©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The cAMP Response Element Binding Protein Synergizes with Other Transcription Factors to Mediate cAMP Responsiveness (*)

(Received for publication, January 25, 1995)

William J. Roesler (§) Janet G. Graham Richard Kolen Dwight J. Klemm (1) Pamela J. McFie

From the Department of Biochemistry, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 0W0 and Division of Basic Sciences, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The cAMP responsiveness of the promoter for phosphoenolpyruvate carboxykinase (EC 4.1.1.32) is mediated by a synergistic interaction between a complex regulatory region, which binds liver-enriched transcription factors, and a typical cAMP response element (CRE). Although a role for the CRE-binding protein (CREB) in the cAMP-responsiveness of this promoter has been generally assumed, some uncertainty remains due to the observations that several C/EBP-related proteins bind with near equal affinity, relative to CREB, to this particular CRE. Thus, a detailed analysis of the involvement of CREB in this synergism was undertaken in HepG2 cells. Gel mobility shift assays demonstrate that a CRE probe is bound by CREB present in HepG2 cells. Furthermore, we show that a dominant repressor of CREB is able to significantly reduce the cAMP responsiveness of the PEPCK promoter in HepG2 cells. Finally, we demonstrate using a GAL4-CREB fusion protein that CREB is able to synergize with the liver-enriched factors bound upstream on the PEPCK promoter to mediate a liver-specific response to cAMP. Examination of several mutant forms of CREB allow us to conclude that the ``synergy'' domain of CREB resides within amino acid residues 83-203, and that residues 83-145 can mediate a partial synergistic response. This study establishes that CREB is able to synergize with liver-enriched transcription factors to mediate a tissue-specific response to cAMP.


INTRODUCTION

Cyclic AMP is an important intracellular second messenger for a number of hormones and mediates the transcriptional induction of many genes. Promoter analysis of some of these genes has identified DNA sequences, termed cAMP response elements (CREs), (^1)which contain a palindromic sequence 5`-TGACGTCA-3` or a minor variation thereof (reviewed in (1) ). A protein which binds to CREs was identified, termed the CRE-binding protein or CREB(2) , and structure/function analysis of this protein has shown that, like most transcription factors, it is modular in nature. CREB has a basic region-leucine zipper (bZIP) DNA-binding motif, as well as transactivation domains involved both in basal and cAMP-inducible transactivation(3, 4, 5, 6) . While in some experimental systems CREB has been shown to independently mediate a cAMP response, many studies have shown that a single molecule of CREB is incapable of mediating a significant response(7, 8, 9) . In these situations, a strong mediating effect of CREB can be observed if the multiple binding sites are incorporated or if additional promoter sequences are included. This suggests that CREB is able to functionally synergize with itself or, perhaps, with other transcription factors. For example, the promoter of the gene encoding the alpha-subunit of glycoprotein hormones, which is cAMP responsive, contains two tandemly repeated CREs(10) . The c-fos promoter apparently utilizes four different cis elements, including CREB/ATF and activator protein (AP)-1 binding sites, to mediate its cAMP responsiveness(11) . The proenkephalin gene requires several cis elements for an optimal response to cAMP, including binding sites for transcription factors AP-1 and AP-2(12) . More recently, Nitsch et al.(13) showed that the cAMP responsiveness of the tyrosine aminotransferase promoter is mediated by a synergistic interaction between a CRE and a binding site for hepatic nuclear factor 4. Despite these observations, no direct evidence exists which demonstrates the ability of CREB to synergize with other transcription factors to mediate this response.

Our laboratory has been examining the cAMP responsiveness of the gene encoding the cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK). This gene is expressed in kidney and liver, but the magnitude of the transcriptional response to cAMP is much greater in the latter tissue(14) . It has been shown that this liver-specific cAMP response unit consists of two components: a typical CRE, and an upstream, complex regulatory element which binds transcription factors which are enriched in liver(8, 9) . Neither component alone displays significant cAMP enhancer activity, but when together, they produce a strong cAMP response unit but only in a liver cell background. It has been difficult to assign an absolute function for CREB in the cAMP responsiveness of the PEPCK promoter, because C/EBP proteins bind with high affinity to this specific CRE(15, 16) , and at least one C/EBP isoform, C/EBPbeta has been suggested to be a possible mediator of a cAMP response(17) .

In the present study, we provide evidence that CREB, through its binding at the CRE, is one of the transcription factors which mediates the liver-specific, cAMP responsiveness of the PEPCK promoter. Furthermore, we demonstrate using a GAL4-CREB fusion protein that the transactivation domain of CREB, in addition to synergizing with other CREB transactivation domains, is also capable of synergizing with liver-enriched transcription factors to mediate the cAMP responsiveness of the PEPCK promoter.


EXPERIMENTAL PROCEDURES

Materials

DNA-modifying enzymes were purchased from New England Biolabs and U. S. Biochemical Corp. Poly[d(I-C)bulletd(I-C)] was purchased from Pharmacia Biotech. [alpha-P]dCTP (3000 Ci/mmol) and [acetyl-^3H]CoA (10 Ci/mmol) were purchased from DuPont NEN. Tissue culture supplies were from Life Technologies, Inc. HepG2 (ATCC HB8065) cells were acquired from American Type Culture Collection. Synthetic oligonucleotides were purchased from either the Regional DNA Synthesis Laboratory (University of Calgary, Calgary, Alberta) or Bio/Can Scientific.

Transfection Experiments

HepG2 cells were grown in Dulbecco's modified essential medium containing 10% fetal calf serum. Four hours before transfection, the cells were plated to approximately 30% confluency in 10-cm plates. DNA transfections were carried out by the calcium-phosphate precipitation procedure as described in Sambrook et al.(18) . pRSV-beta-galactosidase (the beta-galactosidase gene driven by the Rous sarcoma virus promoter) was co-transfected in all experiments to monitor transfection efficiency. The amount of DNA used in the various transfection experiments was maintained at 25 µg per plate by the addition of the phagemid pTZ18R. Descriptions of the expression vectors for DBP, C/EBPalpha, CREB, KCREB, C/EBP, CHOP, and the catalytic subunit of protein kinase A have been previously described(9, 19, 20) . Cell extracts were prepared 48 h after transfection. beta-Galactosidase assays were carried out as previously described(18) , and CAT assays were performed using the [acetyl-^3H]CoA-based assay(21) .

DNA Protein-binding Assays

The gel mobility shift assays were carried out as described previously(22) . Nuclear extracts from HepG2 cells were produced by the method of Shapiro et al., (23) . The CRE oligonucleotide used in these assays has been described previously (7) and was radiolabeled using [alpha-P]dCTP and the Klenow fragment.

Construction of CAT Reporter Vectors

The construction of -490 PCK-CAT, -68 PCK-CAT, and G4-PEPCK have been described previously(6, 9) . pDR4/GAL4X1 was constructed in a two-step strategy. First, PEPCK promoter 5` deletion mutant -68 PCK-CAT was digested with XbaI, and into this site was ligated a 25-mer double-stranded oligonucleotide containing the recognition site for yeast transcription factor GAL4(24) . This vector was then cut with SmaI, which cuts just upstream of the GAL4 recognition sequence, and into this was cloned a blunt-ended NdeI/Bsu36I fragment of the PEPCK promoter containing the liver-specific region (LSR) (sequences -355 to -205) (25) .

The synthetic promoters -68/GAL4X1 and X3 were created by ligating one and three copies of the GAL4 oligonucleotide described above, respectively, into the XbaI site of the PEPCK promoter 5`-deletion mutant -68 PCK-CAT.

Construction of GAL4/Fusion Protein and Synergism Inhibitor Expression Vectors

The vector expressing a GAL4-CREB fusion protein was created by fusing the DNA-binding domain of GAL4 (amino acids 1-147) (26) to the transactivation domain of rat CREB (amino acids 3-203)(27) , with expression driven by the SV40 early promoter. The GAL4-CREB mutant, termed 133 mut, contains a serine-to-alanine mutation of amino acid 133 of CREB and was produced by site-directed mutagenesis using the Kunkel (28) method. GAL4-CREB mutants Delta82, N145, and kinase-inducible domain (KID) were constructed by introducing restriction sites by site-directed mutagenesis at appropriate positions in the CREB coding sequence. GAL4-ATF-1 was created by fusing the ATF-1 cDNA encoding amino acids 4-225 to the coding region of the DNA binding domain of GAL4. This region of ATF-1 essentially contains all domains except for the bZIP domain(29) . Verification that similar levels of appropriately sized GAL4 fusion proteins were synthesized was carried out by performing Western blot analysis of cell extracts prepared from transfected HepG2 cells as described elsewhere(30) , using a GAL4 antibody (Upstate Biotechnology, Inc.).

Construction of expression vectors for the subdomains of CREB was performed using the expression vector used for the GAL4 fusion expression above but which had the GAL4 coding region replaced by the indicated domain of CREB. The subdomain KID also contained CREB residues 1-6 at the amino terminus in addition to residues 83-145.


RESULTS

Fig. 1shows a schematic of the two components of the PEPCK promoter that are required for robust cAMP responsiveness in hepatoma cells. The LSR contains, and requires for its activity, multiple binding sites for C/EBP proteins as well as a binding site for AP-1(15, 22) . The CRE consists of an imperfect palindromic sequence to which recombinant CREB binds(15) . C/EBP proteins also bind to this particular CRE with high affinity, although they exhibit a much reduced binding affinity to perfect palindromic CREs(9, 15) . This observation has led to some debate as to whether CREB is involved in the cAMP responsiveness of the PEPCK promoter in liver. Indeed, it has been argued that the liver-specific nature of the cAMP induction suggests that a C/EBP protein, several isoforms of which are enriched in liver, rather than the ubiquitously expressed CREB, is the factor mediating the effect through the CRE(8, 16) .


Figure 1: A schematic of the two components of the cAMP response unit of the PEPCK promoter. The cAMP response element consists of a single protein binding site extending from -95 to -75 in the PEPCK promoter. The liver-specific region consists of multiple protein binding sites which mutational analyses and reconstitution experiments have shown are all required for the activity of this region.



To address this question, we initially identified the transcription factors present in HepG2 cells that could bind to the CRE. Gel mobility shift assays demonstrated that two complexes were formed when a radiolabeled CRE probe was incubated with HepG2 nuclear extracts (Fig. 2). The specific nature of both complexes was demonstrated by the ability of an unlabeled CRE oligonucleotide to compete for both complexes, while an oligonucleotide corresponding to a yeast transcription factor GAL4 binding site did not compete for either complex (Fig. 2). Immuno-neutralization of CREB in the HepG2 nuclear extracts prior to addition of the probe eliminated formation of complex I but had no effect on complex II formation (Fig. 2). However, immuno-neutralization of C/EBP proteins resulted in the elimination of complex II formation. Additionally, addition of an unlabeled OPT oligonucleotide, which represents an optimal DNA sequence for C/EBP binding(22) , effectively competed for complex II formation (Fig. 2). These results indicate that CREB and C/EBP proteins exist in HepG2 cells which can specifically interact in vitro with the CRE.


Figure 2: HepG2 nuclear extracts contain both CREB and C/EBP protein(s) which bind to the cAMP response element of the PEPCK promoter. Gel mobility shift assays were performed using a radiolabeled CRE oligonucleotide and HepG2 nuclear extracts. In the oligonucleotide competition experiments, nuclear extracts were preincubated with a 100-fold molar excess of unlabeled oligonucleotide for 10 min at room temperature, followed by the addition of the radiolabeled probe and further incubation at room temperature for another 15 min. In the immuno-depletion experiments, polyclonal anti-CREB or anti-C/EBP antiserum was preincubated with nuclear extracts for 10 min at room temperatue before addition of the radiolabeled probe. The binding reactions were resolved on a 6% (49:1) polyacrylamide gel using a Tris-glycine (25 mM:190 mM, pH 7.9) buffer system. I and II refer to the two major complexes formed.



Recently, a dominant repressor of CREB was identified that could be used to examine the role of CREB on specific promoters(20) . This repressor, termed KCREB, differs from wild-type CREB at a single amino acid position in the basic region. When expressed in cells, KCREB is able to form dimers with wild-type CREB which, due to the mutation, are unable to bind to their cognate DNA sequence. We used this repressor to examine the role of CREB in mediating the cAMP responsiveness of the PEPCK promoter.

While KCREB has been shown to be a repressor of CREB, it was important for us to verify the specificity of its inhibitor action. In particular, we needed to ascertain that it was not capable of inhibiting C/EBP-like proteins, thereby allowing us to assess the relative roles of CREB and C/EBP proteins at the CRE. We utilized the ability of C/EBPalpha and DBP, both members of different bZIP families (31) , to transactivate the PEPCK promoter to assess KCREB's specificity. Similar to that observed previously(31) , both C/EBPalpha and DBP were shown to transactivate the PEPCK promoter in HepG2 cells (Table 1). Co-expression of KCREB along with either activator had no significant inhibitory effect (Table 1). However, we did observe that expression of CHOP, a previously described dominant repressor of C/EBP proteins(19) , was able to inhibit the transactivation produced by C/EBP but showed no inhibitory activity against DBP (Table 1). This latter observation is consistent with the incompatible bZIP domains of C/EBP and DBP(32) . Taken together, these results suggest that KCREB's repressor activity is sufficiently specific to allow it to be used to distinguish between C/EBP and CREB proteins.



As observed previously, co-transfection of a PEPCK promoter-CAT vector along with an expression vector for PKA resulted in an approximate 50-fold stimulation of transcription from this promoter (Table 2). However, in the presence of KCREB expression, the fold activation of this promoter by PKA was drastically decreased. This level of inhibition is consistent with the observations that mutation or elimination of the CRE from PEPCK promoter constructs resulted in an almost complete inhibition of PKA responsiveness(8, 9) . These results suggest that CREB is involved in mediating the cAMP responsiveness of the PEPCK promoter, presumably through the CRE since in vitro DNase I footprinting analysis with recombinant CREB indicates that the CRE is the only site on the PEPCK promoter to which CREB binds(15) .



One approach that has been extensively utilized to ascertain, and to further characterize on a structure/function level, the role and properties of a specific transcription factor has been to use GAL4 fusion methodology(26) . By fusing suspect domains of CREB to the DNA-binding domain of GAL4, the latter which binds to DNA sequences not recognized by nuclear factors present in mammalian cells, one can assess the role of CREB on specific promoters. Such analysis has already been used to identify domains of CREB responsible for its inherent basal and cAMP-inducible activities(6, 33) . However, to date no information is available as to CREB's ability to synergize with other transcription factors, nor in what domain of CREB this synergizing activity resides.

The GAL4-CREB protein we created contained amino acids 3-203 of rat CREB, which has been shown to contain all of the inherent transactivation properties of wild-type CREB(6) . Initially, we examined the ability of this fusion protein to mediate cAMP responsiveness of the PEPCK promoter. A PEPCK promoter mutant, G4-PEPCK, was obtained which had the CRE replaced by a GAL4 recognition site(6) . The PKA responsiveness of this promoter when the DNA-binding domain of GAL4, GAL4(147), was expressed was minimal at 3.8-fold induction (Fig. 3). This is similar to the fold induction observed in earlier studies in promoters where the CRE was mutated(8) . However, the fold induction of G4-PEPCK was strongly induced by PKA when GAL4-CREB was expressed (Fig. 3). Indeed, the absolute degree of activation (approximately 70-fold) was very similar to that observed with the wild-type PEPCK promoter(8, 9) .


Figure 3: The transactivation domain of CREB mediates the cAMP responsiveness of the PEPCK promoter in hepatoma cells through synergism with transcription factors bound upstream in the LSR. Panel A, a schematic of the GAL4 DNA binding domain and GAL4-CREB fusion proteins tested in this experiment. Not shown is the mut 133 form of GAL4-CREB, which has a serine-to-alanine mutation at amino acid position 133 of rat CREB. Panel B, HepG2 cells were transfected with 1 µg of RSV-betagal plasmid, 7 µg of promoter-CAT vector, 1 µg of PKA expression vector, and 1 µg of either GAL4(147), GAL4-CREB, or 133 mut expression vector as described under ``Experimental Procedures.'' Values shown are the averages ± SE of at least four experiments.



We next examined the ability of this GAL-CREB fusion protein to synergize with both itself and with the protein(s) bound to the other component of the cAMP response unit in the PEPCK promoter, the LSR. First, we observed that GAL-CREB was unable to mediate activation of a promoter containing a single GAL4 binding site, but did mediate a strong activation on a promoter containing three GAL4 binding sites (Fig. 3). These findings are highly analogous to earlier observations from our laboratory showing that a minimal promoter containing a single CRE responded poorly to PKA, while a promoter containing three tandem CREs was highly responsive(9) . Mutation of serine 133 to an alanine completely eliminated GAL-CREB from synergizing with itself to mediate responsiveness to PKA (Fig. 3), consistent with a previous study which demonstrated the critical role of this serine residue(3) .

Previously we had shown that the two components of the cAMP response unit of the PEPCK promoter, the CRE and the LSR, are both required for mediating optimal responsiveness to PKA, and further that each component alone is a poor enhancer(9) . The promoter DR4 contains the LSR alone linked to a minimal promoter (Fig. 3, and see (9) ). This promoter showed a small fold activation by PKA and, as expected due to its lack of a GAL4 binding site, no additional increase in PKA-responsiveness when GAL-CREB was expressed (Fig. 3). However, the promoter DR4/GAL4X1 was highly responsive to PKA when GAL4-CREB was expressed, although it showed only weak responsiveness when either GAL4(147) or 133 mut was expressed (Fig. 3). These data indicate that the region of CREB contained within amino acid residues 3-203 contains the domain(s) which is able to synergize with the transcription factors bound at the LSR to mediate cAMP responsiveness. Consistent with the liver-specific nature of this response, no synergism between the LSR and the GAL4-CREB binding site was observed when the experiment was performed in the choriocarcinoma Jeg-3 cell line. (^2)

Quinn (6) recently showed that CREB mutants lacking the first 86 amino acids or containing only the amino-terminal 141 amino acids displayed full ability to mediate cAMP responsiveness, although the latter mutant had a reduced ability to mediate basal transcription. We tested whether similar deletion mutants of CREB were capable of mediating the synergism with the LSR. Delta82 is a GAL4-CREB mutant which lacks the amino-terminal 82 amino acids, while N145 contains CREB residues 3-145. The ability of these mutant CREBs to self-synergize to mediate a PKA response was tested using the CAT vector -68/GAL4X3. It should be noted that neither mutant was able to mediate a PKA response when tested using the reporter vector containing a single GAL4 binding site (data not shown), similar to that observed for GAL4-CREB (see Fig. 3B). However, the CREB mutant Delta82 displayed self-synergizing activity similar to that of wild type CREB (denoted as GC) in mediating a PKA response, while the N145 mutant had a greatly reduced ability to mediate a PKA response through self-synergism (Fig. 4A). Thus it appears that amino acid residues 146-203 are required for CREB to mediate a strong transactivation in response to PKA through synergism with other CREB molecules.


Figure 4: Ability of CREB deletion mutants to synergize with the LSR. The ability of CREB mutants (linked to the DNA-binding domain of GAL4) to mediate PKA responsiveness of two different promoters was examined. GC refers to the GAL4-CREB fusion protein shown in Fig. 3B. delta 82 is a GAL4-CREB fusion protein which contains CREB amino acid residues 83-203, N145 contains CREB residues 3-145, and GAL4-KID contains CREB residues 83-145. GAL4-ATF-1 contains ATF-1 amino acid residues 4-225. Transfections were performed as described in the legend to Fig. 3. Fold inductions are calculated as the CAT activity produced in the presence of PKA and GAL4-CREB protein expression divided by the CAT activity produced in the presence of the GAL4-CREB protein expression alone. Values shown are the averages ± SE of at least three experiments. Panel A, GAL4-CREB mutants were compared with GC for their ability to mediate the PKA-responsiveness of the promoter -68/GAL4X3, a minimal promoter containing three binding sites for GAL4 (see Fig. 3B). Panel B, GAL4-CREB mutants were compared with GC for their ability mediate a PKA response through the promoter G4-PEPCK (see Fig. 3B).



We next examined these same mutants for their ability to synergize with the LSR in the context of the PEPCK promoter, using the G4-PEPCK reporter vector. The Delta82 mutant displayed activity nearly equivalent to that of GC in its ability to mediate the PKA responsiveness of the G4-PEPCK promoter (Fig. 4B). Thus CREB residues 1-82 are not involved in mediating this synergism. The N145 mutant was also able to synergize with the LSR, mediating a 20-fold response to PKA (Fig. 4B), significantly larger than the fold activation of the G4-PEPCK promoter by PKA in the absence of GAL4-CREB expression (see Fig. 3B). However, the magnitude of the response mediated by N145 is only 40-50% of that mediated by GC, which contains residues 3-203 (Fig. 4B). Thus, residues 146-203, while not necessary for synergism, appear to contribute to the ability of CREB to fully synergize with the LSR.

Since CREB residues 1-82 appear not to be involved in mediating the synergistic response to cAMP and residues 146-203 are not absolutely required, we hypothesized that CREB residues 83-145, termed KID(6) , by themselves might be able to mediate the synergism. When such a GAL4-CREB mutant (KID) was overexpressed in HepG2 cells, an approximately 20-fold activation of the G4-PEPCK promoter in response to PKA was observed, similar to the magnitude mediated by the N145 mutant (Fig. 4B). This result confirms the importance of this small domain of CREB in mediating the synergistic response to PKA on the PEPCK promoter.

ATF-1 is a member of the CREB/ATF protein family that is capable of mediating cAMP responsiveness(29) . This protein possesses a consensus phosphorylation site for PKA and shares significant homology with CREB around this phosphorylation site, although it lacks the region corresponding to CREB residues 83-103 (Fig. 5). The carboxyl-terminal domain of ATF-1 is also highly homologous to that of CREB, but it lacks the glutamine-rich domain found in the amino terminus of CREB. We decided to use these similarities and dissimilarities between ATF-1 and CREB to further examine the CREB residues involved in mediating the synergistic response to PKA. We constructed an GAL4-ATF-1 expression vector, which contained the coding region for ATF-1 residues 4 to 225, which essentially contains all of the protein except for the bZIP domain(29) . In the presence of GAL4-ATF-1, the G4-PEPCK promoter was not significantly activated by PKA over that observed in the absence of this fusion protein (Fig. 4B). This finding indicates that ATF-1, unlike CREB, is unable to synergize with the LSR to mediate PKA responsiveness, suggesting that amino acid residues 83-103 of CREB likely play a critical role in mediating the synergism. It should be noted that GAL4-ATF-1 did not increase the basal transcriptional activity of the G4-PEPCK promoter,^2 consistent with previous reports that ATF-1 is a poor activator of basal transcription(29) .


Figure 5: Amino acid sequence of the synergism domain of CREB and the corresponding sequences in ATF-1. Shown is the amino acid sequence of KID (CREB residues 83-145), which has been shown in Fig. 4to be sufficient to mediate a synergistic response with the LSR. Aligned above this sequence is the sequence of the ATF-1 domain spanning residues 34-73, with perfect matches with CREB being indicated by the presence of an asterisk (*).



The data presented in Fig. 3and Fig. 4indicate the importance of CREB residues 83-145 in mediating the synergistic response to PKA. Presumably, this domain of CREB makes physical contact with another factor(s) which results in a change in the rate of transcription initiation. This protein-protein interaction might be susceptible to competition by the overexpression of the appropriate CREB subdomain. This hypothesis was tested experimentally, and the results are shown in Table 3. Overexpression of CREB 1-82, which consists of the domain of CREB shown not to be necessary for mediating the synergistic response to PKA (Fig. 4B), had no effect on the induction of the PEPCK promoter by PKA in HepG2 cells (Table 3). However, overexpression of the KID subdomain significantly reduced the PKA responsiveness of the promoter, consistent with the hypothesis that mediation of the synergistic effect requires CREB's interaction with a limiting factor within the cell. Neither subdomain had any significant effect on the basal activity of the PEPCK promoter (Table 3).



CREB has been shown to mediate basal transcription in addition to cAMP-inducible transcription(6) . Thus, we examined whether the synergism we observed between CREB and the protein(s) bound to the LSR regarding the PKA response was also apparent at the level of basal transcription of the PEPCK promoter. In Table 4, it is shown that on a synthetic promoter containing a single GAL4 binding site (-68/GAL4X1), the GAL4-CREB fusion protein exhibited no ability to mediate basal transcription. However, the basal transcriptional activity of CREB was observed on a promoter containing three binding sites for GAL4-CREB, indicating that CREB synergizes with itself to mediate basal transcription. No synergism between CREB and the proteins bound to the LSR was observed, with the basal promoter activity of DR4/GAL4X1 being approximately unchanged in the presence of GAL4-CREB expression (Table VI). Thus, synergism between CREB and the LSR-bound protein(s) is apparently limited to the cAMP-inducible portion of PEPCK promoter transcription.




DISCUSSION

In the present study, we have used several approaches to obtain data which together support a role for CREB in the cAMP responsiveness of the PEPCK promoter. Antibodies raised against CREB were able to block binding of a protein in HepG2 nuclear extracts which bound to a CRE probe. A dominant repressor of CREB was able to inhibit the cAMP responsiveness of the PEPCK promoter in HepG2 cells. Next, we showed that a GAL4-CREB fusion protein, was able to synergize with the proteins bound to the liver-specific region of the PEPCK promoter to mediate the cAMP responsiveness of this promoter. Indeed, our findings using the GAL4-CREB fusion protein along with promoters where the CRE was replaced by a GAL4 binding site, almost exactly mimicked previous observations concerning the relative PKA responsiveness of promoter constructs containing CREs. For example, it has been a consistent observation by us and others that PEPCK promoter constructs containing the single CRE located at -85 in the promoter show only a marginal response to cAMP or PKA expression(7, 8, 9, 22) . Similarly, we showed that in the presence of GAL4-CREB, a promoter containing a single GAL4 binding site was essentially unresponsive to PKA (Fig. 3B). A synthetic promoter containing multiple CREs, however, routinely shows significant responsiveness(9) , which again was observed in this study using GAL4-CREB with a promoter containing multiple GAL4 binding sites. Finally, and probably most importantly, previous studies out of our laboratory have shown that the CRE is able to synergize with the upstream LSR to mediate a strong activation in response to cAMP or PKA (9, 22) . Our synthetic promoters where a GAL4 binding site is linked to the LSR also demonstrated this synergism when GAL4-CREB was present (Fig. 3B). Taken together, the evidence indicates that the robust cAMP responsiveness of the PEPCK promoter in liver is mediated by a synergistic interaction of the transactivation domain of CREB (amino acids 83-203) with liverenriched protein(s) bound upstream.

While our studies have been carried out using a heterologous system, i.e. analyzing a rat promoter in a human hepatoma cell line, the conclusions above concerning the involvement of the CRE and the LSR in the cAMP responsiveness of the PEPCK gene have been supported by the findings of other groups and are consistent with the tissue-specific nature of the hormone response of this gene. For example, it is well documented that administration of cAMP to rats strongly induces PEPCK gene transcription in liver but has no or little effect on expression in kidney(14) . This tissue-specific response of the PEPCK gene has been observed in tissue culture as well, with significant cAMP induction of PEPCK promoter-CAT vectors being observed only in liver-derived cell lines(9) . These observations suggested that perhaps liver contains transcription factors not present in kidney which mediate the hormonal response. In support of this hypothesis, subsequent in vitro DNase I footprinting of the PEPCK promoter demonstrated that rat liver nuclear extracts contained proteins which bound to the LSR while kidney extracts lacked detectable binding activity against this region(25) . The in vitro footprinting pattern obtained was recently shown to be similar to the in vivo footprint pattern over the PEPCK promoter in rat hepatoma H4IIE cells, a cell line in which the PEPCK gene displays appropriate hormonal regulation(34) . Probably the most compelling data obtained to date to support a role for the LSR in cAMP responsiveness has come from transgenic mouse studies. Hanson and colleagues (35) created transgenic mice which contained a chimeric PEPCK promoter-reporter gene cassette with mutations in specific regulatory domains. They observed that a mutation in either the CRE or the LSR eliminated the ability of the transgene to be induced by cAMP. Thus, our observations made concerning the regulation of the PEPCK promoter in human hepatoma cells closely mimics the observations made in in vivo models.

At present, it is difficult to determine the precise nature of the interaction that CREB is making with the factor(s) bound to the upstream LSR. Recent studies from our laboratory suggest that three C/EBP binding sites plus one AP-1 binding site form the basis for the cAMP-mediating activity of the LSR(22) . C/EBP exists in a number of isoforms(36, 37, 38, 39) , and we have yet to determine which isoform(s) is/are involved in the response. As discussed below, while several cases of transcriptional synergism identified to date involve direct protein-protein contact between the factors involved, such an interaction is not a prerequisite for synergism to be observed(40) . Thus, CREB's synergistic interaction with the proteins bound to the LSR is not necessarily indicative of a physical interaction.

The transactivation domain of CREB contains several domains which mediate basal and cAMP-stimulated transcription(3, 4, 5, 6, 33) . The transactivation domain contains a subregion termed KID (residues 86-141) which can mediate PKA responsiveness when linked to a heterologous DNA binding domain(6) . However, it should be noted that this finding was made using a promoter containing multiple binding sites, and we have observed that a single GAL4-KID molecule does not mediate a detectable transcriptional response in the presence of PKA in HepG2 cells.^2 However, the KID domain is able to synergize with the LSR-bound proteins, although maximal synergism also requires CREB residues 146-203 (Fig. 4B). Our data also suggest that subdomains within the complete synergism domain of CREB differ in the degree to which they participate in a particular synergistic interaction. For example, residues 146-203 appear to be significantly more important for CREB to mediate a PKA response in synergism with other CREB molecules (i.e. self-synergism) than when it mediates the response in synergism with the LSR (compare Fig. 4, A and B). Possession of several domains through which synergism can occur may provide an additional mechanism whereby CREB can differentially modulate the response to cAMP in a gene-specific manner.

CREB physically interacts with a protein termed the CREB-binding protein (CBP)(41) , and possesses features of a CREB-coactivator(42) . CBP does not bind to DNA but instead binds directly to the phosphorylated, but not dephosphorylated, form of CREB. CBP, like CREB, possesses a transactivation domain which can mediate basal transcription as well as PKA-mediated transcriptional activation, the latter most likely due to the fact that it possesses a PKA-phosphorylation site. It has been hypothesized that phosphorylation of CREB leads to the binding of CBP which itself becomes phosphorylated and in turn mediates a transcriptional response. Since CBP binds only the phosphorylated form of CREB, and the synergism we have observed between the proteins bound at the LSR and CREB is observed only under PKA-stimulating condition and can be competed by overexpression of KID, it is interesting to speculate that CBP may be involved in this synergistic response. Under one possible scenario, the proteins bound to the LSR could augment CREB's ability to recruit CBP to the promoter i.e. stabilize the CREB-CBP interaction.

Transcription factors are being shown in ever increasing numbers to interact with other transcription factors to synergistically activate transcription. In some cases, synergism is produced through cooperative binding of the proteins involved, suggestive of direct protein-protein contacts. Several pieces of data would suggest that this is not the mechanism in operation on the PEPCK promoter. First, the binding affinity of the proteins to the CRE and the LSR have been shown not to be affected by deletion of any one site i.e. their binding activities are independent of each other(8, 25) . Second, most studies indicate that the phosphorylation of CREB does not alter its binding affinity to CREs(3, 4) , and nuclear extracts from cAMP-treated hepatoma cells do not show altered binding characteristics over the PEPCK promoter compared to untreated cells.^2 Furthermore, Faber et al.(34) recently showed that the in vivo DNase I protection pattern of the PEPCK promoter does not change upon cAMP treatment. However, these observations do not rule out a role for protein-protein contacts between the transcription factors involved, because such interactions could mediate synergism without an apparent effect on DNA-binding. In a recent review article, Herschlag and Johnson (40) presented energetic descriptions and hypothetical physical models of transcriptional synergism. Their models suggest that in situations where a greater than multiplicative effect on transcription is observed in the presence of multiple factors, direct protein-protein contacts between the DNA-bound factors are predicted. In these situations, a direct interaction may lead to synergistic activation by forming a complex which can more efficaciously interact with the general transcriptional apparatus to increase the frequency of initiation. Based on this model, we believe that protein-protein contacts are made between the transcription factors mediating the cAMP responsiveness of the PEPCK promoter, either through direct contacts or perhaps indirectly through simultaneous interactions with a common target such as CBP. The CRE alone mediates a 2-fold induction of transcription in response to PKA; likewise, the LSR mediates a 2-5-fold induction. When these two components are placed together, at least a 50-fold level of induction is observed, significantly greater than a multiplicative effect(9) . Even though five cis elements (the CRE plus four cis elements within the LSR) appear to be involved in the cAMP response, mutation and promoter reconstitution experiments have indicated that elimination of any one element results in a severe decrease in cAMP responsiveness(8, 9, 22) . Thus, the synergistic mechanism appears to involve every element, and thus likely every associated transcription factor, making up the cAMP response unit in the PEPCK promoter. It is interesting to note that activation of the PEPCK promoter by thyroid hormone (triiodothyronine) also requires an element within the LSR. Since cAMP and triiodothyronine synergistically activate transcription of the PEPCK gene(43) , it seems apparent that the LSR serves to integrate information from different signaling pathways. Future studies will be aimed at further characterizing the molecular biology of the LSR.


FOOTNOTES

*
This work was supported by operating grants from the Medical Research Council of Canada and the Canadian Diabetes Association (to W. J. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 306-966-4375; Fax: 306-966-4390.

(^1)
The abbreviations used are: CRE, cAMP response element; PEPCK, phosphoenolpyruvate carboxykinase; CREB, cAMP response element binding protein; C/EBP, CCAAT/enhancer binding protein; DBP, D-site binding protein; LSR, liver-specific region; AP-1, activator protein-1; bZIP, basic region-leucine zipper; CAT, chloramphenicol acetyltransferase; KID, kinase-inducible domain; PKA, protein kinase A; CBP, CREB-binding protein.

(^2)
W. J. Roesler, unpublished observations.


ACKNOWLEDGEMENTS

We thank Marc Montminy, G. Stanley McKnight, Richard Hanson, Bob Rehfuss, David Ron, Patrick Quinn, Uehli Schibler, Steve McKnight, and Ivan Sadowski for plasmids, and Steve McKnight for the C/EBP antiserum. We also thank Gerald Davies for performing the Western blot analyses.


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