©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Design of a C/EBP-specific, Dominant-negative bZIP Protein with Both Inhibitory and Gain-of-function Properties (*)

(Received for publication, September 27, 1995; and in revised form, November 22, 1995)

Michelle Olive (§) Simon C. Williams (1)(¶) Christine Dezan (§) Peter F. Johnson (1) Charles Vinson(§)(**)

From the Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 and the ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, P. O. Box B, Frederick, Maryland 21702-1201

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have developed a bZIP protein, GBF-F, with both dominant-negative (DN) and gain-of-function properties. GBF-F is a chimera consisting of two components: the DNA binding (basic) region from the plant bZIP protein GBF-1 (GBF) and a leucine zipper (F) designed to preferentially heterodimerize with the C/EBPalpha leucine zipper. Biochemical studies show that GBF-F preferentially forms heterodimers with C/EBPalpha and thus binds a chimeric DNA sequence composed of the half-sites recognized by the C/EBP and GBF basic regions. Transient transfections in HepG2 hepatoma cells show that both components of GBF-F are necessary for inhibition of C/EBPalpha transactivation. When the C/EBPalpha leucine zipper is replaced with that of either GCN4 or VBP, the resulting protein can transactivate a C/EBP cis-element but is not inhibited by GBF-F, indicating that the specificity of dominant-negative action is determined by the leucine zipper. All known members of the C/EBP family contain similar leucine zipper regions and are inhibited by GBF-F. GBF-F also exhibits gain-of-function properties, since, with the essential cooperation of a C/EBP family member, it can transactivate a promoter containing the chimeric C/EBPGBF site. This protein therefore has potential utility both as a dominant-negative inhibitor of C/EBP function and as an activator protein with novel DNA sequence specificity.


INTRODUCTION

The determination of the function of individual members of the C/EBP (^1)family of bZIP transcription factors is complicated by their possible redundant roles. Vertebrate gene expression is regulated by an abundance of transcription factors, many of which exist as members of gene families whose products have similar DNA-binding properties and thus have potentially overlapping target genes and biological functions. Several methods exist to address this problem. Gene disruption (1) and replacement (2) by homologous recombination provide a powerful genetic approach to assess the in vivo functions of a particular transcription factor. However, several examples have been reported recently in which the functional redundancy within a gene family allows other family members to substitute, at least partially, for the disrupted gene. A well documented example is the MyoD family of bHLH transcription factors (3) and the Src family of tyrosine kinases(4) . The fundamental problem with this method is that it obscures tissue specific and developmentally later functions. To study these later functions, homologous recombination using the CRE LOX system activated in a tissue or stage specific manner can be used(5) .

An alternative approach which is pharmacological is to use a dominant-negative (DN) system to inhibit the function of one or several family members(6) . Expression of antisense RNA, using either the conserved or nonconserved part of mRNA as a target to inactivate one or all family members, respectively, is an attractive possibility. Lane's (7) group has used this approach to demonstrate that C/EBPalpha is critical for the differentiation of 3T3L1 cells into adipocytes. Expression of DN proteins is another method to inactivate gene function. A number of different DN inhibitors of bZIP protein function have been described, both as naturally occurring members of transcription factor families or as experimentally designed proteins. These DN molecules can be divided into two general classes. The first class binds the same DNA target but lack an activation domain(8, 9, 10, 11) . This class of DNs acts as competitive inhibitors, forming either non-activating homodimers that compete with the target protein for binding to its cognate binding sites or heterodimers with reduced transcriptional activity. By virtue of their mode of action, these DN proteins must be expressed in excess relative to their target for efficient inhibition. The second class dimerizes with the endogenous bZIP protein but which either lack a DNA-binding domain or contain a DNA-binding domain with different binding specificity from the target protein(12) . The second class potentially functions more efficiently as DNs, since heterodimers formed with these proteins do not bind to DNA and are therefore completely inactive.

In this study we describe the design of an efficient DN molecule with specificity for the C/EBP family of bZIP transcription factors. There are four major proteins in the C/EBP family, to which we refer using the nomenclature C/EBPalpha, C/EBPbeta, C/EBP, and CRP1 (reviewed in (13) ). C/EBP proteins have been implicated in a variety of regulatory functions, including activation of constitutive and acute phase responsive genes in the liver, control of adipocyte differentiation, and regulation of inflammatory cytokine genes and other activities of monocytic cells(13) . C/EBP family members can heterodimerize in all combinations and recognize a common palindromic DNA motif (consensus: ATTGCGCAAT). Each monomer is a long (70 amino acids) bipartite alpha-helix when bound to DNA(14, 15, 16, 17) . The carboxyl-terminal half of the alpha-helix is amphipathic with the two helices interacting along their hydrophobic surfaces to create a dimeric leucine zipper coiled coil. The NH(2)-terminal half of the motif interacts with the major grove of DNA in a sequence specific fashion and becomes alpha-helical upon DNA binding(18, 19) . DNA binding stabilizes the dimer complex, which complicates the design of non-DNA binding DNs to bZIP proteins.

As a first step in developing a DN with specificity for C/EBP proteins, we used a previously designed a leucine zipper (the F zipper) that preferentially heterodimerizes with C/EBPalpha as a result of substitutions in charged amino acids occupying the e and g positions of the zipper alpha-helix(20) . We have appended to this F zipper the basic region from the plant bZIP protein G-box binding factor-1 (GBF-1)(21) , which recognizes a DNA sequence highly divergent from that recognized by C/EBP proteins. This chimeric protein creates a dominant-negative protein (GBF-F) that inhibits C/EBP mediated transactivation. Both the heterodimerizing leucine zipper and the novel basic region are critical for optimal activity of the designed DN. In addition to its ability to inhibit C/EBP transactivation of a canonical C/EBP site, we show that GBF-F also has gain-of-function properties as a GBF-F:C/EBP heterodimer which can transactivate the non-palindromic site composed of C/EBP and GBF half-sites.


EXPERIMENTAL PROCEDURES

Transient Transfections and Western Blotting

All transient transfections were carried out in HepG2 cells maintained at 37 °C in Dulbecco's modified Eagle medium with high glucose (4.5 gm/liter), supplemented with 10% fetal calf serum and antibiotics. Cells grown in 100-mm dishes were transiently transfected with 20 µg of DNA consisting of 10 µg of reporter plasmid and 0.3 µg of transactivator C/EBPalpha, -beta, or -, and 0.3-5 µg of DN and salmon sperm DNA. The cells were transfected by the calcium phosphate procedure (22) using the calcium phosphate transfection kit (Life Technologies, Inc.). The DNA precipitate was left in contact with the cells for 18 h, then the cells were washed and refed. After 2 days the cells were harvested and assayed for chloramphenicol acetyltransferase (CAT) activity(23, 24) . CAT activities were normalized for protein concentration (25) and are represented as fold activation over the reporter plasmid alone. For nuclear extract preparation, 17 µg of DNA was used per transfection in 150-mm dishes, consisting of 2 µg of pMEX C/EBPalpha, 5, 10, or 15 µg of pRG GBF-F, and the appropriate amount of pRG. Cells were harvested and nuclear extracts prepared as described previously(26) . Immunodetection of C/EBPalpha in HepG2 nuclear cell extracts was performed as described previously(26) . 30 µg of nuclear extract was electrophoresed through 12% SDS-PAGE, transferred to Immobilon-P membrane (Millipore), and immune complexes detected using the Amersham ECL kit.

Purification of Bacterially Expressed Proteins

C/EBP chimeric proteins were constructed by the four primer polymerase chain reaction mutagenesis method (27) and cloned into the prokaryotic expression vector pT5 as NdeI-HindIII fragments (28) . DNA sequencing was performed on double-stranded templates using the Sequenase kit (U. S. Biochemical Corp.)(29) . Proteins were synthesized in Escherichia coli using the phage T7 expression system(30) . 400-ml bacterial cultures were grown to an optical density of 0.6 at 600 nm and then induced with 1 mM isopropyl-beta-D-thiogalactopyranoside for 2 h. Cells were recovered by centrifugation, resuspended in 6 ml of lysis buffer (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM benzamidine, 1 mM dithiothreitol, and 0.2 mM phenylmethysulfonyl fluoride), frozen, thawed, and gently brought to 1 M KCl by the addition of 2 ml of 4 M KCl. The sample was centrifuged at 25,000 rpm in a Beckman T42 rotor and the supernatant was recovered. The supernatant was heated to 65 °C for 10 min and clarified by centrifugation. Proteins were dialyzed to 100 mM KCl and loaded onto a heparin-agarose column. The column was washed with lysis buffer containing 100 mM KCl, followed by a 300 mM KCl wash, and protein was eluted with buffer containing 1 M KCl. Proteins were dialyzed to 150 mM KCl, 12.5 mM phosphate buffer, pH 7.4, 1 mM EDTA, 1 mM dithiothreitol.

DNA Binding Assay

Purified DeltaC/EBPalpha and GBF-F proteins were mixed in various ratios and added to a DNA-binding reaction in which either a perfect C/EBP binding site oligonucleotide (26) or a chimeric C/EBPGBF binding site oligonucleotide 5`-GGGGGATCGATTGCGTGGAATCGGGGGG-3` was present. (Throughout this article, the dash signifies the junction between the basic region and the leucine zipper, a colon separates the names of the individual proteins in a heterodimer, and a vertical line separates cis-element half-sites.) Reaction conditions were as described(26) . Bound and free complexes were resolved on 6% polyacrylamide gels and visualized by autoradiography. For DNA-binding assays with HepG2 nuclear extracts, approximately 30 µg of protein was used per reaction. In antibody supershift assays, anti-C/EBPalpha antisera C3 (31) was added to nuclear extracts and incubated on ice for 30 min before exposure to labeled oligonucleotides.

Eukaryotic Reporter Plasmids

All reporter plasmids utilize the bacterial CAT gene(23) . The reporter plasmid pAT2-CAT contains the albumin promoter (sequences -787 to +8 of the mouse albumin gene) linked to CAT(32) . The reporter plasmid pAT2/DEI-CAT contains the albumin promoter with point mutations at the C/EBP protein binding site (DEI) that abolish C/EBP binding(33) . The minimal promoter construct (p-35 Alb CAT) contains nucleotides -35 to +22 of the albumin promoter in which the TATA box is located between nucleotides -33 to -29(34) . The C/EBP-dependent version of this promoter has a perfect C/EBP consensus site (5`-GATCGAGCCCCATTGCGCAATCATAGA-3`) inserted into the BglII site at -41 of p-35 Alb CAT. The C/EBP consensus site is in bold type. The chimeric reporter plasmid GBFC/EBP promoter has the following sequence inserted into the BglII site at -41 (5`-GATCGAGCCCCTCCACGCAATCATAGA-3`). In all transfection experiments, 10 µg of reporter plasmid was used.

Eukaryotic Activator Protein Expression Plasmids: Murine Sarcoma Virus Promoter

The eukaryotic expression plasmids for the three C/EBP family members, pMEX C/EBPalpha, pMEX C/EBPbeta, and pMEX C/EBP, are driven by the murine sarcoma virus promoter and have been described elsewhere(35) . In order to monitor the presence of C/EBPalpha in transfected cells, we introduced either the flag (DYKDDDK) or the hemagglutinin (YPYDVPDYA) epitope at the NH(2) terminus of C/EBPalpha by inserting oligonucleotides into the non-unique NcoI site of pMEX C/EBPalpha. The upper strand oligonucleotides used for the insertion of the flag and the hemagglutinin epitopes were 5`-C ATG GAC TAC AAG GAC GAC GAT GAC AAG CA-3` and 5`-C ATG GCG TAT CCC TAC GAC GTG CCC GAT TAT GCC CA-3`, respectively. The transactivating properties of epitope-tagged C/EBPalpha were similar to that of the unmodified protein (data not shown). The chimeric C/EBPalpha-VBP (vitellogenein binding protein(36) ) construct consists of C/EBPalpha sequences from the initiator methionine to Glu-316 (located at the junction of the basic region and leucine zipper) joined to the leucine zipper of VBP (Leu-278 to the COOH terminus). Construction of this chimera was accomplished with a triple ligation of the leucine zipper of VBP as a XhoI-HindIII PCR-generated fragment, a MluI-XhoI fragment of C/EBPalpha (amino acids 191-316) and the pMEX C/EBPalpha vector cut with MluI and HindIII. The C/EBPalpha-GCN4 chimera contains C/EBPalpha sequence from Met-1 to Glu-316 and the leucine zipper of GCN4 (Leu-253 to the COOH terminus) and corresponds to the C2G2 construct previously described(37) .

Eukaryotic Dominant-negative Expression Plasmid: Murine Sarcoma Virus Promoter

Dominant-negative coding sequences (DeltaC/EBP, C/EBPLZ, F, C/EBP-F, GBF-C/EBP, and GBF-F) were cloned by polymerase chain reaction as NdeI/HindIII fragments into the eukaryotic expression vector pMEX previously describe(34) . DeltaC/EBP expresses an 86-amino acid protein that is a NH(2)-terminal truncation of C/EBPalpha, which starts at Lys-273 and includes the entire bZIP domain but lacks activation domains. C/EBP LZ contains only the leucine zipper of C/EBPalpha; 3 Gly at the NH(2) terminus are followed by C/EBPalpha sequences between Thr-310 and the COOH terminus. F is a leucine zipper designed to heterodimerize with the wild type C/EBPalpha zipper(20) . C/EBP-F is a fusion of the DNA basic region of DeltaC/EBP (Lys-273 to Glu-309) and the F zipper. GBF-C/EBP and GBF-F contain the basic region of the plant GBF-1 protein (21) comprising residues Pro-216 to Gln-249 joined to either the C/EBP (Thr-310 to the COOH terminus of C/EBPalpha) or F leucine zipper. The constructs (C/EBPbeta)GBF-F or (VP16)GBF-F consist of GBF-F fused to the activation domains (AD) of either C/EBPbeta or VP16 (in the context of a C/EBPbeta/VP16 fusion) cloned into the pMEX vector. (C/EBPbeta)GBF-F was constructed by isolating a BamHI-HindIII fragment containing the pMEX vector and amino acids 1-162 from pMEX CRP2 (Delta163-191) (34) (CRP2 = C/EBPbeta) and joining it to a BamHI-HindIII polymerase chain reaction fragment consisting of GBF-F. (VP16)GBF-F was constructed in a similar fashion; however, two-thirds of the C/EBPbeta AD (amino acids 48-80) are substituted by the VP16 AD(34) .

Rous Sarcoma Virus Promoter

pRG is a Rous sarcoma virus promoter-driven expression vector(32) . pRG GBF-F was generated by introducing GBF-F as an NdeI-HindIII fragment containing the hemagglutinin epitope at the COOH terminus of the protein. These constructs were used for the transfections to generate the nuclear extracts of Fig. 4.


Figure 4: C/EBPalpha:GBF-F heterodimers bind preferentially to a chimeric C/EBPGBF site in vivo. A, nuclear extracts from HepG2 cells transfected with pMEX C/EBPalpha and different concentrations of pRG GBF-F were prepared and assayed by EMSA using either the consensus C/EBP cis-element (top panel) or a C/EBPGBF chimeric cis-element (bottom panel) as probes (lanes 1-6). The positions of bands corresponding to specific homo- and heterodimeric complexes are indicated by arrows. The positions of C/EBPalpha:GBF-F heterodimers are indicated by an asterisk and the labeled arrow. A supershift assay using a C/EBPalpha-specific antiserum was used to confirm the identity of specific bands (lanes 7-9). DNA concentrations were as follows: lane 1, no DNA; lane 2, 2 µg of pMEX C/EBPalpha, 15 µg of pRG; lane 3, 2 µg of pMEX C/EBPalpha, 5 µg of pRG GBF-F, 10 µg of pRG; lane 4, 2 µg of pMEX C/EBPalpha, 10 µg of pRG GBF-F, 5 µg of pRG; lane 5, 2 µg of pMEX C/EBPalpha, 15 µg of pRG GBF-F; lane 6, 15 µg of pRG GBF-F; lane 7, no DNA; lane 8, 2 µg of pMEX C/EBPalpha, 15 µg of pRG; lane 9, 2 µg of pMEX C/EBPalpha, 5 µg of pRG GBF-F, 10 µg of pRG. B, Western blot analysis of C/EBPalpha expression levels. An immunoblot of the same nuclear lysates used in panel A was probed with anti-C/EBPalpha antiserum to determine the level of C/EBPalpha protein expression in each sample.




RESULTS

The Heterodimerizing F Zipper Does Not Inhibit C/EBPalpha Function

C/EBPalpha has previously been shown to function as a transactivator in HepG2 hepatoma cells in a cis-elementdependent manner(33) . We therefore used this system to test the ability of dominant-negative (DN) molecules to inhibit C/EBPalpha transactivation. Two reporter constructs were examined; one was the rat albumin promoter driving the bacterial CAT gene, while the other contained a single C/EBP consensus binding site inserted 5` of a minimal promoter. Transfection conditions were optimized in order to ensure that DN effects would be readily apparent. Increasing concentrations (0.01-10 µg) of the C/EBPalpha expression plasmid were cotransfected into HepG2 cells with a constant amount of a reporter plasmid consisting of the bacterial CAT gene driven by the albumin minimal promoter with or without the C/EBP consensus site (data not shown). At high levels of C/EBPalpha a large nonspecific effect was observed, as the control promoter lacking a C/EBP site was also significantly activated. Thus, in subsequent studies we used a concentration of C/EBPalpha expression plasmid (0.3 µg) at which transactivation is more specific for the C/EBP-driven promoter. A similar dose dependence was obtained using a 1-kilobase fragment of the albumin promoter, again in a C/EBP cis-element-dependent fashion (data not shown) (33) .

Using this optimized transactivation assay, we examined the inhibition of C/EBPalpha activity by four potential DN proteins: 1) a C/EBPalpha bZIP domain lacking an activation domain (DeltaC/EBP), 2) a bZIP domain containing the C/EBPalpha basic region and the F zipper (C/EBP-F), 3) the C/EBPalpha leucine zipper without a basic region (C/EBP LZ), and 4) the F leucine zipper without a basic region (F LZ) (Fig. 1). In cells transfected with equal amounts of plasmids expressing C/EBPalpha and each of the four DNs no inhibition of C/EBPalpha transactivation was observed (data not shown). Therefore we used a C/EBPalpha to DN plasmid ratio of 1:15 to ensure that sufficient DN was expressed relative to C/EBPalpha. Fig. 1shows transactivation levels of the C/EBP-dependent and minimal promoters as histograms below schematic representations of each putative heterodimer. Under these conditions co-expression of DeltaC/EBP or C/EBP-F resulted in 50% inhibition of C/EBPalpha activity. When the basic regions of DeltaC/EBP and C/EBP-F were deleted, the resultant DNs suppressed C/EBP activity slightly less efficiently (60% of C/EBPalpha activity). Using specific antibodies, we analyzed the expression levels of C/EBPalpha and the various DNs in transfected cells (data not shown). The levels of DeltaC/EBP and C/EBP-F were not significantly higher than that of C/EBPalpha.


Figure 1: Inhibition of C/EBPalpha transactivation by four potential DNs. The structures of a wild-type C/EBPalpha dimer and heterodimers between C/EBPalpha and each DN bound to DNA are represented schematically. HepG2 cells were transfected with 0.3 µg of C/EBPalpha expression plasmid alone or with C/EBPalpha and a potential DN expression plasmid and a CAT reporter plasmid. CAT activities are shown in histograms as fold activation (±standard deviation from three independent experiments) relative to the activity of the reporter plasmid alone. Transfections were carried out using 10 µg of the albumin minimal promoter reporter (nucleotides -35 to +22), either with or without a single consensus C/EBP binding site. A 1:15 molar ratio of C/EBPalpha to DN expression plasmids was used.



Addition of the Plant bZIP Basic Region to the F Zipper Creates GBF-F That Binds a Chimeric Site with C/EBP

A potentially more effective strategy for DN design is to create a protein that displaces C/EBPalpha from its normal palindromic cis-element onto a new DNA site, thereby inhibiting C/EBPalpha-mediated transactivation. We therefore constructed a protein that would heterodimerize with C/EBPalpha via the F zipper, but would bind to a novel cis-element through a heterologous basic region. A comparison of the consensus recognition sequences of several bZIP protein families (Fig. 2A) shows that plant GBF recognize a DNA sequence that shares only one base with the five bases of C/EBP half-site. Since the GBF sequence is the most divergent from a canonical C/EBP site, we chose the basic region from the GBF-1 protein (21) for construction of a bZIP chimera (designated GBF-F).


Figure 2: C/EBPalpha and GBF-F heterodimerize and bind a chimeric site. A, comparison of consensus DNA binding sites of four bZIP protein families. The 4- or 5-bp half-sites are shaded. The C/EBP half-site shares four positions with the PAR site(59) , 2 positions with the ATF/CREB (60) and Fos/Jun(61, 62) sites, but only one position (the G at +1) with the GBF site(21) . To date, the GBF binding site is the most divergent from that of C/EBP among bZIP protein families. B, the bZIP domains of C/EBPalpha and GBF-F were overexpressed in bacteria and purified to homogeneity. The two proteins were mixed in the indicated molar ratios and binding to oligonucleotides containing either the C/EBP consensus site ATTGCGCAAT (left panel) or an idealized chimeric element, ATTGCGTGGA, was assayed by EMSA. The positions of the relevant homo- and heterodimeric species are indicated.



Heterodimers between C/EBP proteins and GBF-F would be predicted to recognize a novel DNA sequence composed of C/EBP and GBF half-sites. In order to test this hypothesis, electrophoretic mobility shift assays (EMSA) were carried out using purified, bacterially-expressed bZIP domains of C/EBPalpha and GBF-F with oligonucleotide probes containing either a consensus C/EBP site or an idealized C/EBP GBF chimeric site (Fig. 2B, left and right panels). DeltaC/EBP alone bound both oligonucleotides, although binding to the chimeric site was noticeably weaker than to the consensus C/EBP site. This finding is consistent with previous studies that demonstrated that C/EBP proteins have the ability to bind to sites which only loosely conform to the consensus site(13) . As the amount of GBF-F in the binding reaction was increased, the amount of DeltaC/EBP homodimers binding to the consensus C/EBP site decreased (Fig. 2B, left panel). At the same time, binding of DeltaC/EBP homodimers to the chimeric sequence was completely abrogated, even at the lowest concentration of GBF-F (Fig. 2B, right panel). Concomitantly, a faster-migrating complex appeared whose intensity increased with additional GBF-F. We conclude that this novel species is a DeltaC/EBP:GBF-F heterodimer, as it displays high affinity for a C/EBPGBF chimeric site.

GBF-F Inhibits C/EBPalpha Transactivation

Having demonstrated that GBF-F can disrupt C/EBPalpha binding to its cognate site, we next investigated whether GBF-F would act as an efficient inhibitor of C/EBPalpha activity in transactivation assays. We compared GBF-F to either C/EBP-F (see Fig. 1) or a chimera consisting of the GBF basic region and the wild-type C/EBPalpha zipper (GBF-C/EBP) in HepG2 cells (Fig. 3A). Both GBF-C/EBP and C/EBP-F decreased C/EBPalpha transactivation approximately 2-fold. Although C/EBPalpha:GBF-C/EBP heterodimers should be inactive since they fail to bind C/EBP sites, the relatively poor inhibition by this DN can be explained by the formation of GBF-C/EBP homodimers that do not compete with C/EBPalpha for binding to C/EBP sites. GBF-F, on the other hand, inhibited C/EBPalpha transactivation approximately 4-fold. The greater inhibitory effect of GBF-F likely results from the extremely inefficient homodimerization of the F zipper(20) ; therefore, the majority of GBF-F molecules form inactive heterodimers with C/EBPalpha. We conclude that both components of GBF-F, the GBF basic region and the F zipper, contribute to the efficient suppression of C/EBPalpha activity.


Figure 3: Inhibition of C/EBPalpha transactivation by GBF-F. A, schematic representations of C/EBPalpha:DN heterodimers are as described in Fig. 1, except the C/EBPalpha:GBF-C/EBPalpha and C/EBPalpha:GBF-F heterodimers are shown binding to the chimeric site, represented by a half-stippled circle. Transactivation assays were carried out in HepG2 cells as described in the legend to Fig. 1using the minimal promoter constructs. CAT activities are shown in histograms as fold activation ± standard deviation from three independent experiments. B, GBF-F inhibits C/EBPalpha transactivation of the complete albumin promoter. Conditions identical to those of Fig. 3A were used, except that the reporter plasmid was pAT2-CAT which contains the albumin promoter (nucleotides -787 to +22).



As shown in Fig. 2, addition of increasing amounts of GBF-F protein to binding reactions containing C/EBPalpha caused an immediate removal of C/EBPalpha from the chimeric site, but only a gradual removal of C/EBPalpha from its consensus binding site. As most C/EBP sites found in vivo conform only loosely to the consensus sequence, we wished to test whether the effect of GBF-F would be more dramatic on a promoter containing a natural, imperfect C/EBP site. The C/EBP element found in the albumin promoter (designated DEI or the D site; ATTTTGTAAT)(38, 39) , is typical of most C/EBP sites identified thus far, consisting of one half-site that conforms fairly closely to the consensus and another, more divergent half-site. EMSA experiments confirm that C/EBPalpha binds to the consensus site approximately 3-fold better than the albumin DEI site (data not shown). We therefore tested the ability of GBF-F to inhibit C/EBPalpha transactivation of the albumin promoter-CAT reporter construct (Fig. 3B). GBF-F was an extremely efficient inhibitor, completely abolishing C/EBPalpha-dependent transactivation under the standard transfection conditions. These data suggest that the dominant-negative properties of GBF-F are more apparent when C/EBPalpha is bound to a weaker cis-element and suggest that GBF-F should act as an efficient DN in vivo.

GBF-F Displaces C/EBP from a Consensus DNA Site in Nuclear Extracts

We next sought to establish that the DN activity of GBF-F in vivo results from the formation of C/EBPalpha:GBF-F heterodimers. Nuclear extracts were prepared from HepG2 cells transfected with various combinations of C/EBPalpha and GBF-F expression plasmids and were tested for binding to the oligonucleotides described in Fig. 2. A high background of endogenous binding activity was observed in untransfected HepG2 cell extracts when the C/EBP consensus oligonucleotide was used as the probe (Fig. 4A, top panel, lane 1). However, these complexes do not contain endogenous C/EBPalpha, as no C/EBPalpha was observed in Western blot analysis of an untransfected HepG2 cell extract (Fig. 4B, lane 1). Nuclear extracts from cells transfected with the C/EBPalpha expression plasmid alone displayed two novel binding species (denoted as C/EBP-specific bands, Fig. 4A), whose identities were confirmed using a C/EBPalpha-specific antiserum in a supershift assay (Fig. 4A, top panel, compare lanes 7 and 8). The upper band consists of C/EBPalpha homodimers and the lower band consists of a heterodimer formed with an unidentified partner(26, 34) . Nuclear extracts from cells transfected with a constant amount of C/EBPalpha expression vector and increasing amounts of GBF-F expression vector showed a dramatic decrease in the intensity of C/EBPalpha-specific binding. This effect cannot be explained by a reduction in C/EBPalpha expression (compare Fig. 4A, top, lanes 2-5 with B, lanes 2-5). Therefore, we infer that GBF-F inhibits C/EBPalpha binding to its cognate site. To confirm the existence of C/EBPalpha:GBF-F heterodimers in vivo, the same nuclear extracts were tested for binding to the idealized C/EBPGBF chimeric binding site (Fig. 4A, lower panel). Weak C/EBPalpha binding was observed with the chimeric site (Fig. 4A, lower, lanes 1 and 2) which was rapidly competed by increasing GBF-F expression. Concomitantly, a novel binding activity appeared (indicated by an asterisk). This novel binding activity increased in intensity with increasing input GBF-F (Fig. 4B, lower, lanes 3-5) and was supershifted with the C/EBPalpha antiserum (Fig. 4B, lower, lane 9), indicating that this complex represents a C/EBPalpha:GBF-F heterodimer. Thus, the in vivo behavior of GBF-F mimicked that observed in vitro (Fig. 2), confirming that GBF-F acts by forming heterodimers with C/EBPalpha that are unable to bind to C/EBP binding sites. For unknown reasons, GBF-F homodimers in nuclear extracts from transfected cells bound to both sites (Fig. 4A, lane 6), while no binding to either site was observed with bacterially-expressed GBF-F (Fig. 2B).

GBF-F Inhibits Other C/EBP Family Members in a Leucine Zipper-dependent Manner

The C/EBP family members share a high degree of amino acid homology in the bZIP region and display related DNA-binding and dimerization specificities. As one of our goals was to develop a DN to efficiently disrupt the function of all C/EBP family members, we tested whether GBF-F was able to inhibit other C/EBP proteins. The synthetic C/EBP-dependent promoter was activated by the three family members examined (C/EBPalpha, C/EBPbeta, and C/EBP) although to slightly different levels (Fig. 5). Co-expression of GBF-F inhibited transactivation by each protein to similar degrees, showing that GBF-F functions as an effective DN for the C/EBP family as a whole.


Figure 5: GBF-F inhibits transactivation by three C/EBP family members in a leucine zipper-dependent manner. Fold activation of the C/EBP-dependent CAT reporter in HepG2 cells by C/EBPalpha, C/EBPbeta, or C/EBP in either the absence (rows 1-4) or presence (rows 5-8) of GBF-F is represented graphically as fold activation ± standard deviation from three independent experiments. Cotransfection of GBF-F did not affect the transactivation properties of C/EBPalpha proteins carrying the leucine zippers of GCN4 or VBP (rows 9-12). Transfection conditions were as described in the legend to Fig. 1.



To confirm that C/EBP-specific inhibition by GBF-F occurs as a result of leucine zipper interactions, we used chimeric C/EBPalpha proteins containing the leucine zipper from two other bZIP proteins, GCN4 and VBP. These hybrid proteins (C/EBP-GCN4 and C/EBP-VBP) activated the C/EBP-dependent promoter to approximately the same level as wild-type C/EBPalpha (Fig. 5). Cotransfection of GBF-F did not affect transactivation by either chimera, indicating that GBF-F specifically inhibits C/EBP family members and is unable to inhibit the activity of proteins containing unrelated leucine zippers.

GBF-F in Conjunction with C/EBP Activate a Chimeric Site Creating Gain-of-function Properties

The inhibition of C/EBPalpha transactivation by GBF-F involves the formation of a C/EBPalpha:GBF-F heterodimer that preferentially binds a chimeric C/EBPGBF site. We tested whether GBF-F in concert with C/EBPbeta is capable of activating the chimeric novel C/EBPGBF site (Fig. 6). The C/EBPGBF site was cloned into the minimal promoter construct described above, in an orientation such that the C/EBP half-site is proximal to the TATA box. GBF-F lacking an activation domain was able to activate the chimeric site only 2-fold when cotransfected with C/EBPbeta (data not shown), suggesting that the presence of an activation domain on both dimeric subunits is essential for efficient transactivation. The activation domains from either C/EBPbeta (Fig. 6A) or VP16 (Fig. 6B) were then joined to the NH(2) terminus of GBF-F and the resulting proteins were expressed in HepG2 cells, alone or with wild-type C/EBPbeta. By themselves, neither of the GBF-F-derived proteins displayed activity on the chimeric site. However, in combination with C/EBPbeta, both GBF-F proteins efficiently activated the reporter. (VP16)GBF-F was slightly more active than (C/EBPbeta)GBF-F in this assay, probably due to the fact that the VP16 activation domain is more potent than that of C/EBPbeta. The decrease in transactivation potential at high C/EBPbeta activation domain concentration has been observed previously(35) . The activation of novel cis-elements by GBF-F in concert with C/EBPbeta suggests the possible use of this molecule in regulating genes containing this chimeric site.


Figure 6: C/EBPbeta and GBF-F containing an activation domain cooperate to activate a chimeric cis-element. A, the AD of C/EBPbeta (residues 1-162) was joined to GBF-F and the resulting protein was tested for its ability to transactivate a reporter gene carrying a chimeric C/EBPGBF element. Various concentrations of GBF-F containing the C/EBPbeta AD were transfected alone or in combination with a fixed concentration of a C/EBPbeta expression plasmid. B, as in A except the VP16 activation domain replaced the C/EBPbeta activation domain to form a VP16-GBF-F fusion.




DISCUSSION

In an attempt to create a potent dominant-negative to C/EBP, we have explored several structurally different protein designs. The first two DNs consisted of bZIP domains from either wild type C/EBPalpha (DeltaC/EBP) or C/EBPalpha containing the F zipper (C/EBP-F), which preferentially heterodimerizes with C/EBPalpha. Since these proteins lack an activation domain, their overexpression could be predicted to competitively inhibit C/EBPalpha activity by either the formation of inactive or less-active heterodimers or, in the case of DeltaC/EBP, competition with C/EBPalpha for binding to its cognate site. Both DeltaC/EBP and C/EBP-F were able to inhibit C/EBPalpha activity by 50%. Similar naturally occurring structures also act as efficient DNs for other bZIP families, including the AP-1 and CREB proteins(9, 10, 11, 40) . A naturally occurring DN molecule analogous to DeltaC/EBP is encoded by the C/EBPbeta gene: translational initiation at an internal AUG codon generates a truncated protein, LIP, that lacks an activation domain (8) . LIP has been reported to exhibit DN properties when co-expressed with the activating form of C/EBPbeta. A similar truncated protein can be generated from C/EBPalpha by internal translational initiation. This shorter form of C/EBPalpha (C/EBP-30) lacks the major activation domain located at the NH(2) terminus(41, 42) . However, unlike C/EBPbeta, C/EBPalpha contains a second activation domain that is retained in C/EBP-30, and, thus, it is likely that this shorter form functions as a weak activator rather than as an inhibitor.

For DeltaC/EBP and C/EBP-F to act as efficient inhibitors, heterodimers between these DNs and C/EBPalpha must be less efficient transactivators than C/EBPalpha homodimers. At present, it is not clear whether these heterodimers are less active. Indeed, the inability of these two DN proteins to efficiently inhibit C/EBPalpha activity suggests that a single activation domain is sufficient for transactivation. In this regard, Goodman's group has been using a heterodimerizing zipper system by mutating amino acids in the e and g positions and substituting a3 asparagine with histidine. They can show that a CREB dimer requires only one phosphorylated activation domain to activate transcription of a CRE-containing promoter(43) .

The second pair of DNs tested consisted of isolated leucine zipper domains. The two C/EBP zippers examined, wild-type and F, were even less effective inhibitors than the bZIP domains, conferring only 40% inhibition of C/EBPalpha activity. We suggest that the lack of efficient inhibition by the F zipper is due to the fact that the F:C/EBPalpha heterodimer is less stable than a C/EBPalpha homodimer. The presence of DNA should drive the equilibrium toward the formation of C/EBPalpha homodimers at the expense of F:C/EBPalpha heterodimers which cannot bind DNA. Zippers designed to heterodimerize even more avidly with C/EBPalpha may not need the basic region for efficient heterodimer formation and, therefore, might function more effectively as inhibitors. However, it is unclear whether a leucine zipper that heterodimerizes with C/EBPalpha more stably than the F zipper could be constructed.

Similar DN molecules with specificity for the Jun/Fos family have been reported. These inhibitors are fusion proteins produced by joining the v-Jun or c-Jun leucine zippers to the bacterial lexA gene (9, 44) . In both cases, the LexA:Jun hybrids efficiently inhibited transactivation by Fos and Jun. Proteins that form a heteromultimer complex which cannot bind DNA has been identified for bHLH (basic helix-loop-helix) proteins. In the bHLH family, the DN proteins Id and extramacrochaetae can multimerize with other bHLH factors, but, because they lack a functional DNA-binding domain, repress the DNA binding activities of their multimerization partners(45, 46, 47, 48) .

GBF-F is a potent DN to C/EBP. The most successful DN design was achieved by altering the DNA binding specificity of the DN:C/EBP heterodimer. The DN thus configured, GBF-F, inactivated sequence-specific DNA binding by C/EBPalpha in vitro as well as transactivation by C/EBPalpha and related family members in HepG2 cells. GBF-F is a chimera, both parts of which are critical for optimal DN properties: one component is the F leucine zipper and the second is the DNA-binding region from the plant bZIP protein, GBF-1(21) . The bipartite bZIP helix permits the exchange of basic regions and, therefore, DNA-binding specificity without changing dimerization specificity(37) . Alone, each component of the GBF-F chimera has modest DN potential (Fig. 3A); in combination, these components exhibit enhanced inhibitory properties (Fig. 3, A and B). Furthermore, the DN activity of GBF-F is specific to bZIP proteins of the C/EBP family, as demonstrated by using C/EBPalpha derivatives containing a heterologous leucine zipper (Fig. 5).

An alternative DN could be generated by extending the leucine zipper in a manner that engenders additional dimerization stability and, simultaneously, prevents the target protein from binding to DNA. One approach, which is currently under development(49) , is the addition of a DNA mimetic in the form of an acidic amphipathic alpha-helical extension to the NH(2) terminus of the F leucine zipper. Such an extension would be predicted to form a heterodimeric coiled-coil interaction with the bZIP basic region helix, thereby stabilizing the dimeric complex and hindering basic region interactions with DNA.

When fused to an activation domain, GBF-F cooperates with C/EBPbeta to transactivate a chimeric DNA site, showing that this DN protein can also exhibit gain-of-function properties (Fig. 6). We show by both biochemical and biological experiments that GBF-F heterodimerizes with C/EBPalpha and binds a chimeric site, consisting of C/EBP and GBF recognition motif half-sites, in preference to a normal C/EBP site. C/EBPalpha:GBF-F heterodimers preferentially bound a C/EBPGBF site in vitro, while in transfected cells, a reporter gene containing the C/EBPGBF chimeric binding site was activated only by the combination of C/EBPbeta and a GBF-F derivative bearing an activation domain. Although the C/EBPGBF sequence is predicted to be the optimal binding site for C/EBP:GBF-F heterodimers, binding site selection experiments currently in progress should reveal whether this is the case.

In vivo, the relative levels of C/EBP proteins and GBF-F should specify the dominant-negative or gain-of-function effects of GBF-F. If GBF-F is expressed at lower levels than endogenous C/EBP, both C/EBP homodimers and C/EBP:GBF-F heterodimers should exist in the cell. Under these conditions, gain-of-function properties should predominate over DN effects. If GBF-F is expressed at greater levels than C/EBP, we expect to observe the activation of chimeric binding sites as well as more complete inhibition of C/EBP homodimer activity. A natural example of a GBF-F-like molecule is CHOP (gadd153), a putative dominant-negative molecule of the C/EBP family(12, 50) . CHOP10 inhibits C/EBP proteins from binding to canonical C/EBP sites, but also recognizes a novel sequence as a heterodimer with C/EBP. (^2)However, the significance and specificity of DNA binding by CHOP10:C/EBP heterodimers is not yet clear. CHOP10 could potentially mediate the activation of a distinct set of genes through its binding with C/EBP family members. In light of its potential to activate natural target genes, CHOP10 may not be an appropriate DN for inhibiting C/EBP function in vivo. GBF-F, on the other hand, confers a novel, presumably non-physiological, DNA-binding specificity to C/EBP and, therefore, is less likely to cause the activation of endogenous genes when expressed in mammalian cells.

C/EBP DN proteins such as GBF-F should be useful for inhibition of C/EBP activity in vivo. For example, two groups have reported the targeted disruption of the c/ebpbeta gene (51, 52) and have shown that this mutation has only minor effects on the expression of inflammatory cytokine genes. These results are surprising in light of previous data showing that C/EBPbeta plays a critical role in the expression of genes such as interleukin-1, interleukin-6, and monocyte chemoattractant protein-1 in cell lines(26, 53, 54, 55) , and suggest that a redundant C/EBP activity compensates for the absence of C/EBPbeta in knockout animals. To demonstrate that the compensatory activity is provided by another member of the C/EBP family, one could create DN transgenic mice that express the C/EBP inhibitor specifically in hepatocytes or monocytic cells. Assuming that these animals are viable, they could be used to examine the requirement for a C/EBP factor in the activation of acute-phase responsive genes or the induction of inflammatory cytokines, respectively.

Numerous experiments indicate that the expression or activation of C/EBP proteins is coincident with terminal differentiation of a particular cell lineage. For instance, C/EBPbeta synthesis is induced during the conversion of HL60 cells or M1 myeloblasts into mature macrophages(56, 57) . Similarly, C/EBP is strongly up-regulated when 32D Cl3 pregranulocytic cells differentiate into granulocytes in response to treatment with granulocyte colony-stimulating factor(57) . In these and other cases, it has not been established whether C/EBP proteins play an essential role in terminal differentiation. Overexpression of C/EBP DNs in the appropriate cell types should provide a straightforward means of determining the requirement for C/EBP-related activator proteins in cell maturation.

The gain-of-function activity observed for the GBF-F protein containing an activation domain suggests the possible use of this molecule in regulating experimental transgenes. When expressed in submolar levels relative to endogenous C/EBP proteins, the GBF-F construct could be used as a tool to activate transfected genes whose transcription is regulated by the C/EBPGBF-F cis-element. Since the activation of these chimeric sites would be dependent on the presence of C/EBP proteins, this approach provides a strategy to control transgene expression by inducing the synthesis or activity of an endogenous C/EBP protein. For example, several mediators of the inflammatory response have been shown to stimulate C/EBPbeta-dependent transactivation(53, 58) . If the same activating mechanism applies to C/EBPbeta:GBF-F heterodimers, stimulation of C/EBPbeta by these cytokines in cells expressing low levels of GBF-F should induce transcription from promoters containing the C/EBPGBF site without significantly disrupting normal gene expression in the cell.


FOOTNOTES

*
This work was supported in part by a grant from the National Cancer Institute under contract with ABL. 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.

Special Fellow of the Leukemia Society of America. Present address: Dept. of Cell Biology and Biochemistry, 3601 4th St., Lubbock, TX 79430.

**
To whom correspondence should be addressed. Tel.: 301-496-8753; Fax: 301-402-3095; vinsonc{at}dc37a.nci.nih.gov

(^1)
The abbreviations used are: C/EBP, CAAT/enhancer-binding protein; CRP, C/EBP-related protein; LZ, leucine zipper; CHOP, C/EBP-homologous protein; bHLH, basis helix loop helix; DN, dominant-negative; GBF-1, G-box binding factor-1; CAT, chloramphenicol acetyltransferase; VBP, vitellogenein binding protein; AD, activation domain(s); EMSA, electrophoretic mobility shift assays; CREB, cAMP response element binding protein.

(^2)
D. Ron, personal communication.


ACKNOWLEDGEMENTS

We thank U. Schindler and A. Cashmore for the GBF-1 plasmid and Ken Zaret for the albumin promoter-containing plasmid pAT2-CAT. We thank V. Vinson, and Marge Strobel for comments, and Claude Klee for support and encouragement.


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