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INTRODUCTION |
In higher eukaryotes, CTP:phosphocholine cytidylyltransferase
(CCT)1 is a rate-limiting and
regulated enzyme in the synthesis of phosphatidylcholine (1-4). The
first mammalian CCT was purified from rat liver (5, 6) and was used for
isolation of the corresponding cDNA encoding a 367-amino acid
(CCT
) protein (7). The predicted CCT
structure consists of a
nuclear localization signal, a catalytic domain, a helical
lipid-binding domain, and a phosphorylation domain (1-4, 8-10).
Recently, cDNAs for two other isoforms, CCT
1 (10) and its
splicing variant CCT
2 (11), were cloned. All isoforms contain a
highly homologous catalytic domain and an amphitropic helical domain
that binds lipids (10). CCT
and CCT
2 also contain a highly
phosphorylated domain at their carboxyl terminus (1-4, 9, 11), whereas
CCT
1 lacks this domain. The roles of individual domains in the
regulation of CCT
enzyme activity have been extensively studied.
CCT
activity is modulated by the binding of specific lipids to the
helical domain (1-4, 12, 13) and by phosphorylation at the
carboxyl-terminal domain (1-4, 9, 14-17).
In addition to regulation at the protein level, CCT
is regulated at
the transcriptional and post-transcriptional levels in various cells
and tissues. CCT
mRNA was increased in colony-stimulating factor
1-stimulated macrophages (18), in hepatic tissues after partial
hepatectomy (19), and during growth and development (20). However, the
question of whether the elevation of CCT
mRNA levels was due to
an increase in transcription or due to stabilization of the produced
transcript needs to be further investigated (21-23).
Tang et al. (24) isolated the murine CCT
gene
(Ctpct) and showed that the exon/intron organization of the
gene closely resembles the functional domains of the enzyme. The gene
is transcribed from two transcriptional start sites. The 5' promoter
region lacks TATA/CAAT boxes, but contains GC-rich regions. Bakovic
et al. (25) demonstrated that three GC regions are
regulatory as follows: a "loose" Sp1 site at
31/
9, a cluster of
three overlapping Sp1 sites (
88/
50), and a canonical Sp1-binding
site (
148/
128). More recently, the same authors showed that
transcription factors Sp1 (26, 27), Sp2 (28), and Sp3 (28) can
competitively bind to these regions and that the promoter activity may
depend on the relative abundance of those three factors (29, 30).
To identify the regulatory elements responsible for CCT
transcription, Bakovic et al. (25) prepared various promoter
deletion constructs of Ctpct linked to the luciferase
reporter. Subsequent functional assays revealed the presence of regions
responsible for basal expression (
52/+38) as well as positive and
negative regulatory elements (
201/
90). Furthermore, gel-shift
assays indicated several unidentified proteins capable of binding to the region
103/
82, (GTTTTCAGGAATGCGGAGGTGG, Eb) and the neighboring region
130/
103.
In this study, we utilized the yeast one-hybrid system (31, 32) to
clone cDNA encoding Eb-binding proteins. One of them was identified
as a member of the transcription enhancer factor (TEF) family of
proteins, TEF-4 (33, 34). TEF-4 is closely related to TEF-1 that was
previously isolated as a regulatory protein of the SV40 enhancer (35).
We report that TEF-4 regulates expression of the CCT
gene in COS-7
and 3T3-L1 cells.
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EXPERIMENTAL PROCEDURES |
Materials--
MATCHMAKER Yeast One-hybrid System for screening
DNA-binding proteins, the Saccharomyces cerevisiae strain
YM4271, and the 11-day mouse embryo MATCHMAKER cDNA library
constructed in the pACT2 vector were purchased from
CLONTECH (Palo Alto, CA). The promoter-less
luciferase vector, pGL3-basic, carrying Photinus pyralis
luciferase, the control pRL-CMV vector, carrying Renilla reniformis luciferase, and the Dual-luciferase Reporter Assay System were obtained from Promega (Madison, WI). FuGENETM6
transfection reagent, Dulbecco's modified Eagle's medium, and fetal
bovine serum were from Roche Molecular Biochemicals, Sigma, and Life
Technologies, Inc., respectively. COS-7 and 3T3-L1 cells were obtained
from the Japanese Cancer Research Resources Bank (Tokyo, Japan).
3-Aminotriazole (3-AT) was from Sigma.
Construction of Plasmids and Yeast Strains Containing the Eb
Sequence--
The triple repeat of Eb (5'-GTTTTCAGGAATGCGGAGGTGG-3')
flanked by an EcoRI site at the 5'-end and by
StuI and XbaI sites at the 3'-end
(5'-GAATTCGTTTTCAGGAATGCGGAGGTGGGTTTTCAGGAATGCGGAGGTGGGTTTTCAGGAATGCGGAGGTGGAGGCCTCTCGAGTCTAGA-3'), and its complementary sequence, were synthesized by the DNA core facility at the University of Alberta. Two strands (500 pmol each) were
annealed in 100 µl of 25 mM Tris-HCl, pH 8.0, 0.5 mM MgCl2, and 25 mM NaCl by heating
to 70 °C for 10 min followed by slow cooling to room temperature.
The annealed sequence (3Eb) was digested and inserted between the
EcoRI and XbaI sites of the yeast pHIS and pLacZ
vectors (CLONTECH) to prepare pHIS-Eb and pLacZ-Eb
plasmids, respectively. The correct orientation of 3Eb was ascertained
by digestion with StuI and sequencing. pHIS-Eb and pLacZ-Eb
were linearized with AflII and NcoI,
respectively, and the linearized products were transformed into YM4271
yeast host. Cells were grown in SD/
His or SD/
Ura selective medium,
and stable integrants for pHIS-Eb and pLacZ-Eb were isolated and named
YHIS-Eb and YLacZ-Eb, respectively. The yeast strain YHIS-Eb was
transformed with the pACT2 mouse embryo cDNA library and incubated
in SD/
His/
Leu selective medium containing 45 mM 3-AT to
suppress completely background growth due to leaky HIS3
expression in this reporter strain. The pACT2 cDNA library
generates fusions of the cDNA for yeast GAL4 activation
domain, the cDNA for a hemagglutinin tag, and a cDNA of encoded
proteins. Positive one-hybrid yeast colonies were collected and
propagated in 1 ml of SD/
His/
Leu medium at 30 °C overnight.
pACT2 plasmids were then isolated using the YEASTMAKER plasmid
isolation kit (CLONTECH) according to the
manufacturer's instructions. The isolated plasmids were amplified
using Electrocomp Transformation Kit (Invitrogen, Carlsbad, CA) and
sequenced. A total of 19 positive pACT2 clones were obtained and
examined. Ten clones were identified to encode TEF-4, presumably an
Eb-binding protein, and two of the TEF-4 positive clones, named pY41b
and pY51b, were used for further investigation.
Colony Lift Filter Assay for
-Galactosidase
Activity--
YLacZ-Eb yeast was transfected with positive TEF-4
plasmids, pY41b and pY51b, or control plasmid pYcont (pACT2 vector) and cultured in SD/
Leu selective media. If bound to the Eb region of the
YLacZ-Eb promoter, TEF-4 expressed from pY41b and pY51b would expected
to initiate transcription of the yeast
-galactosidase. Formed
colonies were streaked on plates with the same selective media and
grown for 2 days. Filter paper was placed on the plates, lifted, frozen
in liquid nitrogen to break the cell walls, and then thawed on a second
filter paper impregnated with
-galactosidase substrate,
5-bromo-4-chloro-3-indol-
-D-galactopyranoside. The appearance of a blue color indicated a positive interaction of TEF-4
protein with the LacZ-Eb promoter. As a positive control for this assay
we used Y53BLUE yeast strain carrying the p53 binding domain in front
of the LacZ promoter transformed with the pGAD53m plasmid carrying the
p53 protein. As negative controls we used a combination of YlacZ-Eb
yeast with pGAD53m and pYcont plasmids or Y53BLUE yeast with pY41b and
pY51b plasmids.
Preparation of Deleted and Mutated CCT
Promoter-Luciferase
Reporters--
Various 5'-deleted CCT
promoter regions, LUC.C6
(
2068/
418), LUC.C7 (
1268/+38), LUC.C8 (
201/+38), LUC.D1
(
90/+38), LUC.D2 (
130/+38), and LUC.D3 (
52/+38), inserted into
the promoter-less luciferase vector pGL3-basic (Promega) were prepared
as described previously (25). To prepare mutated promoters, the ATG
core of the TEF-4 binding region (Eb) was mutated to GCT or AGC (36). These mutations were created from a single-stranded
1268/+38 promoter
isolated from LUC.C7 after ligation into pBluescript II SK (
) vector
(Stratagene, La Jolla, CA) as described (37). The mutated promoter was
either directly ligated back into pGL3-basic vector to prepare LUC.C7m
and LUC.C7m2 or digested before ligation to prepare LUC.C8m as reported
(25).
Construction of TEF-4 Mammalian Expression Plasmids and TEF-4
Deletion Mutants--
TEF-4 mammalian expression plasmid pcTEF-4 was
prepared by digesting pY41b plasmid, encoding the full-length TEF-4,
with EcoRI and XhoI and ligating the fragment
into pcDNA3.1/V5-His vector (Invitrogen, Carlsbad, CA). To prepare
a TEF-4 deletion mutant, the EcoRI/XhoI fragment
obtained from pY41b was ligated into pBluescript II SK(
) to obtain
TEF-4 SK(
). TEF-4 SK(
) was then digested with NcoI and
AatII, and the sticky ends were blunted by T4 DNA polymerase
and ligated to make a deleted TEF-4. This construct contained the
TEA-DNA binding domain, encoding the first 171 amino acids plus 5 additional amino acids, CTTRR, at its carboxyl terminus. The sequence
was then digested from the pBluescript vector and ligated into the
pcDNA3.1/V5-His mammalian expression vector to make a pcmTEA(+)
plasmid. To prepare a second TEF-4 mutant, TEF-4 SK(
) was digested
with BalI and BamHI and then the sticky ends were
blunted and ligated. This procedure generated a construct of TEF-4
without the TEA-DNA binding domain, i.e. lacking
amino-terminal amino acids 5-133. The deleted TEF-4 fragment was
isolated from the vector using EcoRI and XhoI and
ligated into pcDNA3.1/V5-His to make the pcmTEA(
) expression plasmid.
Tissue Culture, Transfection, and Luciferase Assay--
COS-7
and 3T3-L1 cells were grown in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum. Cells (1 × 105) were plated on 35-mm plates (Falcon-Becton Dickinson
Labware, Franklin Lakes, NJ) and grown overnight. Three µl of
FuGENETM6 was suspended in 100 µl of serum-free medium
and mixed with 0.5 µg of the CCT
promoter-luciferase constructs
(see above), 0.001 µg of pRL-CMV Renilla vector as a
transfection control, and 0.5 µg of either pcTEF-4, pcmTEA(
),
pcmTEA(+) or pcDNA control. Transfection was initiated by dropwise
addition of DNA suspensions to the cell culture. Forty eight hours
later cells were harvested, lysed in 200 µl of the Passive lysis
buffer (Promega), and 10 µl of the cell lysate was used for the
dual-luciferase assay according to the manufacturer's instructions.
Luciferase activity was normalized for transfection efficiency by using
the ratio of the activities obtained with the CCT
promoter deletion
constructs (see above) and the pRL-CMV construct carrying the
cytomegalovirus promoter-luciferase fusion.
Preparation of Cell and Nuclear Extracts--
TEF-4-positive
yeast, Y41b, was cultured in 15 ml of SD/
His/
Leu medium containing
45 mM 3-AT. After 48 h, 12 ml of the late log-phase
culture was transferred into 100 ml of the same medium and cultured for
an additional 36 h. The cells were collected by centrifugation at
600 × g for 10 min and resuspended in 1 ml of
homogenization buffer (0.1 M Tris-HCl, pH 7.5, 0.2 M NaCl, 5 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, 0.01 M
-mercaptoethanol, 20% glycerol). After vortexing with glass beads to break the cell walls, the cells were subjected 3 times to freezing and thawing and
centrifuged at 10,000 × g for 5 min to remove the cell
debris (38). The yeast transformed with pYcont were cultured in
SD/
Leu medium and treated in a similar manner to obtain control cell extracts. Nuclear extracts from mammalian cells were prepared according
to Andrews and Faller (39) with minor modification (25).
Preparation and Purification of Glutathione S-Transferase TEF-4
Fusion Protein (GST-TEF-4)--
Full-length TEF-4 cDNA was
digested from pY41b with EcoRI and XhoI and
ligated into the pGEX-KG (Promega) vector to obtain the pGST-TEF-4
expression plasmid. pGST-TEF-4 or control vector were transformed into
Escherichia coli strain HB101 and grown in Luria broth
overnight. The cultures were subsequently transferred to 10 volumes of
growth media and grown for 1 h.
Isopropyl-
-D-(
)-thiogalactopyranoside was then added
to the culture to a final concentration of 0.5 mM, and the
incubation was continued for 3 h. Cells were collected by
centrifugation, resuspended in 0.5 ml of lysis buffer (25 mM Hepes-NaOH, pH 7.5, 20 mM KCl, 2.5 mM EDTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride and 0.02% Triton X-100), sonicated, and centrifuged at 10,000 × g for 15 min.
To obtain purified proteins, the supernatant was applied to a
glutathione-Sepharose 4B column (Amersham Pharmacia Biotech). GST-TEF-4
and GST proteins were eluted according to the manufacturer's instructions.
Electromobility Gel-shift and Supershift Assays--
The
opposite strands of Eb, mutated Eb
(5'-GTGGTTTTCAGGAGCTCGGAGGTGGCATT-3', Ebm) and the SV-40 GT-IIC
enhanson (5'-ACCAGCTGTGGAATGTGTGTCGA-3'), 500 pmol each, were annealed
(70 °C, 10 min) in 100 µl of 25 mM Tris-HCl, pH 8.0, 0.5 mM MgCl2, and 25 mM NaCl and
then cooled to room temperature. An aliquot (10 pmol) of the
double-stranded oligonucleotides was 5'-end-labeled with
[32P]ATP (Amersham Pharmacia Biotech) and T4
polynucleotide kinase and purified on a Sephadex G-25 column (Amersham
Pharmacia Biotech). A DNA protein-binding reaction was performed for 30 min at room temperature in 40 µl of 1× binding buffer (40 mM Tris-HCl, pH 7.9, 4 mM MgCl2, 2 mM EDTA, 100 mM NaCl, 2 mM
dithiothreitol, 200 µg/ml bovine serum albumin, 20% glycerol, and
0.2% Nonidet P-40) containing 1 µg of poly(dI-dC) (Amersham
Pharmacia Biotech), 1 µl of the radiolabeled probe (50,000-80,000
cpm), and one of the following: yeast cell extracts, mammalian nuclear
extracts, purified GST-TEF-4 or GST proteins. In some cases, unlabeled
double-stranded Eb (100-fold molar excess), anti-hemagglutinin antibody
(Roche Molecular Biochemicals), or anti-GST antibody (Amersham
Pharmacia Biotech) was included in the incubation mixture. The reaction was stopped by addition of 4 µl of 6× DNA loading buffer. The labeled probe was separated from DNA-protein complexes by
electrophoresis on 6% nondenaturing polyacrylamide gels in 0.5× Tris
borate/EDTA buffer (44.5 mM Tris-HCl, pH 8.3, 44.5 mM boric acid, and 1 mM EDTA) at 4 °C until
the xylene cyanol dye reached 5 cm from the bottom of the gel.
Autoradiography was carried out by exposure of the gel to Kodak X-Omat
XAR2 film with an intensifying screen at
70 °C for 16-48 h.
Reverse Transcription-Polymerase Chain Reaction
(RT-PCR)--
COS-7 cells were transformed with pcTEF-4 or pcDNA
as described above. After being cultured for 48 h, the cells were
treated with Isogen (Nippon Gene, Toyama, Japan) according to the
manufacturer's instructions, and total RNA was extracted. One µg of
total RNA was reverse-transcribed at 60 °C for 30 min and then
subjected to 20 cycles of amplification (94 °C for 1 min, 56 °C
for 1 min, and 60 °C for 1.5 min) using the one-step RT-PCR kit
(Toyobo, Osaka, Japan). The primers used for CCT
were
5'-ATGCACAGTGTTCAGCCAA-3' (sense) and 5'-GGGCTTACTAAAGTCAACTTCAA-3'
(antisense). Primers for glycero-3-phosphate dehydrogenase (G3PDH) were
5'-TCCACCACCCTGTTGCTGTA-3' (sense) and 5'-ACCACAGTCCATGCCATCAC-3'
(antisense). The intensities of the CCT
bands were normalized to
those of the G3PDH bands using the Quantity One software (PDI,
Huntington Station, NY).
Statistical Analysis--
All values are expressed as means ± S.D. Group means were compared by Student's t test or
the Cochran-Cox test after analysis of variance to determine the
significance of difference between the individual means. Statistical
significance was assumed at p < 0.05.
 |
RESULTS |
Cloning of the Putative Regulatory Proteins That Bind to the Eb
Element of the CCT
Promoter--
A yeast integrant (YHIS-Eb)
carrying three repeats of Eb in the front of the his3 coding
region was constructed and used to screen for the Eb-binding proteins
and promoter activation by the one-hybrid expression system. The
Yhis-Eb yeast strain was transformed with the pACT2 mouse embryo
cDNA library encoding fusions of the yeast gal4
activation domain, a cDNA of an unknown protein, and a
hemagglutinin tag at its 5'-end. The yeast transformation efficiency
was 1 × 104 colony-forming units/µg as determined
on SD/
Leu selective medium. After screening 1.5 × 106 colonies, 19 positive clones were obtained that were
able to grow on the SD/
His/
Leu selective medium containing 45 mM 3-AT. Of the cDNAs obtained from those yeast clones,
10/19 encoded transcriptional enhancer factor-4 (33, 34); 1/19 encoded
ribosomal protein L22 (40); 1/19 encoded ribosomal protein S4 (41);
1/19 encoded phosphatidylinositol phospholipase C
(42); 1/19 encoded
a mouse ortholog of the human FRAP-related protein (43); 1/19 encoded DNA polymerase I (44); and 4/19 encoded unknown cDNAs. Thus, the
most frequently isolated clones were those encoding transcriptional enhancer factor TEF-4. Since we found that the TEF-binding consensus (35), 5'-(A/T)(A/G)(A/G)(A/T) ATG (C/T)(G/A)-3' was present in the Eb
promoter region, we decided to focus on TEF-4 and further analyze its
role in CCT
gene transcription. We selected the clones pY41b and
pY51b, isolated from the yeast Y41b and Y51b, that encoded the
full-length or almost full-length TEF-4 (pY41b from the position
24
and pY51b from the position +94 relative to the translation start codon).
To confirm that TEF-4 clones can drive transcription of other yeast
genes carrying the Eb sequence in their promoters, the yeast integrant
YLacZ-Eb carrying a 3-fold repeat of Eb followed by the
-galactosidase lacZ gene was transformed with
pY41b, pY51b plasmids, or pYcont (pACT2 vector), cultured on SD/
Leu
selective medium, and tested for the expression of
-galactosidase.
Formed colonies were examined for
-galactosidase by the colony-lift filter assay. The colonies of YLacZ-Eb transformed with pY41b and pY51b
expressed
-galactosidase and turned blue in the presence of the
-galactosidase substrate. However, the colonies of YLacZ-Eb transformed with either pGAD53m encoding a fusion of p53 with the
GAL4 activation domain (which does not bind to the Eb
region) or the pYcont empty vector, did not yield blue colonies, as
expected. We next used Y53BLUE yeast strain that carried the
p53-binding sequence in front of lacZ as a positive control.
Y53BLUE transformed with pGAD53m plasmid expressed
-galactosidase
and the colonies turned blue. When the Y53BLUE yeast was transformed
with plasmids pY41b and pY51b encoding TEF-4, which does not bind to
the p53 sites, or with the empty vector pYcont, no blue color was
obtained. The untransformed YLacZ-Eb or Y53BLUE yeast produced a
similar negative response. Together, these results indicate that pY41b and pY51b encodes TEF-4 that binds to the Eb region of heterologous yeast promoters and drives transcription of the his3
and lacZ genes. The results also confirm the validity of the
TEF-4 clones and their expression.
Evaluation of TEF-4 Binding to the Eb Promoter Element--
To
confirm the binding of TEF-4 to the Eb region, we performed gel-shift
analysis using the radiolabeled Eb oligonucleotide as a probe. The
labeled Eb fragment was incubated with cell extracts isolated from the
yeast expressing TEF-4 (Y41b) or control (Ycont), and DNA-protein
complexes were separated by electrophoresis and visualized by
autoradiography (Fig. 1A).
When Y41b extract was used, two slower migrating bands in addition to
the faster moving probe were observed (Fig. 1A, lane
2). The intensities of the two slower bands were markedly
decreased after competition with a large excess of unlabeled probe
(Fig. 1A, lane 4), suggesting that both
originated from a protein that binds to Eb. However, the lower band was
also visible in the experiments with control yeast extracts (Fig.
2A, lane 3)
indicating that only the upper band was specific for TEF-4/DNA binding.
This was further confirmed by a competition experiment with an antibody
specific for the hemagglutinin tag, which was part of the TEF-4 fusion
protein. When various amounts of the anti-hemagglutinin antibody were
added to the reaction mixture, the intensity of the upper band
decreased, whereas the intensity of the lower band was not affected
(Fig. 1B). These results clearly support the notion that the
upper band was specific for the interaction of the TEF-4 fusion protein
with Eb.

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Fig. 1.
Gel-shift and supershift analysis for the
TEF-4/hemagglutinin tag or purified TEF-4/GST fusion protein binding
with the Eb promoter element. A, the end-labeled Eb
probe was incubated with 65 µg of the extract from yeast carrying
pY41b (lanes 2 and 4) or pYcont (lane
3). A 100-fold molar excess of unlabeled probe was used for
competition with the labeled Eb probe (lane 4).
B, the end-labeled Eb probe was incubated with 65 µg of
the extract from yeast carrying pY41b. Cell extracts were incubated
without (lane 1) or with 2 (lane 2), 4 (lane 3), or 8 µg (lane 4) of
anti-hemagglutinin antibody. C, the end-labeled Eb
(lanes 1-7) or GT-IIC probe (lanes 8 and
9) were incubated with 1 µg of purified GST-TEF-4
(lanes 2, 4, 6, and 8) or purified GST
(lanes 3, 5, 7, and 9). In lanes 4 and
5, the purified proteins were incubated with 5 µg of
anti-GST antibody. In lanes 6 and 7, a 100-fold
molar excess of unlabeled Eb probe was added. Bovine serum albumin was
used so that the protein content in each lane was equal. The
arrows indicate the positions of specific DNA-protein
complexes. Each experiment was repeated twice with similar
results.
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Fig. 2.
Gel-shift analysis using TEF-4 expressed in
COS-7 cells or purified GST-TEF-4 with Eb or mutated Eb (Ebm)
probe. A, the end-labeled Eb probe (lanes
1-5) or Ebm probe (lanes 6-8) was incubated with 20 µg of nuclear extract from COS-7 cells transfected with vector
control (lanes 2, 4, and 7) or pcTEF-4
(lanes 3, 5, and 8). A 100-fold molar excess of
unlabeled probe was used for competition with labeled Eb (lanes
4 and 5). B, the end-labeled Eb probe
(lane 1) or Ebm probe (lanes 2 and 3)
was incubated with 1 µg of purified GST (lane 2) or
purified GST-TEF-4 (lanes 1 and 3). The
arrows indicate the positions of specific DNA-protein
complexes. Each experiment was repeated twice with similar
results.
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Antibodies specific for TEF-4 are not available. To confirm that the
binding to Eb is specific for TEF-4 protein regions and not for the
hemagglutinin tag, we cloned and purified a fusion protein, glutathione
S-transferase-TEF-4 (GST-TEF-4). We also made pure GST and
used it as a negative control in parallel gel-shift assays. As shown in
Fig. 1C, migration of the Eb band was retarded only in the
presence of purified GST-TEF-4 (lane 2) and not in the
presence of GST (lane 3). The addition of an anti-GST
antibody (lane 4) or cold Eb competitor (lane 6)
prevented formation of the retarded bands. Purified GST-TEF-4 also
caused a band retardation of the SV40 enhancer element GT-IIC
(lane 8) that is known to be regulated by TEF proteins.
Furthermore, GST-TEF-4 did not bind to the CCT
basal promoter
fragment
90/+38 that does not contain a TEF-4 consensus sequence
(data not shown). Taken together, we conclude that TEF-4 is responsible
for the formation of the protein complexes within the Eb region of the
CCT
promoter.
DNA Binding Properties of TEF-4 after Its Expression in Mammalian
Cells--
To test whether or not TEF-4 could also bind to the Eb
promoter element after expression in mammalian cells, we prepared a mammalian TEF-4 expression plasmid, pcTEF-4, and transfected COS-7 cells with pcTEF-4 or control vector pcDNA. We isolated cell
nuclear extracts and performed gel-shift analysis using Eb and mutated Eb (Ebm in which the TEF-4 ATG-binding core was mutated to GCT) as
probes. Nuclear extracts from COS cells transfected with pcTEF-4 produced two slowly migrating bands in addition to the rapidly migrating Eb band (Fig. 2A, lane 3). The
intensities of the two retarded bands markedly decreased after
competition with an excess of cold Eb probe (lanes 4 and
5). The upper band was also visible in the experiments with
control nuclear extracts (lane 2), indicating that some
endogenous nuclear proteins or the protein complex of TEFs with some
other factors also bind to Eb. The migration of the lower band,
however, coincided with the migration of the purified GST-TEF-4,
suggesting that this band originated from the binding of TEF-4
overexpressed in COS-7 cells. When the mutated element Ebm was used
instead, no protein binding was detected with nuclear extracts from
TEF-4-expressing cells (Fig. 2A, lane 8) nor did the
purified GST-TEF-4 fusion protein bind to the mutated probe (Fig.
2B, lane 3). These results suggested that both overexpressed TEF-4 and an endogenous nuclear protein from COS-7 cells can bind to
the Eb promoter element by recognizing the same ATG core sequence within the TEF binding consensus.
Activation of Chimeric CCT
Promoter-Luciferase Reporters by
TEF-4--
To examine whether or not TEF-4 can mediate transcription
of the CCT
gene, we prepared various CCT
promoter deletion
constructs linked to the luciferase reporter and transfected either
COS-7 or 3T3-L1 cells with pcTEF-4 or pcDNA control. The expression of luciferase activity was determined by dual-luciferase assays and
normalized for transfection efficiency after cotransfection with
pRL-CMV Renilla vector. The Renilla luciferase
activity of the CMV promoter-driven controls was constant and was not
affected by TEF-4 throughout the experiments. As shown in Fig.
3A, when cells were
transfected with CCT
promoter-luciferase constructs the luciferase
activity was highest for the shortest promoter construct,
52/+38
(LUC.D3), and was lower for the longer
90/+38 (LUC.D1) construct.
Luciferase activity increased again with the construct containing
longer promoter region (
130/+38, LUC.D2) and with the construct
201/+38 (LUC.C8). These results suggested that important regulatory
regions for CCT
transcription in COS-7 and 3T3-L1 cells resided at
positions
52/+38 and
202/
52. The core promoter region
52/+38
was responsible for the constitutive expression (25), and further
upstream regions (
202/
52), which were shown to be regulatory (25),
were both negatively and positively modulated in those two cell lines.
When the cells were cotransfected with TEF-4 expression plasmid, the
luciferase activities for all promoter-reporter constructs increased
2-2.5-fold relative to the controls. Surprisingly, the maximal
stimulatory effect of TEF-4 was observed with the core promoter
construct LUC.D3 (
53/+38) lacking the consensus binding sites for
TEF-4. The actual TEF-4-binding site Eb is located further upstream in
the sequence between
103 and
82. These results indicate that the
primary stimulatory effect of TEF-4 on the CCT
promoter was through
its action on basal transcription and that the further upstream TEF-4
consensus binding site, if functional, might play a role that was not
obvious from the above deletion analysis.

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Fig. 3.
Effect of TEF-4 on the expression of various
CCT gene promoter-luciferase reporter
constructs. A, truncated CCT promoters (LUC.D1,
LUC.D2, LUC.D3, LUC.C6, LUC.C7, and LUC.C8) and mutated CCT
promoters (LUC.C7m and LUC.C8m in which the ATG core in the Eb region
was mutated to GCT; and LUC.C7m2 in which the ATG core in the Eb region
was mutated to AGC) were cloned into the luciferase reporter vector
pGL3-basic. Luciferase plasmids (0.5 µg) and pRL-CMV (0.001 µg)
were transfected into COS-7 cells with pcDNA (0.5 µg)
(white bar) or pcTEF-4 (0.5 µg) (black bar).
Reporter activities were measured 48 h after transfection and
normalized for transfection efficiency as described under
"Experimental Procedures." TEF-4 significantly enhanced the
luciferase activity of each luciferase construct compared with vector
control (p < 0.05). * and ** represent
p < 0.03 and p < 0.01, respectively,
for the LUC.C8m to LUC.C8, and LUC.C7m and LUC.C7m2 to LUC.C7 compared
with the identical pcDNA or pcTEF-4 treatment. N.D.
represents not detectable. B, LUC.D3 or LUC.C7 (0.5 µg)
and pRL-CMV (0.001 µg) were transfected into COS-7 cells with
pcDNA (0.5 µg) (lane 1, white bar), pcTEF-4
(0.5 µg) (lane 2, black bar), or mutated TEF-4,
pcmTEA( ) (lane 3, hatched bar) or pcmTEA(+)
(lane 4, striped bar). * and ** represent
p < 0.03 and p < 0.01, respectively,
compared with vector control. C, luciferase plasmids
(LUC.D3, LUC.D1, LUC.D2, LUC.C8, and LUC.C8m) (0.5 µg) and pRL-CMV
(0.001 µg) were transfected into 3T3-L1 cells with pcDNA (0.5 µg) (white bar) or pcTEF-4 (0.5 µg) (black
bar). TEF-4 significantly enhanced the luciferase activity by each
luciferase construct compared with vector control (p < 0.05). * represents p < 0.05 for LUC.C8m compared with
LUC.C8 with the same pcDNA or pcTEF-4 treatment. Values are the
means ± S.D. from three independent dishes. Each experiment was
repeated three times with similar results.
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|
To substantiate the notion that TEF-4 functionally interacts with the
Eb region in mammalian cells, we prepared luciferase reporter
constructs containing point mutations in the Eb region (ATG to GCT) of
the LUC.C8 (
201/+38) and LUC.C7 (
1268/+38), named LUC.C8m and
LUC.C7m, respectively. We also prepared a luciferase reporter
containing point mutations in the Eb region (ATG to AGC) of the LUC.C7
(
1268/+38), named LUC.C7m2, to which TEF-5 was reported not to bind
(36). When COS-7 cells were transfected with these mutated Eb
constructs the luciferase activity increased above that of the
wild-type constructs. Furthermore, TEF-4 expression enhanced the
luciferase activity of the mutated constructs relative to that of the
wild-type constructs (Fig. 3A). These results suggest that
TEF-4 functionally binds to the Eb region of CCT
promoter where it
acts as a transcriptional repressor. Taken together, deletion and
mutation analyses show that TEF-4 can act as both a positive and a
negative modulator of the CCT
promoter activity; the two functions
combined predominantly produce a stimulatory effect.
To establish which domain(s) of TEF-4 is responsible for its
stimulatory activity and binding to the CCT
promoter, we expressed TEF-4 deletion mutants pcmTEA(+) and pcmTEA(
) in COS-7 cells and
measured luciferase activity from the LUC.D3 core promoter construct
(stimulatory effect) and LUC.C7 (stimulatory and inhibitory effects)
construct. The TEF-4 deletion mutant containing only the so-called
TEA-DNA binding domain, pcmTEA(+), retained its stimulatory effect on
luciferase expression from both LUC.D3 and LUC.C7 promoter-reporter
constructs (Fig. 3B, lane 4). A second TEF-4
deletion mutant, lacking the DNA binding domain but containing the
other regions of the protein, did not have any significant effect on
the promoter activity (Fig. 3B, lane 3).
These results suggest that the DNA binding domain of TEF-4 is critical
and sufficient for its function as a transcriptional modulator of the
CCT
promoter which, as demonstrated in Fig. 3A, was
predominantly through its effect on basal transcription. On the other
hand, the full-length GST-TEF-4 fusion protein containing the TEA
domain did not bind to the core promoter region (
90/+38) since this
region does not have a consensus binding site for TEF-4 (data not
shown). Thus, it appears that TEF-4 does not stimulate the basal
activity through direct binding to the core promoter. Instead, we
postulate that TEF-4 stimulates the expression of some other
transcription factor(s) necessary for the basal CCT
gene
transcription and/or directly interacts through its TEA domain with a
nuclear protein(s) that is critical for CCT
transcription.
To confirm that the multiple effects of TEF-4 on the CCT
promoter
activity are not restricted to COS cells, we performed similar
functional assays in 3T3-L1 cells. The results are shown in Fig.
3C. The exogenous TEF-4 expressed in 3T3-L cells enhanced the basal activity of the deletion promoter series and of the TEF-4
consensus site mutant LUC.C8m as was the case in COS-7 cells.
TEF-4 Expression Increases CCT
mRNA Abundance--
COS-7
cells were transfected with pcTEF-4 or its control vector pcDNA to
determine whether or not TEF-4 could also increase the amount of CCT
mRNA by acting on the natural promoter as it did on the
promoter-luciferase reporter chimeras. As shown in Fig.
4, TEF-4 increased the CCT
mRNA
level about 1.4-fold compared with that in control cells. These results
confirm that TEF-4 can modulate the abundance of CCT
transcripts in
cultured cells.

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Fig. 4.
TEF-4 increases CCT
mRNA abundance. A, COS-7 cells were
transfected with pcDNA (0.5 µg) or pcTEF-4 (0.5 µg). Total
mRNA was obtained 48 h after transfection, and 1 µg of total
RNA was used for RT-PCR analysis of CCT and G3PDH. B, the
band intensities in A were quantified as described under
"Experimental Procedures." The values for G3PDH mRNA were used
to normalize the band density of CCT mRNA. * represents
p < 0.05 compared with the vector control. Values are
means ± S.D. from three independent dishes. Each experiment was
repeated three times with similar results.
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 |
DISCUSSION |
The Physiological Role of TEF-4 Is Unknown--
In the
transcription enhancer factor family, TEF-1 was initially purified from
HeLa cells, based on its binding affinity for the SV40 enhancer element
GT-IIC (45), and then subsequently cloned and characterized (35). TEF-1
consists of 426 amino acids forming four separate structural domains as
follows: a DNA-binding domain (TEA/ATTS domain) at its amino terminus,
proline-rich and serine-threonine-tyrosine-rich domains in the middle
region, and a zinc finger motif at its carboxyl terminus. The TEA
region directly interacts with consensus DNA elements, and the
proline-rich and zinc finger domains modulate TEF-1 binding to these
elements (46). TEF-1 also contains potential phosphorylation sites for
calmodulin-, cAMP-, and cGMP-dependent protein kinases (47)
that could also modulate its function as a transcription factor.
TEF-4, initially named as the "embryonic TEA
domain-containing factor" or ETF, was identified in neural precursor
cells by subtraction screening with adult mouse brain mRNA (33).
Based on its level and pattern of expression, TEF-4 has been implicated in the developmental regulation of neural (33), kidney (34), and other
tissues (34), but its actual physiological role remains unknown. TEF-4
contains 66% sequence identity to TEF-1, mostly in the TEA region,
hence other domains may be important for TEF-4-specific activity (33).
Two other members of the family, TEF-3 (34) and TEF-5 (36), were
identified by PCR amplification using degenerate primers corresponding
to the highly conserved TEA domain. TEF factors activate promoters via
binding to the consensus sequence (5'-(A/T)(A/G)(A/G)(A/T)ATG(C/T)(G/A)-3') containing a conserved ATG
core. Although it was known that TEF nuclear factors could activate
viral promoters by binding to GT-IIC and
SphI/SphII enhancer elements (48), until recently
the actual targets for TEFs in the mammalian genome were largely
unknown. TEF-1 was reported to stimulate transcription of
-myosin
heavy chain in myocytes (49) and TEF-5 to regulate the cell-specific
expression of chorionic somatomammotropin-B in placenta (36). There
were no reports of promoters that could be targeted for regulation by
TEF-4. Therefore, this is the first report demonstrating that a
mammalian gene, CCT
, can be regulated by TEF-4 through modulation of
its basal transcription and through binding to the TEF consensus site
within the distal enhancer region.
TEF-4 Has Both Stimulatory and Repressive Actions on the CCT
Promoter--
Our initial studies demonstrated that unknown nuclear
factors bound to the
103/
82, or Eb, region of the mouse CCT
promoter and modulated transcription (25). In the present study, we
isolated an Eb-binding nuclear factor, TEF-4, by functional cloning
using the yeast one-hybrid system. The sequence alignments suggested that the primary structure of the TEA domain of TEF-4 is
indistinguishable from that of TEF-1 (Ref. 34 and data not shown).
Indeed, we showed (Fig. 1C) that purified GST-TEF-4 binds to
the GT-IIC motif present in the SV40 enhancer as previously shown for
TEF-1 (35). Consequently, we might expect that other TEF members,
TEF-1, TEF-3, and TEF-5, would also bind to the Eb sequence. However,
only TEF-4 cDNA was functionally cloned in the present study
suggesting that if the cDNAs for other TEFs were present in the
library, the TEA domain was not the sole determinant of TEF-4 DNA
binding affinity and function (46). The gel-shift and supershift
experiments with yeast and mammalian proteins and purified GST-TEF-4
fusion protein clearly demonstrated that the Eb region within the
CCT
promoter represents a true binding site for TEF-4. However, we did not test other members of the TEF family for their binding and
regulatory properties. Thus, we do not exclude the possibility that
other TEF members may also be relevant for the regulation of expression
of CCT
.
Previous reports showed that overexpressed TEF-1 represses the
expression of the GT-IIC-containing reporter genes in HeLa cells due to
the presence of a limited amount of a putative coactivator (35, 50).
Transfection of TEF-4 failed to induce any obvious effect on the
luciferase gene expression from a tetramerized GT-IIC oligonucleotide
ligated upstream of the thymidine kinase promoter-reporter (33).
However, we report in this paper for the first time that TEF-4 is a
functional transcription factor that is able to enhance the activity of
the CCT
promoter-luciferase reporters and increase the level of
CCT
mRNA in transfected cells. Previous studies have shown that
the level of CCT
mRNA is increased upon stimulation of cells by
colony-stimulating factor-1 (18), after partial hepatectomy (19), or
during tissue growth and differentiation (20). Our data suggest that
TEF-4, and perhaps other members of the family, might play a positive
regulatory role in transcription of the CCT
gene under these
stimulatory conditions.
The present results demonstrate that overexpression of TEF-4
significantly enhances the CCT
promoter activity but that the activation was moderate compared with the vector control. We
demonstrated that TEF-4 might play multiple roles in the regulation of
the CCT
promoter. We established that TEF-4 stimulates basal
transcription since overexpressed TEF-4 increases the activity of the
basal promoter construct
52/+38 that lacks the TEF-4 consensus
binding site. By preparing mutated TEF-4 proteins, we demonstrated that the TEA-DNA binding domain is important and sufficient for its stimulatory activity. These results suggest that either TEF-4 stimulates transcription of a basic transcription factor(s) or that
TEF-4 directly interacts via its TEA domain with pre-existing protein(s) or cofactor(s) required for basal transcription. Recently, it was found that the p160 family of nuclear receptor coactivators, SRC1, TIF2, and RAC3, act as bona fide coactivators of TEF-4
and other members of the TEF family of transcription factors (51). These coactivators are potential targets for the action of TEF-4 on the
CCT
promoter.
Unexpectedly, when the TEF-4 binding region Eb within the CCT
promoter was mutated, the overexpressed TEF-4 did not decrease but
further enhanced the luciferase activity (Fig. 3A). These data suggest that after binding to the Eb region TEF-4 acts as a
transcriptional repressor and might explain why transactivation by
TEF-4 was attenuated in longer promoter constructs and why the effect
of TEF-4 on CCT
transcription was relatively modest. Repression of
transcription by TEF proteins is known to occur. The cause for TEF-1
repression has been suggested to be due to titration by the
overexpressed TEF-1 of another limiting transcriptional coactivator
(35). It is well established that the E-box motif for TEF-1 and the
M-CAT motif for Max are both required for expression of the cardiac
-myosin heavy chain gene in rat myocytes (52). Low concentrations of
either TEF-1 or Max alone modestly activate expression of the gene and
repress the gene at higher concentrations. However, when TEF-1 and Max
were cotransfected, a synergistic transactivation of the
-myosin
heavy chain gene occurred, which was explained not only by TEF-1 and
Max binding to their respective DNA motifs but also by protein-protein
interactions between them. In this respect it is noteworthy that TEF-1
repressed the human chorionic somatomammotropin B promoter in BeWo
choriocarcinoma cells (53) via direct protein-protein interactions with
TATA-binding proteins. However, it was established that another member
of the family, TEF-5, specifically binds to several functional motifs in the human chorionic somatomammotropin-B gene enhancer and thus stimulates its activity (36).