From the Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec H3G 1Y6, Canada
Received for publication, April 6, 2001, and in revised form, May 1, 2001
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ABSTRACT |
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Ectopic expression of DNA methyltransferase 1 (DNMT1) has been proposed to play an important role in cancer.
dnmt1 mRNA is undetectable in growth-arrested cells but
is induced upon entrance into the S phase of the cell cycle, and until
now, the mechanisms responsible for this regulation were unknown. In
this report, we demonstrate that the 3'-untranslated region (3'-UTR) of
the dnmt1 mRNA can confer a
growth-dependent regulation on its own message as well as a
heterologous DNA methylation is a covalent modification of DNA that can
modulate gene expression and is now recognized as a major component of
the epigenome (1, 2). The methylation of cytosines in vertebrates
occurs when they are found 5' to deoxyguanosine in the sequence CpG.
80% of CpGs are methylated (3), and they are distributed in a pattern
that is unique in each tissue and is inversely correlated with gene
expression (3).
The pattern of methylation is faithfully maintained during cell
division by the enzyme
DNMT1,1 the maintenance DNA
methyltransferase, (4) which catalyzes the transfer of a methyl group
from S-adenosyl-methionine to the 5-position of the cytosine
ring (5, 6). DNMT1 has a bilateral relationship with DNA replication
and cell growth (7). DNA methylation occurs concurrently with DNA
replication (8), and the expression of dnmt1 is tightly
coordinated with the cell cycle (9), whereas inhibition of DNMT1
inhibits DNA replication (10), leading to senescence and inhibition of
cell growth (11, 12). Deregulated expression of dnmt1 was
previously suggested to play a causal role in cellular transformation
(13, 14). Several lines of evidence are in accordance with this
hypothesis. First, elevated levels of dnmt1 mRNA and
activity were reported in tumors and cancer cell lines (15-17).
Second, ectopic expression of dnmt1 results in cellular
transformation (18, 19). Third, dnmt1 expression is
regulated by nodal proto-oncogenic signaling pathways (20-22). And
fourth, inhibition of DNMT 1 by 5-azacytidine (a pharmacological inhibitor of DNMT1) or a reduction in DNMT1 levels by either antisense mRNA or antisense oligonucleotides reverses tumorigenesis
(23-25).
The mechanism by which DNMT1 influences cellular transformation is
unclear. It has been proposed that high levels of DNMT1 can lead to
ectopic methylation and silencing of tumor suppressor genes (26).
However, a clear correlation between general dnmt1 overexpression and tumor suppressor hypermethylation has not been established (27, 28). It has recently been suggested that deregulated
expression of dnmt1 during the cell cycle might be critical
for the effects of DNMT1 on cell growth (29, 30). Previous nuclear
run-on experiments have demonstrated that although both growth-arrested
and cycling cells transcribe dnmt1 mRNA at a similar
rate, the mRNA is only detected in cycling cells (9). This suggests
that the cell cycle regulation of dnmt1 occurs at the
posttranscriptional level. It stands to reason that the mechanisms involved are linked to basic control points of the cell cycle, and
potentially linked to cellular transformation. To test this hypothesis,
the factors responsible for regulation of dnmt1 expression with the cell cycle must be identified. One potential element is the
AU-rich 3'-untranslated region (3'-UTR) of the dnmt1
mRNA, because it is well documented that this type of element can
regulate mRNA levels (31).
Regulation of mRNA stability by AU-rich elements (AREs) is an
important mechanism involved in orchestrating the expression of
critical genes in development (32, 33) and cell cycle (34, 35), early
response genes involved in cellular growth such as c-myc
(36) and c-fos (37), and cytokines such as granulocyte macrophage colony-stimulating factor (GM-CSF) (38) and interleukin 3 (39). These regions often modulate mRNA levels in response to a
change in cell environment by growth factors (40, 41), developmental
factors (42), and hormones (43). Although the mechanism by which AREs
act has not been fully elucidated, it appears to involve the binding of
various protein factors, several of which have been well characterized,
such as AUF1 (44, 45) and the embryonic lethal abnormal vision
family members (46, 47).
In this study, we test the hypothesis that the 3'-UTR of the
dnmt1 mRNA plays a role in regulating its levels with
the cell cycle and that deregulation at this level has an effect on
cellular transformation by DNMT1. We show that a 54-nucleotide highly
conserved element within the 3'-UTR can confer a
growth-dependent regulation on both homologous and
heterologous mRNAs in living cells and that this region can
destabilize mRNA in vitro. This element interacts with a
40-kDa protein, the binding of which is inversely correlated with
dnmt1 mRNA levels throughout the cell cycle. Finally, we demonstrate that the 3'-UTR influences the cellular changes observed upon overexpression of DNMT1 in NIH-3T3 cells, thus
providing a link between dnmt1 regulation, cell cycle, and oncogenesis.
Cell Culture and Flow Cytometry--
Balb/c-3T3 cells
(ATCC) were maintained as a monolayer in Dulbecco's modified Eagle's
medium (Life Technologies, Inc.) containing 10% calf
serum (Colorado Serum Co). To arrest cells at
G0/G1, confluent Balb/c 3T3 cells were cultured
in a medium containing 0.5% calf serum for 48 h. The cells were
induced to enter the cell cycle by replacing the growth medium with
medium containing 10% calf serum. Cells at various stages of the cell
cycle were obtained by harvesting them at varying lengths of time after
serum induction. To determine the percentage of cells at different
stages of the cell cycle, cells were stained with propidium iodide and the DNA content was measured by flow cytometry (48).
Plasmid Construction--
For the construction of
pCRII-DNMT1 3'-UTR, human DNMT1 3'-UTR (5090-5408)
was amplified by reverse transcription-PCR from 1 µg of total RNA
prepared from human lung cancer H446 cell line (ATCC). Reverse
transcription was performed using random primers (Roche Molecular
Biochemicals) and Superscript reverse transcriptase as recommended by
the manufacturer (Life Technologies, Inc.). For PCR amplification with
Taq polymerase (CLONTECH), 50 ng of the
sense primer (5'-TCTGCCCTCCCGTCACCC-3') and antisense primer 5'-GGTTTATAGGAGAGATTT-3') was used to amplify the 3'-UTR from 5 µl of
reverse-transcribed cDNA in the presence of 1 mM dNTPs, and the manufacturer's amplification buffer was supplemented to 2 mM MgCl2. Cycling conditions were as follows:
95 °C for 1 min, 50 °C for 1 min, and 72 °C for 1.5 min for 30 cycles. The amplified fragment was subcloned into pCR II using the TA
cloning kit (Invitrogen) as recommended by the manufacturer. To
generate pRBG-DNMT1 3'-UTR, the human DNMT1 3'-UTR was excised from
PCRII-DNMT1 3'-UTR with BamHI and XbaI and
ligated into a XbaI-SalI digested pRBG-GC (38). The incompatible ends were then filled using Klenow DNA polymerase (Roche Molecular Biochemicals) and the blunt ends were joined using T4
DNA ligase (MBI Fermentas).
For the construction of pRBG-DNMT1
pSK-
For the construction of pUD-hDNMT1 and pUD-hDNMT1
For the construction of pAd-DNMT1, pAd-DNMT1 Preparation and Analysis of RNA--
Total RNA was prepared by
the guanidinium isothiocyanate method (50), and mRNA levels were
determined by Northern blot analysis. Approximately 10 µg of RNA was
electrophoresed on a 1.2% denaturing agarose gel and then transferred
to Hybond-N+ membrane (Amersham Pharmacia Biotech). Blots were probed
with the indicated 32P-labeled cDNA probes synthesized
using a random priming labeling kit (Roche Molecular Biochemicals). The
membranes were hybridized at 68 °C for 4-6 h in a buffer containing
0.5 M sodium phosphate, pH 6.8, 1 mM EDTA, 7%
SDS, and 0.2 mg·ml herring sperm DNA. Following hybridization the
membranes were washed twice for 10 min in a 5% SDS, 0.04 M
sodium phosphate, pH 6.8, 1 mM EDTA solution and then four
times for 10 min in the same solution containing 1% SDS. The following
probes were used: rabbit
The level of expression of the different mRNAs was quantified by
densitometric scanning of the relevant autoradiogram. Each experiment
was normalized for the amount of total RNA by hybridization with a
32P-labeled 18-s ribosomal RNA oligonucleotide probe (51),
with the exception of the adenoviral experiments, which were normalized to GFP, which is expressed by the adenoviral vector.
Stable Transfections--
To create the pRBG stable lines, 5 µg of the following plasmids were introduced into Balb/c 3T3 cells by
DNA-mediated gene transfer using the calcium phosphate protocol (52):
pRBG-AT (bearing the 3'-UTR of human GM-CSF and a neomycin resistance gene as a selectable marker), pRBG-GC (control) (both plasmids were a
kind gift of Dr. G Shaw) (38), pRBG-DNMT1 3'-UTR, and pRBG-DNMT1
Stable lines expressing pUD-hDNMT1 and pUD-hDNMT1
Growth rates were determined following plating the cells in six-well
culture dishes at a density of 25,000 cells/well. Cells were then
counted on six subsequent occasions throughout the span of 11 days, and
phase contrast photography was performed on the last day. An average of
four counts was performed for each time point.
Adenoviral Infections--
Balb/c cells were grown to confluence
in six-well plates and were then transferred to 0.5% serum medium for
48 h to arrest cell growth. Cells were then infected for 4 h
in serum-free medium with pAd-DNMT1, pAd-DNMT1 In Vitro RNA Synthesis--
To generate RNA transcripts for the
gel mobility shift, UV cross-linking, and in vitro
degradation assays, the following plasmids were linearized at the
indicated restriction sites: pCRII-DNMT1 3'-UTR (BamHI),
pSK- Preparation of Whole Cell Extracts--
Balb/c 3T3
cells were harvested and resuspended in extraction buffer (20 mM Tris-HCl, pH 7.5, 0.4 M KCl, 20% (v/v)
glycerol, 2 mM dithiothreitol, and 1×
CompleteTM protease inhibitor (Roche Molecular
Biochemicals)). The suspension was frozen immediately at RNA Mobility Gel Shift Assays and UV Cross-linking--
Reaction
mixtures were incubated on ice for 1 h in a 20-µl mixture
containing 50 µg of whole cell extract, 1,000,000 cpm of probe), 10 µg of tRNA (as a nonspecific RNA competitor), and the following
buffer: 10 mM Hepes, pH 7.6, 3 mM
MgCl2, 40 mM KCl, 5% glycerol, 1 mM dithiothreitol. Following incubation, free unbound 32P-labeled RNA was removed by treating the reaction
mixture with 1.5 µl of RNase T1 (1 unit/ml) and 1.5 µl of RNase A
(10 mg/ml) for 10 min at room temperature. Heparin 5 µg/ml was added,
and the reaction was incubated for an additional 10 min at room
temperature. The resulting complexes were resolved on a 10%
nondenaturing polyacrylamide gel. For UV cross-linking assays, the same
reaction mixture was subjected to incubation on ice for 1.5 h
under UV light (254 nm). Cold RNA competitors were used at a
concentration of 1000×. RNases were added as above, and the reaction
mixture was incubated for an additional 30 min at 37 °C. 5 µl of
5× loading buffer (1.05 M Tris-Cl, pH 6.8, 36% glycerol,
100 mg/ml SDS, 0.2% In Vitro RNA Degradation Assay--
RNA decay rates were
assessed using the protocol described in Ref. 42 with the following
modifications. 10 µg of whole cell extract and 2.5 × 105 cpm of in vitro synthesized RNA transcript
were used, and the reactions were carried out for 0.25-4 h at
37 °C. The concentration of RNA at each time point was quantified by
densitometry of the autoradiogram and is presented in the figures as
the percentage of RNA remaining at each time point relative to the
concentration of RNA at time 0. The half-life of the different RNAs was
calculated from the logarithmic decay plots.
Quantification of hDNMT1 mRNA Expression by Competitive
Reverse Transcription-PCR--
Total RNA (1 µg) from TetOff NIH 3T3
stable lines was reverse-transcribed with M-MuL-V reverse
transcriptase and a random primer (MBI) using the manufacturer's
protocol in the presence of 25 µCi of 35S-labeled dATP
(1250 Ci/mmol) to quantify the efficiency of reverse transcription.
Equal amounts of cDNA (30000 cpm as determined by
[35S]dATP incorporation) were subjected to
DNMT1 PCR amplification using Taq polymerase
(MBI) in the presence of decreasing concentrations (10
To further confirm that equal amounts reverse-transcribed cDNA were
being used, we amplified Time Course of dnmt1 mRNA Increase following Serum Stimulation
of Arrested Balb/c-3T3 Cells--
Previous studies have demonstrated
that DNMT1 is regulated with cell growth (9, 29, 55, 56). To correlate
the levels of dnmt1 mRNA with different stages of the
cell cycle in our system, Balb/c-3T3 cells were stimulated with serum
for varying times following growth arrest, and concurrent FACS and
Northern blot analyses were performed. As seen in Fig.
1, A and C, the
majority of cells were arrested at G0/G1
(70%) after serum deprivation for 48 h (t = 0).
Addition of serum-containing medium induced entry of the cells into the
S phase of the cell cycle, which peaked 24 h poststimulation
(S = 51%). dnmt1 mRNA was barely detectable in
arrested cells and was induced late in G1 (8-12 h),
reaching its maximum levels at the G1-S boundary (20 h)
(Fig. 1, B and C). dnmt1 mRNA
remained elevated throughout S, and then began to decrease as cells
continued into G2-M. Because nuclear run-on transcription
experiments have previously shown that dnmt1 is transcribed
at a constant rate throughout the cell cycle (9), the variation in
mRNA levels must occur at the posttranscriptional level. This mode
of regulation has already been reported for dnmt1 in several
cases, such as in myoblast differentiation (57), F9 cell
differentiation (58), and dnmt1 up-regulation by T-antigen (22).
The 3'-UTR of the dnmt11 mRNA Contains a Highly Conserved
AU-rich Element--
Because the 3'-UTR of an mRNA is often
implicated in mediating posttranscriptional regulation, we examined
this region of dnmt1 for the presence of any potential
mRNA regulating motifs. We first reasoned that an important
regulatory element should remain conserved throughout evolution. As can
be seen in Fig. 2, an alignment of the
3'-UTR of the dnmt1 mRNAs from human, mouse, rat,
Xenopus, chicken, and zebrafish (daniorerio) identifies a 54-nucleotide region that is 100% conserved between human and chicken,
and a smaller region of 36 nucleotides that is conserved among all the
species. Also shown are several AU-rich sequences that may act as
potential RNA destabilizing motifs (Fig. 2, boxed and
underlined) (59). Such a homology in a noncoding region is
unusual (less than 30% would be expected without selective pressure
(60)) and strongly suggests an important regulatory role for this RNA
element. The 54-nucleotide sequence is unique to dnmt1, and
no other example was found in the human genome by a BLAST search of the
human genome data bank. Many mRNAs that are regulated
posttranscriptionally contain highly conserved 3'-UTRs, such as
c-fos, bcl2, fibronectin (61), B-Ar (62),
p21 (35), and IgfII (40). As many of these
regions often modulate mRNA levels in response to varying
environmental and growth conditions, it is highly probable that this
region of the dnmt1 mRNA is involved in its cell cycle
regulation.
The DNMT1 3'-UTR Can Down-regulate the Stable The 54-Nucleotide Highly Conserved Region of the 3'-UTR Is Required
to Down-regulate the DNMT1 mRNA in Growth-arrested Cells--
We
then assessed whether the DNMT1 3'-UTR could regulate its
own mRNA in a similar manner, by generating adenoviral vectors expressing human DNMT1 containing deletions within the
3'-UTR (Fig. 3D). The adenovirus system is advantageous
because sufficient levels of transient expression of the exogenous
constructs are obtained, thus avoiding the tedious process of
generating stable cell lines. In addition, the adenoviral vector
contains the gene for GFP, which is under control of the same promoter
and poly(A) signal as the gene of interest and can serve as an ideal
internal control for infection efficiency, as well as any variations in promoter activity due to changes in growth conditions.
Fig. 3E displays a representative Northern blot of RNA from
Balb/c cells infected with the different adenoviral constructs. We used
a human DNMT1 cDNA probe to differentiate between the exogenous human DNMT1 deletions and the endogenous mouse
dnmt1, and under our conditions of hybridization, the
endogenous dnmt1 was undetected. Once the results were
quantified and normalized to GFP (Fig. 3F), they
demonstrated that only the DNMT1 construct bearing the full
3'-UTR (DNMT1) was down-regulated in arrested cells. By removing the
3'-UTR ( A Specific Protein (p40) Binds the Conserved Region of the DNMT1
3'-UTR and Is Up-regulated in Growth-arrested Cells--
The 3'-UTR
can mediate its regulatory effects through interaction with various
RNA-binding proteins, such as AUF1 (44, 45), and the embryonic lethal
abnormal vision proteins (46, 47). Many other 3'-UTR mRNA
binding activities have been found (64-67). However, most of these
have not been well characterized as of yet. In many instances, the
expression or binding activity of the proteins themselves is modified
in response to various stimuli (46, 59) and is correlated with either
up- or down-regulation of the associated mRNAs (68, 69).
We therefore addressed the question of whether the growth regulation of
dnmt1 mRNA levels involves the formation of growth phase-specific RNA-protein complexes. To answer this, we performed an
RNA gel mobility shift assay using a 32P-labeled
DNMT1 3'-UTR RNA transcript (Fig.
4A, 3'-UTR) and whole cell
extracts prepared from Balb/c cells that were growth-arrested and then
serum-stimulated for specific lengths of time (as in Fig. 1). The
RNA-protein complexes were subjected to RNase digestion to remove free
unbound RNA prior to resolving of the complex on a nondenaturing
polyacrylamide gel. As observed in Fig. 4B, the profile of
RNA protein complexes within the 3'-UTR changed as passage into the
cell cycle occurred. Most strikingly, a specific complex (indicated by
arrow) was only observed in extracts prepared from
growth-arrested cells. The fact that these complexes were formed in the
presence of excess of nonspecific tRNA suggests that these interactions
are specific.
UV cross-linking analysis was then utilized to further study
RNA-protein interactions within the 54-nucleotide element, as this
region appears to be critical for dnmt1 mRNA
down-regulation. The UV cross-linking method allows one to observe the
binding of individual proteins to the RNA, and the molecular
mass of the proteins can be approximated. As can be seen in Fig.
4C, the interaction of
The above findings, together with the results in Fig. 3, suggest that
p40 may be involved in the mRNA down-regulation mediated by the
54-nucleotide element in arrested cells. To provide further support to
this theory, we performed a UV cross-linking with extracts prepared at
various time points after serum stimulation of arrested cells. A
representative UV cross-linking is shown in Fig. 3F, and the
average results of three quantified experiments are illustrated in Fig.
3G. We can clearly see that p40 binding was decreased between 0 and 6 h after cell growth is stimulated, and other
proteins interacted with the 54-nucleotide element as cells progressed through the cell cycle. When we overlaid the time course of p40 binding
with dnmt1 mRNA levels following serum stimulation, we observed an inverse correlation between p40 binding and
dnmt1 mRNA levels; dnmt1 mRNA increased
shortly after p40 levels fell and remained elevated while p40 binding
was low. Although these results do not prove that p40 is responsible
for the change in dnmt1 mRNA levels, the facts that p40
binding is highly induced in growth-arrested cells and that it is
specific for the same region that is required for mRNA
down-regulation are consistent with this hypothesis.
The Rate of mRNA Decay in Vitro Is Accelerated in the Presence
of the 54-Nucleotide Region and Arrested Cell Extracts--
Because
mRNA posttranscriptional regulation can occur at multiple levels,
we addressed the question of how the DNMT1 3'-UTR down-regulates mRNA levels in growth-arrested cells. We decided to
investigate the role of the DNMT1 3'-UTR in mRNA decay
for two reasons. First, the 3'-UTR contains several AREs, which have been shown to modulate the stability of a wide variety of mRNAs, and second, alterations in mRNA half-life involving AREs have been
found to involve changes in RNA-protein interactions.
We used an in vitro or cell-free RNA degradation assay (42)
to assess the decay rates of the various DNMT1 3'-UTR
deletion constructs. This technique measures the decay of in
vitro synthesized radiolabeled RNA transcripts that are incubated
with extracts prepared from cells grown under various conditions. This
technique has several advantages over other methods of measuring
mRNA decay (31), the most important being that it avoids the
in vivo use of transcriptional inhibitors such as
actinomycin D and dichlororibofuranosylbenzimidazole, both of
which have been found to alter the stability of certain mRNAs
(70-73) and would therefore confound any half-life measurements. In
our hands, actinomycin D stabilized the dnmt1 mRNA and
therefore could not be used. We also tried using stable lines
expressing tetracycline-repressible DNMT1 constructs;
however, all the cell lines we obtained expressed background levels of
DNMT1 in the presence of the repressor.
Fig. 5, A and B,
shows the results of the decay experiment performed with the various
3'-UTR deletions in the presence of extracts prepared from arrested
Balb/c cells (arrested) or in decay buffer alone (control). We observed
that the entire 3'-UTR and the 54-nucleotide element (
To test whether this decay is affected by cell growth, we compared the
stability of The 3'-UTR Inhibits DNMT1-induced Transformation of NIH-3T3
Cells--
It has been well documented that dnmt1 is
overexpressed in a variety of tumors and cancer cell lines (15-17) and
that ectopic expression of DNMT1 can transform both NIH-3T3
cells (18) and human fibroblasts (19). However, only the coding region
of DNMT1 was used in these latter two studies.
DNMT1 interacts with various cell cycle regulators, such as
proliferating cell nuclear antigen (74) and Rb-E2F (75), and recent data suggest that the cell cycle regulation of DNMT1 is disrupted in colorectal cancer cells (76) and in estrogen
receptor-negative breast cancer cells (56). We therefore tested
the hypothesis that the loss of this cell cycle regulation is the main
reason for the transforming properties of DNMT1 and that
expression of a DNMT1 construct with its full 3'-UTR, which
should be properly regulated, would not result in changes in cellular identity.
We generated stable NIH-3T3 cell lines expressing human
DNMT1 with the full 3'-UTR (hDNMT1) or without the 3'-UTR
(hDNMT1
We first examined whether the constructs were differentially regulated
with cell growth as expected. Fig.
6A depicts the results of a
competitive reverse transcription-PCR performed to quantify the levels
of hDNMT1 mRNA in transfectants that were either
serum-starved or serum-stimulated for 20 h. Because the PCR
primers used amplify both the mouse and human DNMT1, we then hybridized
our PCR with a human-specific DNMT1 32P-labeled
oligonucleotide probe to specifically detect the hDNMT1 constructs. Similar to the results obtained with the adenovirally expressed DNMT1 and the
We then studied in detail the growth characteristics of the different
clones with respect to cell cycle distribution, growth rates, and
morphology. Fig. 6B displays a growth curve of
representative hDNMT1 and hDNMT1 Because hyperactivation of DNMT1 has been implicated in cellular
transformation (14), understanding how this enzyme is regulated with
cell growth is of obvious interest.
In this paper, we first confirm previous findings that the
dnmt1 mRNA is regulated with the cell cycle (9, 29).
dnmt1 mRNA is essentially absent in arrested cells and
then increases as cells are induced by serum and reaches its maximum
level just before the peak of S phase; this coordination would ensure
that sufficient DNMT1 protein is synthesized so as to carry out its maintenance function during replication. Second, we demonstrate that
the 3'-UTR of DNMT1 confers a growth-dependent
regulation on both its own mRNA and a heterologous stable
Whereas we were able to show that the 3'-UTR down-regulates
dnmt1 mRNA in growth-arrested Balb/c cells, we were not
able to demonstrate that this occurs by destabilization of
dnmt1 mRNA in living cells. Actinomycin D is routinely
used to block de novo synthesis of RNA in order to measure
mRNA decay. However, we found that actinomycin D stabilizes the
dnmt1 mRNA. This is not uncommon, and it has been
reported for several other mRNAs (70-73). We also could not obtain
repressible DNMT1 transfectants that show zero DNMT1 expression in the repressed state, which could be used
for measuring stability of mRNA in cells. However, the fact that
the same element that reduces the presence of dnmt1 mRNA
in arrested cells also destabilizes dnmt1 mRNA in
vitro supports the conclusion that the 3'-UTR acts as a cell
cycle-regulated destabilizing element.
Many studies have shown that mRNA posttranscriptional regulation
plays an important role during development (32, 33) and cell cycle
progression (34, 35) and exerts an important control over expression of
proto-oncogenes (36, 37) and cytokines (38, 39). Several pathological
states are associated with aberrant regulation at this level, including
various immune and inflammatory diseases and several types of cancer
(78). One potential mechanism by which the 3'-UTR may function is by
the differential binding of trans-acting factors during the
cell cycle; these RNA-protein complexes could then either increase or
decrease mRNA levels. Studies involving several mRNAs, such as
parathyroid hormone (79), GM-CSF (68), and CYP7A1
(73) have found that changes in mRNA stability are correlated with
the binding of proteins within the 3'-UTR. In accordance with this
postulated mechanism, we show, using an RNA mobility gel shift assay,
that a specific RNA-protein complex is formed within the 3'-UTR
sequence only in growth-arrested cells. In addition, our UV
cross-linking experiments reveal a protein of ~40 kDa (p40) that
exhibits specific binding to the conserved 54-nucleotide element. This
binding is highly induced in arrested cells and decreases at the same
time as dnmt1 mRNA increases during cell cycle
progression. Although our data do not demonstrate a causal link between
the p40-3'-UTR interaction and dnmt1 mRNA
down-regulation, our results are in accordance with this hypothesis.
Further purification and characterization of p40, which is currently
under way in our laboratory, may provide us with a better understanding
of its mechanism of action.
It has been recently suggested that the deregulation of
dnmt1 with the cell cycle rather than its overall levels is
important for cellular transformation by dnmt1 (7, 29, 30).
The data presented here are consistent with this hypothesis. In
agreement with previous studies (18, 19), we found that expression of an exogenous human DNMT1 construct comprising only the
coding region, which is expressed under conditions of serum
deprivation, is able to alter the growth properties of NIH 3T3 cells;
the cells grow at a faster rate, and a higher percentage of cells are
in S phase. They also exhibit a loss of contact inhibition and do not
reach saturation even at high density. In contrast, cells expressing a
DNMT1 construct that includes the 3'-UTR and is not expressed in serum-starved cells maintain the growth properties of the
control cells.
The mechanisms responsible for cellular transformation by DNMT1 are
still unresolved. However, one proposed hypothesis is that elevated
levels of DNMT1 lead to the hypermethylation and inactivation of tumor
suppressor genes (80). It is possible that ectopic expression of
dnmt1 in arrested cells results in de novo
methylation and silencing of a critical tumor suppressor gene. However,
if this is the mechanism, it is not clear why such a gene is methylated
only when dnmt1 is expressed in arrested cells. An
alternative hypothesis is that ectopic expression of dnmt1
causes cell transformation by interfering with cell cycle regulatory
circuits through DNMT1 protein-protein interactions. Because DNMT1
forms a complex with Rb and E2F (75) as well as histone deacetylase 1 and 2 (81, 82), it can inhibit the expression of tumor suppressors by a
mechanism that does not involve DNA methylation, as has been previously
shown (83). DNMT1 can also displace p21 from proliferating cell nuclear
antigen and allow replication to occur (74), and inhibition of DNMT1
has been found to inhibit DNA replication (10, 11). Thus, aberrant expression of dnmt1 during the G0/G1
phase may override the silencing of tumor suppressors and eliminate
normal arrest signals, leading to the inappropriate entry into the cell
cycle that is observed in cancer cells, as has been previously
suggested (84).
Although further studies are required to confirm this hypothesis,
our findings clearly illustrate the importance of the dnmt1 3'-UTR in regulating its expression with the state of cell growth.
-globin mRNA. Our results indicate that a
54-nucleotide highly conserved element within the 3'-UTR is necessary
and sufficient to mediate this regulation. Cell-free mRNA decay
experiments demonstrate that this element increases mRNA turnover
rates and does so to a greater extent in the presence of extracts
prepared from arrested cells. A specific RNA-protein complex is formed
with the 3'-UTR only in growth-arrested cells, and a UV cross-linking
analysis revealed a 40-kDa protein (p40), the binding of which is
dramatically increased in growth-arrested cells and is inversely
correlated with dnmt1 mRNA levels as cells are induced
into the cell cycle. Although ectopic expression of human
DNMT1 lacking the 3'-UTR can transform NIH-3T3 cells,
inclusion of the 3'-UTR prevents transformation. These results
support the hypothesis that deregulated expression of DNMT1
with the cell cycle is important for cellular transformation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
5'259 and
pSK-
5'259, sense and antisense oligonucleotides coding for bases
5349-5405 bearing 5' XbaI and 3' SalI overhangs
were annealed and ligated into the XbaI and SalI
sites of both pRBG-GC and pBluescript SK.
3'56 and pSK-
3'158 were generated by PCR amplification using
10 ng of pCRII-DNMT1 3'-UTR as a template, the 3'-UTR sense primer as
above, and antisense primers 5'-GTCGACTTAATTTCCACTCATACAGTGGTAG-3' for
pSK
3'56 and 5'-GTCGACTTAGTTGATAAGCGAACCTCACACA-3' for pSK
3'158.
The amplified sequences were cloned into PCRII and then removed from
the vector by SalI digestion and ligated into pBluescript-SK cut with SalI and SmaI.
UTR, the pUD1
vector (a gift from Dr. L. Chasin) was digested with EcoRI and HindIII (to remove the DHFR gene) and either blunt-ended
or modified with the following linkers: 5'-AATTCTAG-3' and
5'-AGCTCTAG-3' to generate an XbaI site. Human
DNMT1 cDNA (360-5085) was inserted into the blunt pUD1
to give pUD-hDNMT1
UTR, whereas hDNMT1 cDNA (360-5408) was inserted into the modified pUD1 to give pUD-hDNMT1.
3'56, pAdDNMT1
3'-UTR,
hDNMT1 cDNA was cloned into the AdEasy shuttle vector pAdTrack cytomegalovirus in the XbaI site. Adenoviral
recombination and preparation of the infectious particles in Hek293
cells was performed as described previously (49). The following
DNMT1 regions were used: 1-5408 (pAd-DNMT1), 1-5350
(pAd-DNMT1
3'56), and 1-5085 (pAd-DNMT1
3'-UTR).
-globin (a 500-bp Xho-Bam fragment from
pRBG-GC), hDNMT1 (a 1.5-kilobase pair fragment from nucleotides
601-2191 of the full-length human DNMT1 cDNA), mDNMT1
(a 1.7-kilobase pair fragment from nucleotides 844-2544 of the
full-length mouse dnmt1 cDNA), green fluorescent protein (GFP) (a 765-bp NheI-HindIII fragment from
pEGFP-C1 (CLONTECH)).
5'259. G418-resistant colonies were cloned and propagated in selective
medium containing 0.25 mg/ml Geneticin (Life Technologies, Inc.).
3'-UTR (the pUD
vector bears a tetracycline-repressible promoter) were obtained by
transfecting 6 µg of each construct together with 6 µg of a puromycin resistance expression vector pBABEpuro (53) into TetOff NIH3T3 cells (CLONTECH) using SuperFect reagent
(Qiagen) according to the manufacturer's protocol. Resistant colonies
were selected in medium containing 3 µg/ml puromycin (Sigma)
and were then further cultured in the presence of 1 µg/ml doxycycline
(Sigma) to repress transcription of the transfected DNMT1.
Northern blot analyses were used to verify expression of the ectopic
hDNMT1 for both sets of transfectants. Growth arrest was
accomplished by culturing in 0.5% serum for 48 h; to induce
growth, arrested cells were cultured in 10% serum-containing medium
for 20 h. Doxycycline was removed 48 h before serum
starvation to induce ectopic hDNMT1 transcription.
3'56, and
pAd-DNMT1
3'-UTR at multiplicities of infection of 25, 50, and 150 respectively. 100% of cells were infected as determined by
visualization of the green fluorescent protein, which is expressed by
the adenoviral vector. Cells were then maintained in 0.5% medium for
an additional 24 h before the medium was replaced with a medium
containing either 10 or 0.5% serum. RNA was harvested after 20 h
and was analyzed by a Northern blot analysis as described above.
5'259 (SalI), pSK-
3'56 (SalI), and
pSK-
3'158 (SalI). In vitro transcription was
then carried out in the presence of 50 µCi of
[
32P]-UTP (3000Ci/mmol, PerkinElmer Life
Sciences) using either T7 polymerase (for DNMT1 3'-UTR) or T3
polymerase (for
5'259,
3'56, and
3'158). The Ambion in
vitro transcription kit was used as recommended by the
manufacturer. Cold competitor RNA transcripts were synthesized using
the Promega RiboMax system according to the protocol supplied by the manufacturer.
80 °C for
1 h, and the whole cell extract was isolated by centrifugation at
10000 × g for 15 min. The supernatant was recovered
and used for RNA binding assays. Protease K treatment was carried out
for 1 h at 37 °C at a final concentration of 1 µg/µl.
-mercaptoethanol, 0.12 mg/ml bromphenol
blue) were added, and the mixture was boiled for 10 min. Protein-RNA
complexes were resolved on a 10% SDS-polyacrylamide gel
electrophoresis gel.
12-10
16
nM) of a competitor DNA fragment. Primers
5'-ACCGCTTCTACTTCCTCGAGGCCTA-3' (sense, starting at 3479) and
5'-GTTGCAGTCCTCTGTGAACACTGTGG-3' (antisense, starting at 3813) were
used to amplify the target sequence, and
5'-CCTCGAGGCCTAGAAACAAAGGGAAGGGCAAG-3' (ending at 3576) was used to
create the competitor, as previously described (54) PCR conditions were
as follows: 96 °C for 1 min, 65 °C for 1 min, and 72 °C for 1 min (33 cycles). To differentiate between the endogenous
mdnmt1 and transfected hDNMT1 mRNA, the PCR
was transferred to Hybond-N+ membrane and hybridized with an
oligonucleotide corresponding to bases 3595-3617 of the full-length
human DNMT1 mRNA (GenBankTM accession
number NM001379.1). The oligo was labeled at the 5' end using 20 µCi of [
-32P]-ATP (3000 Ci/mmol, PerkinElmer Life
Sciences) and T4 polynucleotide kinase (MBI Fermantas) according to the
manufacturers' protocols. The membranes were incubated for 16 h
at 42 °C in the following hybridization buffer: 1% SDS, 5× SSC,
5× Denhardt's solution (0.5 mg/ml Ficoll, 0.5 mg/ml
polyvinylpyrrolidone, 0.5 mg/ml bovine serum albumin), 0.1 mg/ml
herring sperm DNA, and then washed twice with 6× SSC + 0.1% SDS for
10 min at room temperature, twice for 10 min at 37 °C, and twice for
10 min at 42 °C.
-actin using the following primers: 5'-GTTGCTATCCAGGCTGTGCTA-3' (sense, starting at 473) and
5'-GCGGATGTCCACGTCACACTT-3' (antisense, starting at 943)
(GenBankTM accession number XM_004814). Touchdown
PCR was used by decreasing the annealing temperature from 66 to
60 °C over four cycles, and then continuing with 96 °C for
30 s, 60 °C for 30 s, and 72 °C for 30 s (21 cycles).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Induction of DNMT1 mRNA by serum
stimulation of growth-arrested Balb/c 3T3 cells. A,
FACS analysis of serum-stimulated cells. Balb/c 3T3 cells were arrested
by serum depletion as described under "Materials and Methods" and
then incubated with serum for the indicated time points and subjected
to a FACS analysis to determine the percentage of cells at different
stages of the cell cycle. M1, apoptosis;
M2, G0/G1; M3, S;
M4, G2/M. B, induction of dnmt1
mRNA by serum stimulation of growth-arrested cells. 3T3 cells were
treated as in A; RNA was then extracted and subjected to a
Northern blot analysis using a mouse dnmt1 cDNA probe
(bp 844-2544). To normalize to the amount of total RNA in each lane,
the blot was then hybridized with an 18-s rRNA oligonucleotide probe.
C, correlation between dnmt1 mRNA levels and
stages of the cell cycle. Northern blots were quantified by
densitometric scanning and the dnmt1 signal was normalized
to that of 18 s. Shown are the dnmt1 mRNA levels
(arbitrary units) and the percentage of cells at each stage of the cell
cycle.
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Fig. 2.
Homology between the dnmt1
3'-UTR of different species. Alignments were performed with
the following cDNA sequences (GenBankTM accession
numbers indicated): mouse (m), 5077-5238 (X14805.1); human
(h), 5249-5405 (NM001379.1); rat (r), 5072-5215
(AB012214.1); Xenopus (x), 4987-5020 (D78638.1);
chicken (c), 4936-4990 (D43920.1); and daniorerio
(dr), 2333-2363 (AF097875). Shaded area denotes
a region of 100% conservation between human and chicken
dnmt1. Underlined and boxed sequences
indicate potential RNA destabilizing motifs. Region in
boldface marks the 54-nucleotide element of
hDNMT1 studied in detail in this report.
-Globin
mRNA in Growth-arrested Cells--
To test the hypothesis that the
regulation of dnmt1 mRNA levels involves
cis-acting sequences within the 3'-UTR, we assayed whether
this region could direct growth-dependent regulation of the
stable rabbit
-globin mRNA. Various AREs, such as those of GM-CSF (38) and type 1 plasminogen activator inhibitor (63), have been shown to destabilize rabbit
-globin mRNA when inserted into its 3'-UTR. We placed either the full 3'-UTR of DNMT1 (pRBG-DNMT1 3'-UTR) or only the 54-nucleotide conserved sequence (pRBG-DNMT1
5'259) in the rabbit
-globin gene 3'-UTR as indicated in Fig. 3A. We used the stable pRBG-GC
construct as a negative control and the pRBG-AT construct bearing the
instability element from GM-CSF as a positive control (38). These were
transfected into Balb/c-3T3 cells, stable transfectants expressing the
different constructs were then selected, and RNA was prepared from
either growth-arrested cells or from cells that had been
serum-stimulated for 20 h (time at which dnmt1 mRNA
peaks; see Fig. 1). The level of globin mRNA under each condition
was determined by Northern blot analysis and normalized relative to
18-s rRNA (Fig. 3, B and C). To take into account
any possible difference in mRNA levels due to a decreased promoter
activity of the RBG vector in arrested versus
serum-stimulated cells, the results presented in Fig. 3C were normalized relative to that of the GC stable control. Our findings
demonstrate that the DNMT1 3'-UTR is able down-regulate the
-globin mRNA in arrested cells but not in serum-stimulated cells. The full 3'-UTR is not required, however, because the chimeric
-globin construct containing the 54-nucleotide element by itself (
5'259) is also able to perform the same function. As expected, the
stable pRBG-GC construct does not show significant variation with the
growth state of the cells, whereas pRBG-AT is constitutively destabilized. These data indicate that the conserved 54-nucleotide region within the DNMT1 3'-UTR is sufficient to
down-regulate a heterologous mRNA in growth-arrested cells.
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Fig. 3.
The DNMT1 3'-UTR
down-regulates mRNA levels in a growth-dependent
manner. A, map of the pRBG constructs. Constructs were
used to create stable transfectants in Balb/c 3T3 cells. The site of
insertion of the DNMT1 3'-UTR, DNMT1 5'259,
GM-CSF destabilizer (AT), and GC control sequences are
indicated. B, representative Northern analysis of chimeric
DNMT1 3'-UTR-
-globin expression. Balb/c 3T3 cells were transfected
with either one of the following plasmids: pRBG-DNMT1 3'-UTR,
pRBG-DNMT1
5'259, pRBG-AT, or pRBG-GC, and stable G418 resistant
transfectants were isolated as described under "Materials and
Methods." RNA was extracted from growth-arrested cells (-) or from
arrested cells that had been incubated in serum for 20 h (+). RNA
was subjected to a Northern blot analysis using standard protocols and
hybridization to a labeled
-globin probe (500-bp Xho-Bam
fragment from pRBG-GC). Blots were then stripped and rehybridized with
an 18-s oligonucleotide probe. C, quantification of
-globin mRNA expressed in the stable transfectants. The amount
of globin mRNA expressed in each of the transfectants was
quantified by scanning densitometry and normalized to the signal
obtained following hybridization with an 18-s probe. All
-globin
levels were then calculated as a percentage of the GC control. The
experiment was repeated with similar results. D, map of the
adenoviral constructs. DNMT1 deletion constructs shown were
cloned into the XbaI site of pAdTrack-CMV. E,
representative Northern blot of infected Balb/c cells. Arrested cells
were infected with the indicated adenoviral constructs and were then
either maintained in serum-deprived conditions (arrested),
or stimulated with 10% serum for 20 h (+serum). RNA
was extracted and subjected to a Northern blot analysis by
hybridization with a labeled hDNMT1 cDNA probe (bp
601-2191). Blots were then stripped and rehybridized with a probe for
GFP (765-bp NheI-HindIII from pEGFP-C1).
F, quantification of hDNMT1 mRNA levels in the infected
cells. The amount of hDNMT1 mRNA expressed in each
infection was quantified and normalized to the signal obtained
following hybridization with the GFP probe. The graph shows an average
of three independent experiments.
3'-UTR), or by simply deleting the conserved 54-nucleotide
sequence (
3'56), we abolished this cell growth-dependent
change in DNMT1 mRNA levels. Taken together, these
results and those from the previous experiment indicate that the
DNMT1 3'-UTR can down-regulate an mRNA in either a
heterologous or homologous context in a growth-dependent
manner and that the conserved 54-nucleotide sequence is necessary and
sufficient for this regulation.
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Fig. 4.
Specific RNA-protein interactions within the
DNMT1 3'-UTR. A, the different
deletion fragments used to synthesize the RNA probes. The solid box
indicates the 54-nucleotide highly conserved region. B, RNA
mobility gel shift assay of arrested Balb/c cells upon serum
stimulation. An RNA gel shift assay was performed using
32P-labeled in vitro transcribed DNMT1
3'-UTR and extracts prepared from cells treated as in Fig.
1A. Time indicates hours after serum stimulation of arrested
cells. The complexes were resolved by nondenaturing polyacrylamide gel
electrophoresis. The complex specific to arrested cells is indicated by
an arrow. C, UV cross-linking assay. Cells were
arrested for 48 h (-) and serum-stimulated for 20 h
(+). Extracts were then prepared and subjected to a UV cross-linking
assay using a 32P-labeled 5'259 probe, and RNA-protein
complexes were resolved on an SDS-polyacrylamide gel electrophoresis
gel. Protease K treatments (+PK) were performed for 1 h
at 37 °C. Control indicates reaction performed in absence of cell
extract. D, competition UV cross-linking. The assay was
performed as above, using extracts prepared from arrested cells. Cold
competitors were synthesized from templates shown in A and
used in a 1000× excess. E and F, time course of
p40 binding. The assay was performed as stated above, using extracts
prepared from cells at various times after serum stimulation. p40
levels were quantified by densitometric scanning and normalized to the
total protein in each lane, which was measured by scanning the gels
after staining with Coomassie Blue. A representative experiment is
shown in E, and an average of three experiments is shown in
F. G, correlation between p40 binding,
dnmt1 mRNA levels, and cell growth. Results from Fig. 1
and the results in F were plotted on the same graph to
compare the pattern of p40 binding to dnmt1 mRNA levels.
In C-E, the molecular masses in kilodaltons are
indicated.
5'259 with a protein of ~40 kDa
(which we will from now on refer to as p40) was found to occur almost
exclusively in arrested cells. A competition experiment was then
carried out, with cold competitors comprising the various 3'-UTR
constructs (Fig. 4, A and D). As expected, both
3'-UTR and
5'259 are effective competitors and displace p40 binding
from the labeled
5'259 probe. However,
3'56 and
3'158 did not
compete, demonstrating that the binding of p40 is specific to the
conserved 54-nucleotide element. No binding activity was observed in
experiments performed with probe alone or with cell extracts
(serum-stimulated and arrested) treated with protease K (Fig.
4C), confirming that the p40 band is indeed a protein.
5'259) decay
with half-lives of 35 and 60 min, respectively. However, the half-lives
of
3'158 and
3'56, both of which lack the conserved sequence,
were significantly longer (100 and 132, min respectively), suggesting
that in the presence of arrested cell extracts, this element can
accelerate the degradation of the DNMT1 3'-UTR in
vitro. No significant decay was observed with any of the RNA
transcripts in buffer alone. The fact that the half-life of the full
3'-UTR is shorter than that of the 54-nucleotide element implies that
other regions upstream of this sequence may enhance its destabilizing
properties.
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Fig. 5.
Differential DNMT1 3'-UTR
decay rates in vitro. A, in
vitro decay assay using 3'-UTR deletions and arrested cell
extract. Radiolabeled 3'-UTR RNA transcripts were incubated with 10 µg of whole cell extract from arrested Balb/c cells
(arrested) or with incubation buffer alone
(control) for various lengths of time and electrophoresed on
an acrylamide gel, and the signals were detected by autoradiography and
quantified by densitometry. The constructs used and their sizes are
indicated beside each experiment. B, half-life measurements.
Experiments in A were quantified and the signal at each time
point calculated as the percentage of RNA remaining relative to the RNA
at time 0. The half-life was calculated as the time point when 50% of
the RNA had been degraded. Squares, 3'56;
diamonds, 3'-UTR; circles,
3'158;
triangles,
5'259. C, in vitro
decay assay using
5'259 and arrested or growth-stimulated extracts.
Radiolabeled
5'259 transcript was incubated with 10 µg of whole
cell extract from arrested or serum-stimulated (+serum)
Balb/c cells for various lengths of time and processed as in
A. D,
5'259 half-life measurements. The
experiment shown in C was quantified, and half-lives were
calculated as in B. Squares, +serum;
diamonds, -serum.
5'259 RNA in the presence of growth-arrested and
serum-stimulated cell extracts (Fig. 5, C and D).
The half-life of the RNA increased from 58 min when it was incubated
with arrested extracts to 105 min when it was incubated with
growth-induced extracts. These data are consistent with the hypothesis
that the likely mechanism by which
5'259 down-regulates mRNA in
arrested cells in vivo (Fig. 3) is by decreasing mRNA stability.
UTR) under the direction of a tetracycline-repressible
promoter. Our first interesting observation was that the hDNMT
UTR
clones (n = 10) grew at a much faster rate than the
hDNMT1 clones. Then, after several passages, many of the transfected
lines exhibited signs of cell death, indicating that expression of
hDNMT1
UTR had toxic effects. This is not surprising, because others
have also observed harmful consequences of DNMT1
overexpression (19, 77). To inhibit expression of the transfected
constructs and prevent this effect, we cultured the transfectants in
the presence of doxycycline until 48 h prior to each experiment.
-globin chimeras (Fig. 3), no
hDNMT1 mRNA was detected in arrested cells expressing
the full hDNMT1 construct (Fig. 6A, left panel). However, in
cells expressing hDNMT1
UTR, mRNA was present at competitor
concentrations of 10
14-10-16nM.
As expected, in growth-stimulated cells, both constructs showed a
similar expression profile (Fig. 6A, right panel).
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Fig. 6.
Effects of the 3'-UTR on cell growth
following ectopic expression of hDNMT1 in NIH-3T3
cells. A, measurement of hDNMT1 mRNA levels by
competitive PCR. 1 µg of RNA from TetOff NIH 3T3 hDNMT1 or
hDNMT1 UTR stable lines, either growth-arrested or serum-stimulated,
was reverse-transcribed, and equal amounts of cDNA (as determined
by [35S]dATP incorporation) were subjected to
DNMT1 PCR amplification in the presence of decreasing
concentrations
(10
12-10
16
nM) of a competitor DNA fragment (upper panels).
To differentiate between the endogenous mouse dnmt1 and
transfected hDNMT1, the PCR was transferred to Hybond N+
membrane and hybridized with an oligonucleotide corresponding to bases
3595-3617 of the full-length human DNMT1 (lower
panels). T, target; C, competitor.
B, growth curves of the stable and parent lines. Cells were
plated in six-well culture dishes at a density of 25,000 cells/well
(day 0). Cells were then counted on 6 subsequent occasions throughout
the span of 11 days. An average of 4 measurements was obtained for each
time point. Squares: hDNMT1
UTR, triangles:
NIH-3T3, diamonds: hDNMT1. C percentage of cells
in S phase of the cell cycle. Serum-starved (-serum) or
growth-stimulated (+serum) cells were harvested and stained
with propidium iodide, and the percentage of cells in S phase was
determined by flow cytometry. D, morphology of hDNMT1
transfectants. Phase contrast light microscopy at increasing
magnifications on day 11 of the growth curve described in B. Top panels, NIH-3T3; middle panels,
hDNMT1; bottom panels, hDNMT1
UTR
UTR expressing clones, as well as
the parental NIH-3T3 cells. Although both the control cells and the
hDNMT1 clone exhibited similar growth rates, which began to level off after 10 days, the hDNMT1
UTR clone grew at a higher rate and did not
reach saturation with high density. Accordingly, a FACS analysis
carried out in the presence or absence of serum (Fig. 6C)
revealed that a higher percentage of hDNMT1
UTR cells are found in S
phase both in the presence and absence of serum. The observation that
the parent cell line grows slightly faster that the hDNMT1 clone could
be because the hDNMT1 clone was maintained in the presence of puromycin
(the selection drug), which we found slowed down cell growth. But
perhaps the most striking are the morphological changes observed in the
hDNMT1
UTR clone. As can be seen in Fig. 6D, although both
the control and hDNMT1 cells formed an organized monolayer and were
contact-inhibited upon reaching confluence, the hDNMT1
UTR clone grew
in a disorganized pattern and continued to so after confluence was
reached, forming large foci. Similar results were observed with six
other clones. These results suggest that the cell cycle regulatory
activity of the 3'-UTR inhibits the cellular changes observed upon
ectopic expression of DNMT1, thus supporting the hypothesis
that deregulation of the cell cycle-dependent expression of
DNMT1 is important for cellular transformation.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-globin mRNA. Third, we identified a highly conserved
54-nucleotide element within the 3'-UTR that is required for mediating
the down-regulation of dnmt1 mRNA in arrested cells.
Thus, cis-acting sequences in the 3'-UTR are likely
responsible for the changes in dnmt1 mRNA which occur
during the cell cycle. Fourth, the 3'-UTR is destabilized in
vitro in the presence of extracts prepared from arrested cells, suggesting that a protein that is present primarily in arrested cells
targets the 3'-UTR for degradation. The decrease in half-life of
5'259 cannot be attributed to the fact that it is shorter than
3'158 and
3'56, because the full 3'-UTR decays at a faster rate.
One conceivable explanation for the reduced destabilizing activity of
5'259 relative to the full 3'-UTR is that AU-rich instability
sequences exist upstream of
5'259. A possible model is that the
54-nucleotide element is responsible for the
growth-dependent stability changes, and region(s) upstream
enhance its destabilizing activity.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. G. Shaw for his kind gift of the pRBG-AT and pRBG-GC plasmids and Dr. L. Chasin for the pUD-1 plasmid. We also thank Andrew Slack for assistance with preparation of the DNMT1 adenoviral constructs.
![]() |
FOOTNOTES |
---|
* This work was supported by a grant from the National Institute of Cancer Canada (to M. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by a fellowship from the Canadian Institute for Health Research.
§ To whom correspondence should be addressed: Dept. of Pharmacology and Therapeutics, McGill University, 3655 Sir William Osler Promenade, Montreal, Quebec H3G 1Y6, Canada. Tel.: 514-398-7107; Fax: 514-398-6690; E-mail: mszyf@pharma.mcgill.ca.
Published, JBC Papers in Press, May 2, 2001, DOI 10.1074/jbc.M103056200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: DNMT1, DNA methyltransferase 1; UTR, untranslated region; ARE, AU-rich element; AU, adenine and uridine; GM-CSF, granulocyte macrophage colony-stimulating factor; PCR, polymerase chain reaction; GFP, green fluorescent protein; bp, base pair(s); FACS, fluorescence-activated cell sorter.
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REFERENCES |
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