.
(1993)
Nature
365,
855-859[CrossRef][Medline]
[Order article via Infotrieve]
|
5.
|
Laurance, M. E.,
Kwok, R. P. S.,
Huang, M. S.,
Richards, J. P.,
Lundblad, J. R.,
and Goodman, R. H.
(1997)
J. Biol. Chem.
272,
2646-2651[Abstract/Free Full Text]
|
6.
|
Ferreri, K.,
Gill, G.,
and Montminy, M.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
1210-1213[Abstract]
|
7.
|
Brindle, P.,
Linke, S.,
and Montminy, M.
(1993)
Nature
364,
821-824[CrossRef][Medline]
[Order article via Infotrieve]
|
8.
|
Loriaux, M. M.,
Brennan, R. G.,
and Goodman, R. H.
(1994)
J. Biol. Chem.
269,
28839-28843[Abstract/Free Full Text]
|
9.
|
Foulkes, N. S.,
Laoide, B. M.,
Schlotter, F.,
and Sassone-Corsi, P.
(1991)
Proc. Natl. Acad. Sci. U. S. A.
88,
5448-5452[Abstract]
|
10.
|
Inada, A.,
Someya, Y.,
Yamada, Y.,
Ihara, Y.,
Kubota, A.,
Ban, N.,
Watanabe, R.,
Tsuda, K.,
and Seino, Y.
(1999)
J. Biol. Chem.
274,
21095-21103[Abstract/Free Full Text]
|
11.
|
Goraya, T. Y.,
Kessler, S. P.,
Stanton, P.,
Hanson, R. W.,
and Sen, G. C.
(1995)
J. Biol. Chem.
270,
19078-19085[Abstract/Free Full Text]
|
12.
|
Hanson, R. W.,
and Reshef, L.
(1997)
Annu. Rev. Biochem.
66,
581-611[CrossRef][Medline]
[Order article via Infotrieve]
|
13.
|
Liu, J.,
Park, E. P.,
Gurney, A. L.,
Roesler, W. J.,
and Hanson, R. W.
(1991)
J. Biol. Chem.
266,
19095-19102[Abstract/Free Full Text]
|
14.
|
Cardinaux, J.-R.,
Notis, J. C.,
Zhang, Q.,
Vo, N.,
Craig, J. C.,
Fass, D. M.,
Brennan, R. G.,
and Goodman, R. H.
(2000)
Mol. Cell. Biol.
20,
1546-1552[Abstract/Free Full Text]
|
15.
|
Park, E. A.,
Gurney, A. L.,
Nizielski, S. E.,
Hakimi, P.,
Cao, Z.,
Moorman, A.,
and Hanson, R. W.
(1993)
J. Biol. Chem.
268,
613-619[Abstract/Free Full Text]
|
16.
|
Matthews, R. P.,
Guthrie, C. R.,
Wailes, L. M.,
Zhao, X.,
Means, A. R.,
and McKnight, G. S.
(1994)
Mol. Cell. Biol.
14,
6107-6116[Abstract]
|
17.
|
Roesler, W. J.,
Crosson, S. M.,
Vinson, C.,
and McFie, P. J.
(1996)
J. Biol. Chem.
271,
8068-8074[Abstract/Free Full Text]
|
18.
|
Krylov, D.,
Olive, M.,
and Vinson, C.
(1995)
EMBO J.
14,
5329-5337[Abstract]
|
19.
|
Zhang, Q.,
Vo, N.,
and Goodman, R. H.
(2000)
Mol. Cell. Biol.
20,
4970-4978[Abstract/Free Full Text]
|
20.
|
Lundblad, J. R.,
Laurance, M.,
and Goodman, R. H.
(1996)
Mol. Endocrinol.
10,
607-612[Abstract]
|
21.
|
Schumacher, M. A.,
Goodman, R. H.,
and Brennan, R. G.
(2000)
J. Biol. Chem.
275,
35242-35247[Abstract/Free Full Text]
|
22.
|
Croniger, C.,
Leahy, P.,
Reshef, L.,
and Hanson, R. W.
(1998)
J. Biol. Chem.
273,
31629-31632[Free Full Text]
|
23.
|
Crosson, S. M.,
and Roesler, W. J.
(2000)
J. Biol. Chem.
275,
5804-5809[Abstract/Free Full Text]
|
24.
|
Gurney, A. L.,
Park, E. A.,
Giralt, M.,
Liu, J.,
and Hanson, R. W.
(1992)
J. Biol. Chem.
267,
18133-18139[Abstract/Free Full Text]
|
25.
|
Roesler, W. J.,
Simard, J.,
Graham, J. G.,
and McFie, P. J.
(1994)
J. Biol. Chem.
269,
14276-14283[Abstract/Free Full Text]
|
26.
|
Mink, S.,
Haenig, B.,
and Klempnauer, K.-H.
(1997)
Mol. Cell. Biol.
17,
6609-6617[Abstract]
|
27.
|
Metz, R.,
and Ziff, E.
(1991)
Oncogene
6,
2165-2178[Medline]
[Order article via Infotrieve]
|
28.
|
Ogryzko, V. V.,
Schiltz, R. L.,
Russanova, V.,
Howard, B. H.,
and Nakatani, Y.
(1996)
Cell
87,
953-959[Medline]
[Order article via Infotrieve]
|
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.