Transcription Cooperation by NFAT (cid:1) C/EBP Composite Enhancer Complex*

The nuclear factor of activated T cells (NFAT) group of transcription factors regulates gene expression in im-mune and non-immune cells. NFAT-mediated gene transcription is orchestrated, in part, by formation of a composite regulatory element. Here we demonstrate that NFAT interacts with transcription factor CCAAT/en-hancer-binding protein (C/EBP) to form a composite enhancer complex, to potentiate expression of the peroxisome proliferator-activated receptor- (cid:1) 2 gene. Formation of a ternary NFAT (cid:1) C/EBP (cid:1) DNA complex is required for the transcriptional cooperation. A similar NFAT (cid:1) C/EBP composite element is found in the regulatory region of the insulin-like growth factor 2, angio-tensin-converting enzyme homolog, and transcription factor POU4F3 genes. Thus, the NFAT (cid:1) C/EBP composite element represents a novel regulatory enhancer to direct NFAT-mediated gene transcription. Antibodies to detect FLAG-NFATc4 (Sigma), NFATc2, and C/EBP (Santa Cruz Biotechnology) were used for immunoblot analysis. Enhanced chemiluminescence was performed to visualize NFAT and C/EBP. and resolved SDS- PAGE, immunoblotting analyses performed to bindings of endogenous NFAT C/EBP

Nuclear factor of activated T cells (NFAT) 1 was first identified as an important regulator for cytokine gene expression (1,2). Subsequent isolation of cDNAs reveals a broad distribution of NFAT in multiple tissues. For example, NFAT regulates expression of type 1 inositol 1,4,5-trisphosphate receptor and is implicated in learning and memory (3). NFAT regulates expression of b-type natriuretic peptide during cardiac hypertrophy (4). NFAT also modulates skeletal muscle fiber type by directing selective gene expression in slow oxidative myofibers (e.g. myoglobin gene and slow fiber-specific troponin I gene) (5). NFAT also regulates expression of fatty acid-binding protein and transcription factor peroxisome proliferator-activated receptor-␥2 (PPAR␥2) during adipogenesis (6,7). Recent microarray analyses have further revealed a wealth of novel NFAT target genes (8 -10). As elucidation of the physiological functions of NFAT progresses, many more novel NFAT target genes will be identified. However, the molecular basis that governs the expression of these NFAT target genes remains to be determined.
NFAT interacts with transcription factor AP-1 (Fos and Jun proteins) to form a cooperative composite enhancer (NFAT⅐AP-1) and to regulate expression of many cytokine genes (11). The cooperative induction is mediated, in part, by protein-protein and protein-DNA interactions. For example, the well characterized antigen receptor-responsive element (ARRE) from the interleukin (IL)-2 gene contains binding sites for NFAT and AP-1 (12)(13)(14). The DNA binding domains of NFAT and AP-1 are required for the cooperative interaction on the ARRE enhancer (15,16). Structural analysis reveals multiple contacts between the DNA binding domains of NFAT and AP-1 (17)(18)(19). Conserved residues are responsible for the protein-protein and protein-DNA interactions. Thus, intimate associations between NFAT, AP-1, and DNA are necessary for the transcription cooperation of the IL-2 gene.
Transcription factor peroxisome proliferator-activated receptor-␥2 (PPAR␥2) plays an important role in adipogenesis (20 -23). Multiple transcription factors have been shown to regulate the PPAR␥2 gene expression. For example, members of the CCAAT/enhancer-binding protein (C/EBP) family induce expression of PPAR␥2 during adipocyte differentiation (24,25). Several C/EBP-binding elements have been identified on the PPAR␥2 gene promoter (26 -30). Recently, two distinctive NFAT-binding sites (proximal and distal) are identified on the PPAR␥2 gene (7). The NFAT-and C/EBP-binding sites are located in the immediate upstream regulatory region of the PPAR␥2 promoter. Whether NFAT cooperates with C/EBP to regulate the PPAR␥2 gene expression remains uncertain.
The significance of the PPAR␥2 NFAT regulatory elements is further implicated by recent DNase I-hypersensitive site studies (31). Two DNase I-hypersensitive sites (DHS1 and DHS2) are found in the PPAR␥2 gene. DNase I-hypersensitive sites represent regions that are "accessible" for transcription factor binding in the nucleosome-packed chromatin (32)(33)(34)(35). Binding of transcription factors to the DNase I-hypersensitive sites may facilitate chromatin rearrangement and/or transcriptional activation of promoters that are actively engaged in gene expression. Thus, identification of proteins that bind to DNase I-hypersensitive sites is important to understand transcriptional regulation. The PPAR␥2 distal NFAT element is located between the DHS1 and the DHS2 DNase I-hypersensitive site. The PPAR␥2-proximal NFAT element is located within DHS1. The proximity of these NFAT elements to the DNase I-hypersensitive sites suggests that understanding the molecular basis of NFAT-mediated transcription will shed new light on the regulation of PPAR␥2 gene.
The purpose of this study is to examine the molecular basis of NFAT-mediated gene expression. We report that NFAT cooperates with C/EBP to regulate PPAR␥2 gene expression. The transcription cooperation is mediated, in part, by the formation of a composite NFAT⅐C/EBP complex on the PPAR␥2-proximal NFAT element. Promoter analysis reveals that a similar NFAT⅐C/EBP composite element regulates transcription of insulin-like growth factor 2, angiotensin-converting enzyme homolog, and transcription factor POU4F3 genes. Thus, the NFAT⅐C/EBP complex represents a novel composite regulatory element to direct NFAT-mediated gene expression.

EXPERIMENTAL PROCEDURES
Cell Culture-HepG2 hepatoma cells were obtained from the Cell Culture Core Facility of the Liver Research Center, Albert Einstein College of Medicine. BHK fibroblasts and HepG2 hepatoma cells were cultured in minimal essential medium. COS and 3T3/L1 cells were cultured in Dulbecco's modified Eagle's medium. All media were supplemented with 10% fetal calf serum, 2 mM L-glutamine, penicillin (100 units/ml), and streptomycin (100 g/ml) (Invitrogen). Cells were transfected by using LipofectAMINE (Invitrogen). 3T3/L1 cells were subjected to differentiation in the presence of insulin (5 g/ml), dexamethasone (1 M), and the phosphodiesterase inhibitor isobutylmethylxanthine (0.5 mM).
Reagents-The PPAR␥2 promoter luciferase reporter plasmids and the expression vectors for calcineurin, C/EBP, and NFATc4 have been described (7, 36 -40). The sequences for the IGF2, ACEH, and POU4F3 genes have been reported (GenBank TM accession number U71085, AF366352, and AF044575, respectively). The IGF2 and ACEH promoters were amplified from genomic DNA and subcloned into pGL3 basic luciferase reporter plasmid using NheI and XhoI sites. Double-stranded oligonucleotides encoding a triple repeat of the POU4F3 NFAT⅐C/EBP element were subcloned upstream of the pGL3 promoter luciferase reporter plasmid. The NFATc4 Rel domain was amplified by PCR and subcloned into the pCDNA3 plasmid using NotI and XbaI sites. Mutations on the NFATc4 Rel domain were generated by overlapping PCR. The NFATc4 Rel domain was also subcloned into pRSET plasmid using PvuII site for recombinant protein expression.
Chromatin Immunoprecipitations-Nuclear factors that were associated with chromatin in differentiated and undifferentiated 3T3/L1 cells were cross-linked to DNA using formaldehyde (1%). Cells were harvested, and cross-linked chromatin was sheared by sonication. Sonicated cell lysate was immunoprecipitated using NFAT or C/EBP antibodies. DNA present in the immunoprecipitated chromatin was isolated after reversed cross-link and proteinase K digestion, and PCR was performed (5Ј-GAATTGGCTGGCACTGTCCT-3Ј; 5Ј-ATAGACTTGTT-GAATAAATC-3Ј) to examine the presence of PPAR␥2 gene promoter.
DNA Precipitation Assays-Double-stranded biotinylated oligonucleotides were incubated with HepG2 or 3T3/L1 cell extract for 12 h before precipitation with 20 l of streptavidin-Sepharose (2 h). After three washes in Triton/lysis buffer, precipitated DNA and its associated proteins were separated in SDS-PAGE, and immunoblot was performed to detect endogenous NFAT and C/EBP. For competition analysis, the excess amounts of non-biotinylated oligonucleotides (100 pmol) were added to the precipitation assays.
Luciferase Assays-A C/EBP expression vector (50 ng) was co-transfected with the PPAR␥2 luciferase reporter plasmid (0.3 g) and the control plasmid pRSV ␤-galactosidase (0.1 g) into BHK cells. Expression vector for constitutive nuclear NFAT (0.3 g), constitutive active calcineurin (0.3 g), or dominant-negative NFAT (0.5 g) was co-transfected as indicated. Luciferase and ␤-galactosidase activities were measured 48 h after transfection. Cells were stimulated with ionomycin (2 M) plus PMA (100 nM) as indicated. The data were presented as relative luciferase activity, calculated as the ratio of the luciferase activity to the activity of ␤-galactosidase (means Ϯ S.E., n ϭ 4).
In Vitro Translation-T7-coupled in vitro transcription-translation (Promega Inc.) was performed in the presence of [ 35 S]methionine and -cysteine (PerkinElmer Life Sciences). Translated NFATc4 was used in binding assays (41) with recombinant GST or GST-C/EBP␤. Wild-type, NFAT-mutated, or C/EBP-mutated PPAR␥2-proximal NFAT oligonucleotides were added as indicated. Bound NFATc4 was visualized by autoradiography and quantitated by PhosphorImager analysis.

NFAT Cooperates with C/EBP to Increase PPAR␥2 Gene
Transcription-We demonstrated previously (7) that NFAT increases PPAR␥2 gene expression. Two NFAT-binding elements (proximal and distal) are located in the upstream regulatory region of the PPAR␥2 gene (Fig. 1A). Several C/EBP-binding sites are found on the PPAR␥2 gene (26 -30) (Fig. 1A). We tested whether NFAT cooperates with C/EBP to regulate the PPAR␥2 gene expression. PPAR␥2 promoter luciferase reporter plasmid was co-transfected with C/EBP expression vectors (C/ EBP␣, C/EBP␤, or C/EBP␦) into BHK cells. Expression of C/EBP increased the PPAR␥2 gene transcription (Fig. 1B). Administration of ionomycin and PMA, to activate the NFAT signaling pathway, also increased the PPAR␥2 gene transcription. Importantly, treatment of ionomycin plus PMA cooperated with C/EBP and led to a further increase in the PPAR␥2 gene transcription. Increased PPAR␥2 promoter activity was blocked by co-expression of the dominant-negative NFAT inhibitor (Fig. 1C), which has been shown to inhibit specifically NFAT-mediated gene transcription (42)(43)(44)(45)(46). Thus, these data indicate that NFAT cooperates with C/EBP to regulate the PPAR␥2 gene transcription.
Calcineurin phosphatase binds to and dephosphorylates NFAT (1,2,47). Dephosphorylated NFAT then translocates into the nucleus to mediate gene transcription. Targets of the calcineurin-mediated dephosphorylation include the conserved Ser phosphorylation sites found in four NFAT members (7, 48 -51). Previous studies (7, 41, 48 -50, 52) showed that expression of constitutive active calcineurin promotes NFAT nuclear localization and increases NFAT-mediated gene transcription. Replacement of the conserved Ser residues with Ala, to prevent phosphorylation, also causes constitutive nuclear localization of NFAT and increases NFAT-mediated gene transcription (7, 48 -51). We tested whether co-expression of activated cal-cineurin or constitutive nuclear NFAT with C/EBP increases PPAR␥2 gene transcription. Expression of activated calcineurin (⌬CN) or constitutive nuclear NFAT (cnNFATc1, cnNFATc3, or cnNFATc4) increased PPAR␥2 gene transcription (Fig. 1, D and E). Co-expression of activated calcineurin or constitutive nuclear NFAT with C/EBP further increased PPAR␥2 gene transcription. These data establish that NFAT cooperates with C/EBP to regulate the PPAR␥2 gene transcription.
Next, we performed deletion analysis on the PPAR␥2 gene promoter to determine the regulatory regions that mediate cooperation between NFAT and C/EBP (Fig. 1A). Deletion to Ϫ273 bp upstream of the PPAR␥2 promoter remained cooperative to NFAT and C/EBP activation (Fig. 1F). Removal of the PPAR␥2-proximal NFAT element, to Ϫ183 bp upstream of the PPAR␥2 gene promoter, reduced the cooperation. These data indicate that the PPAR␥2-proximal NFAT element is important for the cooperation between NFAT and C/EBP.
NFAT and C/EBP Interact with the PPAR␥2 Promoter in Vivo-Sequence analysis indicated that a C/EBP-binding site is located immediately adjacent to the PPAR␥2-proximal NFAT site (Fig. 1). The configuration of the NFAT⅐C/EBP element on the PPAR␥2-proximal NFAT site is analogous to the established composite NFAT⅐AP-1 enhancer found on the IL-2 gene (see Fig. 4). Because the proximal NFAT element is critical for the transcription cooperation of the PPAR␥2 gene and is located within the DHS1 hypersensitive site, we hypothesized that NFAT interacts with C/EBP, to form a novel composite enhancer, to mediate transcription cooperation of the PPAR␥2 gene.
To examine in vivo binding of NFAT and C/EBP to the PPAR␥2 promoter, we performed chromatin immunoprecipitations ( Fig. 2A). 3T3/L1 cells were subjected to differentiation in the presence of insulin, dexamethasone, and IBMX. After 6 days in differentiation media, 3T3/L1 cells became lipid-laden adipocytes. Chromatins from undifferentiated and different stages of differentiating 3T3/L1 cells, after cross-linked with formaldehyde, were harvested. Isolated chromatins were immunoprecipitated with C/EBP␤, NFATc2 or NFATc4 antibodies. The PPAR␥2 promoter in the precipitates was detected by PCR amplification. Chromatin immunoprecipitations indicated that neither C/EBP␤ nor NFAT interacted with the PPAR␥2 promoter in undifferentiated (day 0) 3T3/L1 cells. Two days after initiation of adipocyte differentiation, the PPAR␥2 promoter was detected in the C/EBP␤, but not NFAT, precipitates. On day four of adipocyte differentiation, both C/EBP␤ and NFAT (NFATc2 and NFATc4) interacted with the PPAR␥2 promoter. Interestingly, interaction of NFAT and C/EBP with . Deletion to remove upstream regulatory regions of the PPAR␥2 luciferase reporter plasmid is also indicated. B and C, BHK cells were transfected with PPAR␥2 promoter (Ϫ1 to Ϫ2000) luciferase reporter plasmid and expression vectors for different C/EBP members (C/EBP␣, C/EBP␤, or C/EBP␦) (B). Expression vector for dominant-negative NFAT (dnNFAT) was transfected as indicated (C). The cells were stimulated without (Untreated) and with ionomycin (I, 2 M) plus PMA (P, 100 nM) for 16 h before harvest. Transfection efficiency was monitored by measurement of ␤-galactosidase activity. D and E, BHK cells were transfected with PPAR␥2 promoter (Ϫ1 to Ϫ2000) luciferase reporter plasmid and expression vectors for different C/EBP members (C/EBP␣, C/EBP␤, or C/EBP␦) and/or constitutive active calcineurin (⌬CN) (D). Expression vectors for different constitutive nuclear NFAT members (cnNFATc1, cnNFATc3, or cnNFATc4) were also transfected as indicated (E). Cells were harvested 36 h after transfection. Luciferase and ␤-galactosidase activities were measured. F, different PPAR␥2 promoter luciferase reporter plasmids were co-transfected with C/EBP␣ in BHK cells as indicated. The cells were stimulated without (Untreated) and with ionomycin (I, 2 M) plus PMA (P, 100 nM) for 16 h before harvest. Transfection efficiency was monitored by measurement of ␤-galactosidase activity. the PPAR␥2 gene promoter precedes the full induction of PPAR␥2 protein expression, which occurs after day four of adipocyte differentiation (20). Nonetheless, these data indicate temporal requirements of NFAT and C/EBP in the PPAR␥2 gene transcription. Association of NFAT with the PPAR␥2 promoter in the early stage supports previous studies that NFAT inhibition blocks adipocyte differentiation (6).
We further examined whether NFAT and C/EBP were present in the proximal NFAT element by DNA precipitation assays. Biotinylated oligonucleotides encoding the PPAR␥2-proximal and distal NFAT elements were incubated with extracts isolated from HepG2 or 3T3/L1 cells, which express NFAT, C/EBP and PPAR␥ endogenously. The biotinylated oligonucleotides and their associated proteins were then precipitated using streptavidin-Sepharose and separated on SDS-PAGE. Immunoblotting analysis indicated the presence of NFATc2 in the DNA precipitates (Fig. 2B). C/EBP␣ and C/EBP␤ were detected in the PPAR␥2-proximal, but not the distal, NFAT complex. These data support that the PPAR␥2-proximal and distal NFAT elements form different NFAT complexes (7).
We also performed competition analysis to demonstrate specificity of NFAT⅐C/EBP interaction (Fig. 2C). Incubation with excess amount of wild-type, but not the mutated, non-biotinylated PPAR␥2-proximal oligonucleotides abolished both NFAT and C/EBP binding. Together, these data indicate that C/EBP binds to the PPAR␥2-proximal NFAT element.
The PPAR␥2-proximal NFAT Composite Element Binds NFAT and C/EBP-Next, we performed gel mobility shift assays to examine binding of C/EBP on the PPAR␥2-proximal NFAT element (Fig. 3). Nuclear extracts were prepared from cells transfected with NFATc4, C/EBP␣, and NFATc4 plus C/EBP␣. Immunoblotting analysis revealed similar expression of NFATc4 and C/EBP␣ in different nuclear extracts (Fig. 3B). NFATc4 or C/EBP␣ interacted with the PPAR␥2-proximal NFAT element and formed distinctive protein-DNA complexes (Fig. 3B). Co-expression of NFATc4 with C/EBP␣ markedly increased the formation of protein-DNA complexes. The NFAT⅐C/EBP⅐DNA complex exhibited slight decrease in electrophoretic mobility as compared with the NFAT⅐DNA complex (see asterisks). Importantly, there is a cooperative interaction between NFATc4 and C/EBP␣ upon binding to the PPAR␥2proximal NFAT element. Antibody supershift analysis further revealed specificity and cooperativity between NFAT and C/EBP in the formation of the NFAT⅐C/EBP⅐DNA complex. These data indicate that NFAT and C/EBP form a cooperative complex on the PPAR␥2-proximal NFAT element.
Previous studies indicated that mutation at the NFAT-binding site of the IL-2 NFAT composite element reduces AP-1 binding (16,(53)(54)(55). These results indicate that although consensus AP-1-binding site is found on the IL-2 NFAT composite element, AP-1 requires the presence of bound NFAT to promote DNA binding. To further examine interaction between NFAT and C/EBP on the PPAR␥2-proximal NFAT element, we performed mutagenesis to abolish either the NFAT-or the C/EBPbinding site (Fig. 3A). Gel mobility shift assays indicated that mutation at the cognate NFAT-binding site abolished NFAT interaction (Fig. 3B). In addition, the NFAT mutated PPAR␥2proximal NFAT element failed to bind C/EBP. These data indicate that C/EBP requires the presence of bound NFAT to stabilize DNA binding.
We also examined formation of NFAT⅐DNA complexes from the C/EBP mutated PPAR␥2-proximal NFAT element (Fig.  3B). Mutation at the cognate C/EBP site abolished C/EBP interaction. In addition, the C/EBP mutated PPAR␥2-proximal NFAT element failed to exhibit cooperation in the formation of NFAT⅐DNA complex. Antibody supershift analysis further supported the loss of cooperation. Together, these data indicate that simultaneous binding of NFAT and C/EBP are required for the cooperative interaction on the PPAR␥2-proximal NFAT element.
Previous studies established that recombinant NFAT and AP-1 proteins support cooperative interaction on the IL-2 NFAT element (14,15,56). To further confirm the cooperative binding of NFAT and C/EBP, we performed gel mobility shift assays using recombinant NFATc4 and C/EBP␤ proteins (Fig.  3C). Recombinant NFATc4 or C/EBP␤ proteins interacted with the PPAR␥2-proximal element (see asterisks). NFATc4 and C/EBP␤ also formed multimers on the PPAR␥2-proximal element (see arrowheads). Combination of minimal amount of the NFATc4 protein with the C/EBP␤ protein markedly increased the formation of NFAT⅐C/EBP⅐DNA complexes. Importantly, the NFAT⅐C/EBP⅐DNA complex exhibited a decrease in electrophoretic mobility as compared with the NFAT⅐DNA or the C/EBP⅐DNA complexes (see asterisks). Also, there is a cooperative interaction between NFATc4 and C/EBP␤ upon binding to the PPAR␥2-proximal NFAT element. These data further indicate that NFAT and C/EBP form a cooperative complex on the PPAR␥2-proximal NFAT element.
Next, we examined the dissociation of the NFAT⅐C/EBP complexes on the PPAR␥2-proximal NFAT element (Fig. 3D). Pre-

FIG. 2. NFAT and C/EBP interacts with the PPAR␥2 promoter in vivo.
A, binding of NFAT and C/EBP␤ to the PPAR␥2 promoter at different stages of adipocyte differentiation. 3T3/L1 cells were subjected to adipocyte differentiation in the presence of insulin, dexamethasone, and isobutylmethylxanthine. Chromatin immunoprecipitations were performed to precipitate NFAT (NFATc2 and NFATc4) and C/EBP␤regulated promoters. DNA isolated was amplified by PCR using oligonucleotides encoding the PPAR␥2 promoter. B and C, extracts prepared from HepG2 or 3T3/L1 cells were incubated with biotinylated PPAR␥2proximal or -distal NFAT oligonucleotides. Protein-DNA complexes were precipitated by streptavidin-Sepharose and resolved in SDS-PAGE, and immunoblotting analyses were performed to detect bindings of endogenous NFAT and C/EBP (B). Excess amount of non-biotinylated PPAR␥2-proximal elements were used in competition analysis to indicate specificity of NFAT⅐C/EBP interaction (C). The NFAT⅐C/EBP Enhancer Complex 15878 assembled NFAT⅐C/EBP⅐DNA complexes were competed with excess amount of unlabeled wild-type, NFAT-mutated, or C/EBP-mutated PPAR␥2-proximal NFAT elements. The PPAR␥2 distal NFAT element and the consensus C/EBP-binding site were also used to contest NFAT and C/EBP binding, respectively. Competition analysis indicated that the wild-type and the NFAT-mutated PPAR␥2-proximal NFAT elements promoted dissociation of NFAT⅐C/EBP complex to a similar extent. Both oligonucleotides required about 10 min to dislodge 50% off the preassembled NFAT⅐C/EBP⅐DNA complex. The consensus C/EBP-binding site required about 5 min to attain 50% dissociation. In contrast, the C/EBP-mutated PPAR␥2-proximal NFAT element and the PPAR␥2 distal NFAT element required over 20 min to remove 50% of the NFAT⅐C/EBP⅐DNA complex. These data indicate that C/EBP determines the dissociation of the NFAT⅐C/EBP complex on the PPAR␥2-proximal NFAT element.
Gel mobility shift assays indicate that both NFAT-and C/EBP-binding sites are required for stable formation of the NFAT⅐C/EBP complex on the PPAR␥2-proximal NFAT element (Fig. 3). In contrast, C/EBP is the determining factor for the dissociation of the NFAT⅐C/EBP⅐DNA complex. Next, we examined the contribution of the NFAT-and the C/EBP-binding site of the proximal NFAT element on the transcriptional cooperation of the PPAR␥2 promoter (Fig. 3E). Mutation at the NFATbinding site of the PPAR␥2-proximal NFAT element abolished transcriptional cooperation. Mutation at the C/EBP-binding site of the PPAR␥2-proximal NFAT element also abolished transcriptional cooperation. These data support that simultaneous binding of NFAT and C/EBP on the proximal NFAT element is required for the transcriptional cooperation of the PPAR␥2 gene.
Preferential Binding of C/EBP and AP-1 on the PPAR␥2proximal and the IL-2 NFAT Elements, Respectively-Co-expression of NFAT and C/EBP markedly increases formation of the NFAT⅐C/EBP⅐DNA complex on the PPAR␥2-proximal NFAT element (Fig. 3), indicating a cooperative interaction of NFAT and C/EBP upon DNA interaction. To examine further cooperative binding between NFAT and C/EBP on the PPAR␥2 NFAT element, we combined nuclear extracts, prepared from NFATc4, C/EBP␣, or c-Jun-expressing cells, prior to performing gel mobility shift assays (Fig. 4A). Increasing the amount of C/EBP␣ enhanced the formation of NFAT⅐DNA complexes on the PPAR␥2-proximal element. However, increasing the amount of c-Jun had minimal effect on the formation of NFAT⅐DNA complexes. Increasing the amount of NFATc4 proteins also increased the amount of NFAT⅐DNA complexes in the presence of C/EBP but not c-Jun. Using the ARRE NFAT site of the IL-2 gene as a probe increased the amount of NFATc4enhanced formation of the NFAT⅐DNA complexes in the presence of c-Jun but not C/EBP␣. Increasing the amount of c-Jun, but not C/EBP␣, also increased formation of NFAT⅐DNA com-plexes on the IL-2 NFAT element. These data indicate that NFAT preferentially interacts with C/EBP on the PPAR␥2proximal NFAT element. Conversely, NFAT prefers to interact with c-Jun on the IL-2 ARRE NFAT site.
To examine further preferential binding of C/EBP and AP-1 on different NFAT elements, we performed competition using unlabeled AP-1 and C/EBP consensus oligonucleotides (Fig.  4B). The C/EBP oligonucleotides reduced formation of NFAT⅐ DNA complexes on the PPAR␥2-proximal NFAT element more effectively than the AP-1 oligonucleotides. Conversely, the AP-1 oligonucleotides markedly reduced formation of NFAT⅐ DNA complexes on the IL-2 NFAT element. These data further reveal preferential interaction of C/EBP and AP-1 on the PPAR␥2-proximal and the IL-2 NFAT element, respectively.
NFAT Interacts with C/EBP-NFAT may interact with C/EBP to promote formation of a ternary complex with the PPAR␥2-proximal NFAT element. We prepared cell extracts from HepG2 or 3T3/L1 cells to examine whether NFAT interacts with C/EBP endogenously. Co-immunoprecipitation assays indicated that NFATc2 interacted with C/EBP␣ or C/EBP␤ in vivo (Fig. 5A). Thus, there are interactions between NFAT⅐DNA, C/EBP⅐DNA, and NFAT⅐C/EBP to promote formation of a ternary complex on the PPAR␥2-proximal NFAT element.
Next, we mapped the C/EBP binding domain on NFAT (Fig.  5B). A series of NFATc4 deletion mutants were co-expressed with C/EBP␣ in COS cells. Immunoblot analysis indicated expression of NFATc4 proteins (Fig. 5B). Co-immunoprecipitation assays indicated that NFATc4 interacted with C/EBP␣. Deletion of the COOH-terminal domain (residues 451-902) of NFATc4 had minimal effect on the interaction with C/EBP␣. Further deletion to residues 365 of NFATc4, to remove the Rel homology DNA binding domain, abolished C/EBP␣ interaction. These data indicate that the Rel homology domain of NFATc4 is important for the interaction with C/EBP␣.
We further generated the NFATc4 mutant that contains the Rel homology DNA binding domain to examine NFAT-C/EBP interactions (Fig. 5C). Co-immunoprecipitation assays revealed the presence of NFATc4 Rel proteins in the C/EBP␣ precipitates. These data confirm that the NFAT Rel homology domain interacts with C/EBP.
We also mapped the NFAT-binding site on the C/EBP proteins. We performed binding assays using recombinant NFAT and in vitro translated C/EBP (Fig. 5D). Deletion analysis indicated that removal of the conserved C/EBP DNA binding domain abolished NFAT interaction. These data indicate that the DNA binding domains of NFAT and C/EBP are required for interaction upon binding to the PPAR␥2-proximal element.
To determine whether NFAT directly associates with C/EBP, we performed binding assays using recombinant C/EBP and in vitro translated NFAT (Fig. 5E). Recombinant GST was used as a control. Binding assays indicated that NFATc4 was detected or both NFAT-and C/EBP-binding sites mutated were also used to examine NFAT⅐DNA complex formation. The positions of the NFAT⅐DNA complexes are indicated by asterisks. The amounts of NFAT⅐DNA complexes were quantitated by PhosphorImager analysis. Expression of FLAG epitope-tagged NFATc4 and C/EBP␣ in transfected COS cells was detected by M2 and C/EBP␣ antibody, respectively. C, cooperative DNA binding using recombinant NFATc4 and C/EBP␤ proteins. Different concentrations of NFATc4 and C/EBP␤ proteins were incubated with the PPAR␥2proximal element in gel mobility shift assays. The positions of the NFAT⅐DNA, C/EBP⅐DNA, and NFAT⅐C/EBP⅐DNA complexes are indicated by asterisks. NFAT and C/EBP also formed multimers on the PPAR␥2-proximal element (arrowheads). The amounts of NFAT⅐DNA complexes were quantitated by PhosphorImager analysis. D, dissociation of the NFAT⅐C/EBP complexes. 32 P-Labeled PPAR␥2-proximal NFAT element was incubated with nuclear extracts prepared from cells expressing NFATc4 and C/EBP␣ for 30 min. Excess amounts of unlabeled oligonucleotides were used as competitors and incubated with the preassembled NFAT⅐C/EBP complexes for the indicated times (0, 2, 5, 10, and 20 min). The NFAT⅐C/EBP complexes were separated by non-denaturing polyacrylamide gel electrophoresis, and the amounts of remaining complexes were quantitated by PhosphorImager analysis and plotted against time of competition. E, mutation at the NFAT-or the C/EBP-binding site on the proximal NFAT element abolished transcription cooperation of the PPAR␥2 gene promoter. Site-directed mutagenesis was performed to abolish (ϫ) NFAT (filled triangle) or C/EBP (open oval) binding to the proximal NFAT element of the (Ϫ1 to Ϫ273) PPAR␥2 promoter. Expression constructs for constitutive nuclear NFATc4 and C/EBP␣ were co-transfected with PPAR␥2 luciferase reporter plasmids to examine transcription cooperation. Cells were harvested 36 h after transfection. Luciferase and ␤-galactosidase activities were measured. in the C/EBP but not GST precipitates. These data indicate that NFAT directly associates with C/EBP. Gel mobility shift assays indicate formation of a cooperative ternary NFAT⅐C/EBP⅐DNA complex on the PPAR␥2-proximal promoter (Figs. 3 and 4). Similar cooperation may be detected when NFAT interacts with C/EBP, in the presence of the PPAR␥2-proximal NFAT element, in protein binding assays. Recombinant C/EBPs were incubated with in vitro translated NFATc4 in the presence or absence of wild-type, NFAT-mutated, or C/EBP-mutated PPAR␥2-proximal NFAT elements (Fig. 5F). Binding assays indicated that the wild-type, but not the NFAT-mutated or the C/EBP-mutated, PPAR␥2-proximal NFAT element augmented the amount of NFATc4 proteins in the C/EBP precipitates. There was an ϳ5-fold increase in NFATc4 binding to C/EBP in the presence of the wild-type PPAR␥2-proximal NFAT element. In conjunction with the gel mobility shift analysis (Figs. 3 and 4), these data demonstrate that formation of a cooperative complex on the PPAR␥2 NFAT element is mediated by NFAT⅐DNA, C/EBP⅐DNA, and NFAT⅐C/ EBP interactions.
The Rel Domain of NFATc4 Is Critical for Interaction with C/EBP␣-Previous structural analysis indicated that Arg 468 , Ile 469 , and Thr 535 on the Rel homology domain of NFATc2 interact with AP-1 (Fos-Jun) to promote formation of a ternary complex on the IL-2 NFAT element (17)(18)(19). Similar amino acid residues are found in the Rel domain of other NFAT members (Arg 455 , Leu 456 and Thr 522 of NFATc1; Arg 488 , Tyr 489 , and Thr 555 of NFATc3; and Arg 474 , Asn 475 , and Thr 541 of NFATc4), suggesting conserved interactions with AP-1 (11,15). Because AP-1 and C/EBP are basic leucine zipper type transcription factors, we tested whether similar amino acid residues on the NFAT Rel domain are required for the interaction with C/EBP (Fig. 6A). Replacement of Arg 474 and Asn 475 with Ala and Thr 541 with Gly of NFATc4 abolished NFAT⅐C/EBP interaction. The R474A/N475A/T541G NFATc4 also exhibited reduced binding to c-Jun in co-immunoprecipitation assays. These data indicate conserved interactions in NFAT⅐C/EBP and NFAT⅐ AP-1 complexes.
Reduced interaction between NFAT and C/EBP by mutation at the Rel domain may affect formation of the ternary NFAT⅐C/ EBP⅐DNA complex. We performed gel mobility shift assays to determine the relative binding affinity of wild-type and R474A/ N475A/T541G NFATc4 to the PPAR␥2-proximal NFAT element (Fig. 6B). Immunoblot analysis indicated similar expression of NFATc4 and C/EBP proteins. Binding of the wild-type and R474A/N475A/T541G NFATc4 to the C/EBP-mutated FIG. 4. Preferential binding of C/EBP and AP-1 on the PPAR␥2proximal and the IL-2 NFAT elements, respectively. A, combination of C/EBP␣ and NFATc4 nuclear extracts increases formation of NFAT⅐DNA complexes on the PPAR␥2-proximal NFAT element. Increasing amounts of C/EBP␣ or c-Jun were premixed with the NFATc4 nuclear extract (left panels). Conversely, increasing amounts of NFATc4 were premixed with the C/EBP␣ or the c-Jun nuclear extracts (right panels). The combined nuclear extracts were incubated with 32 P-labeled PPAR␥2-proximal (top panels) or IL-2 (bottom panels) NFAT oligonucleotides to examine formation of the NFAT⅐DNA complex in gel mobility shift assays. B, the PPAR␥2-proximal and the IL-2 NFAT elements were incubated with HepG2 nuclear extracts. Excess amounts of unlabeled oligonucleotides encoding the canonical binding site for AP-1 or C/EBP were used as competitors. Gel mobility shift assays were performed to examine formation of NFAT⅐DNA complexes.
PPAR␥2-proximal NFAT element was also similar, supporting previous studies (15,19) that distinct residues on the Rel domain interact with Fos, Jun, and DNA. Saturation binding assays, however, indicated that the wild-type NFATc4 exhibited about 4-fold higher affinity than the R474A/N475A/T541G NFATc4 toward binding to the PPAR␥2-proximal NFAT element. Increased amounts of NFAT⅐C/EBP⅐DNA complexes were also detected when increasing the amount of the wildtype, but not the mutated R474A/N475A/T541G, NFATc4 was combined with C/EBP prior to gel mobility shift assays (Fig.  6C). Together, these data confirm that formation of a cooperative complex on the PPAR␥2-proximal NFAT site requires protein-protein interaction between NFAT and C/EBP.
We further examined transcription activity mediated by R474A/N475A/T541G NFATc4 (Fig. 6D). Constitutive nuclear NFATc4 cooperated with C/EBP␣ and increased PPAR␥2 gene transcription ( Figs. 1 and 6D). Co-expression of the constitutive nuclear NFATc4 with C/EBP␣ also increased reporter gene activity mediated by multiple repeats of the PPAR␥2-proximal NFAT composite element. To facilitate nuclear accumulation of the C/EBP binding defective R474A/N475A/T541G NFATc4, conserved Ser residues (Ser 168 and Ser 170 ) were replaced with Ala to prevent phosphorylation and to promote nuclear localization. In the absence of C/EBP, expression of S168A/S170A/ R474A/N475A/T541G NFATc4 directed NFAT-mediated activity to a similar extent as the constitutive nuclear NFATc4. However, S168A/S170A/R474A/N475A/T541G NFATc4 had minimal effect to further increase C/EBP␣-activated PPAR␥2 FIG. 5. NFAT interacts with C/EBP. A, endogenous C/EBP␣ and C/EBP␤ were immunoprecipitated (IP) from HepG2 or 3T3/L1 cell extracts without (Control) and with C/EBP␣ and C/EBP␤ antibodies. NFATc2 in the immunoprecipitates was detected by immunoblot (IB) analysis. Expression of NFATc2 in cell lysate was also shown. B, deletion analysis to map the C/EBP binding domain on NFATc4. The structure of NFATc4 is illustrated schematically. FLAG epitope-tagged NFATc4 corresponding to residues 1-902, 1-853, 1-581, 1-450, 1-365, 1-260, and 1-160 were co-expressed with C/EBP␣ in COS cells. Expression of NFATc4 proteins was detected by immunoblot analysis of cell lysates with M2 monoclonal antibody. NFATc4 proteins were immunoprecipitated from COS cell extracts in the presence (ϩ) or absence (Ϫ) of M2 antibody (anti-NFAT). C/EBP␣ in the immunoprecipitates was detected by immunoblot analysis. Expression of C/EBP␣ in cell lysate (L) was also shown. C, the Rel homology domain of NFATc4 interacts with C/EBP␣. Expression vector for the Rel homology domain of NFATc4 (NFATc4 Rel) was co-transfected with a C/EBP␣ expression plasmid into COS cells. C/EBP␣ was immunoprecipitated from COS cell extracts without (Ϫ) and with (ϩ) rabbit polyclonal C/EBP␣ antibody (anti-C/EBP␣). NFATc4 Rel in the immunoprecipitates was detected by immunoblot analysis. Expression of NFATc4 Rel in cell lysate was also indicated. D, deletion analysis to map the NFAT binding domain on C/EBP. The structures of C/EBP␣ and C/EBP␤ are illustrated schematically. Recombinant NFAT was incubated with 35 S-labeled C/EBP prepared by in vitro translation. Proteins in the lysate and bound to the immobilized NFAT were detected by autoradiography. E, NFATc4 interacts with C/EBP in vitro. Recombinant GST and GST-C/EBP␤ were incubated with 35 S-labeled NFATc4 prepared by in vitro translation. Proteins in the lysate and bound to the immobilized GST-C/EBP␤ were detected by autoradiography. F, the PPAR␥2-proximal NFAT element increases NFAT⅐C/EBP interaction. Recombinant GST-C/EBP␤ was incubated, in the presence or absence of the PPAR␥2-proximal NFAT element, with 35 S-labeled NFATc4 prepared by in vitro translation. Oligonucleotides (Oligo) with mutated NFAT-binding site or mutated C/EBP-binding site were also used to examine the NFAT⅐C/EBP interaction. Proteins in the lysate and bound to the immobilized GST-C/EBP␤ were detected by autoradiography and quantitated by PhosphorImager analysis. gene transcription. Together, these data establish that transcription cooperation of the PPAR␥2 gene by NFAT and C/EBP requires interactions between NFAT⅐DNA, C/EBP⅐DNA, and NFAT⅐C/EBP. NFAT⅐C/EBP Composite Elements Regulate Other Gene Expressions-The NFAT⅐C/EBP composite enhancer element represents a novel regulatory motif to direct NFAT-mediated gene transcription. Similar NFAT⅐C/EBP-binding elements are present in the regulatory regions of the IGF2, ACEH, and transcription factor POU4F3 (POU4F3) genes (Fig. 7A). Gel mobility shift assays revealed binding of NFAT and C/EBP on these regulatory elements (Fig. 7, B and C). Importantly, either coexpression or combination of NFAT and C/EBP further increased formation of NFAT⅐DNA complexes, indicating a cooperative effort of NFAT and C/EBP in gene regulation.
We also examined the function of these NFAT⅐C/EBP complexes using reporter gene assays (Fig. 7D). Expression of constitutive nuclear NFAT or C/EBP increased IGF2 and ACEH gene transcription. Co-expression of constitutive nuclear NFAT and C/EBP further increased IGF2 and ACEH gene transcription. Co-expression of constitutive nuclear NFAT and C/EBP also increased gene transcription mediated by a triple repeat of the POU4F3 NFAT⅐C/EBP composite element (Fig. 7D). Together, these data indicate that the NFAT⅐C/EBP composite element represents a novel regulatory enhancer to direct NFAT-mediated gene transcription.

DISCUSSION
NFAT Partners-NFAT mediates gene transcription in immune and non-immune cells. Characterization of the antigen receptor-response element (ARRE) indicated that a cytoplasmic (NFATc) and a nuclear (NFATn) component cooperate together to regulate IL-2 gene transcription (13,45). The NFATc component was subsequently identified as a family of transcription factors (NFATc1, NFATc, NFAT2; NFATc2, NFATp, NFAT1; NFATc3, NFAT4, NFATx; and NFATc4, NFAT3) (1, FIG. 6. The Rel domain of NFATc4 is critical for interaction with C/EBP␣. A, the structure of NFATc4 is illustrated schematically. Replacement of Arg 474 and Asn 475 with Ala and Thr 541 with Gly of NFATc4 are indicated. FLAG epitope-tagged wild-type (WT) and mutated R474A/N475A/T541G NFATc4 were co-expressed with C/EBP␣ or c-Jun in COS cells. NFATc4 proteins were immunoprecipitated (IP) from COS cell extracts without (Ϫ) and with (ϩ) M2 monoclonal antibody (anti-NFAT). C/EBP␣ or c-Jun in the immunoprecipitates was detected by immunoblot (IB) analysis. Expression of NFATc4 proteins is also indicated. B, NFAT⅐C/EBP interactions are important for the formation of a cooperative complex. FLAG epitope-tagged wild-type and mutated R474A/N475A/T541G NFATc4 were co-expressed with C/EBP␣ in COS cells. Nuclear extracts were prepared and incubated with increasing amounts of 32 P-labeled PPAR␥2-proximal NFAT element (40, 80, 120, 160, and 200 fmol). The amount of NFAT⅐C/EBP complexes and remaining probe were quantitated by PhosphorImager analysis. The ratio of bound/free was plotted against the amount of bound to assess relative binding affinity. Expression of NFATc4 and C/EBP␣ is shown. Binding of wild-type and mutated R474A/N475A/T541G NFATc4 to the C/EBP mutated PPAR␥2-proximal NFAT element is also indicated. C, increasing amounts of wild-type and mutated R474A/N475A/T541G NFATc4 was premixed with the C/EBP␣ nuclear extracts. The combined nuclear extracts were incubated with 32 P-labeled PPAR␥2-proximal NFAT oligonucleotides to examine formation of NFAT⅐DNA complex in gel mobility shift assays. Expression of NFATc4 proteins is also shown. D, NFAT⅐C/EBP interactions are important for transcription cooperation of the PPAR␥2 gene. Expression vectors for constitutive nuclear NFATc4 (cnNFATc4) and C/EBP␣ were co-transfected with the PPAR␥2 (Ϫ1 to Ϫ273) luciferase reporter plasmid to examine transcription cooperation. Luciferase reporter plasmid containing a triple repeat of the PPAR␥2-proximal NFAT⅐C/ EBP element was also examined. The effect of R474A/N475A/T541G mutation on cnNFATc4-mediated transcription was also examined. Cells were harvested 36 h after transfection. Luciferase and ␤-galactosidase activities were measured. p values are compared with cnNFATc4-mediated transcription activity.
2). The NFATc group of proteins translocates into the nucleus in response to elevated intracellular calcium. NFATc then cooperates with NFATn to mediate gene transcription. The Fos and Jun groups of transcription factors (AP-1 proteins) were identified as the NFATn component (12,14). Sequence analysis indicates a close proximity of the NFAT-and the AP-1-binding sites on the ARRE element. Molecular and structural analyses further demonstrate intimate associations to promote cooperative binding of NFAT and AP-1 (15,(17)(18)(19). Thus, NFAT and AP-1 forms a composite enhancer complex to mediate IL-2 gene transcription.
In this report, we demonstrate that NFAT interacts with C/EBP to form a composite element to regulate several gene transcriptions. Thus, C/EBP represents a novel NFATn component. Interaction between NFAT and C/EBP is analogous to the NFAT⅐AP-1 complex. For example, the C/EBP-and the AP-1-binding site are located immediately adjacent to the NFAT-binding site. The ability of C/EBP and AP-1 to bind DNA, however, requires the presence of bound NFAT. Upon DNA binding, C/EBP and AP-1 promote formation of a cooperative complex with NFAT. Indeed, similar amino acid residues on the NFAT Rel domain are required for the interaction with C/EBP or AP-1. These data demonstrate an intimate association between NFAT, C/EBP, and DNA. Further structural analysis will reveal whether a ternary complex is formed by NFAT⅐C/EBP⅐DNA. C/EBP and AP-1 are both bZIP-type transcription factors. Conserved interactions of C/EBP and AP-1 with the NFAT Rel domain suggest that other bZIP transcription factors may complex with NFAT to mediate transcription cooperation. These observations also suggest that the DNA sequence immediately adjacent to the NFAT site may govern preferential formation of NFAT⅐C/EBP, NFAT⅐AP-1, or NFAT⅐bZIP complexes. Among the bZIP group of transcription factors, ATF2 and c-Maf coop- FIG. 7. NFAT⅐C/EBP composite elements regulate other gene expressions. A, sequence comparison of the NFAT⅐C/EBP composite elements found on the PPAR␥2, the IGF2, the ACEH, and the transcription factor POU4F3 genes. Canonical NFAT-(filled box) and C/EBP (shaded box)-binding sites are indicated. B and C, co-expression or combination of NFAT and C/EBP increases formation of NFAT⅐DNA complexes from the NFAT⅐C/EBP composite elements of the IGF2, ACEH, and POU4F3 genes. Nuclear extracts prepared from COS cells transfected with NFATc4 and/or C/EBP␣ were used in gel mobility shift assays (B). Combination of NFATc4 and C/EBP␣ nuclear extracts, prior to performing gel mobility shift assays, was also used to examine formation of NFAT⅐DNA complexes (C). D, co-expression of NFAT and C/EBP increases IGF2 and ACEH gene transcription. Co-expression of NFAT and C/EBP also increases transcription mediated by a triple repeat of the POU4F3 NFAT⅐C/EBP composite element. IGF2 promoter (Ϫ1 to Ϫ1400), ACEH promoter (Ϫ1 to Ϫ450), or a luciferase reporter plasmid containing a triple repeat of the POU4F3 NFAT⅐C/EBP element was co-transfected with constitutive nuclear NFATc4 (cnNFATc4) and/or C/EBP␣ into BHK cells. Cells were harvested 36 h after transfection. Luciferase and ␤-galactosidase activities were measured. NFAT-(filled triangles) and C/EBP (open ovals)binding sites on the IGF2, ACEH, and POU4F3 reporter plasmids are illustrated. erate with NFAT to regulate gene expression (57)(58)(59). Whether NFAT interacts with ATF2, c-Maf, or other bZIP factors to form a similar cooperative complex will require identification of optimal binding sequence for their DNA interaction. Alternatively, these bZIP factors may heterodimerize with Fos, Jun, or C/EBP to promote DNA binding and thus circumvent the requirement of a cognate binding sequence.
Other transcription factors, including Oct1, MEF2, GATA, and Sp1, have been reported to cooperate with NFAT to mediate gene transcription (4, 60 -64). Their mode of cooperation with NFAT, however, seems to be different from the NFAT⅐C/ EBP or the NFAT⅐AP-1 complex. Although NFAT interacts with Oct1, MEF2, GATA, or Sp1, cooperative association of NFAT with these factors upon DNA binding has not been demonstrated. The lack of cooperative interaction is probably because they are further apart in their cognate binding sequences. Delineating the molecular mechanisms of these NFAT cooperative associations remains to be ascertained.
NFAT⅐C/EBP and NFAT⅐AP-1 Composite Regulatory Elements-NFAT was first identified as an important regulator in IL-2 gene expression (12,45,54). Many other genes were subsequently identified as NFAT targets (1,2,47,65). Cooperative interaction between NFAT and AP-1 is critical for expression of these genes. Multiple members of the Jun (c-Jun, JunB and JunD) and the Fos (c-Fos, FosB, Fra1, and Fra2) group of transcription factors are indicated in the formation of the NFAT⅐AP-1 complex (12,14,53). Homo-and heterodimerizations between different Fos and Jun members further provide a wide selection of AP-1 proteins to interact with NFAT. Thus, the NFAT⅐AP-1 regulatory element is likely composed of different AP-1 dimers.
The C/EBP group of transcription factors (C/EBP␣, C/EBP; C/EBP␤, NF-IL6, LAP, CRP2; C/EBP␥, Ig/EBP; C/EBP␦, CRP3; C/EBP⑀, CRP1; and C/EBP, CHOP, Gadd153) is expressed in a variety of tissues (66 -71). Homo-and heterodimerizations of C/EBP lead to transcriptional regulation in multiple biological processes. Alternative usage of different translational start sites further generates transcriptional activators or repressors of C/EBP (72,73). Analogous to the NFAT⅐AP-1 complex, the NFAT⅐C/EBP complex is probably composed of a wide array of C/EBP homo-and heterodimers. Interactions with C/EBP transcriptional activators or repressors suggest an additional level of regulation in NFAT⅐C/EBP-mediated gene transcription. Thus, different partners modulate NFAT-mediated gene transcription.
The NFAT-dependent DNA binding of C/EBP (or AP-1) indicates that activation of both NFAT and C/EBP (or AP-1) is required for gene transcription. Dephosphorylation mediated by the calcium-activated calcineurin phosphatase is important for the NFAT activation (1,2,47). Conversely, phosphorylation regulates C/EBP and AP-1. The extracellular signal-regulated kinase-mitogen-activated protein kinase pathway, including the extracellular signal-regulated kinase-activated p90 ribosomal S6 kinase, phosphorylates C/EBP␣ and C/EBP␤ (74 -78), whereas the c-Jun NH 2 -terminal kinase-mitogen-activated protein kinase pathway is important for the c-Jun activation (79 -81). The p38 mitogen-activated protein kinase pathway regulates C/EBP CHOP Gadd153 (82). Integration of multiple signaling pathways that lead to the activation of NFAT, C/EBP, or AP-1 suggests that NFAT⅐C/EBP-or NFAT⅐AP-1mediated gene transcription is critical in multiple biological processes. These observations also suggest that NFAT is a ubiquitous regulator.
It is likely that the NFAT⅐C/EBP and the NFAT⅐AP-1 composite regulatory elements will direct transcription of different subsets of NFAT target genes. These NFAT target genes may be differentially transcribed because of restricted tissue expression of C/EBP and AP-1. In addition, temporal expression of C/EBP and AP-1 may provide another level of regulation. For example, different C/EBP members regulate gene transcription at various stages of adipocyte differentiation (24,25). Different signaling pathways may feed into the NFAT⅐C/EBP or the NFAT⅐AP-1 complex to accomplish differential expression of NFAT target genes as well. Further studies to identify the distinct pools of NFAT targets that are transcribed under cooperation with C/EBP or AP-1 will shed light on the molecular basis of NFAT-mediated gene transcription.
In this study, we identify IGF2, ACEH, and POU4F3 are novel targets of NFAT. Sequence analysis indicates that the NFAT⅐C/EBP-binding site is located in close proximity to a DNase I-hypersensitive site on the IGF2 gene (83,84). DNase I-hypersensitive sites on the ACEH and the POU4F3 genes remain to be determined. Because the NFAT⅐C/EBP complex of the proximal NFAT element is located within the DHS1 of the PPAR␥2 gene (Fig. 1), it is tempting to speculate that NFAT and C/EBP are common residents of DNase I-hypersensitive sites. Future studies to test this hypothesis are warranted.
In conclusion, we have identified C/EBP as a new NFATn component. Formation of NFAT⅐C/EBP composite enhancer complex expands the repertoire of gene transcription mediated by NFAT.