Functional Interaction of bZIP Proteins and the Large Subunit of Replication Factor C in Liver and Adipose Cells*

The transcription factor CCAAT/enhancer-binding protein- a (C/EBP a ) has a vital role in cell growth and differentiation. To delineate further a mechanism for C/EBP a -mediated differentiation, we screened C/EBP a interacting proteins through far-Western screening. One of the strongest interactions was with RFC140, the large subunit of the replication factor C complex. C/EBP a specifically interacted with RFC140 from rat liver nuclear extract as determined by a combination of affinity chromatography and co-immunoprecipitation. Subsequent far-Western blotting showed that the bZIP domain of C/EBP a interacted with the DNA-binding region of RFC140. Overexpression of RFC140 in mamma-lian cells increased the transactivation activity of C/EBP a on both minimal and native promoters. Consistent with the enhanced transactivation, a complex of C/EBP a and RFC140 proteins with the cognate DNA el-ement was detected in vitro . The specific interaction between C/EBP a and RFC140 was detected in the terminal differentiation of 3T3-L1 preadipocytes to adipocytes. The synergistic transcription effect of these two proteins increased the promoter activity and protein expression of peroxisome proliferator-activated receptor- g , which is a main regulator of adipocyte differentiation. SDS-polyacrylamide chemiluminescent ECL reagent. Transient Transfection Analysis— Subconfluent Fao cells (kindly provided transfected by the stand- ard calcium phosphate technique. 48 h after transfection, cells were washed twice with phosphate-buffered saline, harvested, and lysed in 250 m l of reporter lysis buffer. Lysates were clarified by centrifugation at 15,000 rpm fo r 5 s in anEppendorf microcentrifuge, and relative luciferase activity was determined on duplicate aliquots of extract using the luciferase assay system (Promega). All transfections included pCMV- b -galactosidase, and relative b -galactosidase activity was determined using the b -galactosidase enzyme assay system (Promega) to normalize results for transfection efficiency.

Transcription factors, regulatory proteins whose activities are tightly controlled, have been referred to as a paradigm for modular proteins. Minimally, these factors encode a DNAbinding domain and a transcription-activating domain. Additional functional modules were defined by studies analyzing the regulation of transcription factor activity. For example, factors in the nuclear receptor superfamily encode a ligand-binding domain that renders their function hormonedependent. The striking feature that is shared among these protein modules is their ability to function when transferred to another protein (1). Furthermore, structure/function ex-periments showed that some protein modules, e.g. transactivation domains, function by providing a surface for interacting with other proteins.
The transcription factor C/EBP␣ 1 contains a bZIP module and is constitutively expressed in fat cells and adult organs such as liver and lung. A C/EBP␣ homodimer binds the DNA major groove through ␣-helices that are held in register by a classical coiled-coil motif, the leucine zipper (2,3). C/EBP␣ activates transcription of genes that typify the differentiated cell phenotype, most notably in liver and fat cells (4 -6). Interestingly, a relationship between expression of C/EBP␣ and cell growth control has been established (7)(8)(9). Fibroblasts transfected with a C/EBP␣ expression plasmid were observed to withdraw from the cell cycle (10). Subsequently, a role for C/EBP␣ in fat cell differentiation was revealed. A hormonal regimen that initiates differentiation of 3T3-L1 cells results in C/EBP␣ expression coincident with conversion of preadipocytes into fat-producing cells in culture (11)(12)(13)(14). In fact, constitutive overproduction of C/EBP␣ induces growth arrests of several fibroblastic cell lines and in some cases is sufficient to promote adipose conversion (15), suggesting that C/EBP␣ is a component of a differentiation switch. An essential role for C/EBP␣ during fat cell differentiation was documented by expressing an antisense C/EBP␣ construct in 3T3-L1 cells that blocked adipose conversion. Furthermore, homozygous disruption of the C/EBP␣ gene is characterized by accumulation of undifferentiated fat cells prior to prenatal death (16). Recent reports demonstrated a requirement for the Rb protein in the differentiation of fat cells and showed that Rb can interact with C/EBP proteins (17)(18)(19). Another report showed that the cyclin-dependent kinase inhibitor WAF1 plays a role in growth arrest following C/EBP␣ expression in a cultured fibrosarcoma cell line (10). To learn more about the role that C/EBP␣ plays in cell differentiation, we took advantage of the modular nature of the protein.
Activator proteins such as C/EBP␣ bind specific DNA sequences located either upstream or downstream of the core promoter. In response to physiological cues, activators stimulate transcription initiation by interacting with general transcription factors, with TATA-associated factors, or with coactivators. We utilized a radiolabeled GST-C/EBP␣ fusion protein to screen C/EBP␣-interacting proteins using an expression cDNA library prepared from rat liver mRNA. One of the cDNAs we isolated encoded the large subunit of RFC. We mapped the interacting domain of each protein and show that transient transfection of RFC140 has an increased effect on the transactivation activities of C/EBP␣. Furthermore, the functional interaction of C/EBP␣ and RFC140 may be necessary for adipocyte differentiation.

EXPERIMENTAL PROCEDURES
Cloning of cDNAs Encoding C/EBP␣-interacting Proteins-A gt11 cDNA library prepared from rat liver mRNA (CLONTECH) was plated at a density of 30,000 plaques/150-mm plate as described (21). Briefly, phage were plated, incubated at 42°C for 4 h, and overlaid with an isopropyl-␤-D-thiogalactopyranoside-impregnated filter for 4 h at 37°C. A duplicate filter was held in place overnight at 37°C. Filters were rinsed in 1ϫ buffer containing 10 mM Tris (pH 7.5), 150 mM NaCl, and 0.05% Tween 20 and blocked for 1 h in Hyb75 (50 mM Tris (pH 8.0), 75 mM KCl, 0.1 mM EDTA, 2.5 mM MgCl 2 , 5 mM 2-mercaptoethanol, and 0.1% Nonidet P-40) containing 5% nonfat milk. Radiolabeled GST-C/ EBP␣ was added to 25 ml/filter Hyb75 containing 5% nonfat milk and 100,000 cpm/ml labeled probe overnight at 4°C. Filters were washed three times for 5 min each at 4°C in 50 ml/filter Hyb75 containing 0.25% nonfat milk. Filters were air-dried briefly and exposed to film. Duplicate positive plaques were purified to homogeneity; the EcoRI inserts were released and subcloned into pBluescript II SK (Stratagene); and the DNA sequence was determined.
Construction, Expression, and Purification of GST Fusion Proteins-The vector pGEX-2T (Amersham Pharmacia Biotech) was modified by the insertion of a protein kinase A phosphorylation site as described (22). In-frame fusions were prepared from C/EBP␣D1-2 (3) by standard cloning techniques. Constructs lacking the leucine zipper and the bZIP domain were prepared by placing in-frame stop codons at amino acids 310 and 272, respectively. MluI digestion and fill in created the fusion to amino acid 192. Constructs expressing amino acids 281-358 and 281-342 have been described (23). Fusion proteins are designated according to the C/EBP␣ amino acids that they contain and are shown schematically in Fig. 2. Detailed construction of these plasmids has been described (23,38). RFC140 fusion proteins were prepared from the full-length mouse RFC cDNA (kindly provided by Dr. Yoshihiko Yamada, National Institutes of Health). The pair of oligonucleotide primers generated a 5Ј-BamHI site and a 3Ј-EcoRI site for cloning. Expression plasmids were transformed into host strain BL21, grown to an A 600 of 0.8, and induced with 1 mM isopropyl-␤-D-thiogalactopyranoside for 2 h. Bacteria were harvested, lysed by sonication, and purified by affinity purification using glutathione-agarose beads (Amersham Pharmacia Biotech) according to the manufacturer's instructions. Equivalent amounts of purified fusion proteins were determined by SDS-polyacrylamide gel electrophoresis and Coomassie Blue staining.
Affinity Chromatography-Equivalent amounts of MBP or MBP-RFC140 were coupled to Sepharose 4B. 100-l columns were equilibrated in Hyb150 (50 mM Tris (pH 8.0), 150 mM KCl, 0.1 mM EDTA, 2.5 mM MgCl 2 , 5 mM 2-mercaptoethanol, and 0.1% Nonidet P-40). 70 g of rat liver nuclear extract was passed over each column, followed by a 15-volume wash with Hyb150. Columns were eluted stepwise with 1.8 volumes of buffer containing 0.25, 0.5, and 1.0 M KCl. Equivalent percentages of the unbound and eluted fractions were loaded onto 15% SDS-polyacrylamide gels, separated, and electroblotted for Western blot analysis. The presence of C/EBP␣ was detected with anti-C/EBP␣ antiserum.
Preparation of Antiserum-The MBP-RFC140 fusion protein was expressed in 1 liter of broth and purified on maltose beads (New England Biolabs Inc.). Purified antigen was excised from a 15% SDSpolyacrylamide gel, and antiserum was prepared according to standard procedures as recommended by Spring Valley Labs.
Western and Modified Western Blot Analyses-Equivalent amounts of purified truncated C/EBP␣ proteins were fractionated on a 12% SDS-polyacrylamide gel and transferred to nitrocellulose (0.45 m; BA85, Schleicher & Schü ll). The filter was blocked for 1 h in binding buffer A (20 mM Tris (pH 8.0), 120 mM NaCl, 0.1 mM EDTA, 2.5 mM MgCl 2 , 0.05% Nonidet P-40, and 1 mM dithiothreitol) supplemented with 3% nonfat milk. Purified GST-RFC140 (1 g) was added to binding buffer A and incubated with rocking for 30 min at room temperature. The solution was decanted, and filters were washed three times with fresh binding buffer A. Bound RFC140 was detected with anti-RFC Ig (12 ng/ml), followed by horseradish peroxidase-conjugated donkey antirabbit IgG (1:5000), and visualized by chemiluminescence using ECL reagent (Amersham Pharmacia Biotech). Filters were exposed to East-man Kodak X-Omat AR film. Similarly, equivalent amounts of purified GST-RFC140 fusion proteins were separated and electroblotted onto nitrocellulose membrane. The filters were incubated with purified GST-C/EBP␣ and washed, and bound C/EBP␣ was detected with specific antiserum essentially as described above.
Immunoprecipitation and Co-immunoprecipitation Analyses-C/ EBP␣ was immunoprecipitated from 200 g of rat liver nuclear extract by incubation with 15 l of anti-C/EBP␣ IgG in a total volume of 500 l of binding buffer B (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, and 10% glycerol). After rocking for 1 h at 4°C, 20 l of protein A-Sepharose beads (1:1 slurry; Amersham Pharmacia Biotech) was added. The sample was rocked for 1 h at 4°C, washed five times with binding buffer B, and solubilized with SDS sample buffer. The eluted proteins were separated on a 10% SDSpolyacrylamide gel and electroblotted, and coprecipitating RFC140 was detected by Western blotting with anti-RFC140 IgG. Secondary detection was carried out with horseradish peroxidase-conjugated donkey anti-rabbit IgG, followed by chemiluminescent development with ECL reagent.
Transient Transfection Analysis-Subconfluent Fao cells (kindly provided by Mary Weiss, Institut Pasteur) were transfected by the standard calcium phosphate technique. 48 h after transfection, cells were washed twice with phosphate-buffered saline, harvested, and lysed in 250 l of reporter lysis buffer. Lysates were clarified by centrifugation at 15,000 rpm for 5 s in an Eppendorf microcentrifuge, and relative luciferase activity was determined on duplicate aliquots of extract using the luciferase assay system (Promega). All transfections included pCMV-␤-galactosidase, and relative ␤-galactosidase activity was determined using the ␤-galactosidase enzyme assay system (Promega) to normalize results for transfection efficiency.
Electrophoretic Mobility Shift Assay-GST fusion proteins were purified according to the protocol described above. Equal amounts of GST-C/EBP␣298 protein (50 ng) were incubated with the 32 P endlabeled CRE oligonucleotides in the presence or absence of GST-RFC140-(1-151) or GST-full-length RFC140. Binding reactions were performed in a 20-l volume containing increasing amounts of RFC140 proteins, 4 l of 5ϫ binding buffer C (20 mM HEPES (pH 7.5), 50 mM KCl, 1 mM dithiothreitol, 2.5 mM MgCl 2 , and 20% Ficoll), 2 g of poly(dI-dC) as nonspecific competitor DNA, 2 g of bovine serum albumin, and 10,000 -15,000 cpm of radiolabeled oligonucleotide. After 30 min of incubation at room temperature, samples were loaded onto an 8% nondenaturing polyacrylamide gel in 0.5ϫ Tris borate/ EDTA buffer (pH 8.3). After electrophoresis, gels were dried and exposed to x-ray film.
Induction of Adipocyte Differentiation-3T3-L1 fibroblasts were differentiated into adipocytes after they reached confluency by the addition of differentiation medium (high-glucose Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 1 mM L-glutamine, 0.5 mM isobutylmethylxanthine, 1 M dexamethasone, and 1 g/ml insulin). After 2 days, the 3T3-L1 cells were transferred to adipocyte growth medium (high-glucose Dulbecco's modified Eagle's medium plus 10% fetal calf serum, 1 mM L-glutamine, and 1 g/ml insulin) and refed every 2 days. Differentiation of fibroblasts to mature adipocytes was confirmed by oil red O staining of lipid vesicles.

RFC140 Associates with C/EBP␣ in Vitro-
We identified proteins that physically associate with C/EBP␣ by interacting a radiolabeled GST-C/EBP␣ fusion protein with proteins expressed from a liver gt11 cDNA library (21). During this screen, which was confirmed through 3ϫ selective far-Western blot hybridization procedures, we purified a cDNA encoding RFC140. The cDNA insert was subcloned for bacterial expression as a fusion to MBP, and specific antiserum was raised. To more rigorously test the specific interaction between RFC140 and C/EBP␣, we chromatographed fresh rat liver nuclear extract over an MBP-RFC140 affinity column. As a specificity control, an MBP affinity column was run in parallel. After extensive washing, bound proteins were eluted, gel-fractionated, and analyzed by Western blotting with anti-C/EBP␣ antiserum. Endogenous C/EBP␣ migrated as a 42-kDa protein and a 30-kDa internal translation initiation product (Fig. 1A, first lane). The MBP-RFC140 affinity column bound most of the C/EBP␣ in the nuclear extract (second lane), and the interac-tion was stable up to 0.25 M KCl, requiring 0.5 M KCl for elution (fifth lane). In contrast, C/EBP␣ was found exclusively in the unbound fraction of the MBP affinity column (ninth lane). These results suggest that the interaction of RFC140 with C/EBP␣ occurs with specificity.
Co-immunoprecipitation of Endogenous RFC140 with C/EBP␣-The association between C/EBP␣ and RFC140 was further characterized by co-immunoprecipitation analysis. C/EBP␣ was immunoprecipitated from freshly prepared rat liver nuclear extracts, gel-fractionated, and analyzed for RFC140 coprecipitation by Western blotting with anti-RFC140 IgG. As shown in Fig. 1B (fourth lane), endogenous RFC140 was detected as a coprecipitant in C/EBP␣ immunoprecipitates. A parallel immunoprecipitate formed with preimmune serum failed to show RFC140 immunoreactivity (second lane). Compared with RFC140 immunoprecipitates (third lane), ϳ10% of endogenous RFC140 protein could be calculated to interact with C/EBP␣. These results are consistent with the interpretation that C/EBP␣ is capable of associating with RFC140 in vitro and in vivo.
The DNA-binding Domain of RFC140 Associates with the bZIP Domain of C/EBP␣-To determine the protein domains mediating association between these nuclear factors, sequential deletion constructs were expressed as GST fusion proteins, purified, and analyzed for protein interaction using a modified Western blot technique. Equivalent amounts of deletion proteins of C/EBP␣ or RFC140 were separated on SDS-polyacrylamide gels and transferred to nitrocellulose membrane. The blot was incubated with purified GST-RFC140 or GST-C/EBP␣, washed extensively, and subsequently developed with RFC140or C/EBP␣-specific antibodies, respectively. As shown in Fig.  2A (second through fourth and seventh lanes), soluble RFC140 bound to membrane-bound C/EBP␣ deletion proteins if the DNA-binding domain and leucine zipper were present. In addition, weaker association was observed when the leucine zipper was removed (sixth lane), suggesting that interaction is not solely mediated by a coiled-coil interaction. The reciprocal strategy was used to delineate the region of RFC140 required for interaction with C/EBP␣. As shown in Fig. 2B, RFC140-(151-360), the original cDNA insert, bound C/EBP␣. Surprisingly, the truncated protein encompassing amino acids 361-545 bound C/EBP␣ more efficiently. This construct encompasses the DNA-binding/DNA ligase-like domain of RFC140 (amino acids 369 -480), and any truncated protein containing this domain bound C/EBP␣ efficiently (third through fifth lanes). In contrast, the amino-terminal (second lane) and carboxyl-terminal (sixth lane) halves of the protein were dispensable for interaction with C/EBP␣. These results indicate that the bZIP domain of C/EBP␣ is involved in the association with RFC140 through the DNA-binding/DNA ligase-like domain.
RFC140 Interacts with Other bZIP Proteins-As shown in Fig. 2, RFC140 interacted with C/EBP␣ through the bZIP domain. This led us to examine whether RFC140 interacts with other bZIP proteins. The N-terminal region (amino acids 1-545) including the C/EBP␣-binding domain and the C-terminal region (amino acids 546 -1148) of RFC140 were purified from Escherichia coli as GST fusion proteins. The proteins were immobilized on glutathione-Sepharose beads and incubated with in vitro translated 35 S-labeled C/EBP␣, C/EBP␤, CREB, activation transcription factor-2, c-Jun, NF-B (p65), and retinoic acid receptor proteins. C/EBP␣ and C/EBP␤ strongly associated with the N-terminal region of RFC140, but not with the C-terminal region (Fig. 3). In addition to these specific physical associations of RFC140 with members of the C/EBP protein family, the N-terminal truncated RFC140 protein interacted with CREB, activation transcription factor-2, and c-Jun. Other structural transcription factors, p65 of NF-B and the retinoic acid receptor, did not interact with RFC140. This suggests that RFC140 specifically interacts with all bZIP proteins.
The RFC140 Subunit Affects Transactivation by C/EBP␣ in a Transient Assay-If C/EBP␣ and RFC140 associate, overproduction of RFC140 might affect C/EBP␣-dependent transactivation activity. To test this, the hepatoma cell line Fao was transfected with expression plasmids encoding C/EBP␣ and RFC140, along with a luciferase reporter driven by the minimal thymidine kinase promoter containing two proximally inserted C/EBP␣-binding sites. As shown in Fig. 4A, transfection of RFC140 alone did not significantly affect basal reporter activity, whereas transfection of C/EBP␣ alone stimulated the minimal promoter construct ϳ9-fold. When cells were cotransfected with a constant amount of C/EBP␣, the activity of the  140) affinity purification of C/EBP␣. MBP-RFC140 and MBP were immobilized on maltose beads, and 70 g of rat liver nuclear extract was loaded onto each column. After extensive washing, bound proteins were step-eluted, and equivalent percentages of each sample were fractionated on a 10% SDS-polyacrylamide gel. Blotted membranes were reacted with anti-C/EBP␣ antiserum, revealing both the 42-and 30-kDa isoforms of C/EBP␣, which were previously described. B, RFC140 coprecipitates with C/EBP␣ from rat liver nuclear extracts. C/EBP␣ and RFC140 were immunoprecipitated (IP) from rat liver nuclear extract by incubation with anti-C/EBP␣ and RFC140 IgG. Antibody complexes were captured on protein A-Sepharose. The beads were washed three times with binding buffer B and eluted into 1ϫ SDS sample buffer. Proteins were fractionated on 8% SDS gels, electroblotted, and developed as described under "Experimental Procedures." minimal promoter increased in an RFC140 dose-dependent fashion. To demonstrate that the enhanced transactivation by RFC140 was dependent upon C/EBP␣ binding, the two C/EBP␣-binding sites in the reporter plasmid were mutated, and the experiments were repeated. Since C/EBP␣ and RFC140 did not show increased transactivation activity with the mutated reporter plasmid (data not shown), this shows that C/EBP␣-mediated co-transactivation by RFC140 is dependent on C/EBP␣ binding to its sequence. Overproduction of RFC140 was confirmed by Western analysis (see Fig. 4C for representative results).
We also determined whether RFC140 had similar effects on transactivation of the native phosphoenolpyruvate carboxykinase promoter (Ϫ275 to ϩ55) in addition to the minimal promoter. Phosphoenolpyruvate carboxykinase expression was high in liver and adipose cells. C/EBP␣ was reported to bind the CRE sites in this promoter, stimulating its transcription (39). As shown in Fig. 4B, transfected C/EBP␣ stimulated transcription from the phosphoenolpyruvate carboxykinase-(Ϫ275 to ϩ55) promoter construct. Upon cotransfection of RFC140, activation increased from ϳ6to ϳ14-fold. Taken together, these results are consistent with the interpretation that the association of C/EBP␣ with RFC140 potentiates the transactivation activity of C/EBP␣.
The RFC140 Subunit Forms a Complex with C/EBP␣ and DNA-The RFC140 subunit can interact with the bZIP domain of C/EBP␣, which apparently correlates with increased C/EBP␣dependent transactivation activity. This suggests that DNA binding by C/EBP␣ may be affected by RFC140. To examine this, we performed electrophoretic mobility shift assays with recombinant GST-C/EBP␣ in the presence or absence of purified GST-RFC140 protein. As shown in Fig. 4C (fifth through seventh lanes), the status of the C/EBP␣⅐DNA shift complex was not affected by the recombinant GST-RFC140-(1-150) protein. When increasing amounts of GST-full-length RFC140 protein were added to the C/EBP␣/CRE reaction mixture, the supershifted band of the complex of C/EBP␣ and RFC140 with the CRE DNA probe (eighth through tenth lanes) was detected,  with the intensity of the supershifted band gradually increasing dependent on the added amounts of the full-length RFC140 protein. The RFC140 protein alone did not bind the CRE probe in the absence of C/EBP␣ (second through fourth lanes). These results suggest that the interaction of the RFC140 subunit with C/EBP␣ can induce a strong complex with the C/EBP␣-binding DNA element, resulting in the increased C/EBP␣-dependent transactivation by RFC140.
C/EBP␣ Specifically Interacts with RFC140 in Differentiating Adipocytes-The RFC140 protein is increased after induction of adipocyte lineage differentiation of 3T3-L1 cells by inducers (35). C/EBP␣ transient expression is known to appear upon the terminal differentiation of 3T3-L1 cells to adipocytes (11). These results suggest a possibility that C/EBP␣ has functional interaction with RFC140 in the adipocyte differentiation procedure. To examine whether the presence of a protein interaction between C/EBP␣ and RFC140 would be found in the differentiation process of adipocytes, we tried to identify the specific interaction of two proteins by co-immunoprecipitation. RFC140 was detected in the co-immunoprecipitated protein fractions by anti-C/EBP␣ antibody using differentiation-induced cell extract at the 5th and 7th days after inducer treatment (Fig. 5). This interaction was not found in undifferentiated cell extract and in the first 3-day extracts after differentiation induction. Fig. 4 results also show that the coexpression of C/EBP␣ and RFC140 increased the promoter activity of phosphoenolpyruvate carboxykinase, which is a representative marker protein for adipocyte maturation. These results suggest that the interaction of C/EBP␣ and RFC140 functions in the terminal differentiation of adipocytes in vivo.
The Synergistic Action of C/EBP␣ and RFC140 Promotes the Promoter Activity and Protein Expression of PPAR␥-Lyle et al. (36) showed that the treatment of antisense oligonucleotides to RFC140 inhibits adipocyte differentiation. In that report, the criteria used for assessment of differentiation included a well defined regulatory event associated with adipogenesis, specifically the differentiation-specific induction of two mRNAs, aP2 and angiotensinogen, both of which were specifically inhibited by antisense RFC140 treatment. The previous study explains that RFC140 may play a critical role in regulating protein expression of an indispensable molecule for adipocyte differentiation. Among many molecules, PPAR␥ has been thought to be one of the most important regulators of differentiation. Interestingly, C/EBP␣ is known to regulate PPAR␥ transcription. These evidences led us to investigate whether the cooperative action of C/EBP␣ and RFC140 affects PPAR␥ expression. For elucidating this, we investigated the promoter activity and protein expression of PPAR␥ after transient transfection of expression plasmids encoding C/EBP␣ and RFC140. The trans-

FIG. 4. RFC140 increases with C/EBP␣-dependent transactivation activity and forms a complex with C/EBP␣ and CRE DNA.
A, Fao cells were transfected with the indicated amounts of expression vectors encoding RFC140 and C/EBP␣. The reporter 2xC/EBP-luciferase (luc) contains the minimal thymidine kinase promoter modified by proximal insertion of two C/EBP-binding sites. The data shown are an average of three independent experiments. B, transactivation activity was also tested on the phosphoenolpyruvate carboxykinase promoter, which can be activated by C/EBP␣. The reporter contains Ϫ275 through ϩ55 of the phosphoenolpyruvate carboxykinase (PEPCK) promoter linked to the luciferase gene. DNAs transfected are indicated beneath the graphs (in micrograms). The data shown are an average of three independent experiments. C, RFC140 forms a complex with C/EBP␣ and DNA. Bacterially expressed and purified GST-C/EBP␣298, GST-RFC140-(1-150), and GST-full-length RFC140 were incubated as indicated, followed by addition of a probe. Mobility shift assay was accomplished for the C/EBP␣binding site (CRE) as a probe with increasing amounts of GST-RFC140 proteins (20, 50, and 100 ng) added to the fixed amount of 50 ng of GST-C/EBP␣. fection of C/EBP␣ and RFC140 increased the promoter activity of PPAR␥ by 8-and 7-fold, respectively. Compared with this, cotransfection of these two expression plasmids showed the synergistic transactivation of the PPAR␥ promoter up to 23fold (Fig. 6, upper panel). This enhanced gene transcription of PPAR␥ was confirmed by protein expression through Western blot analysis with PPAR␥-specific antibody. The synergistic action of C/EBP␣ and RFC140 increased PPAR␥ protein expression distinctly (Fig. 6, lower panel). These results suggest that the cooperation of these two proteins has an essential role in adipocyte differentiation through regulation of PPAR␥ expression. DISCUSSION We isolated the cDNA for RFC140 as an expressed product that interacts with C/EBP␣. RFC is a five-subunit complex composed of 140-, 40-, 38-, 37-, and 36-kDa subunits (24). Although RFC140 is primarily responsible for DNA binding, each of the subunits shares a conserved domain that is referred to as the PCNA interaction region (25). In yeast, each individual subunit is an essential gene product that functions in the RFC complex to "load" PCNA onto DNA prior to genome duplication. Together, DNA polymerase-␦, PCNA, and RFC compose the replicative holoenzyme complex (26,27). It is intriguing that C/EBP␣, a transcription factor that was reported to induce cellular differentiation of adipocytes and myelocytes, interacts with a component of the DNA replication apparatus.
The association we observed by affinity chromatography and co-immunoprecipitation analysis (Fig. 1) required the bZIP domain of C/EBP␣ and the DNA-binding/DNA ligase-like domain of RFC140 (Fig. 2). Cotransfected RFC140 had a positive impact on transactivation by C/EBP␣, whether assayed on a synthetic reporter or on a more native promoter construct, suggesting that DNA-binding activity may be affected. This was confirmed by determining the relative gel shift activity of nuclear extracts from C/EBP␣-transfected cells titrated by cotransfection with RFC140. These results show that RFC140 expression induces the tight complex with C/EBP␣ and the cognate binding DNA. From these results, we provisionally conclude that interaction of C/EBP␣ with RFC140 specifically affects the DNA-binding and transcriptional activation activities of C/EBP␣. Caution regarding interpretation of these results is warranted, as RFC is a multisubunit complex. As these results were obtained under conditions in which only the RFC140 subunit was overproduced in isolation, we are aware that perturbation of the stoichiometry of the RFC subunits may have pleiotropic effects on cellular metabolism. It is interesting to examine whether other subunits affect this synergistic effect of C/EBP␣ and RFC140.
The mechanism(s) by which C/EBP␣ affects the cell cycle and differentiation is not fully understood. Recently, the Rb protein was shown to be essential for adipocyte differentiation and to associate with C/EBP family proteins, augmenting their DNAbinding and transactivation activities. The involvement of Rb is plausible, as it has been reported to function in the differentiation of several cell types, whereas the role of C/EBP␣ in fat cell differentiation is well documented. What is not clear is how interaction of Rb with the activation domain of C/EBP augments transactivation, whereas interaction of Rb with the activation domain of E2F-1 inhibits E2F-1 activity.
Another protein that regulates the cell cycle and often increases upon cellular differentiation is the cell division kinase inhibitor p21 WAF1/CIP1 . The steady-state level of p21 was shown to increase dramatically following induced expression of C/EBP␣ (10). Although the mechanism was shown to be primarily post-translational, it was concluded that p21 was responsible for C/EBP␣-dependent growth arrest based upon the observation that antisense p21 expression resulted in reentry of C/EBP␣-expressing cells into the division cycle (28). Although this may in part explain the observed growth arrest, it cannot be the complete story, as p21 WAF1 is not an essential gene product.  During development, acquisition of the differentiated phenotype involves expression of function-specific genes along with entry of the cells into a quiescent phase of the cell cycle. C/EBP␣, as well as MyoD, NF-B, Zta, and JunD, was observed to inhibit cell division and to induce cell differentiation (29,30). This is consistent with a differentiation model whereby cells first stop dividing and subsequently express transcripts that reflect the differentiated cell state. Interestingly, the antiproliferative effects exerted by certain transcription factors can be separated from transcription activation activity. For example, expression of MyoD in fibroblasts inhibits cell division without inducing expression of muscle-specific gene products (31).
The interaction of C/EBP␣ with RFC140 presents another mechanism by which C/EBP␣ could contribute to cell proliferation and differentiation. The association of C/EBP␣ with RFC140 could inhibit cell cycle progression by interfering with the loading of a PCNA clamp onto DNA. This is a plausible hypothesis, as it was previously shown that association of PCNA with p21 WAF1 resulted in inhibition of the cell cycle (40). This occurred because the PCNA sliding clamp is essential for processive movement of the replicative DNA polymerase-␦ complex. By binding to PCNA, p21 impedes processive DNA synthesis, but not short repair DNA synthesis. By analogy, it is possible that association of RFC140 with C/EBP␣ interferes with the DNA-binding activity of the RFC complex, with the folding of the pentameric complex, or with the ability of the RFC complex to load the PCNA clamp onto DNA, thereby leading to cell cycle arrest and further terminal cell differentiation. Currently in our laboratory, we are planning to examine whether the interaction of C/EBP␣ with RFC140 disrupts the DNA replication system and induces cell growth arrest.
Although the molecular mechanism(s) by which transcription factors affect growth control is not firmly established, there are insights. For example, overproduction of the Epstein-Barr virus product Zta leads to post-transcriptional induction of the p53 tumor suppressor protein and the cyclin-dependent kinase inhibitors p21 WAF1 and p27 Kip (41). Similarly, the halflife of p21 WAF1 was observed to increase in cells growth-arrested after C/EBP␣ expression (10). The reciprocal outcome may be obtained as well, as the transactivation activity of NF-B was reported to increase after transfection of a p21 WAF1 expression plasmid (42). Thus, blocking the cell cycle may have a positive effect on the transactivation function of certain transcription factors as well.
Clement and co-workers (32) showed that RFC140 is more abundant in nuclear extracts from hepatoma cells treated with butyrate, which blocks the cells in the G 1 phase of the cell cycle, than in those from routinely cultured cells and that nuclear distribution of the large subunit of RFC changes during the cell cycle. The RFC140 subunit has homology to CDC44, which is a putative nucleotide-binding protein required for cell cycle progression (33). In addition to this, Farmer and co-workers (34) showed that the DNA-binding activity of the C/EBP␣ protein is regulated in the G 1 phase of the hepatocyte cell cycle. From these observations, since the function of RFC140 and C/EBP␣ is co-localized in the same cellular compartment dependent on the G 1 phase of cell cycle, it is suggested that RFC140 can affect the regulatory role of C/EBP␣ in the G 1 phase necessary for cell differentiation.
McGehee and Habener (35) showed that RFC140 is cloned as an important molecule for adipocyte differentiation and increases transcriptionally following the induction of differentiation. In addition to this, the treatment of antisense oligonucleotides to RFC140 inhibits adipocyte differentiation (36). It has remained likely that RFC140 serves a role in regulating transcription in some manner, either directly by as yet unknown cofactors or through a DNA-modifying activity that allows transcription to proceed. Here we showed RFC140 increased PPAR␥ expression through activation of its promoter activity together with C/EBP␣. The expression of PPAR␥ has been shown to be sufficient to induce growth arrest as well as to initiate adipogenesis in exponentially growing fibroblast cell lines, demonstrating its critical role in the regulation of adipocyte differentiation (20,37).
Our data demonstrate that the important transcription factor C/EBP␣ and the general DNA replication factor RFC140 functionally and physically interact. This observation, along with those made previously, highlights a unique mechanism by which the levels of the general replication factor can strongly modulate the functional activity of the specific transcription factor as a coactivator in non-proliferating cells, especially in a differentiation procedure.