 |
INTRODUCTION |
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 (NFAT·AP-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 (12-14). 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 (17-19). 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 (32-35). 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 NFAT·C/EBP complex on the PPAR
2-proximal NFAT element. Promoter analysis reveals that a similar NFAT·C/EBP composite element
regulates transcription of insulin-like growth factor 2, angiotensin-converting enzyme homolog, and transcription factor POU4F3
genes. Thus, the NFAT·C/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 µg/ml) (Invitrogen). Cells were transfected by using
LipofectAMINE (Invitrogen). 3T3/L1 cells were subjected to
differentiation in the presence of insulin (5 µg/ml), dexamethasone (1 µM), 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 (GenBankTM
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 NFAT·C/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 (5'-GAATTGGCTGGCACTGTCCT-3'; 5'-ATAGACTTGTTGAATAAATC-3')
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 µl 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.
Gel Mobility Shift Assays--
Nuclear extracts were prepared
from cultured cells as described (7). Double-stranded oligonucleotides
[PPAR
2-proximal NFAT, 5'-ATTACAGGGAAAATATTGCCACACTGTCTC-3';
NFAT mutated PPAR
2-proximal NFAT,
5'-ATTACAGCGATTATATTGCCACACTGTCTC-3'; C/EBP
mutated PPAR
2-proximal NFAT,
5'-ATTACAGGGAAAATATTCGCAGTCTGTCTC-3'; both NFAT
and C/EBP mutated PPAR
2-proximal NFAT,
5'-ATTACAGCGATTATATTCGCAGTCTGTCTC-3'; interleukin-2 ARRE NFAT, 5'-AGAAAGGAGGAAAAACTGTTTCATACAGAAGG-3'; insulin-like growth factor 2 (IGF2) NFAT,
5'-TCAAACATTAGGCAATATTTTCCAGG-3'; angiotensin-converting enzyme homolog
(ACEH) NFAT, 5'-TTTGGAAGGGAAAATGTTGCCCAAGTAGAG-3'; transcription
factor POU4F3 (POU4F3) NFAT,
5'-TGGCGCTGGGAAAATATTGCAGAAGGGCGG-3'; AP-1-binding site,
5'-AATTTTCCGGCTGAGTCATCAAGCG-3'; C/EBP-binding site,
5'-TGCAGATTGCGCAATCTGCA-3'] for gel mobility shift assays were
labeled with [
-32P]dCTP. The binding reactions were
carried out at room temperature in gel-shift buffer (1 mM
CaCl2, 1 mM MgCl2, 10 mM Hepes (pH 7.9), 50 mM NaCl, 15 mM
-mercaptoethanol, 10% glycerol, 0.1 mg/ml bovine serum albumin, and 1 mg/ml poly(dI-dC) for 30 min). The protein-DNA complexes were separated in 5% non-denaturing polyacrylamide gels in
Tris/glycine/EDTA buffer (25 mM Tris, 200 mM
glycine, and 1 mM EDTA) and were visualized by
autoradiography. Recombinant proteins for gel-shift analysis were
prepared as described previously (41). For competition analysis,
unlabeled oligonucleotides (50 pmol) were incubated with the labeled
probe before addition of nuclear extract. For supershift analysis,
antibody was preincubated with nuclear extract for 30 min at room
temperature before addition of the labeled probe. For dissociation
analysis, the excess amounts of unlabeled oligonucleotides (50 pmol)
were added to the preassembled NFAT·C/EBP complex for the indicated
times. The amount of remaining complex was quantitated by
PhosphorImager analysis. For saturation analysis, increasing
amounts of labeled probe (40, 80, 120, 160, and 200 fmol) were
incubated with constant amounts of nuclear extract. The amounts of
NFAT·C/EBP complexes and unbound probe were quantitated by
PhosphorImager analysis. The ratio of bound/free was plotted against
the amount of bound to assess relative binding affinity.
Luciferase Assays--
A C/EBP expression vector (50 ng) was
co-transfected with the PPAR
2 luciferase reporter plasmid (0.3 µg)
and the control plasmid pRSV
-galactosidase (0.1 µg) into BHK
cells. Expression vector for constitutive nuclear NFAT (0.3 µg),
constitutive active calcineurin (0.3 µg), or dominant-negative NFAT
(0.5 µg) was co-transfected as indicated. Luciferase and
-galactosidase activities were measured 48 h after
transfection. Cells were stimulated with ionomycin (2 µM)
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 ± S.E.,
n = 4).
In Vitro Translation--
T7-coupled in vitro
transcription-translation (Promega Inc.) was performed in the presence
of [35S]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.
Co-immunoprecipitations--
Cell extracts prepared using
Triton/lysis buffer (20 mM Tris (pH 7.4), 137 mM NaCl, 2 mM EDTA, 1% Triton X-100, 25 mM
-glycerophosphate, 1 mM sodium vanadate,
2 mM sodium pyrophosphate, 10% glycerol, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin) were incubated (5 h at 4 °C) with antibodies prebound to 20 µl of protein
G-Sepharose. After three washes with Triton/lysis buffer, the bound
proteins were separated in SDS-PAGE and electrotransfered to a
polyvinylidene difluoride membrane (Millipore). 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.
 |
RESULTS |
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 (42-46). Thus, these
data indicate that NFAT cooperates with C/EBP to regulate the PPAR
2
gene transcription.

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 1.
NFAT cooperates with C/EBP to increase
PPAR 2 gene transcription. A,
schematic representation of the PPAR 2 gene promoter. Identified
NFAT- (filled triangles) and C/EBP (open
ovals)-binding sites are illustrated. DNase I-hypersensitive sites
(DHS1 and DHS2) are also shown (shaded
boxes). 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
( 1 to 2000) 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 µM) 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 ( 1 to 2000) 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 µM) plus PMA (P,
100 nM) for 16 h before harvest. Transfection
efficiency was monitored by measurement of -galactosidase
activity.
|
|
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 calcineurin 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
273 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
183 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 NFAT·C/EBP element on the
PPAR
2-proximal NFAT site is analogous to the established composite
NFAT·AP-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 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).

View larger version (44K):
[in this window]
[in a new window]
|
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 2-proximal 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 NFAT·C/EBP interaction
(C).
|
|
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
NFAT·C/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 NFAT·C/EBP·DNA complex exhibited
slight decrease in electrophoretic mobility as compared with the
NFAT·DNA complex (see asterisks). Importantly, there is a
cooperative interaction between NFATc4 and C/EBP
upon binding to the
PPAR
2-proximal NFAT element. Antibody supershift analysis further
revealed specificity and cooperativity between NFAT and C/EBP in the
formation of the NFAT·C/EBP·DNA complex. These data indicate that
NFAT and C/EBP form a cooperative complex on the PPAR
2-proximal NFAT
element.

View larger version (52K):
[in this window]
[in a new window]
|
Fig. 3.
The PPAR 2-proximal
NFAT composite element binds NFAT and C/EBP. A and
B, co-expression of C/EBP with NFATc4 increases formation
of NFAT·DNA complexes on the PPAR 2-proximal NFAT element. The
NFAT- (filled box) and the C/EBP (shaded
box)-binding sites on the PPAR 2-proximal NFAT composite element
are shown (A). Mutations to abolish NFAT or C/EBP binding
are also illustrated (underlined). Nuclear extracts prepared
from cells expressing NFATc4, C/EBP , or NFATc4 and C/EBP were
incubated with 32P-labeled double-stranded oligonucleotides
encoding the PPAR 2-proximal NFAT composite element (B).
Gel mobility shift assays were performed in the presence (+) or absence
( ) of antibody against NFAT or C/EBP . Oligonucleotides with
mutated NFAT-binding site, mutated C/EBP-binding site, or both NFAT- and C/EBP-binding sites mutated were also used to
examine NFAT·DNA complex formation. The positions of the NFAT·DNA
complexes are indicated by asterisks. The amounts of
NFAT·DNA 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 2-proximal element in gel
mobility shift assays. The positions of the NFAT·DNA, C/EBP·DNA,
and NFAT·C/EBP·DNA complexes are indicated by asterisks.
NFAT and C/EBP also formed multimers on the PPAR 2-proximal element
(arrowheads). The amounts of NFAT·DNA complexes were
quantitated by PhosphorImager analysis. D, dissociation of
the NFAT·C/EBP complexes. 32P-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 NFAT·C/EBP complexes for the indicated times (0, 2, 5, 10, and 20 min). The NFAT·C/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 (×) NFAT (filled
triangle) or C/EBP (open oval) binding to the proximal
NFAT element of the ( 1 to 273) 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.
|
|
Previous studies indicated that mutation at the NFAT-binding site of
the IL-2 NFAT composite element reduces AP-1 binding (16, 53-55).
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/EBP-binding
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
2-proximal 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 NFAT·DNA 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 NFAT·DNA 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 NFAT·C/EBP·DNA complexes. Importantly, the NFAT·C/EBP·DNA complex exhibited a decrease in electrophoretic mobility as compared with the NFAT·DNA or the C/EBP·DNA 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 NFAT·C/EBP complexes on the
PPAR
2-proximal NFAT element (Fig. 3D). Preassembled NFAT·C/EBP·DNA 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
NFAT·C/EBP complex to a similar extent. Both oligonucleotides
required about 10 min to dislodge 50% off the preassembled
NFAT·C/EBP·DNA 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 NFAT·C/EBP·DNA complex.
These data indicate that C/EBP determines the dissociation of the
NFAT·C/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 NFAT·C/EBP complex on
the PPAR
2-proximal NFAT element (Fig. 3). In contrast, C/EBP is the
determining factor for the dissociation of the NFAT·C/EBP·DNA 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 NFAT-binding 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
2-proximal and the IL-2 NFAT Elements,
Respectively--
Co-expression of NFAT and C/EBP markedly increases
formation of the NFAT·C/EBP·DNA 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 NFAT·DNA complexes on the
PPAR
2-proximal element. However, increasing the amount of c-Jun had
minimal effect on the formation of NFAT·DNA complexes. Increasing the
amount of NFATc4 proteins also increased the amount of NFAT·DNA
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
NFATc4-enhanced formation of the NFAT·DNA 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 NFAT·DNA complexes on the IL-2
NFAT element. These data indicate that NFAT preferentially interacts
with C/EBP on the PPAR
2-proximal NFAT element. Conversely, NFAT
prefers to interact with c-Jun on the IL-2 ARRE NFAT site.

View larger version (48K):
[in this window]
[in a new window]
|
Fig. 4.
Preferential binding of C/EBP and AP-1
on the PPAR 2-proximal and the IL-2 NFAT
elements, respectively. A, combination of C/EBP and
NFATc4 nuclear extracts increases formation of NFAT·DNA 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
32P-labeled PPAR 2-proximal (top panels) or
IL-2 (bottom panels) NFAT oligonucleotides to examine
formation of the NFAT·DNA 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 NFAT·DNA complexes.
|
|
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 NFAT· 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 NFAT· 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 NFAT·DNA, C/EBP·DNA, and NFAT·C/EBP to
promote formation of a ternary complex on the PPAR
2-proximal NFAT
element.

View larger version (27K):
[in this window]
[in a new window]
|
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 (+) or
absence ( ) 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 ( ) and
with (+) 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 35S-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 35S-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 NFAT·C/EBP
interaction. Recombinant GST-C/EBP was incubated, in the presence or
absence of the PPAR 2-proximal NFAT element, with
35S-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 NFAT·C/EBP interaction. Proteins in the lysate and bound
to the immobilized GST-C/EBP were detected by autoradiography and
quantitated by PhosphorImager analysis.
|
|
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 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
NFAT·C/EBP·DNA 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 ~5-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 NFAT·DNA, C/EBP·DNA, and
NFAT·C/EBP interactions.
The Rel Domain of NFATc4 Is Critical for Interaction with
C/EBP
--
Previous structural analysis indicated that
Arg468, Ile469, and Thr535 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 (17-19).
Similar amino acid residues are found in the Rel domain of other NFAT
members (Arg455, Leu456 and Thr522
of NFATc1; Arg488, Tyr489, and
Thr555 of NFATc3; and Arg474,
Asn475, and Thr541 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
Arg474 and Asn475 with Ala and
Thr541 with Gly of NFATc4 abolished NFAT·C/EBP
interaction. The R474A/N475A/T541G NFATc4 also exhibited reduced
binding to c-Jun in co-immunoprecipitation assays. These data indicate
conserved interactions in NFAT·C/EBP and NFAT· AP-1
complexes.

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 6.
The Rel domain of NFATc4 is critical for
interaction with C/EBP . A, the
structure of NFATc4 is illustrated schematically. Replacement of
Arg474 and Asn475 with Ala and
Thr541 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 ( ) and with (+) 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, NFAT·C/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 32P-labeled
PPAR 2-proximal NFAT element (40, 80, 120, 160, and 200 fmol). The
amount of NFAT·C/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 32P-labeled PPAR 2-proximal NFAT oligonucleotides to
examine formation of NFAT·DNA complex in gel mobility shift assays.
Expression of NFATc4 proteins is also shown. D, NFAT·C/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 ( 1 to 273) luciferase reporter plasmid to examine
transcription cooperation. Luciferase reporter plasmid containing a
triple repeat of the PPAR 2-proximal NFAT·C/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.
|
|
Reduced interaction between NFAT and C/EBP by mutation at the Rel
domain may affect formation of the ternary NFAT·C/EBP·DNA 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 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
NFAT·C/EBP·DNA complexes were also detected when increasing the
amount of the wild-type, 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
(Ser168 and Ser170) 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 gene
transcription. Together, these data establish that transcription
cooperation of the PPAR
2 gene by NFAT and C/EBP requires
interactions between NFAT·DNA, C/EBP·DNA, and NFAT·C/EBP.
NFAT·C/EBP Composite Elements Regulate Other Gene
Expressions--
The NFAT·C/EBP composite enhancer element
represents a novel regulatory motif to direct NFAT-mediated gene
transcription. Similar NFAT·C/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 co-expression or combination of NFAT and C/EBP further increased formation of NFAT·DNA complexes, indicating a
cooperative effort of NFAT and C/EBP in gene regulation.

View larger version (44K):
[in this window]
[in a new window]
|
Fig. 7.
NFAT·C/EBP composite elements regulate
other gene expressions. A, sequence comparison of the
NFAT·C/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 NFAT·DNA
complexes from the NFAT·C/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 NFAT·DNA 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 NFAT·C/EBP
composite element. IGF2 promoter ( 1 to 1400), ACEH promoter ( 1 to
450), or a luciferase reporter plasmid containing a triple repeat of
the POU4F3 NFAT·C/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.
|
|
We also examined the function of these NFAT·C/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 NFAT·C/EBP composite element (Fig.
7D). Together, these data indicate that the NFAT·C/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, 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, 17-19). 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 NFAT·AP-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
NFAT·C/EBP·DNA.
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 NFAT·C/EBP, NFAT·AP-1, or NFAT·bZIP complexes. Among
the bZIP group of transcription factors, ATF2 and c-Maf cooperate with
NFAT to regulate gene expression (57-59). 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 NFAT·C/EBP or the NFAT·AP-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.
NFAT·C/EBP and NFAT·AP-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 NFAT·AP-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 NFAT·AP-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 NFAT·AP-1 complex, the NFAT·C/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 NFAT·C/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 NH2-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 NFAT·C/EBP- or NFAT·AP-1- mediated gene transcription is
critical in multiple biological processes. These observations also
suggest that NFAT is a ubiquitous regulator.
It is likely that the NFAT·C/EBP and the NFAT·AP-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 NFAT·C/EBP or the
NFAT·AP-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 NFAT·C/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 NFAT·C/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 NFAT·C/EBP composite enhancer complex expands the
repertoire of gene transcription mediated by NFAT.