From the
The 5`-upstream sequence of the phospholipase C-
Many kinds of extracellular stimuli trigger the target cell
responses through activation of phospholipase C (PLC)
As
expected from their sequence differences, the various PLC isozymes
appear to be activated by distinct groups of receptors through
different mechanisms(3) . PLC-
PLC-
The
transcriptional rate of each gene is largely controlled by the
interactions of various regulatory factors to the transcriptional
control regions or by the modification of regulatory
factors(22) . Therefore, identification and characterization of
these cis- and trans-acting factors are needed to understand how
transcription of a gene is regulated. We have previously reported the
cloning of the promoter of PLC-
In this study, the GPE1 region,
which might be important for the transcriptional regulation of the
PLC-
The expression of the PLC-
By DNase I footprinting
and EMSA, the proteins bound to GPE1 were identified and their binding
sites were determined. Formation of four different complexes with F1
probe was observed and named complexes A, B, C, and D. Complex B
appeared to be formed by interaction of the protein to the sequence,
-533 GGAGGGGGCG -524, designated as GES1. Complexes C and D
seem to be generated with the same DNA sequence, -512 TGTCACTCA
-504, designated as GES2. The two GES2 binding proteins might be
two different proteins derived from different genes or two different
forms of a protein from a single gene. The two GES2 binding proteins
shared several physical properties, including optimal temperature and
optimal salt concentration for DNA binding, and thermostability (data
not shown). It is, therefore, more likely that GES2 binding proteins
are the products of a single gene, which could be generated by an
alternative splicing or protein modification.
The complex A is
considered to be a multicomplex, which includes GES1 and GES2 binding
proteins, by the following reasons: 1) complex A was detected only
under the conditions that both GES1 and GES2 sites are occupied.
Complex A disappeared when binding of either GES1 or GES2 was hindered (Fig. 3A). 2) When the amount of nuclear protein
increased gradually in EMSA, more complex A was formed. However, the
formation of complex B, C, and D reached the peak at 5 µg and then
decreased with additional nuclear proteins (data not shown). 3)
Inhibition of the protein binding to GES1 site by competition with F1a
increased complexes C and D, compared with the reaction without
competitors (Fig. 3A). Inhibition of the binding on GES2
site, on the other hand, increased formation of complex B.
GES1 site
is similar to AP2 and Sp1 sites. Purified Sp1 proteins recognized the
GES1 site, and GES1 binding protein also bound to the oligonucleotide
containing the Sp1-binding site. An oligonucleotide containing the
AP2-binding site, on the contrary, was not recognized by GES1 binding
protein. These data suggested that C2C12 nuclear protein factor binding
to the GES1 site shared sequence specificity with Sp1 protein. Sp1
enhances the transcription of epidermal growth factor
receptor(35) , and in cases with the overexpression of Sp1, the
mRNA of epidermal growth factor receptor was expressed in high levels
in human gastric carcinomas(36) . Interestingly, it has been
shown that PLC-
On the other hand, although the GES2 site is
weakly homologous to the AP1 site, the GES2 binding protein did not
bind to the AP1 site. This result suggests that GES2 binding protein
might be a novel DNA-binding protein. The identity of GES3 binding
protein is also obscure. As the GES3 site is similar to the CCAAT box,
it could be one of the CCAAT box binding proteins or could be a novel
kind of transcription factor. Among CCAAT box binding proteins, NF1/CTF
does not seem to be the GES3 binding protein, since the GES3 binding
protein was not able to bind to the NF1/CTF site.
GPE1 region could
stimulate the transcription from the heterologous promoter, E1b-TATA
box (Fig. 1), even though the promoter of PLC-
The function of each protein binding site at the GPE1
region was assessed by site-directed mutagenesis. GES3 alone can
activate the transcription from the PLC-
Recent studies showed that human
primary breast carcinomas and colorectal carcinomas contained
considerably higher levels of PLC-
In conclusion, we have identified and
analyzed at least three sites (GES1, GES2, and GES3), to which several
transcription factors bind, in the GPE1 region of PLC-
We thank Drs. C. B. Chae and S. K. Jang (POSTECH) for
critical review of this manuscript, and Dr. S.-J. Kim (NIH) for advice.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
1
(PLC-
1) gene contains several transcriptional regulatory regions.
We have studied one of the regions (-551 to -480, named
GPE1) which exhibits a strong positive regulatory activity. GPE1
stimulated the transcription when fused to heterologous TATA element in
an orientation-dependent manner. The region between -536 and
-470 was identified as the protein binding site in GPE1 by the
DNase I footprinting method. Electrophoretic mobility shift assays with
several competitors revealed three protein binding sites in this
region, designated as GES1, GES2, and GES3. The binding sites were
-535 GGAGGGGGCG -524, -512 TGTCACTCA -504, and
-491 CAATCCA -485, respectively. Mutational analyses
suggested that GPE1 binding proteins cooperate with each other to
activate the transcription of the PLC-
1 gene. Additionally,
immunoblot analyses revealed that the level of PLC-
1 expression
was considerably higher in 9 of 11 colorectal carcinomas than in
adjacent normal colorectal tissues. In 7 of 9 cases of colorectal
carcinomas which express higher level of PLC-
1, the DNA binding
activities to GES1, GES2, and GES3 sites also increased when compared
with normal tissues. These results suggest that the GPE1 binding
proteins might be attributed to the elevated expression of PLC-
1
in colorectal carcinomas and may play important roles in proliferation
of colorectal carcinoma cells.
(
)which hydrolyzes phosphatidylinositol
4,5-bisphosphate into two second messengers, inositol
1,4,5-trisphosphate and diacylglycerol(1, 2) . At least
nine distinct isozymes were cloned from mammals. Comparison of the
deduced amino acid sequences has indicated that PLCs are divided into
three types (PLC-
, PLC-
, and PLC-
) and each type
contains more than one subtype(1, 3) . Although there
are two regions with significant similarity in amino acid sequence,
designated as X and Y, the different PLC types have characteristic
features in their primary structures(1) . Especially, src homology domains, SH2 and SH3, and split pleckstrin homology
domains reside between X and Y regions of the PLC-
type(4, 5, 6) . These domains have been found in
a growing number of proteins that are involved in the regulation of
cell proliferation and differentiation(6, 7) .
is known to be activated by
G
q class (8, 9) as well as
subunit of
G-protein(10, 11) . PLC-
, on the other hand, is
activated through a direct interaction with growth factor receptor
tyrosine kinases, such as epidermal growth factor receptor,
platelet-derived growth factor receptor, and fibroblast growth factor
receptor(3) .
1 was also suggested to be involved in
the process of cellular transformation: (a) microinjection of
PLC-
1 into NIH 3T3 cells caused a dose-dependent transformation,
but injection of anti-PLC-
1 antibody blocked the serum- or
ras-induced transformation(12, 13) ; (b)
PLC-
1 has been found to be overexpressed in human breast
carcinomas(14) , human colorectal cancer (15), familial
adenomatous polyposis(16) , and human skins in
hyperproliferative conditions(17) . In addition,
immunohistochemical studies suggested that expression of PLC-
1 was
regulated during development and differentiation(18) . The
expression of PLC-
1 changed during the differentiation of
F9(19) , C2C12(20) , and U937 cells(21) . Although
the expression of PLC-
1 was shown to be linked to various
physiologic events such as cell growth and differentiation, little is
known about the mechanisms controlling PLC-
1 expression.
1 gene(23) . Deletion
analysis identified several regions which affect transcriptional
activity of the promoter: positive regulatory regions from -551
to -480 (GPE1) and from -90 to -52 (GPE2), a negative
regulatory region from -371 to -305 (GNE1) relative to the
transcriptional initiation site.
1 gene during the physiologic events such as proliferation,
has been further characterized. DNase I footprinting and
electrophoretic mobility shift assay revealed three protein binding
sites (GES1, GES2, and GES3) in this region. Mutational analyses of the
protein binding sites revealed that GES1, GES2, and GES3 binding
proteins cooperate each other to activate the transcription through the
GPE1 region. GES1, GES2, and GES3 binding proteins seemed to be
overexpressed in colorectal carcinomas, suggesting that these proteins
might be attributed to the elevated level of PLC-
1 in cancer
tissues.
Cell Culture Conditions
C2C12 cells (mouse
skeletal myoblast line) were maintained at low density to prevent
fusion in Dulbecco's modified Eagle's medium (Life
Technologies Inc., Gaithersburg, MD) supplemented with 15% fetal bovine
serum (HyClone Laboratories Inc., Logan, UT) and 1 mM sodium
pyruvate (Life Technologies Inc.). Cells were grown in a humidified
incubator at 37 °C in 5% CO.
Construction of CAT Plasmids
The XhoI-ApaI fragment which contains the GPE1 region was
isolated from pPLC551CAT plasmid (23) and blunt-ended with
T4 DNA polymerase (New England Biolabs Inc., Beverly, MA). It was then
inserted into the upstream E1b-TATA box of E1bCAT (24) in both
orientations and named pGPE1F and pGPE1R. The construction of
pPLC
551CAT and pPLC
497CAT was described by Lee et al. (23). Oligonucleotides, F1, F2, F2M, were inserted into the
upstream E1b-TATA box of E1bCAT. F1 was inserted into the XhoI
site. F2 and F2M were inserted into the HindIII site. The
resulting plasmids were named pF1, pF2, pF2M, respectively. F1
oligonucleotide was inserted into the XhoI site of pF2 to
construct pF1F2.
DNA Transfection and Chloramphenicol Acetyltransferase
(CAT) Assay
Reporter CAT plasmids were transfected into C2C12
cells (approximately 30-50% confluent) by the calcium phosphate
coprecipitation method(25) . Ten µg of plasmid DNA was
cotransfected with 3 µg of pCH110 (Pharmacia LKB Biotechnology
Inc., Uppsala, Sweden), a -galactosidase expression vector which
was used as an internal control to normalize transfection efficiency.
After exposure to the DNA precipitate for 12-16 h, cells were
washed and fresh media was then added. The cells were harvested 48 h
later, and CAT activities were measured in the cell
lysates(26) . One-tenth of cell lysate was used to determine
-galactosidase activity. For each CAT reaction, cell extract was
combined with 4 µl of [
C]chloramphenicol
(Amersham Corp., Buckinghamshire, United Kingdom, 55 mCi/mmol, 25
µCi/ml), 10 µl of 20 mg/ml acetyl-coenzyme A, and 250 mM Tris, pH 7.8, to a final volume of 150 µl. The mixtures were
incubated at 37 °C for 2 h, and the reactions were stopped by
extraction with 1 ml of ethyl acetate. Acetylated and nonacetylated
chloramphenicol were separated by thin-layer chromatography.
Preparation of Nuclear Extract
Nuclear proteins
were extracted according to the procedure described by Dignam et
al.(27) . Cells were harvested in buffer A (10 mM HEPES, pH 7.9, 1.5 mM MgCl, 10 mM KCl, and 0.5 mM DTT) and lysed using Dounce homogenizer.
The homogenate was centrifuged to collect nuclei, and the pellet was
resuspended in buffer C (20 mM HEPES, pH 7.9, 25% glycerol,
420 mM NaCl, 1.5 mM MgCl
, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM DTT), incubated at 4 °C for 30 min with gentle stirring, and
centrifuged for 30 min at 15,000 rpm in a Sorvall SS-34 rotor. The
supernatant was dialyzed several hours against 200 volumes of buffer D
(20 mM HEPES, pH 7.9, 20% glycerol, 0.1 M KCl, 0.2
mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 0.5
mM DTT), frozen in liquid nitrogen, and stored at -70
°C.
Electrophoretic Mobility Shift Assay (EMSA)
From
the sequence around the footprinted region, double-stranded DNA
fragments were prepared by annealing of complement oligonucleotides.
The sequences of sense strands of the double stranded oligonucleotides
used in this study are: F1:
5`-CTGTGGGGAGGGGGCGTGGCGGCGTGCTGTCACTCACT-GCCC (-539/-498);
F1M: 5`-CTGTGGTTCATATCACTGGCGGCGTGCTGTCACTCACTGCCC; F1a:
5`-CTGTGGGGAGGGGGCGTGGC (-539/-520); F1b:
5`-TGGCGGCGTGCTGTCACTCACTGCCC (-523/-498); M1:
5`-TTTATGCGTGCTGTCACTCACTGCCC; M2: 5`-TGGCGGATGTCTGTCACTCACTGCCC; M3:
5`-TGGCGGCGTGCGTGAACTCACTGCCC; M4: 5`-TGGCGGCGTGCTGTCAAGACCTGCCC; M5:
5`-TGGCGGCGTGCTGTCACTCACGTAAC; F2:
5`-TGCCCGCTAGCCAA-TCCACGGGTGCGCCCCTCCCCGGAGAGGG
(-502/-460); F2M:
5`-TG-CCCGCTAGCGTCCGTTCGGGTGCGCCCCTCCCCGGAGAGGG; GES1:
5`-TGGGGAGGGGGCGTGG (-536/-521); GES2: 5`-TGCTGTCACTC-ACT
(-515/-502); GES3: 5`-TAGCCAATCCACG
(-495/-483); Sp1: 5`-ATTCGATCGGGGCGGGGCGAGC; AP2:
5`-GATCGAACTGACCGCCCGCGGCCCGT; AP1: 5`-CGCTTGATGAGTCAGCCGGAA; NF1/CTF:
5`-CCTTTGGCATGCTGCCAATATG. Mutated sequences are underlined and the
locations of the sequences in the PLC-1 gene are indicated at the
end of each sequence. Oligonucleotides were labeled with
[
-
P]dCTP by filling in reaction using
Klenow fragment. F1a and F1b were 5`-end labeled by T4 polynucleotide
kinase. The binding reactions for gel shift assay were preincubated for
5 min on ice in a total volume of 13 µl containing 12% glycerol, 12
mM HEPES, pH 7.9, 4 mM Tris, pH 7.9, 60 mM KCl, 1 mM EDTA, 1 mM DTT, 1 µg of
poly(dI-dC)
poly(dI-dc) as nonspecific competitor, and 1-2
µg of nuclear extract with or without specific competitor
indicated. After addition of
P-labeled probe (2
10
cpm), the mixtures were incubated for an additional 30
min at 15 °C, then loaded onto 5% polyacrylamide nondenaturing gels
and subjected to electrophoresis for 2 h at 150 V in 0.25
Tris
borate-EDTA buffer(28) .
DNase I Footprinting
A 510-base pair DNA fragment
corresponding to the positions from -644 to -135 in the
PLC-1 gene was 3`-end labeled with
[
-
P]dCTP using Klenow fragment on the
antisense strand. In the binding reaction, 20-50 µg of C2C12
nuclear extract and 5
10
cpm of
P-labeled probe were used in the same condition as EMSA in
a total volume of 100 µl. The reactions were chilled on ice and
treated with 1-2 units of DNase I (Ambion Inc., Austin, TX) in
100 µl of 2
assay buffer containing 80 mM Tris, pH
7.4, 12 mM MgCl
, 4 mM CaCl
,
and 100 mM KCl for 2 min on ice. DNase I reactions were
terminated by adding 50 µl of stop solution (1 mg/ml proteinase K,
0.5% SDS, 125 mM EDTA, and 0.25 mg/ml tRNA) and incubated at
55 °C for 1 h, and then extracted with phenol/chloroform (1:1,
v/v). Resulting DNA fragments were analyzed on 6% denaturing
polyacrylamide gels with M13 sequencing ladder as size marker. For the
footprinting with Sp1 protein, we used vaccinia-expressed and purified
human Sp1 protein (Promega Corp., Madison, WI).
Mutagenesis at Protein Binding Sites
To prepare
the templates in a phagemid form, the XhoI/HpaI
fragment of pPLC551CAT which contains the -551/+75
region of the PLC-
1 gene and CAT gene were inserted into
pBluescriptII KS
(Stratagene, La Jolla, CA) digested
by XhoI and EcoRV. The plasmid was named pKS551 and
used for preparing single stranded template. pKS479 was constructed
with the fragment from pPLC
479CAT by the same strategy. To
generate the mutations at protein binding sites in GPE1, the
oligonucleotides, F1M, M3, and F2M, were used as mutagenic
oligonucleotides for mutations at GES1, GES2, and GES3 sites,
respectively. Uracil-containing DNA templates were prepared by the
method of Kunkel(29) . Mutagenic oligonucleotides were
phosphorylated at 5`-ends by T4 polynucleotide kinase (New England
Biolabs Inc.) and annealed with templates. In vitro primer
extension was performed using 1 unit of T4 DNA polymerase (New England
Biolabs Inc.) and 3 units of T4 DNA ligase (Amersham Corp.) for 5 min
on ice, for 5 min at room temperature, and 90 min at 37 °C. After
the incubation, Escherichia coli strain DH5
was
transformed by 1/20 of the reaction mixture. Double and triple
mutations were generated using single and double mutated templates,
respectively. Mutations were confirmed by DNA sequence analysis.
Preparation of Whole Cell Extract from Tissue Specimens
and Immunoblot Analysis
Surgical total colorectomy specimens
were obtained from the Department of Surgery at the Seoul National
University Hospital and the Korea Cancer Center Hospital. Fresh
specimens of normal and neoplastic tissues were immediately frozen in
liquid nitrogen and stored at -80 °C for further study. For
preparation of whole cell extract, tissue specimens were minced with a
razor blade and resuspended in extraction buffer (20 mM HEPES,
pH 7.9, 0.35 M NaCl, 20% glycerol, 1 mM MgCl, 1% Nonidet P-40, 1 mM DTT, 0.5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml
leupeptin, 1 µg/ml aprotinin) and incubated on ice. After
centrifugation, supernatant was frozen in liquid nitrogen and stored at
-80 °C. For immunoblot analysis, the whole cell extract was
separated by 8% SDS-polyacrylamide gel electrophoresis. The separated
proteins were transferred onto nitrocellulose membranes and were probed
with the anti-PLC-
1 monoclonal antibody (B-16-5, 1
µg/ml) (30) for 4 h. Immunoreactive bands were visualized by
ECL system (Amersham Corp.) using peroxidase-conjugated goat anti-mouse
IgG + IgA + IgM antibody.
GPE1 Activates Heterologous Promoter in an
Orientation-dependent Manner
We have previously reported the
5`-region of the rat PLC-1 gene and demonstrated that it contains
promoter activity. The promoter regions important for gene expression
were found to be located at -551 to -480, -371 to
-305, and -90 to -52 base pairs from the
transcriptional initiation site, designated as GPE1, GNE1, and GPE2,
respectively(23) . The ability of GPE1 to activate a basic
heterologous promoter was investigated by inserting it upstream of the
E1b-TATA box which is a basic promoter of the adenovirus E1b gene. When
the activity was measured in C2C12 cells, GPE1 was observed to
stimulate the transcription directed by the E1b-TATA box (Fig. 1). This activity of GPE1 is orientation-dependent, since
the activation disappeared when it was reversely oriented.
Figure 1:
Effect of GPE1 on heterologous
promoter. GPE1 region was isolated from the PLC-1 gene and
inserted into the upstream E1b-TATA box in right (pGPE1F) and reverse
(pGPE1R) orientations. Transient transfection assays were performed in
C2C12 cells. For each transfection, 20 µg of plasmids were used and
after 48 h of expression, the CAT activities were measured. The graph
represents fold increase relative to the activity of E1bCAT. Similar
results were obtained from several independent
experiments.
Several Proteins Bind to GPE1 Region
Nuclear
extracts from C2C12 cells conferred a pattern of DNase I-protection
encompassing the region from -536 to -470 base pairs (Fig. 2A) which roughly matches with GPE1 region. Two
double-stranded oligonucleotides, F1 and F2 which correspond to the
sequences, [-539 to -498 and -502 to -460,
respectively, were used for the competition assays. F1 and F2 competed
for binding of nuclear proteins to the corresponding regions (Fig. 2B).
Figure 2:
Identification of GPE1 binding protein.
DNA fragment corresponding to the region -644/-135 of
PLC-1 gene was labeled at lower strand and used as a probe for
DNase I footprinting analysis. A, the probe was incubated with
20 or 50 µg of C2C12 nuclear extract, and then 1 or 2 units of
DNase I was treated for 2 min on ice. The resulting fragments were
analyzed on 6% denaturing acrylamide gel. Lanes A and C represent M13 sequencing ladder. The sequence of protected region
and its position are shown on the left. B, the cold
oligonucleotides (100-fold molar excess), F1 and F2, were added to the
binding reactions as competitors. The sequences of the oligonucleotides
are shown in Fig. 3.
To analyze the interactions of GPE1 with
nuclear factors in more detail, several oligonucleotides were designed
and used in electrophoretic mobility shift assay (Fig. 3). F1
covers the 5`-part of the DNase I-protected region and F1M contains a
mutation around the GC-rich sequence which is related to AP-2 and
Sp1-binding sites(23) . F2 covers the 3`-part of the protected
region and F2M contains a mutation in the CCAAT box-like sequence. In
the presence of C2C12 nuclear extracts, F1 forms four different
DNA-protein complexes, A, B, C, and D from the most slowly-migrating
complex as shown in Fig. 3A. Addition of excess
unlabeled F1 competitor abolished formation of the complexes with
labeled probe. To determine which regions of F1 are responsible for the
formation of the protein-DNA complexes, F1M, F1a, and F1b were used as
competitors. F1a and F1b correspond to the 5`- and the 3`-part of F1,
respectively. In the presence of F1M, only complex B was formed with
labeled F1 probe, which suggests that the sequence, -533
GGAGGGGGCG -524, is responsible for the formation of complex B.
Consistently, unlabeled F1a which contains the sequence, -533
GGAGGGGGCG -524, but not the 3`-part of F1 abolished the complex
B. On the other hand, F1b containing the 3`-part of F1 but not the
5`-part, sequestered the factors forming complexes C and D with the F1
probe. To further localize the site responsible for formation of
complexes C and D, P-labeled F1b probe and M1-M5
competitors were used in competition assay. Whereas F1b, M1, M2, and M5
worked as effective competitors for the F1b probe, M3 and M4
(especially M3) did not (Fig. 3B). These results suggest
that the factor(s) forming the complexes C and D bind to the sequence,
-512 TGTCACTCA -504. Only a single shifted band, complex E,
was detected in the assay with F2 probe (Fig. 3A).
Complex E persisted in the presence of excess unlabeled F2M, whereas
cold F2 itself effectively competed for the formation of complex E.
Therefore, as summarized in Fig. 4, the protein binding sites in
the GPE1 region appear to be -533 GGAGGGGGCG -524,
-512 TGTCACTCA -504, and -491 CAATCCA -485
which were designated as GES1, GES2, and GES3, respectively.
Figure 3:
Determination of the protein binding sites
in the GPE1 region. The sequences of the oligonucleotides which were
used as probes and/or competitors in EMSA are shown below. The mutated
sequences are shown in boxes. Two µg of C2C12 nuclear
extracts were incubated with 2 10
cpm of probe and
analyzed on 5% acrylamide gel. The DNA-protein complexes are indicated
by an arrowhead.
Figure 4:
Summary of protein binding sites in GPE1
region. The sequence of the protected region in DNase I footprinting is
displayed. The protein binding sites identified by EMSA are shown in boxes and the names of the binding sequences are shown above the boxes.
GES1 Binding Protein Might be a Member of the Sp1
Family
GES1 site is homologous to Sp1- and AP2-binding
sites(23, 31, 32) . Therefore, we examined
whether GES1 binding protein was able to bind to Sp1- or AP2-binding
sites by adding the oligonucleotide competitors containing Sp1- or
AP2-binding sequences to EMSA. Sp1 competitor abolished formation of
complex on the GES1 site (Fig. 5A), whereas GES2 and
GES3 binding proteins did not bind to the Sp1 site (data not shown).
The specificity of the interaction between GES1 binding protein and Sp1
sequence appeared to be high, since 50 molar excess of
competitors was enough for complete competition. In contrast, the AP2
sequence could not compete with the GES1 site (Fig. 5A).
It was also examined whether the Sp1 protein was capable of binding to
the GES1 site. As revealed by DNase I footprinting, purified Sp1
protein bound to the GES1 site in proportion to the amount of the
protein added (Fig. 5B). These results suggest that GES1
binding protein specifically binds to the Sp1 site and also that Sp1
protein binds to the GES1 site.
Figure 5:
Sp1 binds to GES1 site. A, EMSA
with various competitors. Two µg of C2C12 nuclear extract were
preincubated without or with the indicated amounts of the
oligonucleotides which contain Sp1, AP2, AP1, or NF1/CTF binding
sequences before adding the labeled probes. The amount of competitor is
written in molar excess. B, DNase I footprinting with purified
Sp1 protein. The -644/-135 probe described in Fig. 3 was
incubated with the indicated amounts of Sp1 proteins. According to the
manufacturer, 1 fpu (footprint unit) is the amount of protein required
to give full protection against DNase I on SV40 early promoter DNA.
After digestion with DNase I, the fragments were analyzed on 6%
sequencing gel. The sequence of the protected region is shown on the left and the GES1 site is underlined.
GES2 site has weak homology to AP1
site(33) . However, in the competitive EMSA, GES2 binding
protein was not able to bind to AP1 site, indicating that GES2 binding
protein may not be related to a family of AP1 transcription factor (Fig. 5A). On the other hand, the GES3 site is
homologous to CCAAT box. From the EMSA using the binding site for
NF1/CTF, one of the CCAAT box binding protein(34) , as a cold
competitor, it was proved that GES3 binding protein did not bind to the
NF1/CTF site (Fig. 5A).
GPE1 Binding Factors Seem to Cooperate with Each Other
Functionally
In order to understand the function of each protein
binding in the GPE1 region, the effects of mutations at the protein
binding sites were assessed in C2C12 cells (Fig. 6). For
site-directed mutagenesis, pKS551 plasmid was used as a template (see
``Materials and Methods''). Transcriptional activities were
denoted as percentage of that of pKS551. The activity of pGES123 which
has mutations at all protein binding sites in GPE1 was 20.2% relative
to that of pKS551. This is consistent with the fact that the relative
activity of pKS479 to pKS551 is 25.2%, implying that GES1, GES2, and
GES3 sites can represent the whole GPE1 region. Mutation at the GES1
(pGES1) or GES2 (pGES2) sites did not reduce the transcriptional
activity, whereas GES3 mutation (pGES3) resulted in a apparent
reduction in CAT expression. In the case of the double mutation in
which the GES3 site was mutated together with GES1 (pGES13) or GES2
(pGES23) sites, the transcriptional activity of the GPE1 region was
completely abolished even though GES2 or GES1 sites still remained
intact in each case. On the contrary, pGES12 which has mutations at
GES1 and GES2 sites showed twice as high activity as pGES13 or pGES23.
From these results, it seems that the GES3 site and at least one of the
GES1 and GES2 sites are required for ultimate activity of the GPE1
region. Therefore, transcriptional activation function of the GPE1
region might arise from functional cooperation of the GPE1 binding
proteins identified in this study.
Figure 6:
Mutational analysis of protein binding
sites in the GPE1 region. The GPE1 region of each plasmid is displayed
on the left. Wild-type protein binding sites are shown as open boxes and mutated binding sites are shown as hatched
boxes. The plasmids were transfected into C2C12 cells and relative
CAT activities are shown on the right. -Galactosidase
expression vectors were cotransfected to normalize the transfection
efficiencies. The activities are expressed as percentage to that of
pKS551 where it is defined as 100. The mean values of at least five
independent experiments are shown.
The functions of GPE1 binding
proteins were further analyzed by oligonucleotide reconstitution in the
upstream basic promoter, E1b-TATA box (Fig. 7). F2, which
contains GES3 site, showed stimulated transcription from E1b-TATA box
about 17-fold as compared to E1bCAT, whereas F1 which possesses GES1
and GES2 sites showed no effect. The stimulation by F2 was eliminated
by the mutation at the GES3 site, which confirmed the involvement of
GES3 binding protein in transcriptional activation. Moreover, pF1F2 was
even more active than pF2, indicating that the activating function of
F2 was further potentiated by F1 oligonucleotide. These results imply
the functional interactions among GPE1 binding proteins.
Figure 7:
Oligonucleotide reconstitution analysis.
The oligonucleotides, F1 which has GES1 and GES2 sites, and F2 which
has GES3 site, were inserted in the upstream E1b-TATA box. The
structures of the plasmids were displayed at the left. These
plasmids were transfected into C2C12 cells. The resulting CAT
activities are shown at the right. Transfection efficiencies
were normalized by the activities from cotransfected
-galactosidase expression vector. The result shown here is
representative of three independent
experiments.
GES1, GES2, and GES3 Binding Proteins Were Highly
Detected in Colorectal Carcinomas
In recent studies, it was
reported that human carcinomas contained higher levels of PLC-1
than normal cells (14, 15). Since the GPE1 region is a strong positive
transcriptional regulatory element, we examined the amount of proteins
bound to GES1, GES2, and GES3 sites in colorectal cancer and normal
tissue extracts to demonstrate whether PLC-
1 expression may be
related to GES1, GES2, and GES3 binding proteins. As observed in
previous studies by Noh et al.(15) , the content of
PLC-
1 was higher in 9 out of 11 cases of colorectal cancer tissues
than in adjacent normal tissues (Fig. 8). Electrophoretic
mobility shift assay revealed that the amounts of GES1, GES2, and GES3
binding proteins increased in 7 of 9 cases where PLC-
1 was
overexpressed, when compared with normal tissues. On the contrary,
there was no significant increase in PLC-
1 content in 2 out of 11
cases and these cases consistently showed no difference in the GES1,
GES2, and GES3 binding proteins compared with normal tissues (data not
shown). These results indicate that the increase of PLC-
1 content
seems to be well associated with the increase of GES1, GES2, and GES3
binding activities in colorectal cancer tissues.
Figure 8:
GES1, GES2, and GES3 binding activities in
human colorectal cancer tissues. A, Western blot analysis of
PLC-1 protein in colorectal cancer and normal tissues. Three
hundred µg of whole cell extracts were separated on 8%
SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose
membrane, and probed by monoclonal anti-PLC-
1 antibody
(B-16-5). Lanes N and C indicate normal and
cancer tissues, respectively. STD indicates the purified
PLC-
1 standard. B, GES1, GES2, and GES3 binding
activities were examined by gel retardation assays using the same
extracts shown in panel A. Fifteen µg of whole cell
extracts were used in each assay.
1 gene is regulated during
various physiologic events such as carcinogenesis and
differentiation(14, 15, 16, 17, 20, 21) .
However, the molecular mechanism underlying the regulation of
PLC-
1 expression is still obscure. In this study, we have
characterized the GPE1 region which is one of the transcriptional
regulatory regions upstream of the PLC-
1 gene along with the
nuclear factors that bind to this region.
1 was overexpressed and colocalized with epidermal
growth factor receptor in human breast cancer(14) . Based on
these findings and the fact that Sp1 can bind to the GPE1 region of the
PLC-
1 gene, it can be suggested that Sp1 may be a transcriptional
regulator of PLC-
1 gene as well as epidermal growth factor
receptor gene. However, several transcription factors which belong to
the Sp1 family have been cloned, and some of them share the binding
specificity with Sp1 protein (37-39). Therefore, it is also
possible that GES1 binding protein might be a member of the Sp1 family
rather than Sp1 itself.
1 gene is
TATA-less. Therefore, we propose that GPE1 is a general transcriptional
activating element. Moreover, activating function of GPE1 is nearly
abolished by orienting it reversely. This suggests elaborate
interactions among GPE1 binding proteins and basic transcriptional
machinery.
1 gene, whereas GES1 or
GES2 alone were not able to activate transcription by itself. However,
GES3 needs to cooperate with GES1 or GES2 for full activity, implying
that GES3 functionally cooperates with the other GPE1 binding proteins.
Therefore, it is likely that the minimum requirement for GPE1 activity
may be GES3 and at least one of GES1 and GES2. In other words, GES1 and
GES2 might be redundant for the transcriptional activity of GPE1 since
only one of them can make up ultimate activity of GPE1 region by
functional cooperation with GES3. In contrast, GES3 seems to be
necessary for full activity of GPE1 since mutation at the GES3 site
caused reduction of transcriptional activity, whereas mutations at GES1
or GES2 did not. The specific roles of these GPE1 binding proteins are
still obscure. One possible explanation is that the GES3 binding
protein may influence the transcription by direct interaction with
basic transcription machinery, whereas GES1 and GES2 binding proteins
may modulate the activity of GES3 binding protein. This explanation is
partly supported by the oligonucleotide reconstitution experiment (Fig. 7). F2 oligonucleotide containing GES3 site could stimulate
E1b-TATA box, whereas F1 oligonucleotide which possesses GES1 and GES2
sites did not augment the transcription by itself. However, F1
oligonucleotide was able to potentiate the transcriptional activation
function of F2 oligonucleotide.
1 than normal
tissues(14, 15) . However, the factors that cause the
overexpression of PLC-
1 in cancer cells remained uncovered. In
this study, we have identified the DNA binding proteins to GPE1, one of
the transcriptional regulatory regions of the PLC-
1 gene, and
examined the PLC-
1 expression and the amount of GPE1 binding
proteins in colorectal cancer tissues and adjacent normal tissues.
Immunoblot analysis and electrophoretic mobility shift assay revealed
the correlation between PLC-
1 expression and the content of GPE1
binding proteins in 9 out of 11 cases; GPE1 binding proteins increased
in 7 of 9 cases where PLC-
1 increased, and in the other 2 cases
where PLC-
1 did not increase, the amount of GPE1 binding proteins
was not changed either. These results suggest that the increase of GPE1
binding activities might be important for the overexpression of
PLC-
1 in cancer cells. The cooperation of GPE1 binding proteins
(GES1, GES2, and GES3 binding proteins) was suggested by mutational
analyses. Since all the binding proteins to GES1, GES2, and GES3 sites
were elevated in cancer tissues, the cooperation of GPE1 binding
proteins might occur in carcinomas to overproduce PLC-
1, which may
result in the amplification of the proliferation signal. The isolation
of each GPE1 binding protein might be an essential step to understand
the process of tumorigenesis.
1 promoter.
GPE1 binding proteins might cooperate with each other to confer
trans-regulation to PLC-
1 expression. The contents of these GPE1
binding proteins were higher in colorectal cancer tissues than in
normal tissues as was that of PLC-
1. Therefore, we propose that
the increase of GPE1 binding proteins and their cooperation might be
attributed to the overexpression of PLC-
1 in carcinomas.
1 gene
positive element; GNE, PLC-
1 gene negative element; SH, src homology; CAT, chloramphenicol acetyltransferase; DTT,
dithiothreitol; EMSA, electrophoretic mobility shift assay.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.