A Short Conserved Motif Is Required for Repressor Domain Function
in the Myeloid-specific Transcription Factor CCAAT/Enhancer-binding
Protein
*
Nicholas D.
Angerer,
Yang
Du,
Demet
Nalbant, and
Simon C.
Williams
§
From the Department of Cell Biology and Biochemistry, Texas Tech
University Health Sciences Center and
Southwest
Cancer Center at University Medical Center, Lubbock, Texas 79430
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ABSTRACT |
CCAAT/enhancer-binding protein
(C/EBP
) is
expressed almost exclusively in the myeloid lineage of the
hematopoietic system and functions during terminal differentiation of
neutrophils and macrophages, and in the regulation of cytokine gene
expression in macrophages and T cells. We have undertaken a series of
structure/function studies on the murine C/EBP
polypeptide to
investigate the mechanism by which C/EBP
activates transcription.
Studies with deletion mutants and fusion proteins consisting of
C/EBP
sequences joined to the Gal4 DNA-binding protein identified
two transcriptional activation domains in C/EBP
. Removal of
sequences between the two activation domains or sequences between the
second activation domain and the C-terminal DNA binding domain
significantly increased the activity of C/EBP
, suggesting the
presence of two separate regulatory domains (designated RD-1
and
RD-2
). RD-1
behaved as a classic active repressor domain being
capable of inhibiting adjacent activation domains irrespective of their
origin and when linked to a heterologous DNA binding domain.
Mutagenesis studies revealed a short motif in RD-1
that appears to
be a target site for protein-protein interactions and is conserved in
repressor domains from C/EBP
, Sp3, c-Fos, and FosB. The
juxtaposition of activation and repressor domains may permit C/EBP
to function as a transcriptional activator or repressor at different
stages of myeloid differentiation or as an inducible transcriptional activator of cytokine genes.
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INTRODUCTION |
Transcription factors play important roles in the commitment of
precursor cells to particular hematopoietic lineages and the terminal
differentiation of specific cell types (1). The CCAAT/enhancer-binding protein (C/EBP)1 family of
basic region/leucine zipper transcription factors consists of six
members that display similar DNA binding specificities and are capable
of homo- and heterodimerization in all combinations (reviewed in Ref.
2). Four family members, C/EBP
, C/EBP
, C/EBP
, and C/EBP
,
are expressed in myeloid cells and appear to play different roles in
differentiating and mature cells (3, 4). C/EBP
appears to be
critical for granulocytic differentiation (5), whereas C/EBP
and
C/EBP
appear to function primarily as regulators of cytokine gene
expression during inflammatory responses in macrophages (6-8).
C/EBP
was first identified based on its relatedness to C/EBP
(9)
and is almost exclusively expressed in myeloid cells, with additional
sites of expression in lymphoid cells and ovary (10-12). C/EBP
expression is essentially undetectable in uncommitted hematopoietic
progenitors but increases during myeloid differentiation resulting in
high level expression in mature neutrophils and macrophages (12-14).
Mice lacking C/EBP
display defects in granulocytic development (15),
a phenotype that is also seen in C/EBP
-deficient mice (5). C/EBP
appears to act later in granulopoiesis than C/EBP
, and its
expression may be activated by C/EBP
in this lineage (16, 17).
Although the primary defects observed in C/EBP
-deficient mice were
in the development of the granulocyte lineage, there is evidence that
C/EBP
also functions in other hematopoietic cell types. C/EBP
mRNA is present in primary murine macrophages and in multiple
immortalized monocytic and macrophage cell lines, and a 34,000 molecular weight C/EBP
polypeptide has been detected in the IC-21
P388D1(IL-1) cell line (12). Ectopic expression of C/EBP
in a
lymphoid cell line activated the expression of a number of genes
normally expressed in macrophages, including lipopolysaccharide-regulated cytokine genes and the macrophage-CSFR (M-CSFR) gene (12). The lack of defects in monocytic development and
function in C/EBP
-deficient mice may be due to functional redundancy
among C/EBP family members expressed in this cell lineage (7). C/EBP
mRNA was also detected in the human Jurkat T cell line (10), and
the expression of some genes normally expressed in T cells, including
interleukin-2 and -4, was diminished or abolished in C/EBP
-deficient
mice (15).
The C/EBP
gene contains at least one intron and alternative splice
sites and promoters permit the production of multiple polypeptides in
humans (14). We have detected a single C/EBP
mRNA in rodent
cells that may direct the synthesis of two forms of C/EBP
due to the
presence of at least two in frame translation initiation codons (12).
We have carried out a structure/function analysis of the murine
C/EBP
polypeptide to identify domains responsible for the
transcriptional activity of C/EBP
. We have identified two activation
domains in C/EBP
, one at the N terminus and a second close to the
center of the protein. In addition, two regions within the C/EBP
polypeptide act as repressor domains and are likely to be sites of
protein-protein interactions that modulate the activity of
C/EBP
.
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EXPERIMENTAL PROCEDURES |
Plasmid Construction--
The murine C/EBP
mRNA contains
two in frame initiation codons separated by 96 base pairs (12). PCR was
used to introduce NcoI sites at the first of these two sites
and a HindIII site following the termination codon, and the
resultant cDNA was inserted into the pMEX eukaryotic expression
vector (9). N-terminal and internal deletion constructs were prepared
by PCR in a manner similar to that previously described for C/EBP
(18). Briefly, PCR was used to introduce either BglII or
BamHI restriction sites at specific positions within the
C/EBP
coding sequence, and the resultant fragments were combined to
generate the required recombinant. The sequences of the
oligonucleotides used in this study are shown in
Table I. Each was designed to insert
restriction sites at or just following a proline residue and each
carried a G-rich sequence at the 5' end to enhance enzyme
digestion.
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Table I
Oligonucleotides used in the synthesis of C/EBP segments
Oligonucleotide primers were synthesized with a G/C-rich clamp sequence
preceding the restriction site to be inserted. Each 5'-oligonucleotide
contained an internal BglII site. Each 3'-oligonucleotide
contains an internal BamHI site, and the amino acid
specified at the beginning of each 5'-oligonucleotide or end of each
3'-oligonucleotide is indicated.
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Gal4 fusion genes were constructed in the Gal0 plasmid, which is
similar to pSG424 (19). This plasmid encodes amino acids 1-147 of the
yeast Gal4 transcriptional activator located downstream of the SV40
early promoter. All C/EBP
- and VP16-derived segments were inserted
downstream of the Gal4 sequences as NcoI/ClaI
fragments as described previously (18). The VP16 activation domain
fragment used here encodes amino acids 429-456 (20). Gal4
C/EBP
-(1-108), Gal4 C/EBP
-(1-83), and Gal4 C/EBP
-(33-64)
(previously named Gal4 CRP1-1-32)) encode the indicated amino acids
from each C/EBP protein and have been described previously (18). The
C/EBP-dependent (DEI)4-35AlbLUC (which consists
of four copies of the DEI element from the serum albumin gene linked to
the minimal promoter region of the same gene) and
Gal4-dependent G5E1bLUC (which consists of five
copies of a Gal4-binding site upstream of the E1b minimal promoter)
luciferase reporter plasmids were described previously (18).
Cell Culture and Transfections--
The human HepG2
hepatocarcinoma and monkey COS-1 kidney cell lines were cultured in
Dulbecco's modified Eagle's medium (Mediatech, Herndon, VI)
supplemented with 10% fetal bovine serum (HyClone, Logan, UT). HepG2
cells were transfected at approximately 30-40% confluency in 3.5-cm
dishes using a standard calcium phosphate co-precipitation method (18).
In a typical experiment 2 µg of reporter and 2.5-5 µg of effector
plasmid of pMEX-based constructs or 2 µg of reporter and 0.6 µg of
effector plasmid for Gal4-based constructs were used. DNA
concentrations were equalized using pBluescript (Stratagene). Although
HepG2 cells do not express their endogenous C/EBP
gene, we have
obtained similar transactivation results in multiple cell lines,
including some that do express C/EBP
.2 Consequently,
HepG2 cells were used in these studies as they are relatively easy to
transfect and yield consistent results. However, we were unable to
detect Gal4 proteins in transfected HepG2 cells and used COS-1 cells
instead for this purpose. COS-1 cells were transfected by
electroporation as
described.3 Statistical
analyses were carried out using a two-tailed Student's t
test, and results that were statistically significant at 95% or 99%
confidence levels are indicated in the figures. Luciferase assays were
carried out as described (12).
Site-directed Mutagenesis--
Point mutations were introduced
into the C/EBP
coding sequence using the Gene Editor kit (Promega)
according to the manufacturer's instructions. Mutagenic
oligonucleotides were synthesized carrying 15 base pairs on either side
of the mutation site, and a restriction site was generally included to
aid in analysis. In all mutagenic reactions the target residue was
changed to an alanine, and introduction of mutations was verified by
sequencing. The names and sequence of oligonucleotides utilized were as
follows (base changes are indicated in lowercase): A82/4 changes
Pro82, Ala83, and Asp84 to
(Ala)3, 5'-AAATGGCCGAGGcgCtGCaGcCAGGTAGTGAGG-3';
A121/3 changes Lys121, Glu122, and
Glu123 to (Ala)3,
5'-GCTGTGGCGGTGgctGcaGcGCCTCGAGGGCCA-3'; A121/2 changes Glu122 and Glu123 to (Ala)2,
5'-GTGGCGGTGAAGGccGcGCCTCGAGGGCCA-3'; and Ala123 changes
Glu123 to Ala, 5'-GCGGTGAAGGAGGcGCCTCGAGGGCCA-3'. The
A121/2 mutation which changes Lys121 and Glu122
to (Ala)2 was generated by repairing the third mutated
position in A121/3 by PCR using the Glu128 oligonucleotide
shown in Table I.
Nuclear Extract Preparation and Western Blotting--
For
transfected HepG2 cells we found that most nuclear proteins were
present in the pellet after centrifugation of the extracts prepared for
luciferase reactions. These were dissolved in Laemmli sample buffer,
and equivalent aliquots of each reaction were separated by 12%
SDS-polyacrylamide gel electrophoresis and transferred to a
nitrocellulose membrane (Micron Separations, Westborough, MA). Nuclear
extracts were prepared from COS-1 cells as described previously (8).
Immune detection was carried out as described previously (22) using
either a C/EBP
-specific rabbit polyclonal antiserum (C-22, Santa
Cruz Biotechnology, Santa Cruz, CA) or a mouse monoclonal antiserum
directed against the DNA binding domain of Gal4 (RK5C1, Santa Cruz
Biotechnology). Immune complexes were detected using the Supersignal
chemiluminescence detection kit (Pierce).
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RESULTS |
The C/EBP
Protein Contains Two Regions That Function as
Transcriptional Activation Domains--
To identify functional domains
that mediate transcriptional activation by C/EBP
, we compared the
ability of wild type C/EBP
and two proteins lacking N-terminal
segments to activate the (DEI)4-35AlbLUC reporter construct
in HepG2 human hepatocarcinoma cells (Fig. 1A). This construct was
activated 45-fold by full-length C/EBP
; however, deletion of either
96 or 139 N-terminal amino acids decreased transcriptional activity to
background levels. Thus, C/EBP
is capable of functioning as a potent
transcriptional activator, and the N-terminal portion of C/EBP
contains most or all of the activation sequences. The N-terminal
sequences of C/EBP
, C/EBP
, C/EBP
, and C/EBP
possess three
segments of significant sequence similarity that correspond to three
activation domain modules (ADM) first identified in C/EBP
(18) (Fig.
1B). We previously demonstrated that amino acids 33-64 of
C/EBP
(which contain ADM2 and -3) functions as a relatively weak
activation domain when fused to the DNA binding domain of the yeast
transcriptional activator protein Gal4 (18). Therefore, we tested
whether inclusion of the first 32 amino acids of C/EBP
, which
contain sequences similar to the ADM1 domain of C/EBP
, would
increase the potency of the N-terminal C/EBP
activation domain
(ADI). The addition of amino acids 1-32 resulted in a slight but
significant (p < 0.01) increase in C/EBP
AD
activity (compare Gal4
-(33-64) to Gal4
-(1-64)), indicating
that the ADM1 region is likely to be a functional component of ADI of
C/EBP
(Fig. 1C). In comparison, the equivalent regions of
the C/EBP
(amino acids 1-108) and C/EBP
-(1-83) proteins were 4.5- and 7.5-fold, respectively, more powerful in this assay. The
C/EBP
polypeptide also shares a second region of homology (amino
acids 140-162) with C/EBP
and C/EBP
which includes sequences identified as a second activation domain in C/EBP
(23-25). This region of C/EBP
functioned as a weak activation domain (referred to
as ADII) when fused to the Gal4 DBD (Fig. 1C).

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Fig. 1.
C/EBP contains two
transcriptional activation domains. A, full-length
C/EBP -(1-281) and two N-terminal deletion mutants initiating at
amino acids 97 and 141 were co-transfected into HepG2 cells with the
(DEI)4-35AlbLUC reporter plasmids. Luciferase activities
were measured and are represented graphically as fold activation
(±S.E.) compared with the reporter plasmid alone. The structure of the
C/EBP proteins is diagrammed, and shaded
regions represent sequence similarity to two activation domains
(ADI and ADII) previously identified in C/EBP
and C/EBP . B, the N-terminal sequences of C/EBP ,
C/EBP , C/EBP , and C/EBP were aligned to identify regions of
similarity. Amino acid coordinates are shown at the ends of each
sequence, and identical amino acids are represented with vertical
bars and conservative substitutions with colons. Three
distinct regions of similarity first identified in C/EBP (named
activation domain modules (ADM) 1, 2, and 3) are indicated.
C, Gal4 fusion proteins containing the indicated C/EBP
coding sequences were tested for their ability to activate a
Gal4-dependent reporter construct in HepG2 cells. Gal4
proteins containing ADI regions from C/EBP and C/EBP were
included for comparison and ADM1, -2, and -3 regions are
shaded in each. The activity of each protein is shown as
fold activation compared with the reporter plasmid alone.
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C/EBP
Contains Two Internal Regulatory Domains--
We next
tested whether the C/EBP
polypeptide contains additional sequences
outside the activation domains that modulate its transcriptional
activity. C/EBP
proteins lacking internal sequences were tested for
their ability to activate (DEI)4-35AlbLUC in HepG2 cells
(Fig. 2A). The removal of
amino acids 65-194 (i.e. all sequences between ADI and the
DBD) resulted in a highly significant (p < 0.01)
6-fold stimulation of transcriptional activity (compare 1-281 and
1-281 (
65-194)). Proteins lacking sequences between ADI and ADII
(1-281(
65-96) and 1-281(
65-139), Fig.
2A) were also more active than the wild type C/EBP
protein in this assay, approximately 4- and 7-fold, respectively,
identifying a region between amino acids 65 and 140 (termed regulatory
domain-1
or RD-1
) that negatively regulates C/EBP
activity.
Removal of amino acids between ADII and the DBD did not significantly
affect C/EBP
activity (1-281(
163-194) Fig.
2A), but combining the deletion of amino acids 163-194 with
the deletion of the RD-1
region resulted in further stimulation of
C/EBP
activity. For example, a protein lacking amino acids 65-139
and 163-194 displayed approximately 1.5-fold greater activity than a
protein lacking amino acids 65-139 alone (Fig. 2A), and
similar results were observed with other constructs lacking sequences
in both regions (data not shown). We conclude that a second negatively
acting domain, named RD-2
, is located between amino acids 163 and
194 which in this context appears to function in combination with
RD-1
. Similar steady-state levels of each recombinant protein were
detected by Western analysis (Fig. 2B) indicating that the
observed effects were not simply due to differences in protein
expression levels or stability.

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Fig. 2.
Identification of two regulatory domains in
C/EBP . A, a series of C/EBP
expression vectors lacking internal sequences were constructed in pMEX
and were tested for their ability to activate
(DEI)4-35AlbLUC in HepG2 cells. The constructs are named
according to the amino acids removed in each case, and their activities
are represented graphically as fold activation compared with the
reporter plasmid alone. Activation and DNA binding domains are labeled
on top, and the approximate location of two regulatory
domains identified by these studies (RD-1 and RD-2 ) are indicated
below the figure. The actual activity value for each
construct shown at the end of each bar represents the average of at
least three independent transfections. Activities that were
significantly different from the wild type protein are indicated at 95 (*) and 99% (**) confidence levels. B, representative
Western blot analysis showing comparable protein levels expressed from
each construct in nuclear extracts of transfected HepG2 cells.
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Fine Mapping of Regulatory Domains Using Gal4 Fusion
Proteins--
To test whether C/EBP
regulatory domains could
function when attached to a heterologous DNA binding domain, we
constructed a series of Gal4-C/EBP
chimeric genes and tested their
ability to activate expression from the Gal4-dependent
G5E1bLUC reporter plasmid in HepG2 cells. Fusion proteins
bearing the first 64, 98, or 116 amino acids of C/EBP
all functioned
as strong transcriptional activators (Fig.
3A). However, a significant
34-fold decrease (compared with Gal4
-(1-64)) in activity was
observed when C/EBP
sequences were extended to amino acid 128, indicating that RD-1
is located N-terminal to this amino acid. A
5-fold increase in activity was observed upon inclusion of sequences up
to amino acid 162, presumably due to the inclusion of ADII, and a
further decrease (3.3-fold) occurred when the C/EBP
sequences were
extended to amino acid 193 to include the RD-2
-containing region.
Finally, we tested whether RD-2
is capable of functioning in the
absence of RD-1
by fusing amino acids 162-193 directly to ADI. The
addition of this region resulted in a 4-fold decrease in activity
(compare Gal4
-(1-64) to Gal4
-(1-64)(162-193) indicating that
RD-2
is capable of repressing ADI function in a position- and
RD-1
-independent fashion in Gal4 fusions. Unfortunately, we were
unable to detect expression of Gal4 fusion proteins in nuclear extracts
of HepG2 cells; however, they were detectable after transient
transfection of COS-1 cells. All Gal4 fusions behaved similarly in
transactivation assays in COS-1 cells2 and were expressed
at similar levels (Fig. 3B) again indicating that the
observed functional differences are not due to variations in protein
levels.

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Fig. 3.
RD-1 and
RD-2 repress AD function independent of DNA
binding. A, various segments of the C/EBP coding
sequence were attached to the DNA binding domain from the yeast
transcriptional activator protein Gal4, and the resultant chimeric
proteins were tested for their ability to activate transcription from
the G5E1bLUC reporter plasmid. The name of each construct
refers to the coordinates of the C/EBP sequences included in each
protein, and the activity of each protein is listed numerically as fold
activation compared with the reporter plasmid alone. A schematic
representation of C/EBP is shown with functional domains labeled.
The repression index was calculated by dividing the activities of
proteins that differ by the presence of RD-1 and/or RD-2 .
Transfections were repeated at least four times for each construct, and
values that were significantly different at the 99% confidence level
are indicated (**). B, Western blot analysis of a subset of
Gal4-C/EBP fusion proteins expressed in COS-1 cells.
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RD-1
and RD-2
Repress a Heterologous Activation
Domain--
To study the activation domain specificity of the two
regulatory domains in C/EBP
, we tested their ability to repress the activity of Gal4 fusion proteins containing the potent activation domain from the herpes simplex virus VP16 protein (Fig.
4A). Initially we tested
whether C/EBP
sequences could repress transactivation when placed
downstream of the VP16AD, i.e. in a similar position relative to the activation domain as in C/EBP
. The activity of the
Gal4VP16 protein on the G5E1bLUC reporter plasmid in HepG2 cells was set at 100%, and the activity of each fusion protein was
calculated relative to this value. The inclusion of amino acids 64-98
or 64-116 did not diminish the activity of the VP16AD. However, a
protein containing amino acids 64-128 displayed only 16% of the
activity of the control, indicating that RD-1
is capable of
repressing the activity of the VP16AD. To map the N-terminal boundary
of RD-1
, three additional constructs containing C/EBP
sequences
initiating at amino acid 97 were tested. Fusion proteins carrying amino
acids 97-162 or 97-193 were 55 and 66% as active as Gal4VP16AD;
however, the attachment of amino acids 97-128 repressed VP16AD
activity to 4% of control levels. These data further refine the
position of RD-1
between amino acids 97 and 128, and the higher
activity of constructs containing amino acids 97-162 and 97-193 is
likely due to the presence of ADII. In the final construct in this
series, RD-2
(amino acids 162-193) was attached to the C terminus
of the VP16AD. This protein displayed only 27% (p < 0.01) of the activity of the control protein, indicating that RD-2
was also capable of inhibiting a heterologous AD. Western blotting
confirmed that all proteins were expressed at comparable levels (Fig.
4B, for example).

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Fig. 4.
RD-1 and
RD-2 are capable of repressing activity of a
heterologous activation domain. A, a series of Gal4
fusion proteins were constructed to test whether the regulatory domains
from C/EBP could inhibit the activity of the heterologous activation
domain from the viral transactivator protein VP16, which is represented
by the hatched box. Each fusion was generated in two forms
that differed in the relative orientation of the VP16 and C/EBP
sequences, and the C/EBP amino acids are indicated in
parentheses in the construct names. Each protein was tested
for its ability to activate G5E1bLUC in HepG2 cells, and
activities are listed relative to the activity of Gal4-VP16, which was
set at 100%. The activities represent the average results of at least
three independent transfections. Protein activities that are
significantly lower than that of Gal4VP16AD at the 95 (*) or 99% (**)
confidence levels are indicated. These data demonstrate that the
minimal RD-1 sequence is located between amino acids 97 and 128 (indicated with a hatched box), and RD-2 is located
between amino acids 162 and 193 (indicated with a stippled
box). B, representative Western blot showing comparable
accumulated levels of proteins with significantly different
activities.
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A second series of chimeric genes was constructed in which the order of
the C/EBP
and VP16 sequences was reversed to test for
position-dependent effects. Although some differences in
the repressive activity of the C/EBP
regulatory domains were
observed in the two sets of proteins, the overall pattern of activity
was similar (Fig. 4A). The minimal RD-1
and RD-2
segments (amino acids 97-128 and 162-193, respectively) both
repressed VP-16AD function, albeit to a slightly lesser but
statistically significant (p < 0.01) extent than in
the previous set of chimeras. In this second arrangement, RD-2
appeared to be an efficient repressor module even in the presence of
ADII of C/EBP
(compare Gal4
-(97-162)-V to Gal4
-(97-193)-V)), which could potentially be explained by its close
proximity to the VP16AD in these proteins.
RD-1
Contains Sequence Motifs That Are Conserved in Other
Repressor Domains--
We next compared the sequence of amino acids
64-128 of C/EBP
to the sequence of repressor domains identified in
other transcription factors. We identified two small conserved motifs
(termed conserved motif (CM) 1 and 2 in Fig.
5) in a subset of factors examined. CM1
is located between amino acids 82 and 86 of C/EBP
and is conserved
in C/EBP
, rat and human C/EBP
, and Sp3 but is absent in chicken
C/EBP
, c-Fos, and FosB. A consensus sequence was derived for CM1 as
Pro-Ala-Asp-X-B, where X is any amino acid and
B is a basic amino acid. CM1 lies outside the region defined
as RD-1
and would not be predicted to be critical for RD-1
function. CM2 is located between amino acids 121 and 128 of C/EBP
(i.e. within RD-1
), and related sequences were present in
all proteins listed. CM2 has the consensus sequence
B-Glu-Glu-X-X-X-Pro-Glu and is most highly
conserved in C/EBP
, C/EBP
(human and chicken), Sp3, and FosB.

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Fig. 5.
C/EBP shares two
conserved motifs with regulatory domains from other transcription
factors. The sequence of regulatory domains from rat C/EBP
(this study), rat C/EBP (RD1 (18)), human C/EBP (46), chicken
C/EBP (21), Sp3 (31), Fos and FosB (ID1 (32)) were aligned to
identify conserved amino acids. Conserved residues in two motifs, named
CM1 and CM2, are shown in reverse print. The
amino acid coordinates are shown for each sequence, and the consensus
sequence for each motif is shown below the alignment.
B, basic amino acid (Lys or Arg); X, any amino
acid.
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We next tested whether mutating residues in CM1 and/or CM2 would
diminish RD-1
function. The mutations focused on the conserved PAD
and KEE motifs in CM1 and CM2, respectively (Fig. 5). Both motifs were
replaced with alanines in one or both of the Gal4
-(1-128) and Gal4
-(64-128)-V fusion proteins (see Figs. 3 and 4). Mutation of the
CM1 sequence had no effect on the activity of Gal4
-(1-128) as
proteins thus configured displayed repressed activities similar to the
wild type protein (A82/4 mutant in Fig. 6A). However, mutating CM2 to
three alanine residues (A121/3 mutants) resulted in significant
increases in the activity of both proteins, 12-fold in Gal4
-(1-128) and 29-fold in Gal4
-(64-128)-V (Fig. 6, A
and B). Mutating pairs of amino acids (A121/2 and A122/3) or
the terminal glutamic acid residue alone (A123) all significantly reduced the activity of the regulatory domain in Gal4
-(1-128) and,
where tested, Gal4
-(64-128)-V indicating that residues 121 and 123 within CM2 are critical for RD-1
function. The contribution of the
glutamic acid at position 122 remains to be verified. Equivalent levels
of each protein were detected by Western blotting (Fig. 6, C
and D).

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Fig. 6.
CM2 is required for RD-1
function. Conserved residues in CM1 and CM2 were changed to
alanine in the Gal4 -(1-128) (A) and Gal4
-(64-128)-V (B) constructs. Each protein was tested for
its ability to activate the G5E1bLUC reporter plasmid in
HepG2 cells. The activities of proteins carrying the indicated
mutations are shown relative to each wild type construct and represent
the average of three independent transfections. Statistical analysis is
represented as in Fig. 2. NT, not tested. C and
D, representative Western blot of recombinant
proteins.
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RD-1
Appears to be a Site for Protein-Protein
Interactions--
Finally, we tested whether co-expression of RD-1
might result in derepression of a Gal4-C/EBP
fusion protein by
competing for binding of a nuclear protein. Increasing amounts of the
C/EBP
-(97-281) expression vector were co-transfected with Gal4
-(1-128) into HepG2 cells, and luciferase activities were measured
(Fig. 7). At the highest level of
C/EBP
-(97-281) expression vector the activity of Gal4
-(1-128)
was 3-fold higher than in the absence of any competitor molecule. To
test whether this effect was attributable to the co-expression of a
molecule containing a functional RD-1
element, a second series of
transfections were carried out using a competitor molecule carrying the
A121/3 mutation, which changes the KEE motif to three alanine residues
and abolishes RD-1
activity (see Fig. 6, A and
B). In this case relief of inhibition was not observed, in
fact luciferase activities decreased as the amount of competitor was
increased which we interpret to be the result of competition between
the promoters in the two expression vectors. Both proteins were
efficiently synthesized as measured by Western analysis (Fig.
7B). Taken together, the results of these assays indicate
that a co-expressed RD-1
-containing protein is able to relieve
inhibition in Gal4
-(1-128) presumably by competing for a cellular
protein that normally binds to RD-1
and mediates the repressive
effect of this domain.

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Fig. 7.
Co-expression of
RD-1 -containing C/EBP
deletion mutant relieves RD-1 -mediated
repression of a Gal4 fusion protein. A, HepG2 cells
were transfected with G5E1bLUC and an expression vector
encoding Gal4 -(1-128) in the absence or presence of increasing
amounts of N-terminal C/EBP deletion mutants encoding amino acids
97-281. Two competitors were used, a wild type protein carrying the
KEE motif at amino acids 121-123 (black bars) and a mutant
(A121/3) bearing three alanines in place of this motif (white
bar). Luciferase activities were measured and are expressed
relative to the activity of Gal4 -(1-128) alone which was set at
1.0. The results represent the averaged values (± S.E.) of a
representative experiment performed in triplicate. B,
Western blot analysis of nuclear proteins from transfected cells,
probed with an anti-C/EBP antiserum. The position of the two
competitor proteins is indicated.
|
|
 |
DISCUSSION |
The individual members of the C/EBP family possess a
highly conserved bZIP DNA binding domain and are consequently capable of binding to identical recognition sequences in the control regions of
target genes (9, 26). Therefore, the unique functions of each protein
are likely to be controlled by domains outside the DNA contacting basic
region, and numerous structure/function studies have been carried out
to identify these domains. In the case of C/EBP
, we have previously
identified domains that control DNA binding, dimerization (9), nuclear
localization (27), and transcriptional activation (12). To understand
more clearly the activity of C/EBP
is regulated we have now
identified additional domains that control the activity of C/EBP
. In
addition to defining sequences that function as transcriptional
activation domains, we also identified two separate regulatory domains,
RD-1
and RD-2
, that repress C/EBP
activity. RD-1
is located
between the two transcriptional activation domains and is wholly
contained in the region spanning amino acids 97-128. RD-2
is
located N-terminal to the DNA binding domain, and its activity in the
context of C/EBP
was only evident when RD-1
was also absent.
However, RD-2
was capable of independently inhibiting a linked
activation domain in Gal4 fusion proteins. Over the past several years,
numerous nuclear proteins have been identified that contain domains
that negatively regulate transcription (reviewed in Ref. 28). Two criteria have been used to define these domains as active repressor domains as follows: the ability to function in a modular fashion when
attached to a heterologous DNA binding domain, and second to act as the
site for interactions with corepressor proteins (29). By these
criteria, RD-1
behaves as an active repression domain as it
inhibited the activity of C/EBP
and VP16 activation domains when
attached to the Gal4 DBD. In addition, our competition data indicate
that RD-1
is a site for protein-protein interactions. RD-2
was
also capable of inhibiting a linked activation domain when attached to
a heterologous DNA binding domain thereby satisfying one criterion for
characterization as an active repression domain.
Specific motifs within repressor domains, often comprised of just a few
amino acids, have been identified that are critical for repressor
domain function. For example, the repressor domains of the
Drosophila proteins Hairy and Enhancer of Split contain the
four amino acid motif, Trp-Arg-Pro-Trp (WRPW), which mediates interactions with members of the Groucho family of corepressors (30).
Our mutation studies identified the sequence Lys-Glu-Glu (KEE) in
RD-1
as being required for repressor domain function and for
protein-protein interactions. This same motif is required for activity
of the repressor domain of Sp3 (31), and similar sequences are present
in RD-1 of C/EBP
(18) and the inhibitory domain (ID-1) of c-Fos and
FosB (32). The corresponding amino acids have not been mutated in
C/EBP
, c-Fos, or FosB; however, the repressor domain of c-Fos has
been shown to be a site for protein-protein interactions (32).
Therefore, KEE or a similar motif may be a signature for repressor
domains from proteins derived from diverse transcription factor
families. The KEE motif is not present in RD-2
; however, as shown in
Fig. 8, RD-2
contains multiple copies
of the consensus minimal recognition sequence for members of the MAP
kinase family, the dipeptide (Ser/Thr)-Pro (33). Therefore, it is
conceivable that phosphorylation of the RD-2
element by a member of
the MAP kinase family might regulate the activity of C/EBP
. Little
is currently known concerning the phosphorylation of C/EBP
; however,
support for this hypothesis comes from studies of other C/EBP family
members, particularly C/EBP
. Comparison of this region of RD-2
to
the corresponding regions of C/EBP
, C/EBP
, and C/EBP
(i.e. the region immediately N-terminal to the basic region)
reveals multiple putative MAP kinase sites in each protein (Fig. 8).
Phosphorylation of the threonine residue at position 238 of human
C/EBP
(amino acid 168 of rat C/EBP
) via a
Ras-dependent MAP kinase pathway in NIH3T3 and P19 cells
has been shown to stimulate C/EBP
activity (34). In addition,
mutation of this residue to a non-phosphorylatable residue decreased
C/EBP
activity in P19 and neuronal cell lines (34, 35). Mutagenesis
studies should begin to define the importance of this region for
RD-2
and C/EBP
activity.

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Fig. 8.
Putative MAP kinase sites in
RD-2 are conserved in
C/EBP , C/EBP , and
C/EBP . Amino acids 174-194 from RD-2
were aligned with the corresponding region of three other C/EBPs in
order to demonstrate the presence of multiple copies of the dipeptide
(Ser/Thr)-Pro (underlined) in each protein. The threonine
residue at position 168 of C/EBP can be phosphorylated by MAP kinase
(34), and it is possible that each C/EBP protein could be targeted by
members of this kinase superfamily.
|
|
Two previous studies proposed that the activity of C/EBP
was
inhibited by intermolecular interactions between the repressor and
activation domains, which served to block access of the activation domain to its target proteins (18, 36). However, the data presented
here suggest that the RD-1
, and probably the RD-2
, repressor
domains of C/EBP
act as sites for interactions with accessory
proteins that act as corepressors. Multiple proteins have been
identified that physically interact with members of the C/EBP family,
although most of these proteins interact via the bZIP domain. These
proteins are generally transcriptional activators that bind to adjacent
sites on promoters and synergize with C/EBP proteins in activating
transcription of the target gene and include NF
B (37, 38), AML1, and
PU.1 (39), glucocorticoid receptor (40), Fos and Jun (41), Nopp140
(42), and Sp1 (43). In addition, a limited number of proteins have been
identified that interact with regions outside the DNA binding domain of
C/EBP proteins, including Sp1 (43) and the retinoblastoma protein (44,
45); however, it is unlikely that any of these proteins interact with
RD-1
. The identification of the KEE motif as a critical component of
RD-1
should facilitate the identification of its cognate binding
proteins and provide clues as to its function in C/EBP
.
Two alternative scenarios might explain the role of these repressor
domains in determining the biological function of C/EBP
. First,
interaction of corepressors with the repressor domains may serve to
maintain C/EBP
in an inactive state until an activating signal is
received. This model would explain the ability of C/EBP
to
participate in the inducible activation of cytokine genes upon lipopolysaccharide stimulation of macrophages and other cell types (12). Further studies will determine whether phosphorylation of
potential MAP kinase sites in RD-2
are involved in regulating C/EBP
activity. Alternatively, it is becoming apparent that
transcriptional repression may be just as important as activation in
regulating cell-specific gene expression during development. Therefore,
C/EBP
could play dual regulatory roles as a transcriptional
activator and repressor, depending on its association with corepressors or coactivators. This model would explain the apparent ability of
C/EBP
to both positively and negatively regulate expression of the
M-CSFR gene (12, 15). Detailed characterization of the exact functions
of each domain in C/EBP
, and their interacting partners, along with
analysis of cellular distributions of putative corepressor molecules
should aid in determining the activity and specific functions of
C/EBP
.
 |
ACKNOWLEDGEMENTS |
We thank Peter Johnson for advice and Curt
Pfarr, Dan Hardy, Elmus Beale, Charles Faust, and Clinton MacDonald for
critical reading of the manuscript.
 |
FOOTNOTES |
*
This work was supported in part by grants from the National
Office and Texas Affiliate of the American Heart Association, the
Association for Research of Childhood Cancer, Texas Tech University Health Sciences Center, and the South Plains Foundation.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom reprint requests should be addressed: Dept. of Cell Biology
& Biochemistry, Texas Tech University Health Sciences Center, 3601 4th
St., Lubbock, TX 79430. Tel.: 806-743-2524; Fax: 806-743-2990; E-mail:
cbbscw{at}ttuhsc.edu.
The abbreviations used are:
C/EBP, CCAAT/enhancer-binding protein; DBD, DNA binding domain; ADM, activation domain module; AD, activation domain; RD, regulatory domain; M-CSFR, macrophage-colony-stimulating factor receptor; CM, conserved
motif; PCR, polymerase chain reaction; MAP, mitogen-activated protein.
2
N. D. Angerer and S. C. Williams,
unpublished results.
3
A. J. Reinhart, S. C. Williams,
B. J. Clark, and D. M. Stocco, submitted for publication.
 |
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