(Received for publication, February 14, 1997)
From the Laboratory of Molecular Structure, NIAID, National Institutes of Health, Rockville, Maryland 20892-1727
We screened an expression cDNA library with a
radiolabeled C/EBP fusion protein and isolated three independent
cDNAs encoding ATF-2, a bZIP protein that binds cAMP response
elements (CRE). This interaction requires the respective bZIP domains,
which form a typical bZIP heterodimer with altered DNA binding
selectivity. C/EBP
and ATF-2 homodimers bind CRE sites, but
ATF-2:C/EBP
heterodimers do not. Heterodimers bind an asymmetric
sequence composed of one consensus half-site for each monomer, and may
thus have a unique regulatory function. As predicted, co-transfection
of ATF-2 with C/EBP
results in decreased activation of transcription
driven from consensus C/EBP-binding sites. In contrast, C/EBP
and
ATF-2 function cooperatively to activate transcription driven by the asymmetric sequence. Both factors are expressed in liver, where immunoprecipitation experiments show that ATF-2 co-precipitates with
C/EBP
. These results are consistent with the interpretation that
C/EBP
and ATF-2 can associate in vivo. Moreover, the
formation of ATF-2:C/EBP
heterodimers suggests that cross-family
dimerization with ATF-2 may be a general property for C/EBP family
proteins.
CCAAT/enhancer-binding protein,
C/EBP,1 was purified as an activity that
bound to consensus enhancer elements and to the CCAAT box motif (1).
C/EBP protein is a member of the basic region-leucine zipper (bZIP)
class of transcription factors (2, 3). The bZIP domain consists of the
leucine zipper, a heptad repeat of leucines, preceded by the basic
region, a sequence with net positive charge (4, 5). A combination of
molecular and structural studies showed that the heptad repeat region
is an amphipathic helix that mediates dimerization by forming a
parallel coiled-coil (4-15). The co-crystal structure of the bZIP
domain of GCN4 bound to DNA showed that the coiled helices of the
leucine zipper separate, positioning one helix from each chain for
sequence specific interaction with DNA (10).
Leucine zippers accommodate both homotypic and heterotypic
dimerization. For example, the first C/EBP protein characterized, C/EBP, is one of at least five gene products comprising the C/EBP gene family (16, 17). These proteins show extensive sequence similarity
that is restricted to the bZIP domain, such that each homodimer binds
the same DNA element. Additionally, all proteins in the C/EBP family
can pair with each other to form DNA binding heterodimers (16, 17).
When not bound to DNA, the subunits of bZIP dimers are in a rapid
monomer:dimer equilibrium such that the lifetime of dimers is estimated
in seconds (18, 19). The ready dissociation of bZIP dimers in
vitro suggests that heterodimers with potentially unique
regulatory properties may form in vivo. In the case of Fos,
a bZIP protein in the AP1 family, no homodimers form. Instead, Fos
forms heterodimers with Jun, another AP1 family protein (20).
Sequence-specific interaction with DNA increases the lifetime of bZIP dimers more than 10-fold (18, 19, 21). However, the apparent DNA affinity constants are lower than might be anticipated (18). Surprisingly, GCN4, a protein that binds the core sequence TGAGTCA, exhibited similar affinity for a core motif containing an extra nucleotide, TGACGTCA (18). The structures of GCN4 and Fos:Jun bound to DNA revealed that most of the amino acids involved in base specific contacts are those that are highly conserved among all bZIP proteins (11), including those with distinct DNA binding specificity. Thus, the basic mechanism by which bZIP proteins interact with DNA is known, while the determinants of sequence discrimination are less clearly understood.
C/EBP activates transcription of several liver and fat cell-specific
genes (22-25). Interestingly, over-expression of C/EBP
in cultured
cells results in growth arrest (26-29), a finding consistent with the
observation that C/EBP
expression commences during the conversion of
3T3L1 preadipocytes into quiescent fat cells in vitro (17,
30, 31). The role of C/EBP
in the adipogenic program was further
verified by antisense experiments (32), and by ectopic expression of
C/EBP
in a variety of fibroblastic cell lines, where efficient
promotion of fat cell differentiation was observed (33).
Support for the notion that C/EBP plays a central role in regulating
energy homeostasis (34) was provided by targeted gene disruption.
Homozygous C/EBP
knockout mice are born with apparently normal blood
glucose levels, but become severely hypoglycemic within minutes (35).
These animals exhibit glycogen storage defects and morphological
anomalies in fat and liver tissues. Although these defects are
consistent with a role for C/EBP
in energy homeostasis, expression
of several putative C/EBP target genes was normal (35). Thus, it has
been difficult to identify genes that are C/EBP
targets in
vivo.
Activator proteins like C/EBP bind specific DNA sequences located
either upstream or downstream of the core promoter. In response to
physiological cues, activators stimulate transcription initiation by
interacting with general transcription factors, with TATA-associated
factors, or with adaptor proteins (36, 37). The observation that
C/EBP
binds a range of DNA sequences, coupled with the observation
that its subunits exchange rapidly, suggests that the monomer may be a
target for regulation. We screened a
gt11 expression cDNA
library for proteins that physically interact with C/EBP
. Three
independent cDNA clones encoding ATF-2, a bZIP protein, were
isolated. We mapped the interacting protein domains to the respective
leucine zippers, and demonstrated formation of a bZIP heterodimer with
restricted DNA binding selectivity, in vitro. C/EBP
and
ATF-2 are expressed in liver, and transient transfection analysis shows
that co-transfected ATF-2 impacts C/EBP
function. Together with
immunoprecipitation results showing that ATF-2 coprecipitates with
C/EBP
, these results are consistent with the interpretation that
functional ATF-2:C/EBP
heterodimers form in vivo.
pGEX-2T (Pharmacia, Uppsala Sweden)
was modified by the insertion of a protein kinase A phosphorylation
site as described (38). pMSV-C/EBP1-2 (24) was used for
construction of N-terminal GST-C/EBP fusions. Truncated constructs
lacking the leucine zipper and the bZIP domain were prepared by placing
in-frame stop codons at amino acids 310 and 272, respectively (39).
MluI digestion and fill-in created the fusion to amino acid
192. Constructs 218-358 and 281-342 have been described (19). A
diagram of the fusion proteins is shown (see Fig. 2A).
Our cDNA clones were identical to rat ATF-2 (GenBank accession
M65148[GenBank]), with the exception of 294 nucleotides inserted at nucleotide
393 from the 5 end. A full-length rat ATF-2 cDNA was generated by
reverse transcriptase-polymerase chain reaction using the primer pair
5
-GGATCCATGAGTGATGACAAACCCTTTCTATGCA-3
and
5
-ATCGATTGCAGGTTTTAATCAACTTCCTGAGGG-3
. ATF-2 fusions to GST were
prepared by polymerase chain reaction. The primers used to prepare
1-323 were 5
-GCGGATCCATGAGTGATGACAAACCCTTTCTATGCA-3
and
5
-CCATCGATTTAACTTGTATTTTGGGTCTGTGGAGT-3
. The primer pairs for
323-492 were 5
-GCGGATCCGGCCGTCGAAGAAGAGCAGCTAATG-3
and
5
-CCATCGATTGCAGGTTTTAATCAACTTCCTGAGGG-3
. The primer pairs for
323-352 were 5
-GCGGATCCGGCCGTCGAAGAAGAGCAGCTAATG-3
and
5
-CCATCGATTTAGCATCTTGAAGCTGCTGCTCTATT-3
. A diagram of the fusion proteins is shown (see Fig. 2B). Polymerase chain
reaction products were verified by sequencing.
pCMX1 was a gift from Catherine Thompson (Carnegie Institution of
Washington) and was used in coupled transcription-translation reactions. C/EBP and ATF-2 were subcloned as BamHI (24)
and BamHI/KpnI fragments, respectively. CREB was
produced by reverse transcriptase-polymerase chain reaction
(BamHI/EcoRI) with the primer pair
5
-GCGGATCCATGACCATGGACTCTGGAGCAGACAA-3
and
5
-CGGAATTCTTAATCTGACTTGTGGCAGTAAAGGTCC-3
. In vitro
translation products were verified by [35S]Met
incorporation and SDS-PAGE analysis.
The reporter vector 2X C/EBP-Luc contains two direct repeats of the
consensus C/EBP-binding site, TGCAGATTGCGCAATCTGCA, and was a gift from P. Rorth (Carnegie Institution of Washington). 2X
C/EBP-Luc has the minimal thymidine kinase promoter (40) and a
BamHI site for proximal insertion of transcription
factor-binding sites. Three iterations of the chimeric binding site
(5
-GCCGTGACGCAATCTC-3
) were inserted into the thymidine kinase
promoter/luciferase vector, producing the reporter 3X Chimera-Luc.
A GST (41) fusion protein
encoding C/EBP amino acids 1-10 fused to amino acids 60-358 (see
Fig. 2A) was radiolabeled with protein kinase A (Sigma) as
described (33). Radiolabeled protein was used for interaction screening
immediately.
A gt11 cDNA library (CLONTECH) prepared from
rat liver mRNA was plated for screening essentially as described
(42). Filters were blocked for 1 h in Hyb75 (50 mM
Tris (pH 8.0), 75 mM KCl, 0.1 mM EDTA, 2.5 mM MgCl2, 5 mM 2-mercaptoethanol,
and 0.1% Nonidet P-40) containing 5% nonfat milk. Radiolabeled
GST-C/EBP
was added at 100,000 cpm/ml in Hyb75, 5% nonfat milk, 25 ml/filter, and incubated overnight at 4 °C with shaking. Filters
were washed 3 × for 5 min each at 4 °C in 50 ml/filter of
Hyb75, 0.25% nonfat milk. Filters were dried briefly and exposed to
film.
Fusion proteins were expressed in Escherichia coli host strain BL21. Log phase cultures were induced at an OD600 of 0.8 for 2 h. Cells were harvested, and GST (41) or MBP (New England Biolabs, Beverly, MA) fusion proteins were purified according to established protocols. Detailed purification of "C/EBP short" was described previously (19).
Preparation of AntiseraThe ATF-2 cDNA
(EcoRI) was subcloned into the vector pMal (New England
Biolabs), for protein expression in bacteria. MBP-ATF-2 was affinity
purified, and the antigen was excised from a 10% SDS-PAGE gel.
Purification of an NH2-terminally deleted C/EBP protein
(C/EBP
short) was described previously (19). This protein was used
to prepare antiserum. Antisera were raised according to standard
protocols, and appropriate reactivity was verified against recombinant
proteins. The specificities of all antisera were verified against
purified recombinant proteins. ATF-2 and C/EBP
antisera do not
cross-react against purified recombinant proteins.
2.5 mg of
MBP-ATF-2 was coupled to CNBr-activated Sepharose 4B (Pharmacia,
Uppsala, Sweden). Following the removal of lipids with Seroclear
(CalBiochem, La Jolla, CA), clarified serum was passed 3 × over a
0.2-ml bed of MBP-ATF-2 equilibrated in phosphate-buffered saline.
Columns were washed with 10 ml of phosphate-buffered saline, and eluted
with 750 µl of 0.1 M glycine (pH 3.0), dripping directly into 750 µl of 0.1 M Na2HPO4 (pH
9.2) for neutralization. Affinity purified antibody was dialyzed into
phosphate-buffered saline to remove glycine, and biotinylated as
described (43). This protocol was adapted for purification and
biotinylation of the antisera against intact C/EBP, kindly provided
by Pernille Rorth, and C/EBP
short proteins.
Proteins from SDS-PAGE were electroblotted to Immobilon P membranes (0.45 µM, Millipore) and developed with polyclonal antiserum. Secondary detection utilized horseradish peroxidase-conjugated donkey anti-rabbit serum (Amersham), which was visualized by chemiluminescence (ECL reagent, Amersham). For biotinylated antibodies, secondary detection utilized streptavidin-conjugated horseradish peroxidase (Amersham) and ECL reagent.
To map protein interaction domains, equal amounts of each truncated protein were separated on 10% SDS-PAGE gels, and transferred to nitrocellulose (0.45 µm, Schleicher & Schuell). Membranes were blocked with 5% nonfat milk, and incubated in Hyb150 containing soluble GST-C/EBP (ATF-2 proteins on the membrane) or soluble GST-ATF-2 (C/EBP proteins on the membrane). After 4 washes, the interaction of soluble protein with membrane-bound protein was probed with specific antiserum.
Affinity ChromatographyEquivalent amounts of MBP or MBP-ATF-2 were coupled to Sepharose 4B. 100-µl columns were equilibrated in Hyb150 (50 mM Tris (pH 8.0), 150 mM KCl, 0.1 mM EDTA, 2.5 mM MgCl2, 5 mM 2-mercaptoethanol, and 0.1% Nonidet P-40). 70 µg of rat liver nuclear extract (RLNE) was loaded onto each column, and washed with 15 volumes of Hyb150. Protein was eluted stepwise with 1.8 volumes of buffer containing 0.25, 0.5, and 1.0 M KCl. Equivalent percentages of the unbound and eluted fractions were loaded onto 12% SDS-PAGE gels for Western blot analysis.
Gel Shift AnalysisProtein-DNA complexes were formed in
standard TBE gels (42). Binding reactions (10 µl) were 10 min at
37 °C in 10 mM Tris (pH 7.5), 1 mM
dithiothreitol, 100 mM KCl, 1 mM EDTA, 10%
glycerol, 1 mg/ml bovine serum albumin, and 0.05 µg/µl poly(dI-dC).
Full-length C/EBP, ATF-2, and CREB were prepared by coupled in
vitro transcription/translation (TNT, Promega, Madison, WI). The
three DNA probes were radiolabeled to comparable specific activities.
One strand of each probe is shown. Consensus C/EBP site:
5-TGCAGATTGCGCAATCTGCA-3
; CRE site: 5
-GATCAGCATTACCTCATCCC-3
, from the jun
promoter (44); chimeric site: 5
-GATCGCCGTGACGCAATCTC-3
. All reactions
were performed in probe excess, but free probe was run off the gels to
improve the resolution of heterodimeric complexes from homodimeric
complexes.
Freshly prepared RLNE (45) was pre-cleared with an irrelevant antiserum and protein A-agarose beads (Life Technologies). After a brief spin, supernatants were separated and adjusted to 50 mM Tris (pH 8.0), 15% glycerol, 0.25 M NaCl, 1 mM MgCl2, 0.1 mM EDTA, 1% Nonidet P-40. 3 µl of affinity purified antibody was added and incubated overnight at 4 °C with rocking. Precipitates were collected onto protein A beads and washed 5 × with 500 µl of 50 mM Tris (pH 8.0), 150 mM NaCl, 1% Nonidet P-40. After SDS-PAGE and electroblotting, proteins were detected with biotinylated, affinity purified antibodies and visualized as described above.
For co-immunoprecipitation, primary precipitates were formed with the first affinity purified antibody (as above), separated by SDS-PAGE, electroblotted, and detected with a second antibody (affinity purified and biotinylated) as above. 20 pmol of double-stranded oligonucleotide-binding sites were included where indicated, and blots were developed as described above.
Transient Transfection AnalysisThe hepatoma cell line Fao
(46) was transfected by the standard calcium phosphate method (42).
pCMX1-Gal, a gift from C. Thompson, was included in all
transfections to normalize the data. Cells were harvested 48 h
later, and relative luciferase (Analytical Luminescence Laboratory, Ann
Arbor, MI) and
-Gal activities (Promega) were determined in
duplicate, and the average was determined. Cells transfected with the
reporter vector alone determined relative luminescence. All results are
the average of three independent experiments.
To search
for proteins that physically interact with C/EBP, a rat liver
expression cDNA library was screened with a radiolabeled GST-C/EBP
fusion protein. From 2 × 106 phage
screened, eight positive cDNAs were isolated. The phage inserts
were subcloned and sequenced, revealing that three of them encoded the
bZIP protein ATF-2, a member of the ATF/CREB transcription factor
family. The interaction between ATF-2 and C/EBP
was specific as both
GST-GCN-4, a yeast bZIP protein, and GST failed to interact with ATF-2
in this assay (not shown).
To test
whether ATF-2 and C/EBP would interact under more stringent
conditions, rat liver nuclear extract (RLNE) was fractionated on a
column displaying ATF-2 (fused to MBP). As a specificity control, a
column displaying MBP alone was run in parallel. Equivalent percentages
of the unbound fraction and the salt eluted fractions were separated by
SDS-PAGE, electroblotted, and probed with C/EBP
antiserum. Multiple
forms of C/EBP
are expressed in the liver, including the full-length
protein (42 kDa) and several internal translation initiation products
(47, 48). The MBP-ATF-2 affinity column, but not the MBP column alone,
selectively binds all forms of C/EBP
present in the nuclear extract
(Fig. 1, compare the eluates from the two columns). Note
that elution of C/EBP
from the ATF-2 column requires 0.5-1.0
M KCl, an indication of a relatively high affinity
interaction.
Association of ATF-2 with C/EBP
To map the domains required for interaction between
C/EBP and ATF-2, a sequential deletion strategy was used. C/EBP
deletion constructs were expressed in E. coli as GST fusion
proteins, and purified on glutathione-agarose. Equivalent amounts of
each protein were separated on SDS-PAGE gels and transferred to
nitrocellulose. The blot was incubated with purified GST-ATF-2, washed
extensively, and subsequently developed with anti-ATF-2 serum. As shown
in Fig. 2A, GST-C/EBP constructs containing
the leucine zipper bound soluble GST-ATF-2 efficiently (lanes 1, 4, 5, and 6). Analogously, truncated versions of ATF-2,
similarly prepared as GST fusions, were tested for the ability to bind
soluble C/EBP
(Fig. 2B). Again, only those constructs
containing the leucine zipper efficiently retained soluble C/EBP
(lanes 1 and 3). The weak binding observed in the
absence of the leucine zippers (Fig. 2, A and B, lanes 2) is likely due to the putative zinc finger motif in ATF-2 and will be addressed under "Discussion." As controls, each blot (Fig. 2, A and B, respectively) was incubated with
antiserum without prior exposure to soluble proteins, showing that the
antisera for C/EBP
and ATF-2 are not cross-reactive (not shown).
These results show that the leucine zippers are sufficient to mediate interaction between these bZIP proteins.
Formation of an
ATF-2:C/EBP heterodimer would bring together different DNA-binding
domains, which may lead to a change in DNA binding selectivity. To
assay for DNA binding heterodimers, three DNA sites were tested in
electrophoretic mobility shift assays (EMSA): a consensus C/EBP-binding
site; a consensus CRE-binding site; and a chimeric site consisting of
one C/EBP half-site directly abutted to one CRE half-site. To
distinguish heterodimeric DNA binding complexes from homodimeric DNA
binding complexes, a truncated C/EBP
protein encompassing the bZIP
domain (19) was used. DNA binding complexes formed with one short
C/EBP
subunit and one full-length C/EBP
, ATF-2, or CREB subunit
migrate intermediate to DNA binding complexes composed of two
full-length or two short subunits (homodimers), and demonstrate subunit
exchange.
As shown in Fig. 3A, both full-length
(lane 4) and short (lane 2) C/EBP homodimers
shift the consensus C/EBP-binding site. When full-length and short
C/EBP
are mixed, a shift of intermediate migration is observed
(lane 3), showing that subunits exchanged. ATF-2 homodimers
do not shift this probe (lane 7), and no ATF-2:C/EBP
heterodimers are evident upon co-incubation with the short C/EBP
protein (lane 6). As expected, CREB homodimers bind the
probe (lane 10), and no heterodimeric complex forms when
CREB and C/EBP
short are mixed (lane 9).
When a radiolabeled CRE site is tested (Fig. 3B), a shift is
observed with C/EBP (lanes 2-4). When C/EBP
short and
ATF-2 are mixed, it is predominantly ATF-2 homodimers that shift the probe (lanes 5-7). Although both homodimers bind, no
heterodimeric DNA binding complex is observed (see lane 6).
Analogous results are obtained when C/EBP
and CREB are mixed
(lanes 8-10), where the predominant shift is by CREB
homodimers, and no heterodimeric complex is detected (lane
9).
Upon EMSA analysis using the chimeric binding site (Fig.
3C), a gel shift complex of intermediate migration is
observed (lane 6), which is consistent with formation of an
ATF-2:C/EBP heterodimer. In fact, C/EBP
, ATF-2, and CREB
homodimers (lanes 4, 7, and 10, respectively) all
bind the chimeric sequence. The failure of ATF-2:C/EBP
heterodimers
to form a gel shift complex on the consensus C/EBP- and CREB-binding
sites suggests that these heterodimers have a restricted DNA binding
selectivity.
Since C/EBP family
proteins dimerize interchangeably, we tested ATF-2 for heterodimer
formation with C/EBP (Fig. 4). As a control, we show
that C/EBP
short and C/EBP
form a heterodimeric DNA binding
complex in the EMSA assay. When C/EBP
was mixed with ATF-2, a
heterodimeric complex formed on the chimeric binding site. Thus, the
capability to form heterodimers with ATF-2 is a property that appears
to be shared among C/EBP family proteins.
Co-transfection of ATF-2 with C/EBP
To
analyze the impact of ATF-2 on C/EBP function, we transfected the
hepatoma cell line Fao with mammalian expression vectors encoding these
proteins. Transcription was analyzed with a simplified luciferase
reporter vector (2X C/EBP Luc) driven by the minimal thymidine kinase
promoter and two copies of the C/EBP-binding site. Hepatoma cells
transfected with ATF-2 alone showed basal levels of reporter gene
activity (Fig. 5, left panel), consistent with the observation that ATF-2 does not bind the consensus C/EBP site.
In contrast, C/EBP
transfectants showed a 7-fold increase in
reporter gene activity. When ATF-2 and C/EBP
were co-transfected, activation levels decreased 43 to 55% (Fig. 5, left panel).
These results are consistent with the interpretation that formation of
ATF-2:C/EBP
heterodimers decreases the pool of C/EBP
homodimers available to bind the reporter construct, resulting in decreased transcription activity.
ATF-2 and C/EBP
The initial
characterization of ATF-2 suggested that the protein lacked
transactivation activity altogether (49-53). Subsequently, transactivation activity was demonstrated, but was subject to tight
control (50, 51). To characterize transcription from the heterodimer
binding site, we cloned 3 copies of the chimeric sequence upstream of
the minimal thymidine kinase promoter (3X Chimera Luc). Surprisingly,
Fao cells transfected with ATF-2 alone showed reporter activity that
was 12-18-fold higher than the control (Fig. 5, right
panel). Similarly, cells transfected with C/EBP alone showed
5-7-fold activation of the reporter. When ATF-2 and C/EBP
were
co-transfected, the reporter gene was activated approximately 26-fold
(Fig. 5, right panel). First, these results show that ATF-2
activates transcription of the minimal promoter in Fao cells, which is
surprising in that ATF-2 activity is repressed in many cell types.
Second, these results are consistent with the interpretation that ATF-2
and C/EBP
form heterodimers that activate transcription from the
chimeric sequence.
Although ATF-2 mRNA is found in essentially all
tissues tested, the protein has been characterized mainly in brain and
thymus. Using affinity purified ATF-2 antibodies, we found that ATF-2 is expressed in rat liver, apparently as a doublet of about 68 kDa
(Fig. 6A, lanes 2 and 4). ATF-2 is
also expressed in thymus, while expression is minimal in L cell and
spleen cell nuclear extracts (not shown). As C/EBP is also expressed
in liver, we tested for co-precipitation of ATF-2 with C/EBP
.
Freshly prepared rat liver nuclear extracts were subjected to
immunoprecipitation with affinity purified C/EBP
antibodies. The 42- and 29-kDa forms of C/EBP
were precipitated by this reagent (Fig.
6A, lane 1). When this immunoprecipitate was blotted for
co-precipitating ATF-2 reactivity, the characteristic doublet at 68 kDa
was observed (lane 3). The same result was obtained when
C/EBP
was immunoprecipitated with a different antibody, one directed
against the COOH-terminal portion of the protein (lane 5).
These results are consistent with the interpretation that ATF-2 and
C/EBP
can form heterodimers in vivo.
Although the half-life of a bZIP dimer is measured in seconds, binding
to a consensus DNA sequence results in a 10-100-fold increase in the
lifetime of the dimer (19). We reasoned that inclusion of a specific
oligonucleotide-binding site during immunoprecipitation would stabilize
heterodimers, facilitating their detection in the co-precipitation
assay. The two binding sites we compared were the consensus C/EBP site
and the chimeric site (see Fig. 3, A and C, for
sequences). As shown previously, ATF-2 is readily detectable in RLNE
(Fig. 6B, lane 1). A control immunoprecipitation with
irrelevant antiserum precipitates neither C/EBP nor ATF-2 (lane 2). Significantly, inclusion of the chimeric binding
site during precipitation with C/EBP
antibodies facilitates
detection of ATF-2 as a co-precipitant (compare lane 3 to
4). When a C/EBP-binding site is included during
immunoprecipitation, the efficiency of ATF-2 co-precipitation
(lane 4) is diminished. These precipitates (lanes
3 and 4) show equivalent reactivity with C/EBP
antibodies (not shown), indicating that enhanced co-precipitation of
ATF-2 in the presence of the chimeric binding site cannot be explained by a simple difference in the amount of C/EBP
precipitated. These results suggest that the subunits of ATF-2 and C/EBP
are in dynamic equilibrium, and that the composition of dimers is a reflection of the
stoichiometry of the individual subunits.
To identify proteins that interact with C/EBP, we screened an
expression cDNA library with a radiolabeled GST-C/EBP
fusion protein. Of the four cDNAs we isolated, three encoded nuclear proteins and one was a novel gene product. Three independently isolated
cDNAs encoded the bZIP transcription factor ATF-2. Neither GST-GCN4, a yeast bZIP protein, nor GST itself, interact with ATF-2
under these conditions, indicating that interaction between ATF-2 and
C/EBP
is specific. The fact that we did not isolate any abundant
cytoskeletal protein that possesses a coiled-coil motif is a further
indication of the stringency of the screen.
Although the protein domains mediating association mapped to the
respective leucine zippers (see Fig. 2, A and B),
weak association of ATF-2 with an immobilized C/EBP fusion protein
containing the DNA-binding domain but lacking the leucine zipper was
consistently observed (Fig. 2A, lane 2). Reciprocally, we
observed weak association of soluble C/EBP
with immobilized ATF-2
constructs containing only the amino-terminal domain of the protein
(Fig. 2B, lane 2). This is likely due to a putative zinc
finger motif located near the amino terminus of ATF-2. This motif
interacts with bZIP domains (54), and may be involved in negative
autoregulation of ATF-2 function (55). Although it is possible that
this motif augmented detection during our initial screen, it is clear
that ATF-2 interacts with C/EBP
primarily by leucine zipper-mediated
dimer formation.
ATF-2:C/EBP heterodimers form a DNA binding complex with a target
specificity that differs from the parental homodimers. The heterodimers
do not bind symmetric DNA elements like consensus C/EBP and CRE sites,
(see Fig. 3, A and B), but bind to an asymmetric sequence consisting of one consensus half-site for each monomer (Fig.
3C). It is noteworthy that ATF-2:C/EBP
heterodimers do not bind the CRE sequence, considering that both parent homodimers bind
this site (Fig. 3B). This suggests that heterodimer
formation results in increased DNA binding selectivity, as C/EBP
homodimers bind all three sites, while ATF-2:C/EBP
heterodimers bind
only one.
Oppositely charged side chains at specific positions in each helix of a
leucine zipper form interhelical electrostatic contacts that stabilize
the dimers (56). Comparison of the charges at appropriate positions
within the C/EBP and ATF-2 leucine zippers revealed the occurrence
of potential stabilizing contacts. EMSA analysis revealed that such
heterodimers can form (Fig. 4), and further suggest that ATF-2 subunits
may be capable of forming heterodimers with all C/EBP family proteins.
Heterodimeric DNA binding complexes involving subunits from different
bZIP protein families have been described previously (7, 58-61). For
example, C/EBP
was reported to form heterodimers with C/ATF, another
CREB/ATF family protein. The heterodimer bound a different asymmetric
CRE element which is found in the promoters of several liver genes (60).
The principle effect of ATF-2 on C/EBP dependent transcription is
transcriptional interference (Fig. 5, left panel).
ATF-2:C/EBP
heterodimers cannot bind consensus C/EBP sites, and thus
decrease the pool of C/EBP
homodimers available to regulate the
reporter gene. Similar results were obtained upon heterodimer formation between C/EBP
and Jun (61), where "repression" of transcription from C/EBP sites was reported. This is in contrast to results obtained
when the C/EBP-binding sites are replaced by the chimeric sequence.
When both ATF-2 and C/EBP
are co-transfected, reporter activity
exceeds that observed with either transcription factor alone (Fig. 5,
right panel). Taken with the results using the C/EBP site
reporter, these findings are consistent with the interpretation that
ATF-2:C/EBP
heterodimers form, and affect transcription activity.
Both ATF-2 and C/EBP are expressed in liver. Using freshly prepared
rat liver nuclear extracts, we showed that immunoprecipitates for
C/EBP
react with affinity purified ATF-2 antibodies, and that the
amount of ATF-2 co-precipitated could be enhanced or diminished in a
predictable fashion by inclusion of specific DNA-binding sites.
Together, the results of transient transfection and immunoprecipitation studies are consistent with the formation of ATF-2:C/EBP
heterodimers in vivo. The fact that ATF-2 can associate with
C/EBP
indicates that this association can probably be extended to
other C/EBP family proteins. One functional consequence of this
interaction is inhibition of transcriptional activity from consensus
C/EBP target sites. It is also likely that heterodimers have a positive impact on transcription from promoters containing chimeric DNA elements. A search of primate promoter sequences in the data base using
the 8-nucleotide chimeric DNA element indicates that the promoters of
the protein disulfide isomerase, glutathione S-transferase, and ornithine decarboxylase genes are candidates for this kind of
regulation.
Heterodimer formation is appealing from a regulatory viewpoint because of the asymmetry that is generated. By selecting for asymmetric DNA elements, heterodimers bind their target in an orientation dependent fashion, presenting distinct surfaces for interaction with proteins bound to adjacent DNA sites. Both the position and the orientation of protein-binding sites within some enhancer elements are important for the formation of what has been termed the "stereospecific" complex (57). Although the function of ATF-2:C/EBP heterodimers is not known, it has been proposed that cross-family heterodimers serve as a common target for the integration of signals arising from different extracellular stimuli. In this model, each subunit of the heterodimer would be modified by a unique protein kinase in response to only one of the signals being transduced. Since each subunit of the heterodimer is independently modified in this scenario, signals from two pathways converge, leading to the appropriate changes in the pattern of gene expression (51, 60, 61).
We thank J. Askins for technical assistance, A. Brooks, R. Ehrlich, C. Hammer, J. Ochoa-Garay, K. Parker, and P. Rorth for review of the manuscript, and A. Friedman, P. Johnson, P. Rorth, and C. Thompson for plasmid reagents. We are indebted to Mary Weiss, Institut Pasteur, for the providing the Fao cell line. J. Shuman would like to acknowledge Dr. Jeffrey Kudlow, Department of Medicine, Division of Endocrinology, University of Alabama at Birmingham for support and encouragement.