Cooperative Mechanism of Transcriptional Activation by a Cyclic AMP-response Element Modulator alpha  Mutant Containing a Motif for Constitutive Binding to CREB-binding Protein*

Daniel M. FassDagger, Johanna C. CraigDagger, 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cyclic AMP-response element modulator alpha  (CREMalpha ) 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). CREMalpha lacks two glutamine-rich Q regions that, in CREB, are thought to be necessary for transcriptional activation. Nevertheless, protein kinase A stimulation induces CREMalpha 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 CREMalpha to allow constitutive binding to the coactivator CREB-binding protein. This mutant, CREMalpha DIEDML, constitutively activated the PEPCK promoter. By engineering the leucine zipper regions of CREMalpha DIEDML and CREBDIEDML to direct their patterns of dimerization, we found that only CREMalpha 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 CREMalpha 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 CREMalpha DIEDML. Furthermore, a GAL4-C/EBPalpha fusion protein and CREMalpha 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 CREMalpha to activate transcription despite its lack of Q regions.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 CREMalpha isoform contains a KID but lacks both Q regions (2). CREMalpha represses transcription induced by PKA-activated CREB at a minimal promoter containing the somatostatin CRE (2). This repression may be mediated by CREB-CREMalpha heterodimers, which are only weak activators, or by CREMalpha homodimers that cannot activate transcription at the somatostatin CRE (8). These data form the bulk of the evidence for the hypothesis that CREMalpha is a repressor of cAMP-induced transcription. However, tests of this hypothesis at native promoters have produced mixed results. For example, CREMalpha 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 CREMalpha . The most direct way to do this was to study the activity of CREMalpha in isolation from other effectors of PKA at the PEPCK promoter. Thus, we developed a constitutively active PKA-independent mutant of CREMalpha , CREMalpha 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 CREMalpha 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 CREMalpha binds with high affinity to the PEPCK CRE. Finally, we used the CREMalpha 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 CREMalpha homodimers can result in the activation of complex promoters containing binding sites for C/EBP.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta -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 CREMalpha (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 CREMalpha 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 CREMalpha ZIP3, lysine replaces glutamate 207, and glutamate replaces lysine 221. In CREMalpha ZIP4, glutamate replaces arginine 202, and lysine replaces glutamate 216. GAL4-C/EBPalpha is Galpha 2 as described previously (17). A-C/EBP is described (see Ref. 18). RSV-beta -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/EBPalpha 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-CREMalpha DIEDML-(1-171) was assayed as described previously (19), using CBP C-20 antibody (Santa Cruz Biotechnology).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 CREMalpha . Because the relevant regions of CREB and CREMalpha are identical, we substituted the residues DIEDML for RRPSYR in CREMalpha to generate the mutant CREMalpha 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 CREMalpha 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, CREMalpha DIEDML produced an ~4-5-fold activation of the PEPCK promoter, whereas wild-type CREMalpha produced only marginal activation. We also performed a glutathione S-transferase pull-down assay to confirm that CREMalpha DIEDML binds CBP (data not shown). Thus, CREMalpha DIEDML constitutively activates PEPCK transcription by recruiting CBP.



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Fig. 1.   Constitutively active CREMalpha mutant, CREMalpha DIEDML, activates the PEPCK promoter under basal conditions in Hep G2 cells. A, comparison of part of the CBP interaction domains in CREB and CREMalpha and in the mutants CREBDIEDML and CREMalpha DIEDML. B, activation of PEPCK-LUC reporter gene by CREMalpha and CREMalpha DIEDML. Hep G2 cells were transfected with 5 µg of PEPCK-LUC and 6 µg of CREMalpha or CREMalpha DIEDML.

We next tested the efficacy and specificity of CREMalpha DIEDML. To measure efficacy, we compared the ability of CREBDIEDML and CREMalpha DIEDML to activate the PEPCK promoter. Fig. 2A shows that CREMalpha DIEMDL produced a slightly greater activation than CREBDIEDML at all doses. Thus, despite lacking the Q regions, CREMalpha appears to be a better activator than CREB in the context of the PEPCK promoter. To test specificity, we measured the activity of CREMalpha DIEDML at the c-fos promoter. Fig. 2B shows that CREMalpha DIEDML had little effect on c-fos reporter gene transcription, whereas CREBDIEDML produced ~9-fold activation. Thus, it appears that CREMalpha DIEDML induces transcription only from promoters at which wild-type CREMalpha can produce activation. These data suggest that CREMalpha DIEDML can be an effective and specific reagent for testing the mechanism of transcriptional activation by CREMalpha .



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Fig. 2.   Comparison of the activities of CREMalpha 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, CREMalpha , or CREMalpha DIEDML (B), or as indicated (A).

In principle, the differences in the activity of CREMalpha 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 CREMalpha , CREB, and C/EBPalpha 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 CREMalpha , CREB, and C/EBPalpha 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.



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Fig. 3.   Binding of CREMalpha , CREB, and C/EBPalpha to the PEPCK CRE. Representative examples of the changes in fluorescence polarization in milliPolarization (mP) units (20) due to the binding of CREMalpha (), CREB (open circle ), and C/EBPalpha (triangle ) are shown. In these experiments, Kd values were 1.2 nM (CREMalpha ), 1 nM (CREB), and 1.5 nM (C/EBPalpha ).

Although it has been presumed that the transfection of CREMalpha DIEDML into Hep G2 cells results in the formation of stable homodimers, it is possible that CREMalpha DIEDML heterodimerizes with endogenous CREB. To test the ability of CREMalpha DIEDML homodimers and heterodimers to activate the PEPCK promoter, we made two leucine zipper mutants of CREMalpha 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 CREMalpha DIEDML homodimer (CREMalpha DIEDMLZIP3/CREMalpha DIEDMLZIP4) can activate the PEPCK promoter to the same degree as CREMalpha DIEDML containing a wild-type leucine zipper. CREMalpha DIEDML/CREBDIEDML heterodimers (CREMalpha DIEDMLZIP3/CREBDIEDMLZIP4 or CREMalpha DIEDMLZIP4/CREBDIEDMLZIP3) produced only marginal activation. Thus, CREMalpha 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.



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Fig. 4.   CREMalpha 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 CREMalpha DIEDML ZIP3 and ZIP4 mutants. In this panel, CREM and CREB refer to CREMalpha DIEDML and CREBDIEDML. Hep G2 cells were transfected with 100 ng of PEPCK-LUC reporter, 100 ng of CREBDIEDML or CREMalpha DIEDML, and 50 ng each of ZIP3 or ZIP4 mutant.

One possible explanation for the context specificity of the function of CREMalpha 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 CREMalpha to produce activation, we tested the ability of CREMalpha 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 CREMalpha DIEDML. In contrast, deleting both groups 3 and 2 greatly diminished activation by CREMalpha DIEDML. Moreover, CREMalpha DIEDML did not activate a PEPCK promoter mutant lacking all regulatory elements upstream of the CRE. Thus, CREMalpha DIEDML must cooperate with transcription factors that bind to the group 2 region to produce full activation of the PEPCK promoter.



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Fig. 5.   PEPCK promoter regulatory group 2 is required for activation by CREMalpha 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 CREMalpha 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 CREMalpha 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 CREMalpha 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 CREMalpha DIEDML to activate the PEPCK promoter. Block mutation of the P3I site partially reduced activation by CREMalpha 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 CREMalpha DIEDML (data not shown). Thus, CREMalpha activates transcription in cooperation with factors that bind to the P3I and P4 sites of the PEPCK promoter.



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Fig. 6.   Two sites in regulatory group 2 of the PEPCK promoter are required for activation by CREMalpha DIEDML. Cells were transfected with 5 µg of wild-type or mutant reporter plasmids and 6 µg of CREMalpha DIEDML. Fold activation values were determined by normalizing the relative luciferase activities in the presence of CREMalpha 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 CREMalpha 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 CREMalpha DIEDML. Thus, C/EBP proteins appear to be required for the activation of the PEPCK promoter by CREMalpha . Of the various C/EBP proteins, C/EBPalpha has been reported to be most critical for activation by cAMP (23). To test whether C/EBPalpha could cooperate with CREMalpha 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/EBPalpha and CREMalpha DIEDML than by each factor alone. These data suggest that the factor that cooperates with CREMalpha to allow activation of the PEPCK promoter is C/EBPalpha .



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Fig. 7.   CREMalpha DIEDML cooperates with C/EBPalpha to activate the PEPCK promoter. A, the dominant negative C/EBP mutant (A-C/EBP) inhibits activation of the PEPCK promoter by CREMalpha DIEDML. Hep G2 cells were transfected with 75 ng of PEPCK-LUC reporter plasmid, 75 ng of CREMalpha DIEDML, and 375 ng of A-C/EBP. B, schematic of the reporter gene (-109G3-CAT) used to test for cooperativity between GAL4-C/EBPalpha and CREMalpha DIEDML. C, CREMalpha DIEDML and GAL4-C/EBPalpha cooperatively activate the reporter gene shown in B. Hep G2 cells were transfected with 7 µg of -109G3-CAT reporter plasmid, 5 µg of CREMalpha DIEDML, and 10 ng of GAL4-C/EBPalpha .



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study, we examined the mechanism by which CREMalpha activates PEPCK transcription. To do this, we developed a constitutively active mutant of CREMalpha , CREMalpha DIEDML. CREMalpha 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 CREMalpha DIEDML (Fig. 1) could have been produced by CREMalpha DIEDML homodimers or by heterodimers of CREMalpha 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 CREMalpha DIEDML and CREBDIEDML to direct their pattern of dimerization. Also, we tested whether activation of PEPCK transcription by CREMalpha DIEDML depended on cooperative interactions with other factors that bind the PEPCK promoter.

Our finding that CREMalpha 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 CREMalpha DIEDML (Fig. 2A), and CREMalpha 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 CREMalpha DIEDML. Thus, the function of the Q regions appears to be highly dependent on promoter context.

CREMalpha 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 CREMalpha is not sufficient for activation of the PEPCK promoter. Rather, it appears that CREMalpha must cooperate with C/EBPalpha to achieve full activation (Fig. 7). Perhaps C/EBPalpha also interacts with CBP, either directly or through an intermediate protein. This interaction might serve to stabilize the binding of CBP with CREMalpha at the PEPCK promoter as has been proposed for C/EBPbeta and Myb at the Mim-1 promoter (26). Further work will be required to elucidate the mechanism of cooperation between CREMalpha and C/EBPalpha at the PEPCK promoter.

Our data with CREMalpha DIEDML led us to propose the model shown in Fig. 8 for the mechanism of activation of the PEPCK promoter. In this model, a CREMalpha homodimer binds to the PEPCK CRE. Direct contacts between this homodimer and the basal transcriptional machinery are not required. Phosphorylation of CREMalpha leads to the recruitment of CBP. In addition, the binding of two C/EBPalpha dimers at the P3I and P4 sites appears to be required for transcriptional activation. C/EBPalpha may help to further stabilize the interaction of CBP with CREMalpha . The model implies that the characterization of CREMalpha as a transcriptional repressor may pertain only to certain promoters. At some promoters, cooperative interactions with factors such as C/EBPalpha may allow CREMalpha to function as a transcriptional activator.



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Fig. 8.   Model of cooperative activation of the PEPCK promoter by CREMalpha and C/EBPalpha . 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 CREMalpha may be able to activate any promoter containing CRE and C/EBPalpha binding sites. However, the c-fos promoter contains a C/EBP binding site (27), and it is not activated by CREMalpha 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 CREMalpha can serve as an activator. Certainly cooperative activation by CREMalpha and C/EBPalpha is very much dependent on promoter context. It is possible, however, that CREMalpha and C/EBPalpha 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/EBPalpha and CREMalpha 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.

Dagger 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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]


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