From the Division of Hematology/Oncology, Department
of Medicine, Cedars-Sinai Medical Center, UCLA School of Medicine, Los
Angeles, California 90048 and the
Division of Pediatric
Oncology, The Johns Hopkins Oncology Center, Johns Hopkins University,
Baltimore, Maryland 21287
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ABSTRACT |
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Human CCAAT/enhancer-binding protein (C/EBP
), a new member of the C/EBP family, significantly
up-regulates both the mim-1 and human myeloperoxidase
promoters, suggesting an important role for C/EBP
in the
transcriptional regulation of a subset of myeloid-specific genes. To
elucidate the structure and function of C/EBP
in transcriptional activation, amino acid residues 1-115, 147-249, or 1-249 of C/EBP
were fused to the yeast GAL4 DNA binding domain. These expression vectors were cotransfected with a chloramphenicol acetyltransferase reporter gene and, in all cell lines tested, only the
GAL-C/EBP
-(1-115) fusion protein significantly activated expression
from the chloramphenicol acetyltransferase reporter gene. Sixteen
deletion mutants of C/EBP
mapped the transactivation domain to amino
acids 1-18 at the N terminus and revealed the presence of a
transcription repression element between amino acid residues 116 and
162. Expression vectors containing the repression domain of C/EBP
strongly inhibited gene transcription from TK, SV40, and adenoviral
major late promoters bearing GAL4 binding sites. Fusion of this
repression domain to the VP16 activation domain inhibited the
transactivation function of VP16. Deletion of this
repression domain increased gene transcription from a neutrophil
elastase promoter-luciferase reporter. Taken together, these data
suggest that C/EBP
regulates transcription by utilizing both
activation and repression functions.
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INTRODUCTION |
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The CCAAT/enhancer-binding protein
(C/EBP)1 family of
transcriptional regulators currently includes six members: C/EBP
(1), C/EBP
(2-7), C/EBP
(8), C/EBP
(3, 9, 10), C/EBP
(11,
12), and C/EBP
(13). This family of transcription factors regulates
several cytokine genes (14), liver-specific genes involved in acute
phase response (7, 15, 16), adipocyte-related genes (17, 18), the
regulatory elements of many viruses (19), and control sequences in the
Ig heavy chain and
loci (20). The C/EBP proteins interact with
other proteins such as the transcription factors c-Myb (21), cAMP
response element-binding protein (22-24), c-Jun (25), glucocorticoid
receptor (26), AML1 (27), NF-
B (28), and the hypophosphorylated form
of the cell cycle regulatory protein retinoblastoma (pRB) (29).
The C/EBP family members share a highly conserved C-terminal basic
amino acid-rich region and a flanking leucine zipper domain that are
essential for DNA binding and dimer formation (30). In contrast, the N
terminus is more diverse. Transcriptional activation domains from
C/EBP (31, 32), C/EBP
(33), and C/EBP
were localized to this
region. However, C/EBP
and C/EBP
lack these N-terminal activation
domains and can function as trans-dominant repressors of
transcription (34, 35). Additional negatively acting regulatory domains
were identified in C/EBP
(32, 36) and C/EBP
(33). These
"attenuator" domains not only regulate transcription but define
cell-specific transcriptional activity. The rat homologue of C/EBP
, CRP1, shares significant homology at its N terminus with other C/EBP
family members (33).
The human C/EBP gene was cloned recently (11, 12). Differential
splicing and the use of alternative promoters generates a total of four
proteins with calculated molecular masses of 32, 30, 27, and 14 kDa
(12, 37). This use of alternative splice sites has also been
demonstrated for the C/EBP
gene (5). Unlike other C/EBP proteins,
C/EBP
is primarily expressed in the myeloid and lymphoid cell
lineages (11, 12, 38, 39). The human C/EBP
protein strongly and
specifically binds to double-stranded DNA containing consensus C/EBP
sites and significantly up-regulates transcription from both the
myeloid-specific c-mim and human myeloperoxidase promoters.
This suggests an important role for C/EBP
in the transcriptional regulation of a subset of myeloid-specific genes that contain these
sites (12).
To understand further the mechanisms of transcriptional regulation of
myeloid-specific genes by C/EBP, we used molecular genetic methods
to perform a systematic structure/function analysis of the C/EBP
protein. The ultimate goal of our experiments was to identify regions
of C/EBP
that contribute to its ability to regulate myeloid gene
expression. In this report, we demonstrate that the transcriptional
activation function of C/EBP
is mediated through a bipartite
activation domain localized in the N terminus. This domain functions as
a transcriptional activator both in the context of the C/EBP
protein
and when fused to the GAL4 DNA binding domain. In addition, we
identified an intrinsic repression domain in C/EBP
that, when
deleted, allowed C/EBP
to enhance transactivation of gene
transcription compared with the wild-type C/EBP
protein. Furthermore, this inhibitory signal conferred its repression function on a heterologous transcriptional activator. We propose a model in
which C/EBP
regulates myeloid-specific gene expression through transcriptional activation and repression that is dependent on its
associated factor(s).
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EXPERIMENTAL PROCEDURES |
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Plasmid Constructions
GAL4-C/EBP Hybrid Constructs--
Cloning vector pGALM (a
gift from Drs. Gregory Kato and Chi Dang) used for all recombinant
plasmids in this study is similar to pSG424 (40) and contains the DNA
binding domain from GAL4 (amino acids 1-147) driven by the SV40 early
promoter and followed by a polylinker containing restriction sites for
subcloning. PCR was used to introduce restriction sites,
EcoRI and SalI, at the beginning and the end,
respectively, of each C/EBP
fragment. The oligonucleotide primers
for introduction of restriction sites consisted of a 5' "clamp"
sequence (6 base pairs), the restriction site, and 18 bases of homology
to the target sequence at the 3'-end. PCR (25 cycles of 1 min at
94 °C, 1.5 min at 55 °C, and 1.5 min at 72 °C) using
Pfu polymerase (Stratagene, La Jolla, CA) was carried out
under standard conditions as recommended by the manufacturer. The
DNA fragments treated with restriction enzymes EcoRI and
SalI were inserted into pGALM. All recombinant
constructs were confirmed by sequencing.
GAL4-VP16-C/EBP Hybrid Constructs--
Restriction sites,
EcoRI/HindIII and
HindIII/SalI, were introduced by PCR into the
VP16 activation domain (AD; amino acids 420-456) (41) and DNA
fragments encoding C/EBP
amino acids 1-45, 1-115, 115-162,
162-199, and 115-199, respectively. After proper treatment with
restriction enzymes, the VP16 AD and the C/EBP
fragments were
inserted into the cloning vector pGALM by a three way ligation and
confirmed by sequencing.
CMV-C/EBP Expression Constructs--
All PCR-derived C/EBP
DNA fragments were digested with the EcoRI/SalI
enzymes before insertion into a CMV expression vector, pCMVSPORT (Life
Technologies, Inc.). A translational initiation codon, ATG, present in
the 5'-primer was introduced into all N-terminal deletions during PCR
reactions. Expression plasmids containing internal deletions of
C/EBP
were made in two ways. (i) For CMV-
116-234, PCR-derived
DNA fragments encoding amino acids 1-70 and 235-249 of C/EBP
were
digested with restriction enzymes SalI and EcoRI and then ligated together after the two fragments were blunted by a
fill-in reaction using Klenow. This produced an in frame fusion of the
two C/EBP
fragments that removed residues 116-234. (ii)
CMV-
71-161, CMV-
116-161, and CMV-
116-184 were
constructed by first synthesizing, by PCR, two targeted DNA fragments
of C/EBP
encoding the corresponding amino acid residues 1-70,
1-115, 162-249, and 185-249, respectively. A HindIII
restriction site, which was included in the primers used in the above
PCR reactions, was introduced at the junction of all internal
deletions. After proper treatment with restriction enzymes, a three way
ligation was performed to insert these DNA fragments into the cloning
vector pGALM.
Reporter Constructs-- Several GAL4-responsive reporter constructs were used in this study: (i) G5E1bCAT, a gift from Drs. Gregory Kato and Chi Dang (42), which contains a chloramphenicol acetyltransferase (CAT) reporter gene regulated by the adenoviral E1b promoter bearing five GAL4 binding sites; (ii) G0TKCAT, G1TKCAT, G5TKCAT, G5SV40CAT, and G5MLPCAT, gifts from Dr. Arnold Berk (43), which contain zero, one, or five GAL4 binding sites upstream of the promoters for herpes simplex virus thymidine kinase (TK), SV40, or adenoviral major late promoter. The neutrophil elastase promoter containing a consensus C/EBP binding site 1 base pair upstream of a Myb binding site (44) was fused to the luciferase gene (NE-Luc) for use as a reporter gene.
Transient Transfections and Reporter Assays
COS-1, CV-1, and 293 cells were maintained in Dulbecco's
modified Eagle's medium containing 10% fetal bovine serum. Cells were
transfected at 50-60% confluency in 60-mm dishes as described (12).
For CAT assays, performed as described (45), a total of 20 µg of DNA
was introduced, consisting of 5 µg of reporter plasmid, 5 µg of
GAL4-C/EBP hybrid constructs, and pUC19 carrier DNA. The plasmid
pCMV-Luc (0.5 µg) was included as a monitor of transfection
efficiency. For luciferase assays, a total of 15 µg of DNA was used,
consisting of 5 µg of NE-Luc reporter plasmid, 5 µg of CMV-C/EBP
constructs, and carrier DNA. The plasmid pCMVSPORT-
-gal (2 µg) was
used as a control of transfection efficiency. Lysates were prepared
48-60 h after DNA was added to the cells.
The HL60 (myeloblast/promyelocytic), U937 (myelomonocytic), and NB4 (promyelocytic) were maintained in RPMI with 20% fetal bovine serum. A total of 50 µg of DNA, including 10 µg each of the reporter and effector plasmids and 5 µg of pCMV-Luc and pUC19 carrier DNA, were introduced into the cells by electroporation as described (12). Cells were harvested 24-48 h after transfection. All effector plasmids were tested at least three times. Levels of transactivation or repression were all compared with that of the reporter plasmid in the absence of any effector plasmid.
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RESULTS |
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The C/EBP Activation Domain Is Localized in the N
Terminus--
We previously demonstrated that C/EBP
can activate
transcription from the myeloid-specific mim-1 and
human myeloperoxidase promoters (12). To map the transcriptional
activation domains as they function in mammalian cells, expression
plasmids were constructed to give the following fusions of the GAL4 DNA
binding domain with C/EBP
: GAL-C/EBP
; GAL-
-(1-115); and
GAL-
-(147-249). Fig. 1A
shows a schematic representation of these constructs. Sequence analysis
revealed that the basic region and the leucine zipper (bZIP) domains
are located between amino acid residues 162 and 235 (11, 12). We used
the adenoviral E1b promoter bearing five GAL4 binding sites that was
linked to the CAT gene as the target promoter.
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Mapping of the C/EBP Regulatory Domains--
To define
regulatory domains within the C/EBP
gene, we constructed two sets of
nested deletions that removed various amino acid residues from either
the N or C terminus of C/EBP
(Fig. 2A). These deletion mutants
were fused to the GAL4 DBD and tested for their abilities to
transactivate the CAT reporter gene in COS-1 cells. The N-terminal
deletions consisted of eight truncations beginning from the first 18 amino acids and extending varying distances toward the C terminus. As
shown in Fig. 2, A and B, all eight hybrid
proteins were completely inactive in transactivation of the CAT
reporter gene. The expression of all the fusion proteins was confirmed
by Western blotting (data not shown).
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Identification of a Repressor Domain in C/EBP--
Our
results suggest that the inability of GAL-C/EBP
to activate the CAT
reporter gene may result from an intrinsic inhibitory domain in the
protein. We observed little transactivation from GAL-
-(1-199),
GAL-
-(1-179), and GAL-
-(1-162) fusion proteins from which the
leucine zipper and basic regions were deleted sequentially (Fig.
2A). Further deletion of amino acids 116-162
(GAL-
-(1-115)), however, produced at least a 10-fold increase in
CAT activity, thus suggesting the presence of a transcriptional
inhibitory element within this segment (residues 116-162) of C/EBP
(Fig. 2A).
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Deletion of the Repression Domain of C/EBP Increases
Transactivational Capability on a Neutrophil Elastase
Promoter--
Our group and others have demonstrated that c-Myb,
together with C/EBP
, C/EBP
, and C/EBP
, synergistically
activates the promoters of a subset of myeloid specific genes including
the neutrophil elastase gene
(44).3 Therefore, we next
investigated the function of the transactivation and repression domains
of C/EBP
in regulating a myeloid-specific gene promoter in the
absence of GAL4DBD. The two full-length forms of C/EBP
(1-249 and
PE32, the latter a gift from Dr. Kleanthis Xanthanopoulos) and various
C- and N-terminal deletions were placed under the control of the CMV
promoter, and four internal deletions were generated that removed the
repression domain and its surrounding residues (Fig.
5A). These constructs, in the
presence and absence of c-Myb, were cotransfected with the reporter
NE-Luc into COS-1 cells, and the activity of the NE promoter was
measured. Both full-length forms of C/EBP
activated the NE promoter
2-fold (Fig. 5B). Each N-terminal deletion mutant failed to
activate transcription from the NE-Luc reporter gene (Fig.
5B). However, two of the internal deletions,
CMV-
71-161 and CMV-
116-161, demonstrated a 2-fold higher
luciferase activity over that seen with the full-length forms. We also
observed that while C/EBP with c-Myb synergistically activated NE-Luc,
deletion of the repression domain further increased transcription
2-fold in the presence of c-Myb (Fig.
5B).3
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DISCUSSION |
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Our results indicate that C/EBP contains a distinguishable
polypeptide element that can function in efficient transcriptional activation both in the context of the C/EBP
protein and when fused
to a heterologous DNA binding domain. Moreover, we have identified an
internal region of the C/EBP
protein that can negatively regulate
gene transcription from three different promoters, as well as
attenuating the function of a known potent transcriptional activator
VP16. Futhermore, when the repression domain was deleted from C/EBP
,
the altered C/EBP
protein enhanced transactivation of gene
transcription 2-fold when compared with that of the wild-type form of
C/EBP
. Taken together, both transactivation and repression domains
exist within the C/EBP
gene (Fig.
6A).
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The activation domain of C/EBP was mapped to the N terminus of the
protein with a minimal requirement of the first 18 amino acids, while
the highest activity of transactivation was conferred by
amino acid residues 1-70. Two predicted activation domains within the
N terminus of CRP1 (rat C/EBP
) were identified based on sequence
comparison between C/EBP family members (33). When the first domain,
amino acids 1-18, was fused to the GAL4DBD, its transactivation
potential retained 80% of the peak activity. Amino acid residues
19-45 of C/EBP
, containing the second domain (amino acids 19-27),
conferred only weak activating potential to the GAL4DBD; however, when
both domains were fused to the GAL4DBD (GAL-
-(1-45)), potent
activity was observed. The potent activity observed with
GAL-
-(1-45) does not reflect synergy between these domains in human
C/EBP
. Deletion of these two domains in the construct
CMV-
-(40-249) abolished the transactivation potential on the NE
promoter and reduced the synergistic stimulation of gene transcription
with c-Myb.
The major difference in the transactivation domains between
C/EBP and other C/EBP proteins is that only one activation domain, amino acids 1-18 of C/EBP
, corresponding to ADM2 of C/EBP
(Fig. 6A), is sufficient to confer a potent transactivation
function to a heterologous GAL4DBD (33). In contrast, the activation domains identified in either C/EBP
or C/EBP
were unable to confer transactivation when fused individually to the GAL4DBD (31, 32, 33,
36). Unlike C/EBP
, combinations of these individual domains resulted
in synergistic activation, indicating that more than one domain was
required for full activating potential to be realized.
Two forms of C/EBP have been identified that differ by 32 amino
acids containing sequences related to the ADM1 region of C/EBP
(11,
33). However, this sequence alone was inactive, and in combination with
the identified activation domains it did not increase the
transactivating potential on the GAL4DBD. Further, both forms activated
the NE promoter to the same level in the absence or presence of c-Myb.
In C/EBP
, two residues, Phe6 and Tyr7,
were identified as being essential for the function of ADM1, and
mutation of these residues yielded a protein with activity similar to a
protein lacking this domain (33). The aforementioned 32 amino acids of
C/EBP
contain tyrosine residues at both sites, suggesting a possible
reason for the lack of activity in this domain.
Among the six C/EBP family members characterized, C/EBP, C/EBP
,
C/EBP
, and C/EBP
, are activators of transcription for a variety
of target genes. However, C/EBP
(32, 36) and C/EBP
(33) both also
contain extra regulatory domains, called the attenuator domain in
C/EBP
and RD1 and RD2 in C/EBP
(Fig. 6A). In C/EBP
, RD1 and RD2 have independent functions. While RD1 modulates the
activation domain of the protein, RD2 controls cell type-specific
transactivation function through regulation of its DNA binding
activity; together, RD1 and RD2 inhibit transcription 50-fold and
decrease VP16 transactivation function by over 95% (33). The
attenuator domain identified in C/EBP
, however, exhibits neither
detectable stimulation nor repression of transcription. When combined
with other activation domains of C/EBP
, the attenuator domain
diminishes their transactivation effect (32, 36). Deletion of these
sequences causes only a modest increase in transcriptional activity
(33). The attenuator domain may actually be a regulatory domain that
allows post-translational modification of C/EBP
, possibly
converting a repressor into an activator or a weak activator into a
strong activator (36).
C/EBP is distinct from other C/EBP family members in that, although
C/EBP
expression occurs exclusively within the myeloid and lymphoid
lineages, the identified domains show little difference in cell
type-specific regulation of transcription. As for C/EBP
and C/EBP
, we have identified a negative regulatory element in C/EBP
localized to amino acids 116-162, downstream of the activation domain
and upstream of the DNA binding and dimerization domain (Fig.
6A). In contrast to C/EBP
, deletion of the C/EBP
repression domain does not give the same magnitude of increase in
transactivation, and this effect may be more similar to the increase
observed with deletion of the attenuator domain of C/EBP
. Also, the
inhibition of VP16 transactivation function by the repression domain of
C/EBP
is significantly less than that mediated by RD1 and RD2 of
C/EBP
. Therefore, the repression domain identified in C/EBP
may
be functionally more similar to the attenuator domain of C/EBP
rather than RD1 and RD2 of C/EBP
. We cannot determine from these
experiments whether the GAL4-C/EBP
fusions repress transcription
directly or whether additional factors interacting with the repression
domain are required.
What role do the transcriptional activation or repression domains play
in the function of C/EBP? Recent studies indicate that it is quite
common for a protein to serve either as an activator or a repressor,
depending on the other gene regulatory proteins present in the cell
(47). In the case of C/EBP
, this may reflect its specific role in
regulating myelopoiesis (48, 49). C/EBP
has been demonstrated to
have a remarkable effect on myeloid cell proliferation (12). Transient
transfection of a promyelocyte cell line (NB4) with a C/EBP
expression plasmid increased cell growth by 7-fold, but proliferation
was significantly decreased upon transfection of the C/EBP
antisense
expression vector (12). Cells may depend on a negatively regulatory
domain (C/EBP
itself) or associated factors that interact with this
domain to control cell proliferation at a certain growth stage.
Therefore, the repression domain (and its associated factors) probably
acts as a buffer for the activation function of C/EBP
.
Our simplified and speculative model shown in Fig. 6B
proposes that C/EBP interacts with different partners in the varied contexts of cellular signaling, which induces exposure of the activation or repression domain. These domains may actually represent oligomerization surfaces that allow C/EBP
to form higher order aggregates either with itself or with other proteins that play a more
direct role in transcriptional regulation of myeloid genes. In support
of this model, our data indicate that Myb is such a potential
activation partner. Many other proteins have been suggested as
potential C/EBP-interacting factors (21-29), and likewise, we anticipate that C/EBP
interacts with other factors. It will be interesting to identify factor(s) that interact with the repression domain to dissect further the repressive function of C/EBP
. Although no common signature has been found within the repression domain of
C/EBP
when searched in the data base, it contains a relatively high
content of prolines and other hydrophobic amino acid residues. Application of the two-hybrid cloning system by using the repression domain of C/EBP
as bait may lead to new information about the network of transcription factors that specifically modulate genes important in hematopoiesis.
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ACKNOWLEDGEMENTS |
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We thank Drs. Gregory Kato and Chi Dang for
kindly providing the cloning vector pGALM; Dr. Arnold Berk for the
generous gifts of G0, G1, G5TKCAT, G5SV40CAT, and G5MLPCAT reporter
plasmids; and Dr. Kleanthis Xanthanopoulos for the generous gift of the plasmid of the full-length C/EBP. We also thank Drs. Carl Miller and
Dorothy Park and members of the C/EBP group in the Koeffler Laboratory
for helpful suggestions.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health and U.S. Army grants as well as the Parker Hughes Fund and the Concern 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.
§ These authors contributed equally to this work.
¶ To whom correspondence should be addressed: Cedars-Sinai Research Institute, UCLA School of Medicine, 8700 Beverly Blvd., B121, Los Angeles, CA 90048. Tel.: 310-855-4609; Fax: 310-659-9741.
** Holds the endowed Mark Goodson Chair of Oncology Research.
1
The abbreviations used are: C/EBP,
CCAAT/enhancer-binding protein ; CMV, cytomegalovirus; PCR,
polymerase chain reaction; CAT, chloramphenicol acetyltransferase; TK,
thymidine kinase; NE, neutrophil elastase.
2 A. M. Chumakov, unpublished observations.
3 W. Verbeek, manuscript in preparation.
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REFERENCES |
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