From the Department of Basic Gerontology, National Institute for Longevity Sciences, 36-3 Gengo, Morioka-cho, Obu, Aichi 474-8522, Japan
Received for publication, December 12, 2000, and in revised form, March 20, 2001
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ABSTRACT |
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Telomerase, the reverse transcriptase that
maintains telomere DNA, is usually undetectable in adult human tissues,
but is positive in embryonic tissues and in cancers. However, in
rodents, several organs of normal adult animals express substantial
amounts of telomerase activity. To elucidate relevant control
mechanisms operating on the tissue-specific expression of telomerase in
rodents, we examined the transcriptional regulation of telomerase
reverse transcriptase (mTERT) gene in muscle cell differentiation.
Reverse transcriptase-polymerase chain reaction analysis showed that
the reduction of telomerase activity was caused by the decrease of mTERT mRNA level during myogenesis. Transfections of mTERT promoter showed that the proximal 225-base pair region is the core promoter responsible for basal transcriptional activity and also participates in
the reduced transcription after muscle differentiation. Electrophoretic mobility shift assays showed that this region contained the GC-boxes, which bind to Sp1 family proteins, and the E-box, which binds to c-Myc.
Furthermore, DNA binding activities of Sp1, Sp3, and c-Myc were
down-regulated during myogenesis. These data suggest that Sp1, Sp3, and
c-Myc have critical roles of TERT transactivation in mouse, and the
lack of these transcription factors cause down-regulation of mTERT gene
expression in muscle cells differentiation.
Telomeres are specialized structures at the ends of chromosomes
composed of DNA and proteins that are essential for maintaining the
stability of the eukaryote genome (1). In vertebrates, they consist of
tandem hexanucleotide repeats, (TTAGGG)n, maintained by a
specialized ribonucleoprotein enzyme, called telomerase, which adds
motif-specific nucleotides using its RNA subunit as a template.
Recently, three major telomerase subunits have been identified. The
telomerase RNA component provides the template for telomere repeat
synthesis (2, 3), the telomerase-associated protein (TEP1) binds to
telomerase RNA and coordinates assembly of telomerase holoenzyme (4,
5), and the most important component responsible for the catalytic
activity of telomerase is telomerase reverse transcriptase
(TERT)1 (6, 7). Previous
studies have shown that TERT is expressed in most malignant tumors but
not in normal human tissues and that expression of TERT is closely
associated with telomerase activity, whereas two other components are
constitutively expressed in both tumors and normal tissues (8, 9).
These findings indicate that TERT is a rate-limiting determinant of
catalytic activity of telomerase. Analysis of the human and mouse TERT
promoters reveals that they are regulated by a number of inducible
transcription factors, including c-Myc and NF- Most telomerase-positive cells are highly regenerative or immortal.
Among normal tissues of adult humans, telomerase activity was almost
undetectable except germ line cells, although very small amounts of
activity are detectable in normal bone marrow, peripheral blood
leukocytes, lymphoid cells, and skin epidermis (15-18). In contrast,
highly regenerative tissues of normal rodents express modest levels of
telomerase even in adult animals (19, 20). In the normal mouse,
telomerase activity exists in colon, liver, ovary, and testis but not
in brain, heart, stomach, and muscle (21, 22). Moreover, cell
differentiation causes reduced telomerase activity in some kinds of
cell types such as murine F9 teratocarcinoma and C2C12 myoblast cells
(23, 24). These results suggest that this remarkable difference among
various tissues in mouse may reflect the different regulation
mechanisms of telomerase expression, which may cause the
tissue-specific features of differentiation and proliferation. However,
the detailed mechanisms that contribute to the telomerase expression
during development and cell differentiation in mouse are largely
unknown. In this study, to understand the mechanisms that reduce mTERT gene expression during muscle cell differentiation, we constructed a
series of reporter plasmids containing the 5'-franking sequence of
mTERT gene and transfected these constructs into mouse cell lines,
including C2C12 myoblasts. Transcriptional activity of mTERT was
dependent on the proximal 225-bp region of promoter, and this promoter
region contained E-boxes and GC-boxes, which bind to bHLH proteins and
Sp1 family proteins, respectively. We attempted to identify the
transcription factors directing mTERT expression and found that Sp1,
Sp3, and c-Myc, but not MyoD, play crucial roles in the regulation of
mTERT transcription during muscle cell differentiation.
Materials--
Cell culture reagents and fetal bovine
serum were obtained from Sigma-Aldrich (St. Louis, MO). Antibodies were
from the following sources: anti-c-Myc, anti-MyoD, anti-Sp1, and
anti-Sp3 from Santa Cruz Biotechnology (Santa Cruz, CA); horseradish
peroxidase (HRP)-conjugated anti-rabbit immunoglobulin G and
HRP-conjugated anti-mouse immunoglobulin G from New England BioLabs
(Beverly, MA). Sp1 and c-Myc consensus oligonucleotides were obtained
from Santa Cruz Biotechnology.
Cell Culture--
Mouse NIH3T3 fibroblasts, C3H10T1/2
fibroblasts, and C2C12 myoblasts were obtained from Riken Cell Bank
(Tukuba, Japan) and maintained in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and
100 µg/ml streptomycin (standard medium). In some experiments,
subconfluent C2C12 cells were induced to differentiate by lowering
fetal bovine serum to final concentration of 0.5% (differentiation medium).
RNA Isolation and Analysis--
Total cytoplasmic RNA was
isolated using the guanidine isothiocyanate method from the cells that
were cultured in standard medium or differentiation medium for 1, 4, 7, or 10 days. Samples of 20 µg of total RNA were denatured, separated
by electrophoresis in a 1% agarose gel containing formaldehyde, and
transferred to GeneScreen membranes (PerkinElmer Life Sciences). The
membranes were prehybridized and then hybridized with cDNA probes
labeled with [ RT-PCR Amplification of a Mouse TERT cDNA Fragment--
The
RT-PCR primers, 5'-AGACTSCGCTTCATCCCCAAG-3' (sense) and
5'-GTCTGGAGGCTGTTCACCTGC-3' (antisense), were constructed
according to the mouse TERT cDNA (GenBankTM accession number
AF073311) (7). Total RNA was isolated from C2C12 cells as
described above. First-strand cDNA was synthesized with 1 µg of
total RNA using a First Strand cDNA synthesis kit (Life
Technologies, Inc., Gaithersburg, MD) in the presence of 1.6 µg of
oligo-(dT)2 primer in a final volume of 25 µl. After
denaturation for 5 min at 94 °C, 4 µl of reaction product was
amplified by PCR for 29 cycles (94 °C for 30 s; 55 °C for
30 s; 72 °C for 1 min). The amplified products were separated
by electrophoresis on either a 2% agarose gel or 8% polyacrylamide
gel electrophoresis (PAGE) and visualized with ethidium bromide. The
DNA sequences of RT-PCR products were confirmed at least once by DNA
sequencing and were found to be identical to the corresponding sequence
of mouse TERT cDNA (nucleotides 1857-2975, data not shown). Each
RT-PCR was performed three times with independent preparations of RNA,
and typical results are shown in Fig. 1. As an internal control, RT-PCR of glyceraldehyde-3-phosphate dehydrogenase was performed for all RNA
samples, using the PCR primers, 5'-ACCACAGTCCATGCCATCAC-3' (sense) and
5'-TCCACCACCCTGTTGCTGTA-3' (antisense). The linearity of RT-PCR with
respect to RNA amount was determined, and the estimation of mRNA
for mTERT was done within a linear range.
Extraction of Telomerase--
C2C12 cells were washed twice with
ice-cold phosphate-buffered saline, scraped off, and transferred to
1.5-ml microtubes. After centrifugation for 3 min at 1000 × g at 4 °C, pellets were suspended in 500 µl of the wash
buffer (pH 7.5) containing 10 mM HEPES, 1.5 mM
MgCl2, 10 mM KCl, 1 mM
dithiothreitol, and centrifuged again for 3 min at 1000 × g at 4 °C. Whole cell extracts were prepared by
suspending cells in 30 µl of CHAPS lysis buffer, containing 10 mM Tris-HCl, pH 7.5, 1 mM MgCl2, 1 mM EGTA, 0.1 mM phenylmethylsulfonyl fluoride,
5 mM 2-mercaptoethanol, 0.5% CHAPS, and 10% glycerol. Cell suspensions were subjected to three cycles of freezing and thawing
using liquid nitrogen. After being placed on ice for 30 min, they were
centrifuged for 30 min at 10,000 × g and the
supernatant was collected (CHAPS extracts). Three independent
experiments were performed to measure telomerase activity of the cells
cultured with differentiation medium.
Assay of Telomerase--
The TRAP (telomere repeat amplification
protocol) assay was employed with minor modifications described by Kim
et al. (25). The cell extract (representing 1 × 105 cells) was incubated for 30 min at 37 °C in a
mixture containing 20 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 63 mM KCl, 0.005% Tween 20, 1 mM EGTA, 50 µM each of dATP, dGTP,
dTTP, 10 µM dCTP, 2 µCi of [ Plasmid Constructs--
The mouse TERT promoter-luciferase
reporter plasmids were constructed by pGL3-basic vector (Promega, WI)
and the DNA, which was obtained by PCR amplification using mouse
genomic DNA according to a published sequence (GenBankTM accession
number AF121949) (11). Various lengths of DNA fragments upstream of the
initiating ATG codon of the mTERT gene were amplified and
inserted into the pGL3-basic vector. For the construction of reporter
plasmids containing substitution mutations in transcription factor
binding sites, site-specific mutagenesis was performed by a PCR-based
protocol. Expression vector for c-Myc protein (pcDNA3-cMyc)
encoding the full-length of c-Myc was a kind gift from
Rónán C. O'Hagan and Ronald A. DePinho, Harvard Medical
School (26). Expression vector pCGN-Sp1, encoding the full-length of
human Sp1, and its backbone vector (pCGN) were kind gifts from Thomas
Shenk, Princeton University (27). pCGN-Sp3, encoding the full-length of
Sp3, was described previously (28).
Stable Transfection--
One microgram of mTERT
promoter-luciferase expression plasmid and 0.5 µg of pSV2neo vector
were cotransfected into C2C12 cells grown in a 12-well plate using
SuperFect reagent (Qiagen, Hilden, Germany) as previously described
(29). The transfected colonies were selected by 400 µg/ml G418 (Life
Technologies, Inc.) for 3 weeks. More than 50 colonies were pooled.
Cell extracts were from the transfectants that were grown in the
presence of 200 µg/ml G418 and differentiated in Dulbecco's modified
Eagle's medium containing 0.5% (v/v) fetal bovine serum with
antibiotics and G418 for 10 days. Luciferase activity of these
transfectants was normalized against the number of cells. A series of
culture experiments were repeated at least three times.
Luciferase Assays--
Transient transfection of luciferase
reporter plasmids was carried out using SuperFect reagent (Qiagen) as
previously described (29). In general, the day before transfection,
cells were plated onto 12-well tissue culture plates at a density of
20,000 cells/well, supplemented with fresh medium before transfection.
A total of 0.825 µg of DNA consisting of 0.75 µg of the indicated
luciferase reporter plasmid and 0.075 µg of the pRL-thymidine kinase
control vector (pRL-TK) (Promega, WI) per well was used for
transfection studies. For cotransfection studies, cells grown in
12-well plates were transfected with 0.75 µg of luciferase reporter
plasmid, 0.075 µg of pRL-TK, and 0.75 or 0.25 µg of the indicated
expression plasmids. PCGN or pcDNA3 was used to adjust the total
amount of expression plasmid DNA. After harvest, the cells were assayed by the Dual-Luciferase Reporter Assay system (Promega, WI), using a
luminometer (EG&G Berthold, Germany). Protein concentrations of the
cell lysates were determined by the method of Bradford with the Bio-Rad
protein assay dye reagent (Bio-Rad, CA). Promoter activities were
expressed as relative activities, normalized against the concentration
of the protein. All transfection experiments were repeated at least
three times.
Nuclear Extract Preparation--
Nuclear extracts were prepared
from the C2C12 cells that were cultured in differentiation medium for
1, 4, 7, or 10 days using the rapid preparation as previously described
(29). The protein concentrations of the nuclear fractions were
determined by the Bradford assay, and all extracts were stored at
Electrophoretic Mobility Shift Assays--
Electrophoretic
mobility shift assays (EMSAs) were performed using 5 µg of nuclear
extract from either untreated or differentiation medium-treated cells.
Synthetic complementary oligonucleotides with a G overhang were
annealed and labeled with [ Western Blot Analysis--
Cells cultured in differentiation
medium for 1, 4, 7, and 10 days were lysed in a buffer consisting of 20 mM Tris-HCl (pH 8.0), 137 mM NaCl, 2 mM EDTA, 10% (v/v) glycerol, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml
aprotinin, 1 mM sodium vanadate, and 1% (v/v) Triton
X-100. Each cell lysate containing 25 µg of protein was run under
reducing conditions in 8% or 10% sodium dodecyl
sulfate-polyacrylamide electrophoresis gels (SDS-PAGE), transferred
onto polyvinylidene difluoride membranes (Millipore, MA), and reacted
with antibodies as described. The primary antibody was detected by
counterstaining with an HRP-linked antibody and visualized by enhanced
chemiluminescence. Nuclear extracts (20 µg) were subjected to
SDS-PAGE and Western blotting.
Decreased Telomerase Activity and mTERT Expression during Muscle
Cells Differentiation--
C2C12 cells in culture were induced to
differentiate by lowering serum concentration. Differentiation of C2C12
myoblasts into myotubes was associated with a diminished activity of
telomerase (Fig. 1A), as
observed previously (23, 24). During the culture in differentiation
medium, mTERT mRNA levels were also gradually decreased without
changing the expression of glyceraldehyde-3-phosphate dehydrogenase
mRNA level (Fig. 1B). However, the culture in
differentiation medium resulted in up-regulation of muscle-specific
genes, MyoD or myogenin (Fig. 1B), in agreement with
previous reports (31-34). Thus, the decrease of mTERT mRNA level
was negatively correlated with the expressions of muscle-specific
genes. These results indicate that the reduction of telomerase activity
during myogenesis is regulated by the reduction of mTERT gene
expression in C2C12.
Identification of cis-Elements in the Core Promoter of the mTERT
Gene Essential for Transcriptional Activation--
To understand the
mechanisms of the mTERT expression in cell differentiation, analysis of
the mTERT promoter is indispensable. As the first step, luciferase
assays were performed using mouse NIH3T3 and C3H10T1/2 as well as C2C12
cell lines. These cell lines were transiently transfected with a series
of 5' terminus-truncated mutants of the mTERT promoter linked to the
luciferase reporter gene. As shown in Fig.
2, a 1.6-kb region of mTERT promoter
(
Although deletions from
From these observations, we defined a 225-bp fragment as the mTERT
minimal promoter and assumed that myogenesis-responsive elements are
located in this region. According to data base analysis, we noticed
that three GC clusters and two E-boxes are located in this region,
which are known to bind with Sp1 family proteins and bHLH proteins,
respectively (Fig. 4). To examine the
role of these cis-elements more precisely, luciferase assays
were performed with reporter plasmids with site-specific mutants, which
were introduced into the mTERT minimal promoter (Fig.
5). One E-box is located at the 5'-end of
the minimal promoter, and the deletion of this site resulted in a 50%
reduction in transcriptional activity in C2C12 cells, as shown in Fig.
2 ( GC-boxes Are Important for Down-regulation of mTERT Gene during
Myogenesis--
As shown in Fig. 3, a core promoter region ( Sp1 and Sp3 Specifically Bind to the GC-boxes in the mTERT Core
Promoter--
To identify the proteins that bind to these GC-boxes
(GC-143 and GC-105) in mTERT gene core promoter, EMSA was performed
with 32P-labeled oligonucleotides corresponding to each
GC-box and nuclear extracts from C2C12 cells cultured in Dulbecco's
modified Eagle's medium supplemented with 10% fetal bovine serum. Two
major DNA·protein complexes were observed by using either
GC-143 or GC-105 for probes (Fig.
7A, lane 1 or
5). The specificity of these complexes was confirmed by
competition assay, using an excess amount of unlabeled Sp1 consensus
oligonucleotides, which almost eliminated detection of complex
formation (Fig. 7B). Given that these complexes appeared similar to those observed in previous reports (35, 36), we investigated
whether the complexes consisted of Sp1 or Sp3. The slower migrated
complex was shifted by the addition of either anti-Sp1 or anti-Sp3
antibodies (Fig. 7A, lanes 2, 3,
6, and 7), whereas the faster migrated complex
was shifted by the addition of anti-Sp3 antibody (lanes 3 and 7). Two major bands were almost eliminated by the
addition of both antibodies (Fig. 7A, lanes 4 and
8). Based on these results, we conclude that the slower migrated band was derived from the Sp1·DNA or Sp3·DNA
complexes, whereas the faster migrated band was derived from
Sp3·DNA complex. However, using GC-88 as probe, no shifted bands were
observed by the addition of either anti-Sp1 or anti-Sp3 antibodies,
although several DNA·protein complexes were detected (data not
shown). To determine whether Sp1 and Sp3 modulate the transcriptional activity of the mTERT promoter, C2C12 myoblasts were cotransfected with
one of expression vectors for Sp1 (pCGN-Sp1), Sp3 (pCGN-Sp3), or null
vector (pCGN) together with reporter plasmid containing mTERT core
promoter ( c-Myc Specifically Bind to the E-box in the mTERT Core
Promoter--
As shown in Fig. 5, E-box at
Then we tested the modulation of mTERT transcription by c-Myc in a
myoblast using the luciferase reporter plasmid. C2C12 myoblasts were
cotransfected with c-Myc expression vector pcDNA3-cMyc, together with a reporter plasmid with mTERT core promoter ( Sp1, Sp3, and c-Myc Are Repressed during Myogenesis--
We found
that Sp1, Sp3, and c-Myc bind to the mTERT gene core promoter in
myoblasts but not in differentiated myotubes (Figs. 3 and 6). To
understand the nature of the mechanisms involved, we determined the
levels of each transcriptional factor in nuclear extracts prepared from
C2C12 cells cultured in differentiation medium. By the Western blot
analysis, the contents of all these transcription factors decreased
gradually during muscle cell differentiation (Fig.
9A). To confirm the binding
activities of these transcription factors to mTERT core promoter,
nuclear extracts obtained from myoblasts cultured in differentiation
medium were subjected to EMSA using GC-143, GC-105, or E-201
oligonucleotides as probe. Sp1·DNA, Sp3·DNA, and c-Myc·DNA
complexes were detected, and each complex was diminished during the
culture under serum-starved condition (Fig. 9, B and
C). These results suggest that the decrease in DNA binding
of Sp1, Sp3, and c-Myc, consistent with the reduction in the
concentration of these transcription factors, resulted in the
elimination of telomerase activity during myogenesis.
In this study, we demonstrated that telomerase activity is
down-regulated along differentiation of muscle cells as a consequence of decrease in the mRNA for mTERT. The cellular content of mTERT mRNA decreased in parallel with the transcriptional activity of mTERT promoter in response to myogenesis, indicating that the alterations in the transcription of the mTERT accounts for the elimination of telomerase activity during myogenesis. The putative mTERT core promoter region, ranging from Two GC-boxes, GC-143 and GC-105, were essential for the mTERT core
promoter activity, whereas GC-88 was not. Mutation in GC-143 or GC-105
led to the remarkable reduction of transcriptional activity (>95% or
>60% decrease, respectively; Fig. 5). As expected, double mutations
in these two GC-boxes resulted in marked reduction of transcriptional
activity (>98% decrease), suggesting that these two elements may be
the most essential cis-elements in the mTERT core promoter.
We have also identified the binding of Sp1 and Sp3 to these GC-boxes in
the region Other groups have also shown that telomerase activity decreases along
muscle cell differentiation (23, 24). It was also shown that myogenesis
decreases the binding of Sp1 and Sp3 to the promoter region of GLUT1
gene in C2C12 cells, in which Sp1 and Sp3 are down-regulated (35, 36).
These results are consistent with our conclusion that both Sp1 and Sp3
directly regulate the expression of mTERT in C2C12 cells by the
interaction with the promoter region of mTERT gene in myogenesis.
Muscle cell differentiation is mediated by a transcription factor MyoD,
which acts as a master regulator leading to the activation of many
muscle-specific genes (32, 45, 46). Recent studies showed that the
overexpression of MyoD leads to the repression of Sp1 and Sp3 (35, 36).
In this context, although MyoD may not take part directly in decreasing mTERT gene transcription in our experimental systems, it might suppress
mTERT gene transcription in vivo by an indirect mechanism, i.e. via repressing genes for Sp1 and Sp3.
c-Myc, a transcription factor encoded by a proto-oncogene, is
suppressed during myogenic differentiation through post-transcriptional mechanisms (47-50). In humans, c-Myc binds to the hTERT gene promoter and plays a critical role in regulation of hTERT expression (10-12). Similar relationships between mTERT and c-Myc were observed during differentiation of mouse erythroleukemia cells and mitogen-stimulated lymphocyte proliferation. These findings suggest there is a potential link between increased c-Myc and up-regulation of mTERT in normal proliferating and transformed cells (8, 11). Consistent to these
observations, we showed that c-Myc enhanced the activity of the mouse
TERT promoter and that transcriptional stimulation by c-Myc was higher
with the reporter plasmid harboring a longer promoter region than with
that contained in the core promoter region (Fig. 8B).
These observations indicate that c-Myc, in addition to Sp1 and Sp3,
also plays an important role in mTERT gene transcription and that there
are other c-Myc-responsive cis-elements outside the core
promoter region. In this context, the decrease of c-Myc expression in
muscle cell differentiation may be responsible for the reduction of
mTERT transcription. It is generally accepted that c-Myc activates
transcription as part of a heterodimeric complex with a number of
interacting partners, including members of the Max and Mad families
(51, 52). Kyo et al. (13) showed recently that human TERT
gene expression is variable depending on the Myc/Max ratio or cell
types used. In our study, although mTERT transactivation by c-Myc was
lower than that by Sp1 or Sp3 (Figs. 7C and 8B).
the expression of appropriate Myc/Max ratio might also lead to
remarkably increased mTERT promoter activity as well as human TERT.
During myogenesis, the promoter activity of the In summary, our results showed that Sp1 and Sp3 have critical roles of
mTERT transactivation in mouse. In particular, two GC-boxes (GC-143 and
GC-105) in the mTERT core promoter are the most essential for
transcriptional activity. In addition to Sp1 and Sp3, c-Myc also
activated mouse TERT gene expression. Our data also
indicated that the repression of these transcription factors causes
down-regulation of the mTERT gene in muscle cell differentiation.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B (10-14). These
transcription factors are likely to contribute to the observed
instances of TERT gene activation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]dCTP using a random primer labeling
system (Amersham Pharmacia Biotech, Buckinghamshire, UK). After
hybridization, the membranes were washed and exposed to x-ray film. All
blots were rehybridized with a glyceraldehyde-3-phosphate dehydrogenase
cDNA probe to normalize for mRNA loading differences. To
quantify the contents of mRNA in the cells, the membranes were
exposed to imaging plates and radioactivities were measured with a
bioimage analyzer, Fijix BAS 1500 (Fuji Film, Tokyo, Japan).
-32P]dCTP,
0.1 µg/µl bovine serum albumin, 2 units of Taq DNA
polymerase, 0.1 µg of TS primer (5'-AATCCGTCGAGCAGAGTT-3'), and 0.1 µg of ACX primer (5'-GCGCGGCTTACCCTTACCCTTACCCTAACC-3'). After the
mixtures were heated at 94 °C for 90 s, they were subjected to
30 cycles of PCR amplification (one cycle consisting of 94 °C for
30 s and 58 °C for 45 s). After the reaction, PCR products
were analyzed by 12.5% non-denaturing PAGE (1-mm thick). The gels were
autoradiographed with the x-ray film (Hyperfilm MP, Amersham Pharmacia
Biotech, Buckinghamshire, UK) at
80 °C for 2 h, and
telomerase activity was assessed on incorporated radioactive substrate
in ladders of product DNA, multiplies of 6 bases corresponding to the
telomere repeat unit.
70 °C.
-32P]dCTP, using the Klenow
fragment. DNA binding reactions were performed as previously described
(29). Molar excess (5-, 20-, 100-fold) of c-Myc, Sp1 consensus
oligonucleotides, MyoD binding element, or oligonucleotides containing
mutated sequences were used as an unlabeled competitor. The following
pairs of oligonucleotides derived from the muscle creatine kinase
promoter were used as MyoD binding oligonucleotides;
5'-GCCCCAACACCTGCTGCCTGA-3' and 5'-GTCAGGCAGCAGGTGTTGGGG-3' (30). For
EMSAs using antibodies, nuclear extracts were preincubated with
antibodies (0.2-0.4 µg per reaction) for 20 min at 4 °C. The
reactions were separated on 7% or 6% polyacrylamide gels. Gels were
dried and subjected to autoradiography. The following pairs of
oligonucleotides were used: E-201 containing the nucleotide
211 to
186; 5'-CCGGGGAACACACCTGGTCCTCATGC-3', 5'-GCATGAGGACCAGGTGTGTTCCCCGG-3', GC-88 containing the nucleotide
97
to
68; 5'-TTCCTCCGTTCCCAGCCTCATCTTTTTCGT-3',
5'-ACGAAAAAGATGAGGCTGGGAACGGAGGAA-3', GC-105 containing the nucleotide
116 to
88; 5'-TCCGCCTGAATCCCGCCCCTTCCTCCGTT-3', 5'-AACGGAGGAAGGGGCGGGATTCAGGCGGA-3', and GC-143 containing the nucleotide
153 to
125; 5'-ATTGCTGCGACCCCGCCCCTTCCGCTACA-3', 5'-TGTAGCGGAAGGGGCGGGGTCGCAGCAAT-3'. The positions of each
oligonucleotide are shown in Fig. 4.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Decrease of telomerase activity during muscle
cell differentiation. A, CHAPS extracts were prepared
from uninduced C2C12 (C) and differentiated myoblasts after
1 week in differentiation medium (D) and assayed for
telomerase activity of CHAPS extracts (representing 1 × 105 cells) by the TRAP method as described under
"Experimental Procedures." At least three independent experiments
were performed, and typical results are shown. B, C2C12
myoblasts were cultured in differentiation medium for the indicated
time. The cells were harvested, and total RNA were isolated and
subjected to RT-PCR for mouse TERT (top). 20 µg of total
RNA was subjected to Northern blot analysis and probed with a MyoD and
myogenin cDNA, which was followed by a glyceraldehyde-3-phosphate
dehydrogenase probe to normalize for loading differences.
1561/+53-Luc) demonstrated the significant transcriptional
activities in all these cell lines. NIH3T3 and C3H10T1/2 conferred
higher transcriptional activity than C2C12. C2C12 exhibited modest
transcriptional activity equivalent to 30-40% of NIH3T3, but this
activity was still significant, because it was 250-fold the activity in
promoterless reporter plasmid (pGL3-basic).
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Fig. 2.
Deletion analysis of the mTERT promoter.
NIH3T3, C3H10T1/2, and C2C12 cells were transfected with a series of
luciferase reporter constructs containing the 5'-flanking DNA of the
mouse TERT gene. The luciferase activity of pGL3-basic plasmid was
normalized to 1, and the relative luciferase activity is shown. The
standard deviations (S.D.) of the means are indicated by the
error bars. The positions of the bases are indicated
relative to the mTERT transcription initiation site (+1). Results are
expressed as the mean ± S.D. of at least three independent
experiments, each performed in triplicate.
1561 to
225 of mTERT gene promoter resulted
in no significant alterations in luciferase activity, truncation of a
further 166 base pairs (from
225 to
59) led to a remarkable
reduction of the transcriptional activity in these three cell lines
(Fig. 2). These findings suggest that the proximal 225 bp is the core
promoter essential for basal transcriptional activation of mTERT.
Subsequently, C2C12 cells were stably transfected with reporter
plasmids and induced to differentiate by the culture in differentiation
medium for 10 days. Transfectants with
1561/+53-Luc,
414/+53-Luc,
or
225/+53-Luc reporter plasmids exhibited the reduction of
transcriptional activities (Fig. 3),
being consistent with the decrease of mTERT mRNA level in muscle
cell differentiation (Fig. 1B). These results suggest that
the core promoter region (
225/+53) is also responsible for reduction
of mTERT transcription in muscle cell differentiation.
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Fig. 3.
Effect of myogenesis on the expression of
mTERT gene promoter constructs in C2C12 cells. C2C12 cells were
stably transfected with 1561/+53-Luc,
414/+53-Luc,
225/+53-Luc,
and pGL3-basic reporter plasmids and were induced to differentiate by
the culture in differentiation medium for 10 days. The promoter
activities were normalized against the number of cells, and the
luciferase activity of the
1561/+53-Luc reporter plasmid was
standardized to 1. The relative luciferase activities are shown. The
standard deviations (S.D.) of the means are indicated by the
error bars. Results are expressed as the mean ± S.D.
of at least three independent experiments, each performed in
triplicate.
225/+53-Luc and
185/+53-Luc). Abrogation
of this E-box (E-201) by substitution mutations also led to a 50%
reduction of transcriptional activity (Fig. 5, Ebox-mt #1),
suggesting that this E-box is essential for transactivation. In
contrast, the mutation of another E-box (E-4) that is
adjacent to the transcription start site led to no significant
alteration of transcriptional activity (Ebox-mt #2).
Although a mutation in the GC-box (GC-88) at
88 bp of
minimal promoter slightly increased transcriptional activity
(GC-mt #3), the mutations in the other two GC-boxes
(GC-143 and GC-105) reduced the transcriptional
activity (GC-mt #1, #2). Especially, abrogation
of the GC-box located at
143 bp (GC-143) of the minimal
promoter led to a remarkable reduction (>95%) of transcriptional
activity (GC-mt #1). Abrogation of the GC-box located at
105 bp (GC-105) also caused a >50% reduction of
transcriptional activity (GC-mt #5). Mutations of all of two E-boxes and three GC-boxes led to a marked loss of transcriptional activity (99.9 ± 0.2% reduction, GC-E-mt), and
mutations of only two GC-boxes (GC-143 and
GC-105) except GC-88 also caused a >98% reduction of
transcription activity (GC-mt #4). These results indicate
that several cis-elements, including GC-143 and -105, as
well as E-201, are important for basal transcriptional activity of
mTERT gene. In particular, two GC-boxes (GC-143 and
-105) are the most essential cis-elements for
basal transactivation of mTERT gene in C2C12 cells.
View larger version (21K):
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Fig. 4.
Sequences of the mouse TERT core
promoter and consensus motifs for factor binding sites. The start
site of transcription is shown by an arrow. 1
indicates the first nucleotide 5' to the start site of transcription,
while +1 indicates the first nucleotide of the mRNA. The
initiating ATG codon is shown in boldface. The sequences of
putative E-boxes and GC-boxes are underlined. The sequences,
which were mutated in the functional analyses of
cis-elements, are indicated by asterisks, and
above these the substituted sequences are indicated in
italics.
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Fig. 5.
Mutation analysis of minimal region of the
mouse TERT promoter. C2C12 cells were transfected with luciferase
reporter constructs containing the substitution mutation of the factor
binding site in the mTERT gene core promoter. The luciferase activity
of the 225/+53-Luc plasmid was normalized to 1, and the relative
luciferase activities are shown. The putative E-boxes (E;
open boxes) and GC-boxes (GC; gray
boxes) are shown, the crossed-out boxes indicate the
mutated sites for binding factors as shown in Fig. 4. The standard
deviations (S.D.) of the means are indicated by the error
bars. Results are expressed as the mean ± S.D. of at least
three independent experiments, each performed in triplicate.
225/+53)
is responsible for reduction of mTERT transcription during myogenesis. To identify the role of cis-elements in this core promoter,
C2C12 cells were stably transfected with reporter plasmids containing mutations in five protein binding sites and induced to differentiate with differentiation medium for 10 days (Fig.
6). Transfectants with GC-mt #1, GC-mt
#2, and Ebox-mt #1 reporter plasmids, which have single mutation among
these cis-elements, reduced transcriptional activity during
myogenesis (75-85% reduction), as well as transfectants with either
1561/+53-Luc or
225/+53-Luc reporter plasmid. Mutation of two
E-boxes (Ebox-mt #2) also caused a decrease of luciferase activity (75% reduction). This result indicates that GC-boxes in the
mTERT core promoter are responsible for differentiational down-regulation of mTERT gene in C2C12 cells. Abrogation of two GC-boxes (GC-143 and GC-105; GC-mt #4)
also caused a slight decrease of transcription activity during
myogenesis (15% reduction, Fig. 6). However, we could not clearly
ascertain whether the rest of the E-boxes in the mTERT core promoter
are indispensable for muscle cell differentiation or not, because there
was not enough gene expression activity to be lost further with GC-mt
#4 reporter plasmid, as shown in Fig. 5. In establishing these stable
transfectants, several clones were shown to have different basal
transcriptional activities from those of the transient transfectants
(Fig. 5), because these results might represent the difference of the
number of reporter gene copies, which were integrated into host
chromosomal DNA.
View larger version (21K):
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Fig. 6.
Identification of
cis-elements for the repression of mTERT gene during
myogenesis. C2C12 cells were stably transfected with luciferase
reporter constructs containing the substitution mutation of the factor
binding site in the mTERT gene core promoter and induced
differentiation by the culture in differentiation medium for 10 days.
The luciferase activity of the 1561/+53-Luc plasmid was normalized to
1, and the relative luciferase activities are shown. The putative
E-boxes (E; open boxes) and GC-boxes
(C; gray boxes) are shown, the crossed-out
boxes indicate the mutated sites for binding factors. The standard
deviations (S.D.) of the means are indicated by the error
bars. Results are expressed as the mean ± S.D. of at least
three independent experiments, each performed in triplicate.
225/+53-Luc). Sp1 and Sp3 induced a large transactivation
of mTERT promoter activity (6.5-fold and 4.2-fold, respectively) in
C2C12 myoblasts (Fig. 7C). These findings suggest that
Sp1 and Sp3 function as potent transactivators of mouse TERT gene.
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Fig. 7.
Sp1 family proteins bind to the proximal
promoter region of mTERT and enhance transcriptional activity.
Nuclear extracts prepared from C2C12 cells interacted with
32P-labeled GC-143 (lanes 1-4), GC-105
(lanes 5-8). A, anti-Sp1 (lanes 2 and
6), anti-Sp3 (lanes 3 and 7), or both
antibodies (lanes 4 and 8) were added to the
extract before the addition of the probe. B, the specificity
of the complexes was examined by the addition of 5-fold (lanes
2 and 6), 20-fold (lanes 3 and
7), or 100-fold (lanes 4 and 8) molar
excess of unlabeled Sp1 consensus oligonucleotides for competitors. The
arrows indicate the Sp1- or Sp3-related complexes.
Representative autoradiograms are shown. C, one of the
expression vectors for Sp1 (pCGN-Sp1), Sp3
(pCGN-Sp3), or null expression vector (pCGN), was
cotransfected with the 225/+53-Luc reporter plasmid into C2C12 cells.
The luciferase activity of reporter plasmid with pCGN was normalized to
1, and the relative luciferase activity is shown. Results are expressed
as the mean ± S.D. of three independent experiments, each
performed in triplicate.
201 to
196 (E-201) of
mTERT promoter was also responsible for the basal transcription. To
identify the proteins that bind to this E-box in mTERT gene core
promoter, EMSAs were carried out with nuclear extracts prepared from
the C2C12 myoblasts (Fig. 8A).
Three bands (bands A-C) were observed by using E-201
oligonucleotides as probe, although band C sometimes diminished. These
three (or two) bands were competed by homologous competitors (Fig.
8A, lanes 4-6), but not by MyoD binding
oligonucleotides nor mutated oligonucleotides in which the E-box
sequences were altered (lanes 7-12). Only band B, but
neither band A nor band C, was eliminated by an excess amount of
unlabeled c-Myc consensus sequences (Fig. 8A, lanes
1-3). Therefore, band B may represent the c-Myc·DNA complex.
The formations of bands A and C were not affected by adding either an
excess amounts of c-Myc consensus sequence (lanes 1-3) or
the anti-c-Myc antibody (data not shown). Therefore, these bands may
not be the complex of Myc·Max nor their degradation products and have
thus remained to be identified.
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Fig. 8.
c-Myc binds to E-box at the minimal region of
mTERT gene promoter. A, nuclear extracts prepared from
C2C12 cells interacted with 32P-labeled E-201. Five
micrograms of nuclear extracts were subjected to the EMSA using the
E-box region at 201 as probe (see "Experimental Procedures") in
the presence or absence of competitor. The specificity of the complexes
was examined by the addition of 20-fold (lanes 2,
5, 8, and 11) or 100-fold (lanes
3, 6, 9, and 12) molar excess of
unlabeled c-Myc consensus oligonucleotides (c-Myc;
lanes 2 and 3), homologous competitors
(homo; lanes 5 and 6), MyoD binding
oligonucleotides (MyoD; lanes 8 and
9), and oligonucleotide containing a mutated E-box site
(mt; lanes 11 and 12) for competitors.
B, one of the expression vectors for c-Myc
(pcDNA3-cMyc; gray boxes) or null
expression vector (pcDNA3; open boxes) was
cotransfected with
225/+53-Luc or
1561/+53-Luc reporter plasmid
into C2C12 cells. Cells grown in 24-well plates were transfected with
0.25 µg of luciferase reporter plasmid, 0.033 µg of pRL-TK, and
0.075 µg of the c-Myc expression plasmids. The luciferase activity of
reporter plasmid with pcDNA3 was normalized to 1, and the relative
luciferase activity is shown. Results are expressed as the mean ± S.D. of three independent experiments, each performed in
triplicate.
225/+53), or with a
longer 1.6-kb upstream region (
1561/+53). As shown in Fig.
8B, expressed c-Myc increased significantly the promoter activities in both cases. Interestingly, the stimulation (1.9-fold) was
higher, with the reporter plasmid harboring a long promoter region of
1561/+53 than with that contained in the core promoter of
225/+53
(1.3-fold). These observations suggest that c-Myc, in addition
to Sp1 and Sp3, also act on the mTERT promoter to enhance transcription
of mTERT gene.
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Fig. 9.
Sp1, Sp3, and c-Myc are repressed during
myogenesis. The C2C12 cells were cultured in differentiation
medium for 1, 4, 7, or 10 days. A, nuclear extracts were
analyzed by immunoblotting with antibodies against Sp1, Sp3, and c-Myc.
B, 5 µg of nuclear extracts was subjected to EMSAs using
GC-box region at 143 (GC-143; lanes 6-10) or
at
105 (GC-105; lanes 1-5) as probe. C, 5 µg
of nuclear extracts was subjected to EMSAs using E-box region at
201
(E-201) as probe. Representative autoradiograms are
shown.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
225 to +53, contains several
GC-boxes and E-boxes. Deletion and mutation of these elements resulted
in a significant loss of transcriptional activity in several cell
lines, including C2C12 myoblasts. We attempted to identify the
cis-elements in the mTERT core promoter for transactivation and found that two GC-boxes (GC-143 and GC-105) bound to Sp1 and Sp3,
and an E-box (E-201) bound to c-Myc but not to MyoD in C2C12 myoblasts.
It was demonstrated that decreased binding of Sp1, Sp3, and c-Myc to
three cis-elements in the mTERT core promoter were well
correlated with the down-regulation of mTERT gene expression during
muscle cell differentiation.
225/+53 of the mTERT gene by EMSAs (Fig. 7, A
and B). Furthermore, overexpression of either Sp1 or Sp3
elevated the level of mTERT transcription in undifferentiated myoblasts
(Fig. 7C). Sp1 is thought to be a ubiquitously expressed transcription factor that plays a primary role in the regulation of a
large number of genes, including constitutive housekeeping genes and
inducible genes (37). Sp3 is considered to be bifunctional such that it
represses Sp1-mediated activity of several promoters (38-41), whereas
it acts as an activator of gene expression in mammalian cells
(42, 43). In this study, we provide evidence that, in mTERT gene
transcription, both Sp1 and Sp3 act as positive regulators (Fig. 9). As
shown in Fig. 2, NIH3T3 and C3H10T1/2 conferred higher transcriptional
activity than C2C12. Consistently, EMSAs showed greater binding
activities of Sp1 and Sp3 in NIH3T3 and C3H10T1/2 than in C2C12 (data
not shown). These results might be caused by different extents of Sp1
and Sp3 expression among cell types (44). In mouse, high levels of Sp1
expression have been found in spermatids, T cells in thymus, epithelial
cells, and hematopoietic cells, whereas Sp1 expressions in heart,
skeletal muscle, and smooth muscle cells of stomach have been shown to be low (44). The different expression levels of Sp1 are consistent with
tissue-specific expressions of telomerase activity in adult mouse.
Telomerase activities are not detected in brain, heart, stomach, and
muscle in mouse (22).
1561/+53 long
upstream region decreased to a greater extent than that of the core
promoter (
225/+53) (Fig. 3). In parallel, expression of mTERT
mRNA largely decreased to a greater extent than the expression of
luciferase conjugated with
225/+53 during the differentiation. Moreover, as shown in Fig. 8B, other c-Myc responsive
cis-elements are probably located in the
1561/+53 long
upstream region. According to data base analysis, we also noticed that
a number of putative cis-elements, including GC-boxes or
E-boxes, are located 5' upstream beyond the core promoter region of
mTERT (data not shown). These putative cis-elements may also
regulate the transcription of mTERT gene by binding transcription
factors such as the bHLH proteins or the Sp1 family. It remains to be
solved whether chromatin remodeling factors and/or nucleosomal
packaging, as well as transcription factors, may also play a role for
mTERT transactivation through the enhancer or silencer region outside
the core promoter sequence. To analyze further the tissue-specific
expression of telomerase in mouse, experiments using an upstream region
of the mTERT core promoter are underway in our laboratory.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Shosei Yoshida (Kyoto University) for providing a mouse MyoD cDNA and myogenin cDNA and Thomas Shenk (Princeton University) for providing pCGN-Sp1 and pCGN. The expression vector for c-Myc (pcDNA3-cMyc) was a kind gift from Dr. Rónán C. O'Hagan and Dr. Ronald A. DePinho (Harvard Medical School). We thank Dr. Shonen Yoshida (Nagoya University) for many helpful comments and suggestions.
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FOOTNOTES |
---|
* This work was supported in part by the Fund for Comprehensive Research on Aging and Health and for Longevity Sciences (Ministry of Health and Welfare 10-03).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.
Recipient of a domestic research fellowship from the Japan Science
and Technology Corporation.
§ To whom correspondence should be addressed: Tel.: 81-562-44-5651; Fax: 81-562-44-6591; E-mail: kenisobe@nils.go.jp.
Published, JBC Papers in Press, March 28, 2001, DOI 10.1074/jbc.M011181200
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ABBREVIATIONS |
---|
The abbreviations used are: TERT, telomerase reverse transcriptase; hTERT, human TERT; mTERT, mouse TERT; bp, base pair(s); HRP, horseradish peroxidase; RT-PCR, reverse transcriptase-polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; TRAP, telomere repeat amplification protocol; EMSA, electrophoretic mobility shift assay; kb, kilobase(s); bHLH, basic-helix-loop-helix.
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