(Received for publication, September 27, 1995; and in revised form, November 22, 1995)
From the
We have developed a bZIP protein, GBF-F, with both
dominant-negative (DN) and gain-of-function properties. GBF-F is a
chimera consisting of two components: the DNA binding (basic) region
from the plant bZIP protein GBF-1 (GBF) and a leucine zipper (F)
designed to preferentially heterodimerize with the C/EBP leucine
zipper. Biochemical studies show that GBF-F preferentially forms
heterodimers with C/EBP
and thus binds a chimeric DNA sequence
composed of the half-sites recognized by the C/EBP and GBF basic
regions. Transient transfections in HepG2 hepatoma cells show that both
components of GBF-F are necessary for inhibition of C/EBP
transactivation. When the C/EBP
leucine zipper is replaced with
that of either GCN4 or VBP, the resulting protein can transactivate a
C/EBP cis-element but is not inhibited by GBF-F, indicating
that the specificity of dominant-negative action is determined by the
leucine zipper. All known members of the C/EBP family contain similar
leucine zipper regions and are inhibited by GBF-F. GBF-F also exhibits
gain-of-function properties, since, with the essential cooperation of a
C/EBP family member, it can transactivate a promoter containing the
chimeric C/EBP
GBF site. This protein therefore has potential
utility both as a dominant-negative inhibitor of C/EBP function and as
an activator protein with novel DNA sequence specificity.
The determination of the function of individual members of the
C/EBP ()family of bZIP transcription factors is complicated
by their possible redundant roles. Vertebrate gene expression is
regulated by an abundance of transcription factors, many of which exist
as members of gene families whose products have similar DNA-binding
properties and thus have potentially overlapping target genes and
biological functions. Several methods exist to address this problem.
Gene disruption (1) and replacement (2) by homologous
recombination provide a powerful genetic approach to assess the in
vivo functions of a particular transcription factor. However,
several examples have been reported recently in which the functional
redundancy within a gene family allows other family members to
substitute, at least partially, for the disrupted gene. A well
documented example is the MyoD family of bHLH transcription factors (3) and the Src family of tyrosine kinases(4) . The
fundamental problem with this method is that it obscures tissue
specific and developmentally later functions. To study these later
functions, homologous recombination using the CRE LOX system activated
in a tissue or stage specific manner can be used(5) .
An
alternative approach which is pharmacological is to use a
dominant-negative (DN) system to inhibit the function of one or several
family members(6) . Expression of antisense RNA, using either
the conserved or nonconserved part of mRNA as a target to inactivate
one or all family members, respectively, is an attractive possibility.
Lane's (7) group has used this approach to demonstrate
that C/EBP is critical for the differentiation of 3T3L1 cells into
adipocytes. Expression of DN proteins is another method to inactivate
gene function. A number of different DN inhibitors of bZIP protein
function have been described, both as naturally occurring members of
transcription factor families or as experimentally designed proteins.
These DN molecules can be divided into two general classes. The first
class binds the same DNA target but lack an activation
domain(8, 9, 10, 11) . This class of
DNs acts as competitive inhibitors, forming either non-activating
homodimers that compete with the target protein for binding to its
cognate binding sites or heterodimers with reduced transcriptional
activity. By virtue of their mode of action, these DN proteins must be
expressed in excess relative to their target for efficient inhibition.
The second class dimerizes with the endogenous bZIP protein but which
either lack a DNA-binding domain or contain a DNA-binding domain with
different binding specificity from the target protein(12) . The
second class potentially functions more efficiently as DNs, since
heterodimers formed with these proteins do not bind to DNA and are
therefore completely inactive.
In this study we describe the design
of an efficient DN molecule with specificity for the C/EBP family of
bZIP transcription factors. There are four major proteins in the C/EBP
family, to which we refer using the nomenclature C/EBP,
C/EBP
, C/EBP
, and CRP1 (reviewed in (13) ). C/EBP
proteins have been implicated in a variety of regulatory functions,
including activation of constitutive and acute phase responsive genes
in the liver, control of adipocyte differentiation, and regulation of
inflammatory cytokine genes and other activities of monocytic
cells(13) . C/EBP family members can heterodimerize in all
combinations and recognize a common palindromic DNA motif (consensus:
ATTGCGCAAT). Each monomer is a long (
70 amino acids) bipartite
-helix when bound to
DNA(14, 15, 16, 17) . The
carboxyl-terminal half of the
-helix is amphipathic with the two
helices interacting along their hydrophobic surfaces to create a
dimeric leucine zipper coiled coil. The NH
-terminal half of
the motif interacts with the major grove of DNA in a sequence specific
fashion and becomes
-helical upon DNA
binding(18, 19) . DNA binding stabilizes the dimer
complex, which complicates the design of non-DNA binding DNs to bZIP
proteins.
As a first step in developing a DN with specificity for
C/EBP proteins, we used a previously designed a leucine zipper (the F
zipper) that preferentially heterodimerizes with C/EBP as a result
of substitutions in charged amino acids occupying the e and g positions of the zipper
-helix(20) . We have appended
to this F zipper the basic region from the plant bZIP protein G-box
binding factor-1 (GBF-1)(21) , which recognizes a DNA sequence
highly divergent from that recognized by C/EBP proteins. This chimeric
protein creates a dominant-negative protein (GBF-F) that inhibits C/EBP
mediated transactivation. Both the heterodimerizing leucine zipper and
the novel basic region are critical for optimal activity of the
designed DN. In addition to its ability to inhibit C/EBP
transactivation of a canonical C/EBP site, we show that GBF-F also has
gain-of-function properties as a GBF-F:C/EBP heterodimer which can
transactivate the non-palindromic site composed of C/EBP and GBF
half-sites.
Figure 4:
C/EBP:GBF-F heterodimers bind
preferentially to a chimeric C/EBP
GBF site in vivo. A, nuclear extracts from HepG2 cells transfected with pMEX
C/EBP
and different concentrations of pRG GBF-F were prepared and
assayed by EMSA using either the consensus C/EBP cis-element (top panel) or a C/EBP
GBF chimeric cis-element (bottom panel) as probes (lanes
1-6). The positions of bands corresponding to specific homo-
and heterodimeric complexes are indicated by arrows. The
positions of C/EBP
:GBF-F heterodimers are indicated by an asterisk and the labeled arrow. A supershift assay
using a C/EBP
-specific antiserum was used to confirm the identity
of specific bands (lanes 7-9). DNA concentrations were
as follows: lane 1, no DNA; lane 2, 2 µg of pMEX
C/EBP
, 15 µg of pRG; lane 3, 2 µg of pMEX
C/EBP
, 5 µg of pRG GBF-F, 10 µg of pRG; lane 4, 2
µg of pMEX C/EBP
, 10 µg of pRG GBF-F, 5 µg of pRG; lane 5, 2 µg of pMEX C/EBP
, 15 µg of pRG GBF-F; lane 6, 15 µg of pRG GBF-F; lane 7, no DNA; lane 8, 2 µg of pMEX C/EBP
, 15 µg of pRG; lane 9, 2 µg of pMEX C/EBP
, 5 µg of pRG GBF-F, 10
µg of pRG. B, Western blot analysis of C/EBP
expression levels. An immunoblot of the same nuclear lysates used in panel A was probed with anti-C/EBP
antiserum to determine
the level of C/EBP
protein expression in each
sample.
Using this optimized transactivation assay, we examined the
inhibition of C/EBP activity by four potential DN proteins: 1) a
C/EBP
bZIP domain lacking an activation domain (
C/EBP), 2) a
bZIP domain containing the C/EBP
basic region and the F zipper
(C/EBP-F), 3) the C/EBP
leucine zipper without a basic region
(C/EBP LZ), and 4) the F leucine zipper without a basic region (F LZ) (Fig. 1). In cells transfected with equal amounts of plasmids
expressing C/EBP
and each of the four DNs no inhibition of
C/EBP
transactivation was observed (data not shown). Therefore we
used a C/EBP
to DN plasmid ratio of 1:15 to ensure that sufficient
DN was expressed relative to C/EBP
. Fig. 1shows
transactivation levels of the C/EBP-dependent and minimal promoters as
histograms below schematic representations of each putative
heterodimer. Under these conditions co-expression of
C/EBP or
C/EBP-F resulted in 50% inhibition of C/EBP
activity. When the
basic regions of
C/EBP and C/EBP-F were deleted, the resultant DNs
suppressed C/EBP activity slightly less efficiently (60% of C/EBP
activity). Using specific antibodies, we analyzed the expression levels
of C/EBP
and the various DNs in transfected cells (data not
shown). The levels of
C/EBP and C/EBP-F were not significantly
higher than that of C/EBP
.
Figure 1:
Inhibition of C/EBP
transactivation by four potential DNs. The structures of a wild-type
C/EBP
dimer and heterodimers between C/EBP
and each DN bound
to DNA are represented schematically. HepG2 cells were transfected with
0.3 µg of C/EBP
expression plasmid alone or with C/EBP
and a potential DN expression plasmid and a CAT reporter plasmid. CAT
activities are shown in histograms as fold activation (±standard
deviation from three independent experiments) relative to the activity
of the reporter plasmid alone. Transfections were carried out using 10
µg of the albumin minimal promoter reporter (nucleotides -35
to +22), either with or without a single consensus C/EBP binding
site. A 1:15 molar ratio of C/EBP
to DN expression plasmids was
used.
Figure 2:
C/EBP and GBF-F heterodimerize and
bind a chimeric site. A, comparison of consensus DNA binding
sites of four bZIP protein families. The 4- or 5-bp half-sites are shaded. The C/EBP half-site shares four positions with the PAR
site(59) , 2 positions with the ATF/CREB (60) and
Fos/Jun(61, 62) sites, but only one position (the G
at +1) with the GBF site(21) . To date, the GBF binding
site is the most divergent from that of C/EBP among bZIP protein
families. B, the bZIP domains of C/EBP
and GBF-F were
overexpressed in bacteria and purified to homogeneity. The two proteins
were mixed in the indicated molar ratios and binding to
oligonucleotides containing either the C/EBP consensus site
ATTGC
GCAAT (left panel) or an idealized chimeric
element, ATTGC
GTGGA, was assayed by EMSA. The positions of the
relevant homo- and heterodimeric species are
indicated.
Heterodimers between C/EBP proteins and GBF-F would be predicted to
recognize a novel DNA sequence composed of C/EBP and GBF half-sites. In
order to test this hypothesis, electrophoretic mobility shift assays
(EMSA) were carried out using purified, bacterially-expressed bZIP
domains of C/EBP and GBF-F with oligonucleotide probes containing
either a consensus C/EBP site or an idealized C/EBP GBF chimeric site (Fig. 2B, left and right panels).
C/EBP
alone bound both oligonucleotides, although binding to the chimeric
site was noticeably weaker than to the consensus C/EBP site. This
finding is consistent with previous studies that demonstrated that
C/EBP proteins have the ability to bind to sites which only loosely
conform to the consensus site(13) . As the amount of GBF-F in
the binding reaction was increased, the amount of
C/EBP homodimers
binding to the consensus C/EBP site decreased (Fig. 2B, left
panel). At the same time, binding of
C/EBP homodimers to the
chimeric sequence was completely abrogated, even at the lowest
concentration of GBF-F (Fig. 2B, right panel).
Concomitantly, a faster-migrating complex appeared whose intensity
increased with additional GBF-F. We conclude that this novel species is
a
C/EBP:GBF-F heterodimer, as it displays high affinity for a
C/EBP
GBF chimeric site.
Figure 3:
Inhibition of C/EBP transactivation
by GBF-F. A, schematic representations of C/EBP
:DN
heterodimers are as described in Fig. 1, except the
C/EBP
:GBF-C/EBP
and C/EBP
:GBF-F heterodimers are shown
binding to the chimeric site, represented by a half-stippled
circle. Transactivation assays were carried out in HepG2 cells as
described in the legend to Fig. 1using the minimal promoter
constructs. CAT activities are shown in histograms as fold activation
± standard deviation from three independent experiments. B, GBF-F inhibits C/EBP
transactivation of the complete
albumin promoter. Conditions identical to those of Fig. 3A were used, except that the reporter plasmid was pAT2-CAT which
contains the albumin promoter (nucleotides -787 to
+22).
As shown in Fig. 2, addition of
increasing amounts of GBF-F protein to binding reactions containing
C/EBP caused an immediate removal of C/EBP
from the chimeric
site, but only a gradual removal of C/EBP
from its consensus
binding site. As most C/EBP sites found in vivo conform only
loosely to the consensus sequence, we wished to test whether the effect
of GBF-F would be more dramatic on a promoter containing a natural,
imperfect C/EBP site. The C/EBP element found in the albumin promoter
(designated DEI or the D site;
ATTTT
GTAAT)(38, 39) , is typical of most C/EBP
sites identified thus far, consisting of one half-site that conforms
fairly closely to the consensus and another, more divergent half-site.
EMSA experiments confirm that C/EBP
binds to the consensus site
approximately 3-fold better than the albumin DEI site (data not shown).
We therefore tested the ability of GBF-F to inhibit C/EBP
transactivation of the albumin promoter-CAT reporter construct (Fig. 3B). GBF-F was an extremely efficient inhibitor,
completely abolishing C/EBP
-dependent transactivation under the
standard transfection conditions. These data suggest that the
dominant-negative properties of GBF-F are more apparent when C/EBP
is bound to a weaker cis-element and suggest that GBF-F should
act as an efficient DN in vivo.
Figure 5:
GBF-F inhibits transactivation by three
C/EBP family members in a leucine zipper-dependent manner. Fold
activation of the C/EBP-dependent CAT reporter in HepG2 cells by
C/EBP, C/EBP
, or C/EBP
in either the absence (rows
1-4) or presence (rows 5-8) of GBF-F is
represented graphically as fold activation ± standard deviation
from three independent experiments. Cotransfection of GBF-F did not
affect the transactivation properties of C/EBP
proteins carrying
the leucine zippers of GCN4 or VBP (rows 9-12).
Transfection conditions were as described in the legend to Fig. 1.
To confirm that C/EBP-specific inhibition by GBF-F occurs
as a result of leucine zipper interactions, we used chimeric C/EBP
proteins containing the leucine zipper from two other bZIP proteins,
GCN4 and VBP. These hybrid proteins (C/EBP-GCN4 and C/EBP-VBP)
activated the C/EBP-dependent promoter to approximately the same level
as wild-type C/EBP
(Fig. 5). Cotransfection of GBF-F did
not affect transactivation by either chimera, indicating that GBF-F
specifically inhibits C/EBP family members and is unable to inhibit the
activity of proteins containing unrelated leucine zippers.
Figure 6:
C/EBP and GBF-F containing an
activation domain cooperate to activate a chimeric cis-element. A, the AD of C/EBP
(residues
1-162) was joined to GBF-F and the resulting protein was tested
for its ability to transactivate a reporter gene carrying a chimeric
C/EBP
GBF element. Various concentrations of GBF-F containing
the C/EBP
AD were transfected alone or in combination with a fixed
concentration of a C/EBP
expression plasmid. B, as in A except the VP16 activation domain replaced the C/EBP
activation domain to form a VP16-GBF-F
fusion.
In an attempt to create a potent dominant-negative to C/EBP,
we have explored several structurally different protein designs. The
first two DNs consisted of bZIP domains from either wild type
C/EBP (
C/EBP) or C/EBP
containing the F zipper
(C/EBP-F), which preferentially heterodimerizes with C/EBP
. Since
these proteins lack an activation domain, their overexpression could be
predicted to competitively inhibit C/EBP
activity by either the
formation of inactive or less-active heterodimers or, in the case of
C/EBP, competition with C/EBP
for binding to its cognate
site. Both
C/EBP and C/EBP-F were able to inhibit C/EBP
activity by 50%. Similar naturally occurring structures also act as
efficient DNs for other bZIP families, including the AP-1 and CREB
proteins(9, 10, 11, 40) . A
naturally occurring DN molecule analogous to
C/EBP is encoded by
the C/EBP
gene: translational initiation at an internal AUG codon
generates a truncated protein, LIP, that lacks an activation domain (8) . LIP has been reported to exhibit DN properties when
co-expressed with the activating form of C/EBP
. A similar
truncated protein can be generated from C/EBP
by internal
translational initiation. This shorter form of C/EBP
(C/EBP-30)
lacks the major activation domain located at the NH
terminus(41, 42) . However, unlike C/EBP
,
C/EBP
contains a second activation domain that is retained in
C/EBP-30, and, thus, it is likely that this shorter form functions as a
weak activator rather than as an inhibitor.
For C/EBP and
C/EBP-F to act as efficient inhibitors, heterodimers between these DNs
and C/EBP
must be less efficient transactivators than C/EBP
homodimers. At present, it is not clear whether these heterodimers are
less active. Indeed, the inability of these two DN proteins to
efficiently inhibit C/EBP
activity suggests that a single
activation domain is sufficient for transactivation. In this regard,
Goodman's group has been using a heterodimerizing zipper system
by mutating amino acids in the e and g positions and
substituting a3 asparagine with histidine. They can show that a
CREB dimer requires only one phosphorylated activation domain to
activate transcription of a CRE-containing promoter(43) .
The second pair of DNs tested consisted of isolated leucine zipper
domains. The two C/EBP zippers examined, wild-type and F, were even
less effective inhibitors than the bZIP domains, conferring only 40%
inhibition of C/EBP activity. We suggest that the lack of
efficient inhibition by the F zipper is due to the fact that the
F:C/EBP
heterodimer is less stable than a C/EBP
homodimer.
The presence of DNA should drive the equilibrium toward the formation
of C/EBP
homodimers at the expense of F:C/EBP
heterodimers
which cannot bind DNA. Zippers designed to heterodimerize even more
avidly with C/EBP
may not need the basic region for efficient
heterodimer formation and, therefore, might function more effectively
as inhibitors. However, it is unclear whether a leucine zipper that
heterodimerizes with C/EBP
more stably than the F zipper could be
constructed.
Similar DN molecules with specificity for the Jun/Fos family have been reported. These inhibitors are fusion proteins produced by joining the v-Jun or c-Jun leucine zippers to the bacterial lexA gene (9, 44) . In both cases, the LexA:Jun hybrids efficiently inhibited transactivation by Fos and Jun. Proteins that form a heteromultimer complex which cannot bind DNA has been identified for bHLH (basic helix-loop-helix) proteins. In the bHLH family, the DN proteins Id and extramacrochaetae can multimerize with other bHLH factors, but, because they lack a functional DNA-binding domain, repress the DNA binding activities of their multimerization partners(45, 46, 47, 48) .
GBF-F
is a potent DN to C/EBP. The most successful DN design was achieved by
altering the DNA binding specificity of the DN:C/EBP heterodimer. The
DN thus configured, GBF-F, inactivated sequence-specific DNA binding by
C/EBP in vitro as well as transactivation by C/EBP
and related family members in HepG2 cells. GBF-F is a chimera, both
parts of which are critical for optimal DN properties: one component is
the F leucine zipper and the second is the DNA-binding region from the
plant bZIP protein, GBF-1(21) . The bipartite bZIP helix
permits the exchange of basic regions and, therefore, DNA-binding
specificity without changing dimerization specificity(37) .
Alone, each component of the GBF-F chimera has modest DN potential (Fig. 3A); in combination, these components exhibit
enhanced inhibitory properties (Fig. 3, A and B). Furthermore, the DN activity of GBF-F is specific to bZIP
proteins of the C/EBP family, as demonstrated by using C/EBP
derivatives containing a heterologous leucine zipper (Fig. 5).
An alternative DN could be generated by extending the leucine zipper
in a manner that engenders additional dimerization stability and,
simultaneously, prevents the target protein from binding to DNA. One
approach, which is currently under development(49) , is the
addition of a DNA mimetic in the form of an acidic amphipathic
-helical extension to the NH
terminus of the F leucine
zipper. Such an extension would be predicted to form a heterodimeric
coiled-coil interaction with the bZIP basic region helix, thereby
stabilizing the dimeric complex and hindering basic region interactions
with DNA.
When fused to an activation domain, GBF-F cooperates with
C/EBP to transactivate a chimeric DNA site, showing that this DN
protein can also exhibit gain-of-function properties (Fig. 6).
We show by both biochemical and biological experiments that GBF-F
heterodimerizes with C/EBP
and binds a chimeric site, consisting
of C/EBP and GBF recognition motif half-sites, in preference to a
normal C/EBP site. C/EBP
:GBF-F heterodimers preferentially bound a
C/EBP
GBF site in vitro, while in transfected cells, a
reporter gene containing the C/EBP
GBF chimeric binding site was
activated only by the combination of C/EBP
and a GBF-F derivative
bearing an activation domain. Although the C/EBP
GBF sequence is
predicted to be the optimal binding site for C/EBP:GBF-F heterodimers,
binding site selection experiments currently in progress should reveal
whether this is the case.
In vivo, the relative levels of
C/EBP proteins and GBF-F should specify the dominant-negative or
gain-of-function effects of GBF-F. If GBF-F is expressed at lower
levels than endogenous C/EBP, both C/EBP homodimers and C/EBP:GBF-F
heterodimers should exist in the cell. Under these conditions,
gain-of-function properties should predominate over DN effects. If
GBF-F is expressed at greater levels than C/EBP, we expect to observe
the activation of chimeric binding sites as well as more complete
inhibition of C/EBP homodimer activity. A natural example of a
GBF-F-like molecule is CHOP (gadd153), a putative dominant-negative
molecule of the C/EBP family(12, 50) . CHOP10 inhibits
C/EBP proteins from binding to canonical C/EBP sites, but also
recognizes a novel sequence as a heterodimer with C/EBP. ()However, the significance and specificity of DNA binding
by CHOP10:C/EBP heterodimers is not yet clear. CHOP10 could potentially
mediate the activation of a distinct set of genes through its binding
with C/EBP family members. In light of its potential to activate
natural target genes, CHOP10 may not be an appropriate DN for
inhibiting C/EBP function in vivo. GBF-F, on the other hand,
confers a novel, presumably non-physiological, DNA-binding specificity
to C/EBP and, therefore, is less likely to cause the activation of
endogenous genes when expressed in mammalian cells.
C/EBP DN
proteins such as GBF-F should be useful for inhibition of C/EBP
activity in vivo. For example, two groups have reported the
targeted disruption of the c/ebp gene (51, 52) and have shown that this mutation has only
minor effects on the expression of inflammatory cytokine genes. These
results are surprising in light of previous data showing that
C/EBP
plays a critical role in the expression of genes such as
interleukin-1, interleukin-6, and monocyte chemoattractant protein-1 in
cell lines(26, 53, 54, 55) , and
suggest that a redundant C/EBP activity compensates for the absence of
C/EBP
in knockout animals. To demonstrate that the compensatory
activity is provided by another member of the C/EBP family, one could
create DN transgenic mice that express the C/EBP inhibitor specifically
in hepatocytes or monocytic cells. Assuming that these animals are
viable, they could be used to examine the requirement for a C/EBP
factor in the activation of acute-phase responsive genes or the
induction of inflammatory cytokines, respectively.
Numerous
experiments indicate that the expression or activation of C/EBP
proteins is coincident with terminal differentiation of a particular
cell lineage. For instance, C/EBP synthesis is induced during the
conversion of HL60 cells or M1 myeloblasts into mature
macrophages(56, 57) . Similarly, C/EBP
is
strongly up-regulated when 32D Cl3 pregranulocytic cells differentiate
into granulocytes in response to treatment with granulocyte
colony-stimulating factor(57) . In these and other cases, it
has not been established whether C/EBP proteins play an essential role
in terminal differentiation. Overexpression of C/EBP DNs in the
appropriate cell types should provide a straightforward means of
determining the requirement for C/EBP-related activator proteins in
cell maturation.
The gain-of-function activity observed for the
GBF-F protein containing an activation domain suggests the possible use
of this molecule in regulating experimental transgenes. When expressed
in submolar levels relative to endogenous C/EBP proteins, the GBF-F
construct could be used as a tool to activate transfected genes whose
transcription is regulated by the C/EBPGBF-F cis-element. Since the activation of these chimeric sites
would be dependent on the presence of C/EBP proteins, this approach
provides a strategy to control transgene expression by inducing the
synthesis or activity of an endogenous C/EBP protein. For example,
several mediators of the inflammatory response have been shown to
stimulate C/EBP
-dependent
transactivation(53, 58) . If the same activating
mechanism applies to C/EBP
:GBF-F heterodimers, stimulation of
C/EBP
by these cytokines in cells expressing low levels of GBF-F
should induce transcription from promoters containing the
C/EBP
GBF site without significantly disrupting normal gene
expression in the cell.