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INTRODUCTION |
Eukaryotic transcription factors commonly occur in families
whose members share similar DNA binding specificities and other functional properties. Many transcription factors are dimeric and can
form homodimers as well as heterodimers with other family members. The
capacity to heterodimerize provides a means of enhancing regulatory
diversity, as the various dimeric species within a protein family may
exhibit distinct functional properties (1-4). Thus, it is important to
elucidate the dimerization status of transcription factors in
vivo to understand the full range of their biological activities
and regulation.
In the present study, we have examined the dimerization properties of
CCAAT/enhancer-binding proteins
(C/EBPs)1 in cells. C/EBPs
are a family of basic leucine zipper (bZIP) DNA-binding proteins (5)
consisting of five core members: C/EBP
, C/EBP
, C/EBP
,
C/EBP
, and C/EBP
(Ig/EBP). C/EBP proteins bind to DNA as dimers
and display highly related DNA binding and dimerization specificities
(reviewed in Refs. 6-8). C/EBPs are involved in the regulation of many
cellular processes. C/EBP, C/EBP, and C/EBP mediate responses
to stress and inflammatory signals, including regulation of acute phase
response genes in hepatocytes and expression of proinflammatory
cytokine genes in monocytic cells (9-13). Overexpression experiments
and analysis of knockout mice demonstrate that C/EBP proteins also
control cell growth and differentiation (6-8, 14). For example, forced
expression of C/EBP and/or C/EBP in precursor cells of the
adipocyte, granulocyte, and keratinocyte lineages causes growth arrest
and induces cellular differentiation (15-17). In other contexts,
C/EBP has been reported to stimulate cell growth (18, 19). Thus,
C/EBPs regulate a variety of cellular phenotypes in a wide range of
cell types.
In addition to forming homodimers, C/EBP proteins are capable of
heterodimerizing with the other family members in vitro
(20-22). Heterodimerization could potentially alter several functional activities of C/EBP proteins, including DNA binding, transactivation potential, responsiveness to signaling pathways, and the ability to
cooperate with other transcription factors. It has been assumed that
heterodimers between C/EBP family members occur in vivo and possess regulatory activities that are distinct from the homodimeric forms. However, there is only limited evidence for such heterodimers in vivo. An association between C/EBP and C/EBP was
observed in transient overexpression experiments (20), and evidence has been reported for C/EBP·C/EBP heterodimers in liver nuclear extracts (23) and monocytic cells (24). In addition, C/EBPs appear to
heterodimerize with proteins from other bZIP subfamilies, including
Fos/Jun (25) and ATF/CREB (26-28).
Here we report that C/EBP proteins in cell and tissue extracts are
found predominantly as heterodimers with C/EBP. C/EBP is a
ubiquitously expressed member of the C/EBP family that was first
identified by its affinity for cis-regulatory sites in the Ig heavy chain promoter and enhancer (29). C/EBP contains a C/EBP-like bZIP region but lacks an amino-terminal transactivation domain (30, 31) and can inhibit transcriptional activation by C/EBP
or C/EBP (31). We show that C/EBP can repress C/EBP- and
C/EBP-mediated transactivation of a reporter gene in fibroblasts in
a leucine zipper-dependent manner, indicating that the
repression by C/EBP involves heterodimerization with its partner.
Interestingly, C/EBP did not repress transactivation by C/EBP or
C/EBP in HepG2 hepatoma cells, nor did it inhibit C/EBP activity
in either cell type. Thus, the ability of C/EBP to inhibit C/EBP
activators is cell-specific and differs for the various C/EBP family members.
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EXPERIMENTAL PROCEDURES |
Cells and Cell Culture--
L cell fibroblasts were cultured in
Dulbecco's modified Eagle's medium (BioWhittaker, Inc.) supplemented
with 10% fetal bovine serum (HyClone, Inc.) in the presence of
kanamycin, streptomycin, and penicillin. HepG2 hepatoma cells (ATCC
HB-8065) were maintained in minimum essential Eagle's medium
(BioWhittaker, Inc.) supplemented with nonessential amino acids, sodium
pyruvate, 10% fetal bovine serum (HyClone, Inc.) in the presence of
kanamycin, streptomycin, and penicillin. Other cell lines used for
analysis of nuclear extracts were C6-2B (rat glioma; Ref. 32), IC-21
(murine macrophage; Ref. 33; ATCC TIB 186), and EMT6 (murine mammary
tumor). P388-C and P388-C-C1 cells are stably transfected P388
lymphoblasts (ATCC CCL 46) expressing C/EBP and C/EBP,
respectively (34, 35). P388 cell lines were grown in RPMI 1640 (BioWhittaker) supplemented with 10% fetal clone I serum (HyClone,
Inc.), glutamine, kanamycin, streptomycin, and penicillin.
Antibodies--
C/EBP antiserum specific for the COOH
terminus (C-19) was obtained from Santa Cruz Biotechnology. Peptide
antisera recognizing the NH2 terminus of C/EBP (20) and
the NH2 terminus of GCN4 (36) have been described. A
polyclonal antiserum against bacterially expressed C/EBP (37) was
kindly provided by K. Calame. Two peptide antisera were raised against
murine C/EBP by immunizing rabbits with synthetic peptides
corresponding to the amino-terminal (Ser-Lys-Leu-Ser-Gln-Pro-Ala-Thr-Thr-Pro-Gly-Val-Asn-Gly-Cys) or
car boxyl-terminal peptide
(Cys-Ile-Ser-Thr-Glu-Thr-Thr-Ala-Thr-Asn-Ser-Asp-Asn-Pro-Gly-Gln). Cysteine residues were included in both peptides for covalent coupling
to carrier protein.
Plasmid Constructs--
The C/EBP coding sequence was
amplified by PCR from a plasmid containing a C/EBP clone of murine
origin (29). Two oligonucleotide primers were used, one overlapping the
initiation codon with an introduced NcoI restriction site
and the second spanning the termination codon with an introduced
HindIII restriction site. The PCR product was digested with
NcoI (partial) and HindIII (complete) and
inserted into the pMEX expression vector to generate pMEX-C/EBP. A
bacterial expression construct, pJL6-C/EBP, was generated by
inserting the digested PCR product into the expression vector pJL6 as
described (38). The GAL4-C/EBP hybrid construct was described
previously (38).
Transient Transfections--
Transfections were carried out
using 30-40% confluent monolayers in 10-cm dishes using
FuGENETM (Roche Molecular Biochemicals). For
co-transfection experiments, a constant amount of C/EBP reporter
plasmid (DE1)4-alb-luc (2.5 µg) (38) and C/EBP expression
constructs pMEX-C/EBP, pMEX-C/EBP-GLZ, pMEX-C/EBP,
or pMEX-C/EBP (0.75 µg) were transfected with varying quantities
of C/EBP expression construct pMEX-C/EBP (0.5-8.5 µg).
pRSV--galactosidase (0.5 µg) was co-transfected to normalize for
transfection efficiency. The total amount of DNA used for transfection
(12.25 µg) was kept constant by adding an appropriate amount of the
pMEX vector. After 48 h, the cells were lysed and analyzed for
luciferase activity using the Enhanced Luciferase Assay Kit (PharMingen
International) and for -galactosidase activity using the luminescent
-galactosidase Genetic Reporter System II
(CLONTECH Laboratories, Inc.).
GAL4-C/EBP transfection assays were conducted using 30-40%
confluent monolayers in 60-mm wells using FuGENETM (Roche
Molecular Biochemicals). One µg of (G)5 E1B-luc reporter plasmid (38), 5 ng of GAL4-C/EBP vector, and 25 ng of Ha-Ras(12V) vector were transfected with varying quantities of the pcDNA3.1 C/EBP expression vector (13.3 ng to 1.25 µg). The
Renilla luciferase vector, pRL-TK (Promega), was
co-transfected as an internal standard for transfection efficiency.
Sixteen hours prior to harvesting the cells, the medium was removed and
replaced with serum-free media. Cells were collected 48 h after
transfection, lysed, and analyzed using the
Dual-Luciferase® assay system (Promega).
Nuclear Extracts--
Nuclear extracts from cell lines and
transfected cells were prepared by a detergent lysis method.
Transfected cells were washed once with phosphate-buffered saline,
scraped, and then divided. 20% of the cells from 10-cm dishes were
used for luciferase assays by resuspension in detergent lysis solution
(100 mM potassium phosphate (pH 7.8), 0.2% Triton X-100, 1 mM dithiothreitol (DTT); CLONTECH
Laboratories, Inc.), incubation at room temperature for 5 min and
centrifugation. The remaining 80% of the cells were used to make
nuclear extracts by resuspension in lysis buffer (20 mM
HEPES (pH 7.9), 1 mM EDTA, 10 mM NaCl, 1 mM DTT, 0.1% (v/v) Nonidet P-40, 0.5 mM
phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin, 5 µg/ml
aprotinin, 5 µg/ml antipain ) and incubation on ice for 10 min.
Nuclei were pelleted by centrifugation at 3,500 rpm for 10 min.
Proteins were extracted from nuclei by incubation in high salt buffer
(25 mM HEPES (pH 7.9), 0.2 mM EDTA, 0.42 M NaCl, 0.2 mM DTT, 25% glycerol, 0.5 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 10 µg/ml aprotinin, 5 µg/ml antipain) at 4 °C for 20 min with
vigorous shaking. Nuclear debris was pelleted by centrifugation at
14,000 rpm for 5 min, and the supernatant was collected and stored at
70 °C. Nuclear extracts from mouse tissues were prepared by an
Nonidet P-40 lysis procedure as described previously (39).
Electrophoretic Mobility Shift Assay (EMSA)--
The following
double-stranded oligonucleotides containing the wild-type consensus
C/EBP site (bold) or a mutant C/EBP site (bold) were used as probes or
competitor DNAs.
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The probe was end-labeled using [32P]dCTP and
Klenow polymerase. DNA-binding assays were carried out in a 25-µl
reaction containing 20 mM HEPES (pH 7.9), 200 mM NaCl, 5% (w/v) Ficoll, 5% (v/v) glycerol, 1 mM EDTA, 50 mM DTT, 0.01% Nonidet P-40, 0.06%
bromphenol blue, 1.75 µg of poly(dI-dC), and 7.5 × 104 cpm probe. After incubation for 20 min at room
temperature, 10 µl of the binding reaction was loaded onto a 6%
polyacrylamide gel in 1× TBE (90 mM Tris base, 90 mM boric acid, 0.5 mM EDTA) and electrophoresed
at 160 V for 2 h. The gel was dried before autoradiography.
Supershift assays were carried out by preincubating the nuclear extract
with 2 µl of rabbit antiserum at 4 °C for 30 min before addition
of the binding reaction mixture. Where appropriate, DNA-protein
complexes were quantitated using a PhosphorImager and ImageQuant
software (Molecular Dynamics). Mixing experiments to form C/EBP
heterodimers were incubated at 45 °C for 20 min in the presence of
95 mM DTT.
Bacterially Expressed Proteins--
His-tagged C/EBP,
C/EBP, C/EBP-GLZ, and truncated C/EBP-(191-276)
were expressed in Escherichia coli and purified as described (38).
Immunoprecipitation--
Approximately 5.8 µg of purified
His-tagged C/EBP was incubated with 5.8 µg of His-tagged C/EBP
or truncated C/EBP-(191-276) at 45 °C for 20 min in the
following buffer: 0.2 M Tris (pH 7.5), 0.2 M
NaCl, 5 mM EDTA, 10 mM DTT, 0.1% Nonidet P-40,
0.5 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml
leupeptin, 5 µg/ml aprotinin, 5 µg/ml antipain. 3 µl of
NH2-terminal C/EBP antiserum and 10 µl of protein
A-Sepharose beads were then added. As a control, His-tagged C/EBP or
truncated C/EBP-(191-276) were incubated alone in the same manner.
After a 3-h incubation at 4 °C, the beads were collected by
centrifugation and washed three times with buffer. 1× Laemmli sample
buffer was added to the beads, and the samples were boiled for 5 min
and centrifuged. The eluted proteins were resolved by SDS-PAGE and examined by Western blotting using Super Signal® West
HisProbeTM kit (Pierce).
Immunoprecipitation experiments using nuclear extracts from transfected
L or HepG2 cells were performed using the SeizeX immunoprecipitation kit (Pierce). Cells were transfected with 5 µg of control plasmid, 5 µg of tagged C/EBP vector, or 5 µg of tagged C/EBP vector with 5 µg of C/EBP vector, and nuclear extracts were prepared. Seventy-five µg of N.E. was added to protein A-Sepharose beads cross-linked to the NH2-terminal GCN4 antibody and
incubated for 3 h at 4 °C. The beads were washed three times
with buffer and eluted with protein sample buffer and the eluted
proteins analyzed by Western blotting.
Western Blotting--
Nuclear extracts were mixed with an equal
volume of 2× sample buffer (40), heated at 95 °C for 5 min, and
loaded on precast 12% or 16% SDS-PAGE gels (Novex). Gels were
transferred to Immobilon-P membranes (Millipore) and blocked with 5%
dry milk in Tris-buffered saline (pH 7.6). Blots were developed using
the enhanced chemiluminescence (ECL) detection system (Pierce).
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RESULTS |
C/EBP Occurs in Cells Predominantly as a Rapidly Migrating EMSA
Complex--
To examine the dimeric state of C/EBP proteins in cells,
we performed EMSA on a series of nuclear extracts using a consensus C/EBP binding site oligonucleotide as the probe. We initially focused
our analysis on C/EBP, because this protein is widely expressed in
cell lines and tissues. We examined nuclear extracts from cell lines
representing glioma (C6-2B), mammary epithelia (EMT6), and macrophages
(IC-21) (Fig. 1A). The cell
line extracts contained multiple species that bound specifically to the
C/EBP motif, as determined by the ability of the unlabeled wild-type probe, but not a mutated oligonucleotide, to compete for binding (Fig.
1B and data not shown). To identify complexes containing C/EBP, we performed supershift analysis using a C/EBP antibody. As shown in Fig. 1A, C/EBP binding activities were
observed in all extracts, and the C/EBP EMSA complexes occurred in
two forms. One complex exhibited the same mobility as a bacterially
expressed C/EBP homodimer (lane 7), whereas a second
species (indicated by asterisks) displayed considerably
faster mobility in the gel. In all cases this faster migrating form was
the predominant C/EBP species. We also analyzed extracts from
P388-C cells, which express C/EBP from a retroviral vector (34,
35). P388 is a lymphoblastic tumor cell that contains very low levels
of endogenous C/EBP proteins except C/EBP (Ref. 41; see below).
P388-C nuclear extracts contained almost exclusively the rapidly
migrating form of C/EBP (Fig. 1C, lane 2).
Thus, ectopic C/EBP in P388 cells is detected predominantly as a
high mobility EMSA species, similar to the results observed for
endogenous C/EBP in the other cell lines (Fig. 1A).
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Fig. 1.
C/EBP proteins in cells occur primarily as
rapidly migrating EMSA complexes. A, EMSA of nuclear
extracts from cell lines. A consensus C/EBP binding site was used as
the probe. Extracts (3-12 µg) were assayed in the presence or
absence of the NH2-terminal C/EBP antibody. Positions of
the rapidly migrating species are indicated by asterisks.
Bacterially expressed C/EBP was analyzed in parallel (lane
7) to show the mobility of the C/EBP homodimer. B,
specificity of C/EBP·DNA complexes. Increasing amounts of unlabeled
C/EBP competitor probe (lanes 7-12) or a mutant
oligonucleotide with a disrupted C/EBP site (lanes 1-6)
were added to binding reactions containing nuclear extracts from L
cells. C, EMSA of nuclear extracts from stably transfected
P388 lymphoblasts. Extracts from P388 cells and a P388 transfectant
expressing C/EBP (P388-C) were analyzed as described in
panel A.
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The Rapidly Migrating C/EBP Complexes Are
Heterodimers--
The fact that the C/EBP antibody used for the
supershift experiments recognizes the amino terminus of the protein
indicates that the rapidly migrating complex contains an intact
C/EBP subunit and is not a truncated isoform, such as LIP, that
contains the COOH-terminal DNA-binding domain (42, 43). The absence of truncated forms of C/EBP in the extracts was confirmed by Western blotting using a COOH-terminal C/EBP antibody (data not shown). To
test whether the rapidly migrating C/EBP complex is a heterodimer with another cellular protein, we performed a mixing experiment in
which purified recombinant C/EBP was combined with nuclear extract
from P388 cells and assayed by EMSA (Fig.
2). This procedure generated a rapidly
migrating complex that co-migrated with the endogenous C/EBP complex
from P388-C cells (compare lanes 1 and 4).
Similar results were obtained using recombinant C/EBP and C/EBP
(data not shown). In contrast, a chimeric C/EBP protein (C/EBP-GLZ) containing a heterologous leucine zipper
from the yeast GCN4 protein did not form the rapidly migrating complex with P388 extract (lanes 5 and 6). These findings
show that the C/EBP leucine zipper is necessary to produce the
rapidly migrating C/EBP complex and indicate that this species
consists of a heterodimer between C/EBP and another nuclear
protein.
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Fig. 2.
Identification of a C/EBP heterodimerizing
factor using an in vitro mixing assay.
Recombinant C/EBP was assayed by EMSA either alone or after mixing
with P388 cell nuclear extract. Nuclear extracts from P388-C cells
or P388 cells are shown in lanes 1 and 2,
respectively. Recombinant C/EBP-GLZ, which contains the
GCN4 leucine zipper, did not form a rapidly migrating complex when
mixed with P388 extract (lanes 5 and 6). The
consensus C/EBP site oligonucleotide was used as the probe.
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Identification of C/EBP as the C/EBP Heterodimeric
Partner--
The rapid mobility of C/EBP heterodimers and their
presence in all cell types examined indicate that the dimeric partner is a small, ubiquitously expressed protein. These features suggested that the partner might be C/EBP (Ig/EBP), a 16.4-kDa protein that
can dimerize with other C/EBP proteins and is expressed at the mRNA
level in many cell types (29, 31). A C/EBP polyclonal antiserum
raised against recombinant C/EBP (31) did not supershift the
C/EBP heterodimer, although it shifted a more rapidly migrating complex that corresponds to a C/EBP homodimer (data not shown). However, biochemical purification of the rapidly migrating complex to
near homogeneity suggested that the heterodimerizing factor may indeed
be C/EBP.2 Therefore, we
generated additional antisera using synthetic peptides corresponding to
the C/EBP amino and carboxyl termini and tested the antibodies in
EMSA supershift experiments. Both C/EBP antisera supershifted a
partially purified, rapidly migrating C/EBP complex isolated from
P388-C/EBP cells, as well as a putative C/EBP homodimer also
present in this preparation (Fig.
3A). Thus, these two peptide
antibodies recognize C/EBP complexes that appear to contain
C/EBP.
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Fig. 3.
The rapidly migrating C/EBP heterodimers
contain C/EBP. A, the C/EBP
heterodimer reacts with two peptide antisera specific for C/EBP. A
partially purified fraction containing the C/EBP heterodimer
isolated from P388-C/EBP cells (data not shown) was analyzed by EMSA
supershift using antisera raised against peptides corresponding to the
amino or carboxyl termini of C/EBP (lanes 2 and
3, respectively). B, upper
panel, nuclear extracts from the cell lines used in Fig. 1
were assayed by EMSA supershift using the COOH-terminal C/EBP
antibody. P388-C-C1 is a P388 derivative expressing C/EBP.
Positions of the C/EBP·C/EBP heterodimers and C/EBP homodimers
are shown on the right. Lower panel,
Western blot analysis of C/EBP in cell extracts. The nuclear
extracts (~30 µg of protein) from the upper panel were analyzed by
Western blotting using the COOH-terminal C/EBP antibody. Nuclear
protein (1 µg) from untransfected (lane 6) or
C/EBP-transfected (lane 7) L cells was analyzed in
parallel to demonstrate that overexpressed C/EBP is identical to the
endogenous protein. C, EMSA of C/EBP complexes in nuclear
extracts from mouse tissues. Each extract was analyzed in the absence
and presence of C/EBP and C/EBP antibodies. Bands corresponding
to C/EBP·C/EBP heterodimers are indicated by
asterisks. The last three
lanes contain extracts from untransfected (lane
13), C/EBP-transfected (lane 14), and C/EBP- plus
C/EBP-transfected (lane 15) L cells. The identities of
the EMSA complexes are indicated on the right.
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To determine whether the rapidly migrating C/EBP complexes in cell
extracts are C/EBP heterodimers, we tested a panel of cell extracts
in antibody supershift assays using the COOH-terminal C/EBP
antibody. Fig. 3B (upper panel) shows
that rapidly migrating C/EBP complexes from glioma cells
(lanes 1 and 2) and mammary epithelial cells
(lanes 3 and 4), as well as from P388-C cells (lanes 7 and 8), were supershifted by the
C/EBP antibody. Similarly, a C/EBP heterodimeric complex from
P388-C-C1 cells (lanes 9 and 10) reacted with
the C/EBP antibody. Interestingly, the rapidly migrating C/EBP
complex from IC-21 macrophages was only weakly supershifted by the
COOH-terminal C/EBP antibody (lanes 5 and 6)
and by the NH2-terminal C/EBP antibody (data not shown),
despite the fact that this complex migrates identically with
C/EBP·C/EBP heterodimers from other cells. C/EBP complexes
from two other monocyte/macrophage cell lines also did not react
appreciably with C/EBP antibodies (data not shown). These findings
indicate that a distinct, but functionally related, protein
heterodimerizes with C/EBP in monocytic cells. Western blot
experiments using the same panel of cell extracts (Fig. 3B,
lower panel) confirmed that a protein of ~18
kDa, identical in size to ectopically expressed C/EBP (lane
9), was detected in all cells examined including macrophages.
Because our analysis of C/EBP dimerization thus far used transformed or
immortalized cell lines, we next wished to determine whether C/EBP
heterodimerizes with C/EBP in normal tissues. We prepared nuclear
extracts from mouse liver, brain, ovary, and spleen and analyzed them
by EMSA and antibody supershift experiments. As shown in Fig.
3C, each extract contained a rapidly migrating C/EBP
complex (denoted by an asterisk) that could be supershifted by the C/EBP and C/EBP antibodies. Nuclear extracts from L cells transfected with C/EBP (lane 14) or C/EBP plus
C/EBP (lane 15) were analyzed on the same gel to confirm
the identities of the homo- and heterodimeric C/EBP and C/EBP
EMSA species. In summary, the experiments of Fig. 3 show that C/EBP
occurs mainly as a heterodimer with C/EBP in cell lines as well as
animal tissues.
C/EBP Causes Cell-specific Repression of C/EBP-mediated
Transcription--
We next investigated whether heterodimerization
with C/EBP affects the transcriptional activity of C/EBP.
Initially, we examined the effect of C/EBP on C/EBP-mediated
transactivation using a C/EBP-dependent promoter-reporter
construct ((DEI)4-alb-Luc; Ref. 38) in HepG2 hepatoma cells
(Fig. 4A, left
panel). C/EBP alone increased reporter expression by
~15-fold. Co-transfecting increasing amounts of a C/EBP expression
vector did not significantly diminish reporter gene expression, even at
the highest dose of C/EBP (8.5 µg), which is an 11.3-fold excess
of C/EBP over the C/EBP vector. This result was unexpected,
because C/EBP was previously found to inhibit the transactivation
function of C/EBP and C/EBP in B lymphoma cells, 3T3 fibroblasts,
and promonocytic cells (31). Therefore, we performed a similar C/EBP
titration experiment in L fibroblastic cells (Fig. 4A,
right panel). In these cells C/EBP clearly
inhibited C/EBP-mediated transactivation of the
(DEI)4-alb-Luc reporter in a dose-dependent
manner. Luciferase activity decreased linearly with the amount of
C/EBP vector added and reached 22% of the control level at the
maximal dose of C/EBP. Similar results were obtained using a
reporter construct containing two copies of a consensus C/EBP binding
site (data not shown). Thus, C/EBP is capable of inhibiting C/EBP
activity in L cells but not in HepG2 hepatoma cells.
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Fig. 4.
C/EBP inhibits
C/EBP activity in a cell-dependent
manner. A, HepG2 and L cells were cotransfected with a
constant amount of a C/EBP reporter plasmid, (DE1)4-alb-luc
(2.5 µg), pMEX-C/EBP expression vector (0.75 µg), and the
indicated amounts of pMEX-C/EBP expression vector. The luciferase
activity in cells transfected with the reporter and C/EBP alone was
set to 100%. Luciferase data (dashed lines)
represent the average (± S.E.) of at least three independent
transfections. C/EBP binding activity (solid
lines) was measured by quantitating the DNA-protein
complexes in EMSAs (panel B) using a PhosphorImager. The
indicated ratios of dimeric complexes were calculated and plotted.
B, EMSA of nuclear extracts from the transfected cells.
Positions of C/EBP homodimers (:),
C/EBP·C/EBP heterodimers (:), and C/EBP
homodimers (:) are shown. Quantitative analysis of the
complexes is presented in panel A. C, Western
blot analysis of C/EBP expression in the transfected cells. 20 µg
of each nuclear extract was loaded onto the gel, and the blot was
developed with a C/EBP antibody.
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To assess the levels of homo- and heterodimeric C/EBP complexes in the
transfected cells, we prepared nuclear extracts and subjected them to
EMSA analysis (Fig. 4B). C/EBP homodimer and heterodimer
levels were increased in the transfected cells. Heterodimers were
observed in cells transfected with C/EBP alone, resulting from
dimerization with endogenous C/EBP (lane 2). The amount of heterodimeric complex increased with the addition of C/EBP vector, as did the levels of C/EBP homodimer. The EMSA complexes were quantitated by phosphorimaging, and C/EBP homodimer levels were
calculated either as the percentage of total C/EBP binding activity
(heterodimer plus the two homodimeric species) or as the fraction of
C/EBP binding activity (C/EBP homodimer plus heterodimer) (Fig.
4A). The proportion of C/EBP homodimer decreased with
added C/EBP, and the dimerization curves were similar in HepG2 cells
(no transcriptional repression by C/EBP) and L cells (repression).
Western blotting showed that C/EBP did not alter C/EBP levels in
the nuclear extracts (Fig. 4C). These experiments show that
C/EBP·C/EBP heterodimers are formed in both cell types, suggesting that heterodimers are transcriptionally active in HepG2 cells but not in L cells.
To further examine whether inhibition of C/EBP by C/EBP requires
heterodimerization, we used the zipper-swap mutant,
C/EBP-GLZ, which is unable to dimerize with C/EBP.
Fig. 5A shows that C/EBP did not repress C/EBP-GLZ activity in either HepG2 or L
cells, whereas in HepG2 cells transactivation was actually enhanced at the highest dose of C/EBP. EMSA (Fig. 5B) verified that
C/EBP-GLZ·C/EBP heterodimers were not formed in the
transfected cells, although homodimers were observed. Thus, the ability
of C/EBP to inhibit C/EBP activity requires heterodimerization
between the two proteins and is not the result of competitive binding
of transcriptionally inactive C/EBP homodimers to the promoter.
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Fig. 5.
Repression of C/EBP
activity by C/EBP requires the
C/EBP leucine zipper. A,
co-transfection experiments were performed in HepG2 and L cells as
described in Fig. 4, except that pMEX-C/EBP-GLZ was used
instead of wild-type C/EBP. Data are the average (± S.E.) of three
experiments. B, EMSA of nuclear extracts from the
transfected cells. Positions of C/EBP-GLZ homodimers and
C/EBP homodimers are indicated.
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Because the DNA binding activity of C/EBP was not repressed by
heterodimerization, it seemed likely that C/EBP inhibits transactivation. To investigate this possibility, we used a
GAL4-C/EBP fusion protein whose ability to activate a
UAS-dependent reporter gene (G5E1b-luc) depends
on the transactivation domain (TAD) of C/EBP (38). Normally the
activity of full-length C/EBP fused to GAL4 is very low because of
strong repression of the TAD by inhibitory sequences located in
COOH-terminal regions of the molecule, including the bZIP domain (38,
44). However, GAL4-C/EBP can be activated by coexpression of
oncogenic Ha-Ras.3 Therefore,
we transfected GAL4-C/EBP with a Ha-Ras(12V) vector and the
G5E1b-luc reporter into L cells along with increasing amounts of the C/EBP vector. As shown in Fig.
6, C/EBP potently inhibited
GAL4-C/EBP activity in a dose-responsive manner. This result
demonstrates that heterodimerization with C/EBP suppresses the
ability of the C/EBP TAD to stimulate transcription, even when
C/EBP is tethered to DNA through a heterologous DNA-binding domain.
C/EBP did not inhibit GAL4-C/EBP activity in HepG2 cells (data
not shown), further supporting the observation that C/EBP repression
is cell-specific.
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Fig. 6.
C/EBP inhibits
transactivation by a GAL4-C/EBP fusion
protein. L cells were transfected with the G5E1b-Luc
reporter (1 µg), GAL4-C/EBP vector (5 ng), pcDNA3-Ha-Ras(12V)
(25 ng), and the indicated amounts of pcDNA3.1-C/EBP. Luciferase
data are the average (± S.E.) of three experiments.
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We next asked whether C/EBP could repress transactivation by other
C/EBP family members (Fig. 7). Neither
C/EBP nor C/EBP was inhibited by C/EBP in HepG2 cells, and, in
fact, C/EBP activity was stimulated nearly 2-fold at the maximal
C/EBP dose. This enhanced activity was associated with increased
expression of C/EBP (data not shown), the mechanism of which is
unknown. Similar to C/EBP, C/EBP transactivation was repressed by
C/EBP in L cells. In contrast, C/EBP activity was unaffected by
C/EBP in L cells (Fig. 7) despite the fact that the two proteins
formed heterodimers (data not shown). Collectively, our data indicate that C/EBP·C/EBP heterodimers are active in L cells, whereas C/EBP·C/EBP and C/EBP·C/EBP dimers are repressed.
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Fig. 7.
Differential repression of the C/EBP family
members by C/EBP. Inhibition of C/EBP-
and C/EBP-mediated transactivation by C/EBP was analyzed in HepG2
and L cells, as described in Fig. 4. Repression of C/EBP activity is
included for comparison (the data are reproduced from Fig.
4A). The data are the average (± S.E.) of three
experiments.
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DNA-independent Association of C/EBP and C/EBP in Vitro and
in Vivo--
The observation that C/EBP proteins in cells occur
predominantly as heterodimers with C/EBP suggested that they might
preferentially dimerize with C/EBP. Using recombinant His-tagged
C/EBP and C/EBP proteins, we performed coimmunoprecipitation
assays to compare the ability of C/EBP to self-dimerize and to
heterodimerize with C/EBP in vitro (Fig.
8A). Self-dimerization was
examined by mixing full-length (p34) C/EBP with a truncated C/EBP
protein (C/EBP-(192-276)) containing only the bZIP portion of the
molecule. Mixtures of full-length C/EBP and either C/EBP or
C/EBP-(192-276) were immunoprecipitated with an antibody directed
against the amino terminus of C/EBP. The immunoprecipitates were
analyzed by Western blotting using a reagent that detects the
polyhistidine tag. In the absence of full-length C/EBP, neither of
the other proteins was immunoprecipitated (lanes 1 and
2), as expected. When full-length C/EBP was added to the
mixtures, C/EBP and C/EBP-(192-276) were detected in the
precipitated fraction (lanes 3 and 4). Both
proteins were immunoprecipitated with similar efficiency, suggesting
that the C/EBP leucine zipper has comparable affinity for itself and
for C/EBP. Thus, the predominance of heterodimers in vivo
may be the result of a molar excess of C/EBP in cells, or,
alternatively, dimerization might be regulated by a cellular mechanism
such as phosphorylation.
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Fig. 8.
DNA-independent association of
C/EBP and C/EBP in
vitro and in vivo. A, purified
His-tagged C/EBP, C/EBP, and truncated C/EBP-(192-276) (5.8 µg each) were mixed in the indicated combinations and subjected to
immunoprecipitation using the NH2-terminal C/EBP
antibody. The immunoprecipitates were analyzed by Western blotting
using a His tag detection reagent. The individual purified proteins
(0.36 µg each) are shown in lanes 5-7. B,
association of C/EBP and C/EBP in transfected cells. Nuclear
extracts (20 µg) from L and HepG2 cells transfected with the
indicated expression vectors were analyzed for expression of C/EBP
and epitope-tagged C/EBP by Western blotting (top
two panels). The same nuclear extracts (75 µg)
were subjected to immunoprecipitation using a GCN4
NH2-terminal antibody (bottom panel).
The immunoprecipitates were analyzed for C/EBP by Western blotting
using the COOH-terminal C/EBP antiserum.
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We next examined the association between C/EBP and C/EBP in
transfected cells using a co-immunoprecipitation assay. Epitope-tagged C/EBP, which contains the NH2-terminal 13 amino acids of
GCN4 fused to its amino terminus, was expressed in L or HepG2 cells, either alone or with C/EBP. Nuclear extracts were prepared and subjected to immunoprecipitation with an antibody recognizing the GCN4
tag (36), followed by Western blotting for C/EBP. As shown in Fig.
8B (bottom panel), ectopic C/EBP
co-immunoprecipitated with C/EBP in L cells and in HepG2 cells
(lanes 3 and 6). Thus, in both cell types
C/EBP and C/EBP are associated in the absence of DNA. These
findings further support the conclusion that impaired heterodimerization does not explain the inability of C/EBP to repress transcription in HepG2 cells.
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DISCUSSION |
Our studies demonstrate that C/EBP activator proteins exist
predominantly as heterodimers with C/EBP in vivo. By
comparing EMSA complexes generated with recombinant C/EBP with those
from nuclear extracts, we observed that C/EBP in cell lines and
tissues occurs mainly as a rapidly migrating heterodimer. Antibodies
specific for the NH2 and COOH termini of C/EBP
supershifted the rapidly migrating C/EBP species, confirming that the
heterodimers contain C/EBP. Our characterization of C/EBP
heterodimers in this study has focused on C/EBP, because this
isoform is expressed in many cell lines. However, C/EBP and C/EBP
also occurred as rapidly migrating heterodimers in P388 transfectants
that stably express these proteins (Fig. 3B and data not
shown), as well as in transiently transfected L cells (data not shown).
Thus, it seems likely that all of the C/EBP activators heterodimerize
with C/EBP in vivo.
Heterodimerization with C/EBP did not detectably alter the
DNA-binding specificity of its C/EBP partner, because homodimers and
heterodimers bound efficiently to a consensus C/EBP element. The major
effect of dimerization with C/EBP was to repress C/EBP transactivation function. In L cells, coexpression of C/EBP
inhibited the ability of C/EBP and C/EBP to activate
transcription from a C/EBP-dependent promoter. A C/EBP
chimera containing the GCN4 leucine zipper that cannot heterodimerize
with C/EBP was resistant to repression. C/EBP also suppressed
transactivation by a GAL4-C/EBP fusion protein. Collectively, these
results indicate that heterodimerization with C/EBP inhibits the
transcriptional activity of C/EBP. At present it is unclear how
heterodimerization with C/EBP suppresses transactivation. C/EBP
lacks a TAD and by itself neither activates nor represses transcription
of target genes (31).2 It is possible that, because C/EBP
heterodimers contain only one activating subunit, they cannot
efficiently stimulate transcription. Alternatively, heterodimerization
with C/EBP might block access to a coactivator protein for which
association with the C/EBP activator involves sequences in the leucine
zipper and/or basic region. This possibility is currently under investigation.
The fact that C/EBP did not repress transactivation by any of the
C/EBPs in HepG2 hepatoma cells is noteworthy. Analysis of the C/EBP
dimeric species expressed in transfected cells showed that heterodimers
were produced and their levels increased in proportion to the amount of
transfected C/EBP vector. A homodimeric C/EBP complex was
observed in both L and HepG2 cells, and this complex did not
appreciably diminish with increased C/EBP expression (Fig.
4B). It is possible that a pool of homodimers exists that is
resistant to heterodimerization, perhaps because of a specific post-translational modification. However, because the occurrence of
these persistent homodimers did not differ in the two cell types, their
presence cannot account for the differential repression by
C/EBP.
Because there was no difference in heterodimer formation in HepG2 and L
cells, at least as assessed by EMSA and co-immunoprecipitation experiments, we postulate that C/EBP·C/EBP heterodimers are
transcriptionally active in HepG2 cells but not in L cells. There are
several potential explanations for this difference in activity.
Heterodimers could be the target of activating kinases in HepG2 cells
but not in L cells, whereas homodimers might be effective substrates in
both cells. Such modifications could occur on either the C/EBP
activator protein or the C/EBP subunit. It is also conceivable that
protein:protein interactions mediated by the bZIP region are necessary
for transcriptional activation and that heterodimeric bZIP domains are
differentially active for this function in the two cell types.
Irrespective of the mechanism, the ability of C/EBP to affect the
transactivation potential of C/EBP activators in a cell-specific manner
represents a novel means of controlling C/EBP activity.
A mouse strain carrying a null mutation at the C/EBP
locus has been developed (45). Homozygous mutant animals show grossly normal embryonic development and are initially viable after birth. However, the majority of mutant mice die within 48 h of postnatal development. Although the cause of mortality was not determined, the
lethal phenotype shows that C/EBP has an essential function in
newborn animals and presumably also in adult mice. It remains to be
determined whether the lethality of C/EBP-deficient mice results
from the absence of C/EBP heterodimers, leading to formation of
homodimers with altered regulatory activities. Considering the
involvement of C/EBP proteins in many biological processes and the
predominance of C/EBP·C/EBP heterodimers in cells, it is not
surprising that deletion of C/EBP would have severe phenotypic consequences. An additional function for C/EBP in lymphoid cells was
revealed by analysis of bone marrow chimeras generated from C/EBP
null donor cells (45). Natural killer cells derived from mutant donors
display reduced cytolytic activity and impaired production of
interferon- in response to interleukin-12 or interleukin-18 stimulation. Nevertheless, the molecular basis for defective
interferon- gene expression in C/EBP-deficient natural killer
cells has not been elucidated.
In another study examining C/EBP function in vivo,
Zafarana et al. (46) created transgenic mice
overexpressing C/EBP in erythroid cells. Animals heterozygous for
the C/EBP transgene displayed increased fetal -globin gene
expression compared with adult -globin expression, indicating that
C/EBP positively regulates -globin transcription. However, when
C/EBP expression was increased further by making the transgenic
allele homozygous, fetal erythropoiesis was eliminated and the embryos
did not survive beyond embryonic day 14.5. These results demonstrate
that C/EBP stoichiometry critically affects development of the
erythroid lineage. We suggest that the developmental defects associated
with high ectopic C/EBP expression may result from decreased levels
of C/EBP homodimers in erythroid precursor cells.
In addition to regulating transcription, C/EBP proteins can induce cell
growth arrest (47-49). In proliferating cell lines, C/EBP proteins
occur primarily as heterodimers, raising the possibility that
heterodimerization with C/EBP mitigates the growth arrest activity
of these proteins. In experiments to create P388 cell lines expressing
the zipper swap mutant, C/EBP-GLZ, only minimal amounts
of the mutant protein were detected in stable transfectants whereas the
wild type protein could be expressed at much higher levels (41). This
result is consistent with the idea that C/EBP must heterodimerize
with C/EBP for its expression to be tolerated in proliferating
cells. Furthermore, HepG2 hepatoma cells express significantly lower
levels of C/EBP and C/EBP than are found in normal, terminally
differentiated hepatocytes (50). We speculate that C/EBP may be
unable to suppress C/EBP-mediated growth arrest in hepatoma cells,
similar to its inability to inhibit C/EBP-dependent transcription in these cells. Thus, conversion of hepatocytes to
proliferating hepatoma cells might require strong down-regulation of
C/EBP and C/EBP expression. In future studies it will be informative to examine the ability of C/EBP to modulate
C/EBP-mediated cell growth arrest in various cellular contexts.
The observation that C/EBP activity can be inhibited by
heterodimerization with C/EBP suggests that C/EBP dimerization might be regulated to control gene transcription. In this regard,
calcium-regulated phosphorylation of a serine residue in the leucine
zipper of C/EBP has been linked to its increased transcriptional
activity (51). Although the molecular mechanism underlying this
activation event has not been elucidated, the authors raised the
possibility that phosphorylation of the C/EBP zipper might control
dimerization. Our studies indicate that C/EBP could be involved in
this putative regulatory mechanism. Although our experiments thus far
have focused on artificial promoters, future studies will address
potential differences between C/EBP homodimers and heterodimers in
activating authentic promoters, in addition to the possibility that
C/EBP heterodimerization is regulated by developmental cues or other physiological signals.
(Ig/EBP)*
,
, and
TOP
ABSTRACT
INTRODUCTION
Supported by Grant-in-aid 9950490N from the American Heart Association.
