From the Burnham Institute, La Jolla Cancer Research
Center, La Jolla, California 92037, the ¶ Buck Institute for Age
Research, Novato, California 94945, the
Sidney Kimmel Cancer
Center, San Diego, California 92121, and the ** Cancer
Center, University of California at San Diego,
La Jolla, California 92093
Received for publication, October 14, 2002, and in revised form, January 31, 2003
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ABSTRACT |
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Egr1, an immediate early transcription factor,
responds to diverse stimuli and affects gene transcription to
accomplish its biological effects. One important effect of Egr1
expression is to decrease the growth and tumorigenic potential of
several tumor cell types. To identify important Egr1 target genes, we
have adapted a methodology involving formaldehyde-induced protein-DNA
cross-linking, chromatin immunoprecipitation, and multiplex PCR. Using
this approach, we report the cloning of a new Egr1 target gene that is
able to account, at least in part, for the growth inhibitory activity of Egr1. We have named this new protein TOE1 for
target of
Egr1.
A common feature associated with the expression of immediate-early
genes is their rapid, transient response to a diverse variety of
extracellular signals. We have been studying the properties of the
early growth response gene, Egr1, which can be transcriptionally induced by a wide spectrum of stimuli including growth factors, cytokines, stresses, depolarizing stimuli, phorbol esters, vascular injury, and irradiation, both ionizing and nonionizing, in a rapid and
transient manner with kinetics mirroring those of c-fos (1). We have previously presented evidence suggesting a role for Egr1 in
suppressing tumor cell growth (2, 3). Specifically, we demonstrated
that overexpression of Egr1 in transformed cells suppresses growth in
soft agar as well as inhibits their tumor formation in nude mice.
Furthermore, it was shown that the DNA-binding domain of Egr1 is
necessary for its ability to suppress tumor formation, highlighting the
importance of its transactivation of downstream genes in this process
(4). Together these results indicate that transformed cells can be
induced to revert to normal growth patterns following the re-expression
of Egr1. These studies suggest that the loss of Egr1 may result in the
loss of cellular homeostasis because of a deficit in Egr1-responsive
genes and that this may play a pivotal role in tumorigenesis.
Clearly, the identification of a genetic profile of Egr1-responsive
genes would constitute a significant step in understanding the
different activities associated with Egr1, including its role in
cellular growth control. Over the past several years there have been
numerous studies identifying various individual Egr1 target genes in
diverse cell and tissue types. Reported Egr1 targets include
TGF- Currently, few techniques are available to address this issue. Both
differential display and subtractive hybridization analyses are aimed
at isolating messages that are up- or down-regulated from pools of RNA
isolated from cells or tissues either positive or negative for the gene
in question. One clear drawback with both of these techniques is that
they select for any RNA message that shows a change in expression
pattern. Therefore, when screening for changes in gene expression
induced by a transcription factor, these methods do not select purely
for direct targets. Recently we and others have described a method for
the direct isolation of protein-bound DNA involving in vivo
chemical cross-linking using formaldehyde followed by
immunoprecipitation from chromatin (ChIP). This method was successfully
used in applications ranging from examining chromatin structures
surrounding the polycomb group proteins during Drosophila
development (6) and the identification of nuclear matrix attachment
sites (7) to the isolation of DNA sequences bound by Egr1 (8). In
addition, the same cross-linking method has been used to examine
nucleosomal structure, transcription factor occupancy of promoter
sites, regions of histone acetylation, and mapping of telomere
silencing protein binding, illustrating its broad application utility
(9-12). Recently, coupling the ChIP approach with hybridization to
genomic or promoter region DNA microarrays has allowed a comprehensive
characterization of in vivo transcription factor DNA binding
patterns (13-16).
In this report we have extended ChIP technology, allowing gene
discovery of Egr1 target genes by multiplex PCR. Moreover, we present
the cloning of a newly identified gene, called TOE1, as an
Egr1 target gene. We have characterized TOE1 as a cell growth inhibitor
by altering the cell cycle through the induction of p21.
Furthermore, we show that the increase in the p21 level is consistent with a mechanism involving TGF- Cells, Transfection, Antibodies, and Growth Assays--
Both the
H4 clone derived from the human fibrosarcoma cell line HT1080 and the
Egr1 stably transfected H4 subclone E9 have been previously described
(4). 293 cells were grown in Dulbecco's modified Eagle's medium
containing 10% fetal bovine serum. All of the DNA transfections were
performed using LipofectAMINE 2000 (Invitrogen), following the
manufacturer's instructions. Antibodies against cdc2,
phospho-cdc2(Y15), and phospho-p53(S15) were from Cell Signaling
Technology. Antibodies against cyclin B1, p21, and p53 were from Santa
Cruz Biotechnology. Anti-actinin and the M2 monoclonal anti-FLAG
antibody were from Sigma. For cell growth assays 20 × 103 control and TOE1 expressing 293 selected and pooled
clones were seeded into 96-well plates in triplicate. At the indicated
times, cell growth was determined using the CellTiter Cell
Proliferation Assay (Promega).
In Vivo Formaldehyde Cross-linking and Chromatin
Immunoprecipitation--
Cross-linking and chromatin
immunoprecipitation were performed as previously described (6, 8).
Briefly, the cells were grown in 150-mm plates to 80-90% confluence
and then cross-linked by the addition of buffered formaldehyde to a
final concentration of 1%. Following exposure to formaldehyde at room
temperature for a period of 30 min, the cells were lysed by sonication
and chromatin purified by centrifugation through a 5-8 M
urea gradient in TE buffer (10 mM Tris, pH 8.0, and
1 mM EDTA). Purified chromatin was dialyzed against 10 mM Tris-HCl, pH 7.5, 25 mM NaCl, 5% glycerol to remove the urea. Samples of 30-60 µg of chromatin were digested with 10 units of EcoRI overnight at 37 °C and then
precleared by the addition of nonimmune rabbit serum and protein
A-Sepharose beads. The precleared samples were immunoprecipitated with
affinity purified anti-Egr1 antibodies and protein A-Sepharose beads
(17). DNA fragments cross-linked and co-precipitating with Egr1 were purified and ligated to EcoRI linkers consisting of
5'-AATTCGAAGCTTGGATCCGAGCAG-3' and 5'-CTGCTCGGATCCAAGCTTCG-3'.
Following ligation, the products were amplified using the 20-mer as
primer. Amplification conditions were 95 °C for 30 s, 65 °C
for 30 s, and 72 °C for 4 min for 30 cycles. For direct
amplification of the ChIP samples, no linker ligation was performed,
and direct amplification from the Egr1 immunoprecipitates was done
using specific primers for TOE1 (see below), TGF- Library Multiplex PCR and TOE1 cDNA Cloning--
Following
amplification of linker-ligated products as described above, the
linkers were removed by EcoRI digestion, and the products
were purified using a PCR product purification kit (Roche Molecular
Biochemicals). Multiplex PCR was performed using 100 ng of PCR products
as the 5' primer mix and a T7 oligonucleotide as the 3' primer, with
100 ng of an excised undifferentiated NT2 cell cDNA library
(Stratagene). 30 cycles of hot start PCR were performed using the
following parameters: 95 °C for 45 s, 55 °C for 30 s,
and 72 °C for 4 min. A 2-kilobase pair band derived from the
multiplex PCR was excised from the gel, eluted, cloned into the pCR3.1
TA cloning vector (Invitrogen), and sequenced. Data base homology
searches were performed using the BLAST program. To confirm the
full-length TOE1 cDNA, we performed 5' rapid amplification of
cDNA ends using the fetal brain Marathon-Ready cDNA kit
(Clontech), following the manufacturer's
instructions. The TOE1 specific primer used for 5' rapid amplification
of cDNA ends was 5'-GTGAGGGGTACAGCTTTGCC-3'. A FLAG-tagged
TOE1 expression vector was generated by PCR using the following
primers: 5'-CCGAAGCTTATGGATTACAAGGACGACGACGATAAGGCCGCCGACAGTGAC-3' incorporating the FLAG epitope tag and
5'-CCGGAATTCTCAGCTACTGCCCCAA-3'. PCR was performed for 30 cycles
of 95 °C for 45 s, 62 °C for 30 s, and 72 °C for 2 min. The PCR product was digested with
HindIII/EcoRI and cloned into the same sites in
pcDNA3. All of the constructs were sequence-confirmed.
Cloning of the TOE1 Proximal Promoter and Luciferase
Assays--
The proximal region of the TOE1 cDNA sequence was
cloned from human genomic DNA using the Advantage-GC genomic PCR kit
(Clontech). Primers used for PCR were
5'-GCCGGTACCCGCTCTTACACC-3' and 5'-CCCGTTAACGACACCGCTCGT-3'. The PCR
parameters used were 95 °C for 45 s, 60 °C for 30 s,
and 72 °C for 1 min for a total of 30 cycles. This reaction
generated a 580-bp product immediately 5' of the initiation codon. The
PCR product was digested with KpnI and HpaI and
cloned into the KpnI and SmaI sites of pGL3basic
(Promega). 293 cells were transfected in 12-well plates with a total of
500 ng of DNA using LipofectAMINE 2000 (Invitrogen). Transfected DNA
consisted of 200 ng of expression vector DNA, 200 ng of reporter DNA,
and 100 ng of cytomegalovirus- Mutagenesis--
To generate the TOE1 expression construct
without the putative nuclear localization signal, QuikChange
mutagenesis (Stratagene) was performed. The primers used were
5'-GCGGCAGAGGACGCTTTATTGAACCTA-3' and
5'-TAGGTTCAATAAAGCGTCCTCTGCCGC-3'. Construction of the correct deletion was confirmed by sequencing.
Gel Shift--
The gel shift assay was performed as previously
described (8) using the 580-bp radiolabeled TOE1 promoter region
described above and recombinant Egr1 protein.
Confocal Microscopy--
Control and TOE1 expressing H4 cells
were dually stained with rabbit anti-FLAG (Affinity Bioreagents) and
mouse anti-nucleolin (Santa Cruz Biotechnology) antibodies. Secondary
labeling was performed using fluorescein isothiocyanate-conjugated goat
anti-rabbit IgG (Santa Cruz Biotechnology) and Texas Red-conjugated
goat anti-mouse IgG (Jackson Immunoresearch).
Flow Cytometry--
The cells were harvested and fixed in 70%
methanol and stored at Northern Blotting--
A human Multiple Tissue Northern blot
(Clontech) was hybridized with a PCR-generated
TOE1-specific 32P-labeled probe using the primers
5'-AAGCGGCGACGGCGACGACG-3' and 5'-GTGAGGGGTACAGCTTTGCC-3' following
the manufacturer's instructions.
RT-PCR--
To detect TOE1 expression following Egr1
transfection, total RNA was harvested from transfected cells using Tri
Reagent (Molecular Research Center). Following DNase I treatment, 2 µg of RNA was used for reverse transcription using Moloney murine
leukemia virus reverse transcriptase (New England Biolabs). TOE1
expression was then assessed by PCR using the same primers described
above for Northern probe preparation, and glyceraldehyde-3-phosphate
dehydrogenase expression was determined as a loading control using the
primers 5'-AACCATGAGAAGTATGACAAC-3' and
5'-GTCATACCAGGAAATGAGCT-3'. Expression of the p21 gene was
determined using the primers 5'-CTCAAATCGTCCAGCGACCTT-3' and
5'-ACAGTCTAGGTGGAGAAACGGGA-3'. TGF-
Real time PCR reactions were performed using the one-step RT-PCR SYBR
green kit from Roche using a Roche Light Cycler instrument. Following
the RT reaction for 30 min, the PCR conditions were 95 °C for
15 s, 55 °C for 15 s, and 72 °C for 30 s for 40 cycles. mRNA quantitation was performed by measuring cyclophilin
mRNA levels against a standard curve measurement of cyclophilin
mRNA from a control sample. The primers used are described above.
In Vitro Kinase Assay--
In vitro phosphorylation
was performed as described (18).
Cloning of TOE1--
We have previously characterized a clone of
HT1080 cells, called H4, as a cell line that does not express either
basal or UV-induced Egr-1. We have also described a series of
stable transfected Egr1 clones (19). We used the clone with the maximum
expression of Egr1, termed E9, to isolate and identify in
vivo Egr1 target genes. We performed formaldehyde cross-linking on
untreated and UV-stimulated cells followed by chromatin
immunoprecipitation as described earlier (8). Because it is generally
accepted that Egr1-binding sites usually occur within the proximal
promoter region of genes, our immunocaptured Egr1-bound sequences are
likely to consist of predominantly promoter regions with extensions
into the 5'-untranslated region and even into the coding region. To identify target gene sequences we performed multiplex PCR using our
immunocaptured Egr1-bound DNA sequences as 5' multiplex primers. As
template we selected a cDNA library and used a T7 primer that anneals 3' to all cDNAs permitting full-length cDNA
amplification. Using DNA captured from E9 cell Egr1 immunoprecipitates,
we found that multiplex PCR-amplified products only in the presence of the multiplex primers, cDNA library, and the 3' T7 primer (Fig. 1A, lane 2). When
multiplex primers derived from UV-treated E9 cells were used, on
occasion we found some self-amplification from the multiplex primers
resulting in a high molecular weight smear (Fig. 1A,
lane 4). However, the addition of cDNA library template
produced a much stronger and distinctly different profile of amplified
products (Fig. 1A, lane 5), suggesting that
cDNAs were obtained from these primers as well. To directly address the question of whether these amplified cDNAs represented
bona fide Egr1 target genes, we isolated and cloned an
individual target gene.
We focused on the distinct DNA band amplified using primers isolated
from E9 cells and migrating with an approximate size of 2 kb (Fig.
1A, lane 2). Cloning and sequencing of this DNA revealed an open reading frame coding for a predicted polypeptide of
510 amino acids and with a predicted molecular mass of ~58 kDa. To
confirm that this clone represented a full-length cDNA, we
performed 5' rapid amplification of cDNA ends. Sequencing
results confirmed that the captured sequence represented a full-length cDNA clone. A data base homology search of the DNA sequence
identified the chromosomal map position on human chromosome 1 (1p34.1-35.3). Comparison of the sequence of this region of chromosome
1 to our cloned cDNA identified an 8 exon gene. BLAST homology
searches (20) revealed no extended homology with any known protein.
However, a potential single zinc finger was noted as well as a possible nuclear localization signal.
To show that the clone represented an expressed gene, a multiple tissue
Northern blot was hybridized and showed intense hybridization to a 2-kb
mRNA species in six of the 12 tissues with the highest level of
expression in placenta, liver, and kidney (Fig. 1B). We
cloned the open reading frame of the cDNA, together with a FLAG
epitope tag, into a mammalian expression vector and transfected the
construct into H4 cells. Western analysis of cells transfected with the
FLAG-tagged expression vector and anti-FLAG antibodies showed that the
expressed protein migrated on SDS-PAGE with a molecular mass of ~60
kDa, in close agreement with its predicted mass of 58 kDa (data not shown).
TOE1 Is a Target for Egr1 Binding and Transactivation--
To
confirm the specificity of Egr1 binding to TOE1 in vivo, DNA
recovered from immunoprecipitates was PCR-amplified to detect the 5'
region of TOE1. As shown in Fig.
2A we were able to amplify TOE1 from E9 but not from H4 immunoprecipitates. We did,
however, confirm the presence of the TOE1 gene in the total
chromatin fraction, thus ruling out the formal possibility that the
TOE1 gene is deleted in H4 cells. Further, the known Egr1
target gene TGF-
Direct binding of Egr1 to the TOE1 promoter region was
assessed by a gel shift analysis using as probe a region spanning 580 bp upstream of the translation start. Using recombinant Egr1 we found
specific binding to the probe (Fig. 2C). When
oligonucleotides representing the consensus Egr1-binding site were used
as competitor, effective competition was also observed (data not
shown). As a test of the functional properties of the complex we
inserted the same 580-bp 5' region upstream of a luciferase reporter.
We observed that this region responds to Egr1 expression by activating
transcription (Fig. 2D). Together, these results are
consistent with in vivo binding of Egr1 to and
transactivation of the TOE1 gene.
Subcellular Localization of TOE1--
To determine the
intracellular localization of TOE1, a FLAG-tagged expression construct
was transfected into H4 cells. As shown in Fig.
3, following immunostaining for the FLAG
epitope, the subcellular localization of TOE1 was distinctly nuclear.
Transfection and staining of H4 and 293 cells (not shown) showed
patterns of concentrated localization within the nucleus. These sites
of concentration appeared to correspond to nucleoli. Dual staining
using anti-FLAG and anti-nucleolin antibodies followed by confocal
microscopy (Fig. 3) showed that most of the expressed TOE1 co-localized
with nucleolin, indicating a predominant nucleolar location for TOE1. In addition to its nucleolar localization we observed intense staining
for TOE1 as multiple nuclear speckles. As noted above, data base
homology searches identified a putative nuclear localization sequence
consisting of KRRRRRRREKRKR located at positions 335-347 in the
510-amino acid protein. Deleting the putative nuclear localization basic stretch of amino acids resulted in the cytoplasmic localization of TOE1 (Fig. 4), suggesting that this
sequence is responsible for TOE1 nuclear targeting.
TOE1 Expression Affects the Growth of 293 and H4 Cells--
To
test whether TOE1 might be involved in mediating the growth effects of
Egr1, we measured the growth rate of cells stably transfected with a
TOE1 expression vector. Fig.
5A shows that the growth rate
of TOE1-expressing cells was severely reduced in comparison with empty
vector control cells. The doubling time for control cells was ~24 h,
whereas a pool of TOE1 expressing clones required 40 h to double
in number. Transfection of the same vector expressing the calcium
binding protein calbindin had no effect on cell growth (data not
shown), suggesting that inhibition by TOE1 was not a nonspecific effect
of protein over expression. Similar results were obtained in H4 cells
(data not shown).
Cell growth inhibition in TOE1-expressing cells was also examined by
performing colony forming assays. Control cells formed numerous rapidly
growing colonies, whereas TOE1-expressing cells were only able to form
30% as many colonies (data not shown). To determine whether the
decrease in cell growth of TOE1-expressing cells represented a
generalized slowing of growth or a cell cycle stage-specific slowing,
we performed flow cytometry on log phase cells. We found a significant
increase in the fraction of cells present in the G2/M
phases of the cell cycle in TOE1-expressing cells (27%), compared with
the control cells, with 13% of the cells in this fraction (Fig.
5B). We found no difference between the mitotic index of
control and TOE1-expressing cells, suggesting that TOE1 was pausing the
cells in the G2 phase (data not shown). In addition, it
should be noted that we found TOE1 expression to be highly influenced
by the growth state of the cells. Specifically, we have found TOE1
expression to be regulated by cell culture density, possibly indicating
a form of activation caused by contact inhibition.2 The expression
of TOE1 in dense cell cultures occurred even in cells that cannot
express Egr1, indicating that although Egr1 can activate expression of
TOE1, the gene must be subject to additional forms of regulation.
TOE1 Causes an Increase in p21 Expression in H4 Cells--
To
investigate the mechanism of TOE1 induced G2 phase delay,
we performed Western blotting on several G2 cell cycle
markers. Fig. 6A shows that
there was no significant change in cyclin B1, cdc2, or phospho-cdc2
levels between control, TOE1, and mutant TOE1-expressing cells (with
the nuclear localization deleted). This suggested that the activation
potential of the G2-specific CDK complex was unaffected by
the expression of TOE1. We therefore examined the possibility that the
activity of the complex might be modulated by its known inhibitor p21.
The level of p21 was dramatically up-regulated in TOE1-expressing cells
but not in either control or TOE1 mutant cells. Because p53 is a known
transactivator of the p21 gene, we examined the level and activation of
p53 in our cells. We were unable to find a significant induction or
activation of p53, at least insofar as serine 15 phosphorylation is
concerned. Further exploration of the induction of p21 using RT-PCR
showed that TOE1-expressing cells up-regulated p21 at the mRNA
level (Fig. 6B). This activation was not seen in cells
expressing non-nuclear mutant TOE1. To demonstrate that the increase in
p21 was functionally associated with an effect on cdc2 activity, we
immunoprecipitated cyclin B1 and measured the associated kinase
activity in vitro with histone H1 as substrate. Fig.
6C shows a significant decrease in kinase activity only in
TOE1-expressing cells, correlating with increased p21 expression in
those cells.
Increased TGF- With these studies we report, for the first time, the application
of chromatin immunoprecipitation to cDNA cloning using a form of
multiplex PCR. We have demonstrated that this technique was successful
not only in cloning transcription factor target genes but also in the
identification of a new target for Egr1. Together our results indicated
that the multiplex amplification produced a genuine cDNA and that
the cloned DNA represented an expressed gene. This newly cloned gene
encodes a 510-amino acid protein that we have shown to be an authentic
Egr1 target gene. To confirm that the gene codes for an endogenously
expressed protein, we have recently raised a polyclonal antibody using
a synthetic peptide epitope derived from the predicted amino acid
sequence. Preliminary testing has shown reactivity against both
recombinant and an endogenous protein of identical molecular mass,
suggesting that the cDNA is expressed at both the mRNA and
protein level.
During the course of these studies an unpublished and unnamed cDNA
generated through a library sequencing effort was deposited in the
GenBankTM data base that was identical to our cloned
cDNA (nucleotide accession number AK024011). Based on the sum of
our observations, we have called this cDNA the HUGO approved name
and symbol TOE1 for target of
Egr1. Expression of TOE1 was detected
in all of the adult human tissues examined but at varying levels,
indicating that the regulation of this gene may vary depending on cell
or tissue type.
Examination of the sequence of TOE1 did not reveal conserved domain
structures apart from a single potential zinc finger and a possible
nuclear localization signal. Immunostaining confirmed that TOE1 was
found localized to the nucleoplasm and nucleolus. Despite the absence
of a recognized DNA-binding domain, we have examined the possibility
that TOE1 might participate in transcriptional regulation. However,
TOE1 cloned as a GAL4 fusion failed to activate a GAL4-binding site
reporter, suggesting that TOE1 alone is not sufficient for
transcriptional regulation. The possibility remains that TOE1 can
participate in transcriptional regulation through protein interactions
and indirect DNA association not recapitulated in the GAL4 fusion
experiments. Although no extended homology to any known gene was noted
by BLAST searches, a limited region of homology to poly(A)-specific
deadenylation nuclease was revealed. We are currently investigating the
possibility that TOE1 may function as a nuclease.
To better understand the biological role of TOE1, we examined the
effects of its expression and noted a dramatic decrease in both the
growth rate and colony growth of H4 cells. We found that this was not
the result of a general decrease in growth rate but rather was due to a
G2 cell cycle phase delay. Furthermore, the
G2-specific cell cycle delay correlated with an increase in the expression of the cyclin-dependent kinase inhibitor
p21. Deletion of the nuclear localization signal abrogated this effect,
suggesting not only that TOE1 could induce cell cycle-specific
G2 pausing but also that its nuclear/nucleolar localization
was critical for this function. The localization of TOE1 in the
nucleolus may provide further evidence for a role in cell cycle
regulation because it has been found that many important cell cycle
proteins can be found in the nucleolus as a means of sequestration,
thereby limiting their function until the appropriate time
(22-24).
Because p21 is also able to inhibit cyclin-dependent kinase
activities controlling passage through the G1 restriction
point, it would be predicted that the TOE1-directed increase in p21
levels would also display a G1 phase pausing. Although we
did not see this in log phase growing cells, when cells were
synchronized in the M phase and then released to pass through
G1, we noted a marked delay in the TOE1-expressing cells
(data not shown). This suggested that the increase in p21 levels was
also active at the G1 check point, but this was only seen
if cells had been synchronized outside of the G2 phase.
Although p21 is well known for its activity in G1 phase
pausing, its role in G2 is being increasingly recognized
(25, 26). These results suggest that the mechanism by which TOE1
affects cell growth is through transcriptional up-regulation of the p21
gene. We have not, however, formally ruled out the possibility that the
increase in p21 levels might be due to an increase in transcript
stability rather than increased expression. Also, we have not
completely ruled out a contributing role for p53 in the up-regulation
of p21 but have demonstrated that p53 levels and serine 15 phosphorylation were not altered. Further, we have provided evidence
that TOE1-dependent TGF- Finally, It is intriguing to note that the chromosomal location of
TOE1 maps to 1p34.1-35.3. Deletion of the distal portion of
1p accounts for a significant proportion of chromosome 1 aberrations and has been observed in brain, breast, ovarian, colorectal, and other
tumor types (27-29). Combined data suggest that chromosome 1p likely
harbors one and possibly multiple tumor suppressor genes, and given the
growth inhibitory effect of TOE1, we are currently investigating the
possibility that TOE1 may also function in this capacity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1,1 platelet-derived
growth factors A and B, basic fibroblast growth factor, tissue
factor, interleukin 2, and CD44 to mention only a few (reviewed
in Ref. 5). These studies have focused on the in vitro
analysis of an individual target gene in a specific cell type under a
defined set of experimental conditions. As a step toward a more
complete understanding of the biological role for a transcription
factor, it would be informative to be able to identify in
vivo target genes.
1.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, and cyclophilin.
The TGF-
primers used were 5'-GGGCTGAAGGGACCCCCCTC-3' and
5'-TCCTCGGCGACTCCTTCCTC-3'. The cyclophilin primers used were 5'-CTCCTTTGAGCTGTTTGCAG-3' and 5'-CACCACATGCTTGCCATCC-3'.
-galactosidase DNA for normalization.
24 h after transfection, the luciferase assays were performed as
described (8).
20 °C until all of the samples were
collected. The cells were collected by centrifugation at 2000 × g for 3 min, and the cell pellets were suspended in
phosphate-buffered saline, digested with RNase A, and stained with
propidium iodide.
1 expression was assessed using the primers 5'-GCCCTGGACACCAACTATTGCT-3' and
5'-AGGCTCCAAATGTAGGGGCAGG-3', and cyclophilin A was amplified
using the primers 5'-CTCCTTTGAGCTGTTTGCAG-3' and
5'-CACCACATGCTTGCCATCC-3'. PCR conditions were 95 °C for
30 s, 56 °C for 30 s, and 72 °C for 1 min for 25 cycles.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Cloning and characterization of the newly
identified Egr1 target gene TOE1. A, multiplex
PCR amplification of Egr1 target genes from a NT2 cDNA library.
Linker PCR amplification products of DNA from Egr1 containing
immunoprecipitates were used as multiplex primers in a PCR reaction
containing a NT2 cell cDNA library template as well as the T7 3'
primer. cDNA amplification products are seen in lanes 2 and 5, where all components are present. Both control and
UV-treated E9 cells produced PCR products. Lane M,
1-kilobase pair DNA markers. B, multiple tissue Northern
blot hybridized with a TOE1 probe shows expression of an
approximate 2-kb message in adult human tissues. The nucleotide sizes
are indicated to the left. Br, brain;
H, heart; Sm, skeletal muscle; C,
colon; Th, thymus; Sp, spleen; Ki,
kidney; Li, liver; SI, small intestine;
Pl, placenta; Lu, lung; PL, peripheral
leukocytes.
was also amplified from E9 cells (21). The lack of
amplification of cyclophilin sequence served as a negative control.
This provided evidence that TOE1 was indeed a target of Egr1
in these cells and that the immunoprecipitated DNA included the 5'
region of the gene. Because E9 cells constitutively overexpress Egr1,
we sought to determine whether TOE1 is an Egr1 target in an alternate
cell type upon transient Egr1 induction. MCF7 cells were stimulated to
express Egr1 by 12-O-tetradecanoylphorbol-13-acetate
treatment, and then the ChIP assay was performed on untreated or
12-O-tetradecanoylphorbol-13-acetate-treated cells. The
results shown in Fig. 2A, TOE1 was also an Egr1 target gene
in these cells. To determine the role of Egr1 in regulating the
transcription of TOE1, we used RT-PCR following transfection with an increasing amount of an Egr1 expression vector and found a
proportional increase in TOE1 expression (Fig. 2B).
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Fig. 2.
Egr1 binds to the 5' region of
TOE1 and activates its expression. A, PCR
amplification of the TOE1 5' region from cross-linked
chromatin. Either total cross-linked chromatin (Input) or
Egr1 immunoprecipitates (Egr1 i.p.) were screened directly
for the presence of TOE1 5' sequences by PCR using primers
designed to amplify a 580-bp fragment 5' of the initiation codon. The
same samples were also used for amplifications using primers for
TGF- 1 and cyclophilin A. The same primers were used to analyze Egr1
immunoprecipitates from untreated or
12-O-tetradecanoylphorbol-13-acetate-treated MCF7 cells.
B, Egr1 expression activates TOE1 expression.
RT-PCR amplification of TOE1 from Egr1 transfected H4 cells. Increasing
amounts of Egr1 (shown above the lane) were transfected into H4 cells,
and total RNA was prepared 24 h later to perform RT-PCR for
TOE1. Primers within the coding sequence of TOE1
were designed to amplify a 454-bp product. An equal RNA loading in the
RT-PCR reaction was determined using primers amplifying
glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
C, Egr1 binds directly to the TOE1 5' region. The 580-bp
region upstream of the initiation codon of TOE1 was used as
a probe in the gel shift assay. Increasing amounts of recombinant Egr1
showed the binding to this region. Specific binding was determined by
adding either unlabeled homologous probe DNA or nonspecific DNA at a
50-fold molar excess. The positions of the free probe (FP)
and Egr1 shift (Egr1) are indicated. D, Egr1
transactivates expression from the TOE1 5' region. The same
580-bp 5' sequence from TOE1 was cloned into the pGL3basic luciferase
reporter. Empty reporter vector or the TOE1 reporter in the presence or
absence of co-transfected Egr1 expression vector were transfected into
293 cells. 24 h later the cells were harvested and analyzed for
luciferase activity. The results have been normalized for transfection
efficiency as determined by
-galactosidase measurements. The results
are plotted as the average values ± standard deviations. The
experiment was repeated three times with similar results.
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Fig. 3.
TOE1 is a nuclear/nucleolar protein.
Control vector and a FLAG-tagged TOE1 expression vector were
transfected into H4 cells. The cells were immunostained with antibodies
to FLAG and to the nucleolar protein nucleolin. Texas Red and
fluorescein isothiocyanate-labeled secondary antibodies were used to
label nucleolin and FLAG, respectively. Confocal microscopy was
performed showing nucleolar co-localization of TOE1 and nucleolin. The
bar in each panel represents 10 microns.
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Fig. 4.
Identification of TOE1 nuclear localization
sequence. H4 cells were transfected with either a FLAG-tagged wild
type TOE1 expression vector (left panel) or a FLAG-tagged
TOE1 expression vector containing a deletion in the putative nuclear
localization sequence (right panel). Following fixation, the
cells were subjected to immunostaining using anti-FLAG
(red). For the cells expressing the TOE1 nuclear
localization sequence deletion, the nuclei were counterstained with
4',6-diamidino-2-phenyl.
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Fig. 5.
TOE1 expression affects cell growth and the
cell cycle. A, TOE1 decreases the growth rate of 293 cells. Pooled clones of empty vector or TOE1-expressing
cells were used to determine their growth rate over a period of 5 days.
Solid squares, control transfected cells; solid
triangles, TOE1-expressing cells. The results are the
averages of triplicate readings, and the experiment was repeated three
times with similar results. B, TOE1 expression
affects the cell cycle. The cell cycle distribution of log phase
growing control and TOE1 expressing clones of H4 cells was determined
by flow cytometry. The calculated percentages of the cell cycle phases
are indicated.
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Fig. 6.
TOE1 affects growth inhibition through
increased p21 expression. A, control, TOE1, and TOE1 NLS
cells were probed by Western blotting with the indicated antibodies.
B, RNA was extracted from cells, and RT-PCR was performed
for the expression of p21 and glyceraldehyde-3-phosphate dehydrogenase
(GAPDH). C, in vitro phosphorylation
assay. Cyclin B1 immunoprecipitates were incubated with histone H1 and
radiolabeled ATP. The products were visualized by SDS-PAGE and
autoradiography.
1 in TOE1-expressing cells--
Because Egr1
expression is known to affect TGF-
1 levels (21), we sought to
determine whether the increase in p21 levels might be mediated by
TGF-
1. Using real time quantitative PCR, we examined the TGF-
1
levels in cells transfected with a TOE1 expression vector. As shown in
Fig. 7, using both MCF7 and H4 cells
lines, we noted an increase in the level of TGF-
1 mRNA in TOE1
transfected cells compared with control transfected cells.
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Fig. 7.
TOE1 expression affects the levels of
TGF- 1 mRNA. A, RT-PCR
measurement of TGF-
1 and TOE1 from MCF7 and H4 cells transfected
with an empty vector control (
) or a TOE1 expression vector (+).
24 h after transfection, RNA was harvested from the cells, and
RT-PCR was performed using the protocol described under "Materials
and Methods" with 25 cycles of amplification. Cyclophilin A
amplification was used to demonstrate the equal RNA amounts included in
each reaction. B, real time quantitative PCR was performed
on MCF7 and H4 cells transfected with either control empty vector or a
TOE1 expression vector. 24 h following transfection, RNA was
collected and subjected to real time PCR for TGF-
1 mRNA
quantitation. The open bars represent the relative quantity
of TGF-
1 level in control cells, and the closed bars
represent that for TOE1 transfected cells. mRNA samples were
normalized to cyclophilin A levels. The results shown are the averages
of four independent experiments showing standard deviations.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 activation may participate in
the increase in p21. However, it also remains possible that TOE1 and
p53 cooperate in the transactivation of p21 either directly or
indirectly. We have preliminary evidence that TOE1 and p53 are able to
interact physically, but the significance and specificity of this
interaction remain to be
analyzed.3 Although the
precise mechanism of action remains to be studied, our results have
shown that expression of TOE1 leads to growth inhibition as well as a
decrease in colony forming ability, likely involving the activation of
p21. Given that these same features are seen following expression of
Egr1, we expect that the downstream target TOE1 plays an important role
in executing this physiological function of Egr1 in its proposed role
as a tumor suppressor.
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ACKNOWLEDGEMENT |
---|
We thank Dr. Erkki Ruoslahti for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants CA67888 (to E. D. A.) and CA76173 (to D. M.), by University of California Grant TRDRP 9KT-0078 (to I. d. B.), by United States Army fellowship DAMD17-99-1-9092 (to I. d. B.), and by a grant from the American-Italian Cancer Foundation (to S. S.).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.
Ian de Belle and Sabina Sperandio dedicate this manuscript to the memory of Ted and Marilyn Crain.
§ To whom correspondence should be addressed. Tel.: 858-646-3100 (ext. 3650); Fax: 858-646-3198; E-mail: idebelle@burnham.org.
Published, JBC Papers in Press, January 31, 2003, DOI 10.1074/jbc.M210502200
2 I. de Belle and J.-X. Wu, unpublished observation.
3 I. de Belle, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: TGF, transforming growth factor; ChIP, chromatin immunoprecipitation; RT, reverse transcriptase.
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REFERENCES |
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