From the Advanced BioScience Laboratories-Basic Research Program, NCI-Frederick Cancer Research and Development Center, National Institutes of Health, Frederick, Maryland 21702-1201
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ABSTRACT |
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CCAAT/enhancer-binding protein (C/EBP) is a
bZIP transcription factor whose expression is restricted to specific
cell types. Analysis of C/EBP
mRNA and protein levels in various
mammalian cells indicates that expression of this gene is controlled
both transcriptionally and post-transcriptionally. We report here that C/EBP
translation is repressed in several cell lines by an
evolutionarily conserved upstream open reading frame (uORF), which acts
in cis to inhibit C/EBP
translation. Mutations that
disrupt the uORF completely abolished translational repression of
C/EBP
. The related c/ebp
gene also contains an uORF
that suppresses translation. The length of the spacer sequence between
the uORF terminator and the ORF initiator codon (7 bases in all
c/ebp
genes and 4 bases in c/ebp
homologs) is precisely conserved. The effects of insertions, deletions,
and base substitutions in the C/EBP
spacer showed that both the
length and nucleotide sequence of the spacer are important for
efficient translational repression. Our data indicate that the uORFs
regulate translation of full-length C/EBP
and C/EBP
and do not
play a role in generating truncated forms of these proteins, as has
been suggested by start site multiplicity models.
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INTRODUCTION |
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The transcription factor CCAAT/enhancer-binding protein
(C/EBP)1 , a member of the
bZIP (basic region leucine zipper) class of DNA-binding proteins (1), has been a useful model for investigating the
role of transcription factors in cellular differentiation. C/EBP
is
primarily expressed in terminally differentiated cells such as
hepatocytes and adipocytes, where it activates transcription of
differentiation-specific target genes (reviewed in Refs. 2 and 3).
Several studies demonstrated that C/EBP
plays an essential role in
the conversion of adipoblasts to mature adipocytes. For example,
overexpression of C/EBP
antisense RNA in the preadipocytic cell line
3T3-F442A inhibited hormonally induced differentiation of these cells
(4). Similarly, Lin and Lane (5) showed that expression of C/EBP
antisense RNA in 3T3-L1 preadipocytes prevents their conversion to
adipocytes and the subsequent expression of fat-specific genes such as
aP2, GLUT4, and SCD1. In the converse type of experiment, expression of an inducible C/EBP
-estrogen receptor fusion protein in 3T3-L1 adipoblasts resulted in
estrogen-dependent cell growth arrest (6). In addition,
3T3-L1 cells stably transfected with a C/EBP
expression vector
exhibited reduced proliferative potential and displayed the
differentiated adipocyte morphology at high frequency (7). Forced
expression of C/EBP
also promoted the adipogenic program in
fibroblastic cell lines such as NIH 3T3, which normally are unable to
differentiate into mature adipocytes (8-10). These studies
demonstrated that C/EBP
is a critical regulator of the
adipogenic program and led to the proposal that C/EBP
plays a
role in controlling the balance between cellular growth and
differentiation (6).
In view of the important role of C/EBP in cellular differentiation,
the mechanisms that regulate the tissue specificity and developmental
timing of C/EBP
expression are of considerable interest. Surveys of
C/EBP
expression show that whereas C/EBP
transcripts are present
at varying levels in many mammalian tissues and cell lines (Refs. 11
and 12; see also Fig. 1), C/EBP
protein occurs in only a subset of
these cell types. Cells in which the C/EBP
protein has been detected
include differentiated hepatocytes (1, 12), adipocytes (13), intestinal
epithelial cells (14), myelomonocytic progenitor cells (15), ovarian follicles (16), and type II cells of the lung (17). The
disproportionate levels of C/EBP
mRNA and protein observed in
certain cells indicates that post-transcriptional regulation plays a
role in restricting C/EBP
expression.
In this study, we have investigated the molecular mechanism underlying
post-transcriptional control of C/EBP expression. We demonstrate
that C/EBP
translation is inhibited in transfected cell lines by a
short, evolutionarily conserved upstream open reading frame (uORF)
located 7 bases upstream of the C/EBP
ORF (18). uORF-mediated
repression was also observed for the related C/EBP family member
C/EBP
. The uORF repressed C/EBP
translation by a
cis-acting mechanism, and inhibition was overcome by
mutations that inhibit translation of the uORF. Certain changes in the
length and sequence of the uORF-ORF spacer region also caused
translational derepression. Interestingly, we did not detect truncated
C/EBP
and C/EBP
isoforms in these experiments. These findings may
necessitate a re-evaluation of models proposing that initiation at
internal start sites produces truncated forms of C/EBP proteins
(19-22).
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EXPERIMENTAL PROCEDURES |
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Cell Culture and Transfection-- HeLa, 3T3-L1, and 293 cells (American Type Culture Collection) were cultured in Dulbecco's modified Eagle's medium (BioWhittaker, Inc.) supplemented with 10% fetal bovine serum (Hyclone Laboratories) in the presence of kanamycin, streptomycin, and penicillin. Differentiation of 3T3-L1 cells was performed as described previously (13). HepG2 cells were cultured as described (12). HeLa and 293 cells (30-40% confluent) were transiently transfected in 10-cm plates by a standard calcium phosphate coprecipitation procedure (23). HeLa cells were transfected with 7.5 µg of test plasmid, 7.5 µg of internal control plasmid, and 5 µg of carrier plasmid (pMEX) for a total of 20 µg of DNA/plate. The cells were incubated in precipitate for 18-24 h, washed twice with unsupplemented Dulbecco's modified Eagle's medium, and then incubated in complete medium for an additional 18-24 h prior to harvesting. 293 cells were transfected with 2.5 µg of test plasmid, 2.5 µg of internal control plasmid, and 15 µg of pMEX plasmid. The cells were incubated in DNA precipitate for 12-15 h, washed twice with unsupplemented Dulbecco's modified Eagle's medium, and incubated in complete medium for an additional 12-15 h prior to harvesting. For the experiment of Fig. 7, 10-cm plates of HeLa cells were transfected using 2.5 µg of DNA and 10 µl of DMRIE-C reagent (Life Technologies, Inc.) according to the supplier's recommendations. Duplicate plates were harvested after 48 h for protein and mRNA, respectively. For all transfection assays, at least two independent isolates of each recombinant plasmid were tested, and the transfections were repeated independently at least three times.
For stably transfected HeLa cells, transfection conditions were identical to those described above, except that 20 µg of test plasmid were used in addition to 2.5 µg of the selectable marker (pMEX.neo). After ~48 h, cells were split into fresh 10-cm plates and allowed to recover for an additional 48 h. Cells were then fed with complete medium supplemented with G418 (0.3-0.5 units/ml; Life Technologies, Inc.). Pools of ~20-50 independent neor transfectants were obtained and analyzed.Plasmid Constructions--
Plasmids used for constitutive
expression of C/EBP and C/EBP
were pMEX-C/EBP
and pMEX-CRP2
(hereafter denoted pMEX-C/EBP
), respectively (12). C/EBP
expression plasmids containing wild-type and mutant 5'-leader regions
were constructed as follows. PCR was used to introduce both a
restriction site immediately upstream of the C/EBP
5'-leader and
mutations within the 5'-leader. PCR (30 cycles of denaturation at
94 °C for 40 s and annealing/extension at 50 °C for 1 min,
20 s) was carried out using the Gene-Amp kit (Perkin-Elmer) under
the conditions recommended by the supplier, except that 5% dimethyl
sulfoxide was included to aid template denaturation. All PCR-derived
fragments were sequenced using Sequenase T7 DNA polymerase and a
7-deaza-dGTP sequencing kit (U. S. Biochemical Corp.) to verify that
no extraneous mutations were introduced. Wild-type and mutant 5'-leader
constructs of C/EBP
were cloned using SphI (a site in the
pMEX vector polylinker) at the 5'-end and NcoI, which
overlaps the c/ebp
initiation codon, at the 3'-end. Oligonucleotides used to generate wild-type and mutant C/EBP
constructs were as follows: 5'-oligonucleotide for the wild type and all other mutants, 5'-GGATCCGCATGCATTCGCGACCCAAAG; wt 3', 5'-GACTCCATGGGGGAGTTAG; uORF(ATG*) 3',
5'-GACTCCATGGGGGAGTTAGAGTTCTCCCGGTGTGGCGAGCCTC; uORF(TAA*) 3',
5'-GACTCCATGGGGGAGTTCGAGTTCTCCCG;
ORF 3',
5'-GACTCCATGGGGGAGGGCGAGCCTCGG; uORF-C/EBP
fusion 3',
5'-GACTCCATGGCGAGTTCTCCCGG; uORF Sp+10 3',
5'-GACTCCATGGGGGGACGCCTGGGAGTTAGAGTTCTCC; uORF+Stu 3',
5'-GACTCCATGGGGGGAGGCCTCCGAGTTAGAGTTCTCC; uORF Sp C*(
4) 3',
5'-GACTCCATGGGTGAGTTAGAGTTCTCC; uORF Sp C*(
3
5) 3',
5'-GACTCCATGGATTAGTTAGAGTTCTCC; uORF Sp+1 3', 5'-GACTCCATGGGGGGAGTTAG; uORF Sp+2 3', 5'-GACTCCATGGGGGGGAGTTAG; uORF Sp+3 3',
5'-GACTCCATGGGGGGGGAGTTAG; uORF Sp
1 3', 5'-GACTCCATGGGGAGTTAG; uORF
Sp
2 3', 5'-GACTCCATGGGAGTTAG; and uORF Sp
3 3',
5'-GACTCCATGGAGTTAG.
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RNA Extraction and Northern Blot Analysis--
Total cellular
RNA was isolated from transfected cells using RNA STAT-60 (TEL-TEST,
Inc.) or Trizol reagent (Life Technologies, Inc.). RNA (15 µg) was
separated by electrophoresis using formaldehyde-containing 1.2%
agarose gels and then transferred to GeneScreen membrane (DuPont)
according to the manufacturer's instructions. Membranes were
hybridized overnight at 42 °C with
[-32P]dCTP-labeled DNA probes. The hybridization
solution consisted of 50% formamide, 5× Denhardt's solution, 5×
SSPE (750 mM NaCl, 50 mM
NaH2PO4·H2O, and 5 mM
EDTA), 1% SDS, and 0.1 mg/ml salmon sperm DNA (27). Following
incubation, blots were washed with 0.1% SDS and 0.5× SSPE at 65 °C
and then exposed to x-ray film (Eastman Kodak Co.). DNA probes for
analysis of RNA from transfected cells by Northern blotting were
prepared by isolating the following fragments: c/ebp
,
350-base pair PstI-SacI fragment from the 3'-end of the coding region of the murine c/ebp
gene; FLAG,
170-base pair XhoI-HpaI fragment from
pMEX-C/EBP
-F (contains the FLAG epitope and 3'-untranslated
sequences from pMEX);
-actin, 2-kilobase HindIII fragment
excised from the plasmid
2000 (28); and cyclophilin, as described by
Danielson et al. (29). DNA fragments were labeled to high
specific activity with [
-32P]dCTP using the Prime-It
II kit (Stratagene).
Cell Extracts and Western Blotting-- Whole cell extracts were prepared by lysing cells directly in radioimmune precipitation assay buffer (30). Nuclear extracts were prepared as described by Lee et al. (31), except that Buffer A also contained 0.1% Nonidet P-40 (Sigma) and 5 µg/ml leupeptin (Boehringer Mannheim), and nuclear extracts were not dialyzed against Buffer D. Nuclear extracts from tissue samples were prepared according to the method of Gorski et al. (32). Protein concentrations were determined either by the Bio-Rad protein assay or by estimating protein following SDS-polyacrylamide gel electrophoresis and Coomassie Blue staining. Samples were denatured in sample buffer at 85 °C and then electrophoresed on 10% SDS-polyacrylamide gels (33). Molecular weights of the proteins were determined by comparison to Rainbow molecular weight markers (Amersham Pharmacia Biotech). SDS-polyacrylamide gels were transferred to nitrocellulose (Schleicher & Schuell) by electroblotting. Blots were blocked in 5% nonfat dry milk and Tris-buffered saline, pH 7.6, for 2-6 h at room temperature. Blots were incubated with primary antibody for 4-16 h at room temperature. The antigen-antibody complex was visualized using the Amersham Pharmacia Biotech enhanced chemiluminescence kit. Pan-CRP antiserum was used as described (26). The FLAG epitope was detected using the anti-FLAG M2 monoclonal antibody (Eastman Kodak Co.).
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RESULTS |
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Post-transcriptional Control of C/EBP Expression--
Fig.
1 shows a survey of C/EBP
mRNA and
protein expression in several mammalian cell lines and murine tissues.
C/EBP
transcript levels were highest in liver and differentiated
3T3-L1 adipocytes, two cell types in which the C/EBP
protein is
abundant. HeLa and HepG2 cells and kidney tissue contained lower but
significant levels of C/EBP
mRNA, whereas they lacked detectable
C/EBP
protein. Liver contained 5-10-fold more C/EBP
mRNA
than kidney (Fig. 1A), but at least 20-fold more C/EBP
protein, as estimated by serial dilution of the protein extract (Fig.
1C). The difference in protein levels is a minimum estimate
since the C/EBP
signal in kidney extracts was below the level of
detection. This disparity between mRNA and protein levels suggests
that a post-transcriptional mechanism contributes to the regulation of
C/EBP
expression.
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Inhibition of C/EBP Translation by a Conserved Upstream Open
Reading Frame--
A comparison of the 5'-leaders of rat, mouse, human
(34), bovine (35), chicken, and Xenopus laevis c/ebp
genes revealed the presence of a short (5-amino acid) conserved ORF
(Fig. 2A) (18). This uORF is
located precisely 7 nucleotides upstream of the C/EBP
initiation
codon in all c/ebp
homologs. The evolutionary conservation of the uORF element and its location in the transcript suggest an important function in regulating C/EBP
expression. We
suspected that the uORF might suppress translation of the C/EBP
ORF
since many eukaryotic mRNAs lack upstream AUG codons, and the
insertion of a single functional initiation codon upstream can severely
inhibit translation from downstream AUG codons (reviewed in Refs.
36-39).
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uORF-mediated Translational Repression Is Exerted in cis--
We
next addressed the possibility that the uORF encodes a peptide product
that inhibits C/EBP translation in trans. The uORF(ATG*) mutant was transfected into HeLa cells together with a construct that
contains the uORF but lacks the C/EBP
ORF. Transfecting increasing
amounts of the uORF expression plasmid did not diminish expression
either from uORF(ATG*)-C/EBP
-F or from the control, pMEX-C/EBP
-F
(Fig. 5). Although we cannot directly
assess synthesis of the uORF-encoded peptide in the cell, the high
expression seen from the uORF-ORF fusion construct (Fig. 2C,
lane 8) demonstrates that translation initiates efficiently
at the uORF in HeLa cells. Thus, the inability of the uORF to repress
C/EBP
translation when expressed from a separate transcript suggests
that the uORF inhibits C/EBP
expression via a cis-acting
mechanism.
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The uORF-ORF Spacer Length and Nucleotide Sequence Are Critical for
Translational Repression--
The spacing between the uORF termination
codon and the C/EBP start site is exactly 7 bases in all
c/ebp
homologs characterized (Fig. 2A),
suggesting that the spacer length is an important feature for C/EBP
regulation. We examined whether mutations that increase or decrease the
spacer length (Fig. 6A) affect
translational repression in 293 cells. Insertion of 1-3 cytosines in
the spacer did not abolish suppression of C/EBP
translation by the
uORF (Fig. 6B, lanes 5-7). In contrast, removing
a single cytosine caused a large increase in C/EBP
translation (Fig.
6C, lane 5), and deleting 2 or 3 of the 5 cytosines preceding the ORF also resulted in significant derepression
(Fig. 6C, lanes 6 and 7). Thus,
decreasing the spacing by 1 nucleotide renders the uORF unable to
inhibit C/EBP
translation, demonstrating that the length and/or
sequence of the spacer is critical for uORF-mediated repression.
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A Conserved uORF Regulates C/EBP Translation--
Expression of
another C/EBP family member, C/EBP
, is also regulated
post-transcriptionally. Descombes et al. (44) reported that
C/EBP
(or LAP (liver-enriched transcriptional
activator protein) transcripts occur in liver,
lung, spleen, kidney, brain, and testis, whereas the protein was
detected only in liver. These results imply that a translational
control mechanism suppresses C/EBP
protein expression in certain
cell types.
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DISCUSSION |
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In this report, we have examined the molecular basis for
post-transcriptional regulation of C/EBP and C/EBP
expression. Mutational analysis revealed that the uORF is a potent inhibitor of
C/EBP
translation. The uORF initiation codon was readily recognized by the translational machinery, and mutations that abolished uORF translation disrupted repression of the C/EBP
ORF. The repressive effect of the uORF on C/EBP
translation was not cell-specific since
the same results were observed in several cell lines using both
transient and stable transfection assays. Although repression was seen
in multiple cell lines, there must be a mechanism to overcome
repression imposed by the uORF in cells that express the C/EBP
protein abundantly, such as terminally differentiated hepatocytes and
adipocytes.
C/EBP also contains a conserved uORF that represses translation
(Fig. 7). This mechanism could control the induction of
p34
translation in response to cell-specific or
physiological cues. For example, the human homolog of C/EBP
, NF-IL6
(45), was shown to be translationally regulated in the pulmonary
alveolar epithelial cell line A549 (46). Protein expression from the
NF-IL6 transcript was induced upon infection of the cells with
respiratory syncytial virus. Enhanced protein expression occurred
without detectable changes in NF-IL6 transcript abundance or size,
indicating the existence of a translational control mechanism. Since
C/EBP
is implicated in transcription of cytokine genes and other
inflammatory mediators in monocytic and epithelial cells (47),
up-regulation of C/EBP
translation may be an important component of
the cellular response to infectious or toxic agents. C/EBP
protein
expression, but not mRNA levels, was also found to be up-regulated
during terminal differentiation of primary keratinocytes in
culture2 and differentiation
of rat Leydig cells at puberty (49). We suggest that induction of
C/EBP
expression in each of these cases involves derepression of
uORF-mediated translational inhibition.
The truncated forms of C/EBP and C/EBP
(p30
and
p20
/LIP, respectively) have been hypothesized to result
from alternative translation initiation (19-22). It has been proposed
that internal initiation within the C/EBP
or C/EBP
ORFs occurs by
a mechanism that requires the 5'-leader region. The resulting truncated
proteins contain a DNA-binding domain, but lack an N-terminal
activation domain. LIP, which lacks activating sequences altogether,
has been proposed to function as an inhibitory isoform of C/EBP
. Internal initiation in the C/EBP
and C/EBP
mRNAs is believed to arise either from "leaky scanning" (19-21) or from termination after uORFs and reinitiation at downstream AUG codons (22, 50).
Our results do not support the hypothesis that C/EBP and C/EBP
translation reinitiates at downstream AUG codons within the ORF. The
presence of the wild-type C/EBP
5'-leader diminished translation of
p42
(Figs. 3-5), but no truncated proteins such as
p30
were produced from any of the C/EBP
constructs.
The absence of truncated proteins was not a function of the cell type
since these proteins were not observed in any cell line tested,
including HeLa, 293, HepG2, and undifferentiated 3T3-L1 cells (data not shown). Similarly, truncated proteins were not expressed from the
C/EBP
constructs (Fig. 7). These results were consistently observed
with two different antibodies (pan-CRP and anti-FLAG M2), indicating
that antibody specificity does not account for the inability to detect
p30
and p20
/LIP. Rather, we have detected
only p30
or p20
/LIP in nuclear cell
extracts from transfected cells under certain cell lysis
conditions.3 Specifically,
the addition of detergent (Nonidet P-40) and the protease inhibitor
leupeptin to the lysis buffer prevented the appearance of truncated
proteins. The level of endogenous p30
protein observed
in differentiated 3T3-L1 cells (Fig. 1B, lane 4)
could also be diminished or eliminated by altering the cell lysis
protocol. In addition, we have found that a calpain protease cleaves
recombinant C/EBP
to generate a product that is indistinguishable in
size from LIP.4 Collectively,
our observations suggest that truncated forms of C/EBP
and C/EBP
may arise in many cases from proteolysis rather than translation
initiation. We propose that the function of C/EBP
and C/EBP
uORFs
is to regulate initiation at the ORFs immediately downstream and not to
generate truncated proteins by internal initiation.
Mutations designed to test the mechanism of uORF repression showed that
the uORF-ORF spacer is critical for inhibition of C/EBP translation.
Removal of 1-3 cytosine residues significantly decreased translational
repression, especially when a single cytosine was deleted. One
explanation for this result is that the C/EBP
spacer sequence
dictates the efficiency of ribosome release after uORF termination.
Support for this notion comes from studies of the yeast gcn4
gene, which encodes a transcriptional activator that regulates the
synthesis of several biosynthetic genes in response to amino acid
starvation. Gcn4 translation is controlled by four uORFs within the
5'-leader and is regulated primarily by the first and fourth uORFs
(41). uORF1 alone inhibits GCN4 translation by ~50% and is also
required for translational derepression in response to amino acid
starvation, whereas uORF4 constitutively represses translation of GCN4.
The sequences surrounding the termination codons of both uORFs are
critical for regulation of GCN4 translation (51). Sequences downstream
of the uORF1 terminator are required for reinitiation at uORF4 when
amino acids are plentiful or at GCN4 under starvation conditions. A
rare proline codon at the 3'-end of uORF4 and sequences just downstream
of the terminator appear to be necessary for efficient ribosome release
following termination, thereby preventing reinitiation at downstream
AUG codons. Thus, sequences 3' of the uORF1 terminator favor
reinitiation downstream, whereas those 3' of uORF4 favor ribosome
release.
C/EBP translational repression could also involve ribosome release
after uORF termination, resulting in weak reinitiation at the C/EBP
AUG codon. Spacer deletions and nucleotide substitution mutants that
up-regulate C/EBP
expression may function by decreasing the
efficiency of uORF termination/release. An alternative possibility is
that these mutations increase initiation efficiency at the C/EBP
start site. However, this explanation seems less likely since the
sequence context of the initiation codon should be the predominant
factor dictating the efficiency of initiation (40, 52, 53), and the
mutations were designed to minimize disruption of the C/EBP
Kozak
sequence. In addition, mutants in which uORF translation is disrupted
express high levels of C/EBP
, showing that the ORF AUG codon is in a
favorable sequence context for initiation. The spacer length may also
affect the efficiency of reinitiation, although the derepression seen
with spacer deletion mutants could be explained by the removal of
nucleotides required for efficient ribosome release. Nevertheless, the
stringent conservation of uORF-ORF spacing in c/ebp
and
c/ebp
genes argues that spacer length per se
is an important feature of translational regulation.
In some uORF-containing genes, repression of downstream ORFs is
dependent on the peptide translated from the uORF. Examples of this
include yeast CPA-1 (54), the human cytomegalovirus gpUL4 (gp48) gene (55, 56), and
AdoMetDC (57, 58). Experimental data suggest that the
nascent polypeptide translated from these uORFs in some way stalls the
translational machinery, possibly during termination of uORF
translation. The peptides encoded by these various uORFs do not contain
any apparent sequence homologies and are variable in size. Like the
C/EBP uORF, all appear to function only in cis. Further
mutagenesis experiments should establish whether the uORF amino acid
sequence is important for repression of C/EBP
and C/EBP
translation.
We speculate that uORF-mediated inhibition of C/EBP translation may
operate to restrict C/EBP
expression to cells that are undergoing
terminal differentiation. C/EBP
possesses potent differentiative and
antimitotic activities (6, 8, 59), and its expression may not be
tolerated by most proliferating cells. Overexpression of C/EBP
was
also found to cause growth arrest in hepatoma cells (59). C/EBP
activates expression of two growth-arrest associated genes: p21
(WAF-1/CIP-1/SDI-1), a member of the
cyclin-dependent kinase inhibitor family that blocks
G1-S cell cycle progression (60), and gadd145, a
growth arrest-inducible gene (61). Thus, stringent regulation of
C/EBP
and C/EBP
expression may be necessary to prevent premature
mitotic arrest, differentiation, and activation of target genes.
uORF-mediated translational regulation may contribute to the control of
C/EBP
and C/EBP
expression during development and could also
modulate C/EBP
protein synthesis in response to cell stress or viral
infection.
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ACKNOWLEDGEMENTS |
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We are grateful to E. Sterneck, R. Schwartz, and R. Kirken for critical reading of the manuscript and L. Sewell for expert technical assistance. We also thank M. Powers for synthesis of oligonucleotides and C. Weinstock and H. Marusiodis for help with preparation of the manuscript.
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FOOTNOTES |
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* This work was supported in part by NCI, Department of Health and Human Services, under contract with Advanced BioScience Laboratories.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.
Present address: Lady Davis Inst., 3755 Côte-ste-Catherine,
Montréal, Quebec H3T 1E2, Canada.
§ Present address: Dept. of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Southwest Cancer Center at University Medical Center, 3601 4th St., Lubbock, TX 79430.
¶ To whom correspondence should be addressed: Advanced BioScience Laboratories-Basic Research Program, NCI-Frederick Cancer Research and Development Center, P. O. Box B, Frederick, MD 21702-1201. Tel.: 301-846-1627; Fax: 301-846-5991; E-mail: johnsopf{at}fcrfv1.ncifcrf.gov.
1 The abbreviations used are: C/EBP, CCAAT/enhancer-binding protein; ORF, open reading frame; uORF, upstream ORF; PCR, polymerase chain reaction; wt, wild-type.
2 Oh, H.-S. and Smart, R. C. (1998) J. Invest. Dermatol., in press.
3 A. J. Lincoln, M. Baer, E. Sterneck, and P. F. Johnson, unpublished data.
4 M. Baer, A. J. Lincoln, E. Sterneck, and P. F. Johnson, unpublished data.
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REFERENCES |
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