(Received for publication, August 25, 1994; and in revised form, November 14, 1994)
From the
Using deletion analysis and site-specific mutagenesis to map the 5` regulatory region of the DNA methyltransferase (MeTase) gene, we show that a 106-bp sequence (at -1744 to -1650) bearing three AP-1 sites is responsible for induction of DNA MeTase promoter activity. Using transient cotransfection chloramphenicol acetyltransferase assays in P19 cells, we show that the DNA MeTase promoter is induced by c-Jun or Ha-Ras but not by a dominant negative mutant of Jun, ∂9. The activation of the DNA MeTase promoter by Jun is inhibited in a ligand dependent manner by the glucocorticoid receptor. Stable expression of Ha-Ras in P19 cells results in induction of transcription of the DNA MeTase mRNA as determined by nuclear run-on assays and the steady state levels of DNA MeTase mRNA as determined by an RNase protection assay. These experiments establish a potential molecular link between nodal cellular signaling pathways and the control of expression of the DNA MeTase gene. This provides us with a possible molecular explanation for the hyperactivation of DNA MeTase in many cancer cells and suggests that DNA MeTase is one possible downstream effector of Ras.
The establishment of a pattern of DNA methylation of CpG
dinucleotide sequences is critical for normal development of
vertebrates(1) . It is well established that regulated changes
in the pattern of DNA methylation occur during development (2, 3) and cellular differentiation(4) , and
that aberrant changes in the pattern of methylation occur during
cellular transformation(5, 6, 7) . We have
previously suggested that one mode of control of the pattern of
methylation is at the level of regulation of the DNA methyltransferase (MeTase) ()gene which
encodes the activity that catalyzes the transfer of methyl groups to
DNA(8, 9) . If the level of DNA MeTase activity in the
cell is an important determinant of DNA methylation patterns, then it
should be coordinated with cell growth and development. As the 5`
region of the DNA MeTase was recently cloned(10) , we
can now address the question whether it contains such regulatory
elements.
Many cancer cell lines express high levels of DNA MeTase
activity (11) and human colon carcinoma cell lines have been
shown to express dramatically higher levels of DNA MeTase mRNA
than normal cells in a manner that correlates with the state of
malignancy of the cell(12) . While some sites have been shown
to be hypomethylated in cancer cells(5) , critical regions of
the genome are hypermethylated, such as CpG islands(13) ,
candidate tumor suppressor genes(14) , cell type-specific
genes(15) , genes responsible for terminal differentiation such
as MyoD(16) , and areas of the genome associated with
tumor suppression(17) . Several lines of evidence suggest that
this methylation plays an important role in the inactivation of genes
in tumor cells and the process of cellular transformation: (a) in vitro methylation of CpG islands results in inactivation of
these genes following transfection (18) and (b)
treatment of cells with the methylase inhibitor 5-azacytidine (14, 19) or expression of an antisense to the DNA MeTase mRNA results in reactivation of this class of genes (20) . Recent experiments have shown that forced expression of
an endogenous DNA MeTase cDNA in NIH 3T3 cells leads to their
transformation (21) and expression of an antisense to the DNA MeTase in the adrenocortical carcinoma cell line Y1, or
treatment of Y1 cells with 5-azacytidine leads to reversal of
transformation. ()The molecular mechanisms responsible for
the increased DNA methylation activity in the process of cellular
transformation are unknown. One possible explanation is that the
regulatory elements of the gene are responsive to some oncogenic
signaling pathways (23) in addition to the cell cycle
regulation of DNA MeTase gene expression which occurs mainly
at the posttranscriptional level(24) .
The DNA MeTase 2-kb 5` upstream region bears one consensus AP-1 site located at -1744 and six AP-1 sites with one or two mismatches(10) . This study tests the hypothesis that overexpression of signaling pathways leading to activation of Jun (25, 26, 27) can result in hyperinduction of the DNA MeTase promoter.
Figure 3: Deletion analysis of the DNA MeTase 5` region. The physical maps of the different deleted pMetCAT+ constructs are shown in the right panel relative to the original pMetCAT+ construct. Regions deleted are indicated by a broken line. The boundaries of the deleted sequence are indicated relative to the transcription initiation site and listed in Table 1. The first number indicates the 5` of the deleted sequence and the second number indicates the 3` boundary. Exons are indicated by filled boxes, CAT sequence is hatched, transcription initiation sites are indicated by horizontal arrows, shaded ovals indicate AP-1 sites (forming DNA protein interactions, Fig. 4), filled ovals are mismatched AP-1 sites. HIII, HindIII recognition site; BII, BglII; RI, EcoRI; RV, EcoRV; BHI/NI is an original NaeI site that was modified by linkers to a BamHI site, BHI, BamHI; S3AI, SauIIIAI. M/Wt, the AP-1 site at -1750, is mutated to TGAGGCA but the one at -1644 is wild type. Wt/M, the AP-1 site at -1750 is wild type but the 3` site is mutated as above. The different constructs (10 µg) were transfected in triplicate into P19 cells with either 4 µg of SK plasmid (Control) or 4 µg of RSV-c-jun (+Jun), harvested after 48 h, and CAT activity was determined as described above. Each experiment was repeated twice using different plasmid preparations. The results are presented as averages of three determinations ±S.D.
Figure 4:
Binding of the AP-1 transcription complex
to sequences in the DNA MeTase 5` upstream region. A, A 21-mer
double-stranded oligomer, Met 5` AP-1, encoding the sequence:
5`-AATGCAGCATGACTCATGCT-3` located at(-1753, -1734) in the
DNA MeTase 5` region (the putative AP-1 recognition sequence is underlined) was end-labeled with
[-
P]ATP (Amersham Corp.) and incubated with
10 µg of P19 nuclear extract. For competition experiments, an
excess of nonlabeled double-stranded oligomers was used. The free
oligomer (gray arrow) and the bound AP-1 complex (black
arrow) were separated on a native polyacrylamide gel. An excess of
labeled substrate was used for all assays. The following competitors
were used: (a), Met 3` AP-1 is located at -299 to
-275 in the DNA MeTase 5` region and encodes the sequence
5`-GTTTTGAGGCAGGATTTTGA-3` (the underlined sequence contains
one mismatch with the consensus AP-1 recognition sequence); (b) AP-1 encodes a consensus AP-1 sequence
(5`-CGCTTGATGAGTCAGCCGGAA-3`) (Promega); (c) Sp1 encodes the
binding recognition sequence of the transcription factor Sp-1
(5`-GATCGATCGGGGCGGGGCGATC-3`). B, an in vitro DNase
footprinting assay was performed on an end labeled 0.6-kb BamHI-Sau3AI fragment containing the 5` AP-1 region
of the DNA MeTase gene (see Fig. 3) which was incubated
with 1 footprint unit (fpu) of a bacterially expressed c-Jun protein
(Promega). The AP-1-reacted fragment and a naked (nonreacted) control
were subjected to cleavage by DNase I (Boehringer Mannheim) which was
followed by electrophoresis and autoradiography. An M13 sequencing
reaction (Pharmacia) was used as as a size marker ladder. The regions
that are protected from DNase I cleavage by AP-1 are indicated. The
footprints at -1744 to -1738 (TGACTCA) and
-1650-1644 (TGACTGA) match the AP-1 consensus recognition
sequence. The footprint at -1718 to -1708 (TGGACGGCTTT)
does not correspond to the consensus AP-1 recognition
sequence.
Figure 1:
Fos and Ras induce the mouse DNA MeTase promoter. A, P19 cells, an embryonic carcinoma
cell line(22) , were transfected with 10 µg of
pMetCAT+ (see Fig. 3for physical map) and increasing
concentrations (1-10 µg) of expression vectors expressing
c-jun (RSV-c-jun) (29) or Ha-ras (pZEM). Cells were harvested after 48 h and
CAT activity was determined as described under ``Materials and
Methods.'' Each experiment was performed in triplicate. Twenty-two
independent experiments using independent cultures of P19 cells at
different times were performed for the Jun expression vector and eleven
experiments with the Ha-ras. The maximal fold induction above
the values obtained with pMetCAT+ was determined per construct for
each experiment. Each value is presented as mean of the values obtained
in all experiments ±S.D. The difference between Jun- and
Ras-induced pMetCAT + expression and the noninduced control is
highly significant p < 0.001. B, P19 cells were
transfected with either pMetCAT+ (4 µg) and SK-,
pMetCAT+ (4 µg) and RSV-cJun (4 µg), or pMetCAT+ (4
µg) and pZEM
(4 µg) and an increasing
concentrations of a vector encoding the transdominant negative mutant
of Jun ∂9-v-Jun which was kindly provided by Dr. B.
Wasylik(32) . The total concentration of plasmid DNA was kept
constant by adding SK plasmid to the transfection mix. The values are
presented as an average of three determinations
±S.D.
To demonstrate that this transactivation of the DNA MeTase promoter by Ras and Jun is dependent on functional Jun activity rather than a nonspecific effect, we cotransfected pMetCAT+ (4 µg/ml) and increasing concentrations of a transdominant negative mutant of Jun, ∂9 which lacks the transactivation domain of Jun but expresses the DNA binding domain(32) . The total concentration of transfected plasmid DNA was maintained constant by adding SK plasmid DNA. As demonstrated in Fig. 1B, ∂9 cannot transactivate the DNA MeTase promoter. Coexpression of ∂9 with Jun (4 µg/ml) results in a dose-dependent inhibition of transactivation by Jun as expected. When ∂9 is coexpressed with Ras (4 µg/ml), the induction of the DNA MeTase promoter by Ras is repressed even at low concentrations of the ∂9 plasmid. This demonstrates that both Ras and Jun induce the DNA MeTase by a common pathway.
Figure 2: Inhibition of Jun-dependent induction of the DNA MeTase promoter by the glucocorticoid receptor. A, Ten µg of pMetCAT+ were cotransfected in triplicate into P19 cells with either SK plasmid (pMetCAT+), SK plasmid and 10 µg of the human glucocorticoid receptor expression vector (HGCRC)(27) , RSV-c-jun (+Jun), or RSV-c-jun and the HGCRC (as indicated in the figure). One set of transfectants was treated with 0.1 µM of dexamethasone (Sigma) for 24 h before harvesting (+dex) and CAT activity was determined in extracts prepared from the different transfected cells 48-h posttransfection as described under ``Materials and Methods.'' The fold induction over pMetCAT+ (10 µg with SK-) was determined. The experiment was repeated three different times using three different cultures of P19 cells. The values are presented as an average of three independent experiments ±S.D. The statistical significance of the difference between Jun and Jun + HGCRC transfectants and Jun as well as the statistical difference between dexamethasone treated HGCRC + Jun and Jun transfectants was determined using a t test, p < 0.05.
To visualize the specific molecular interactions between Jun and the AP-1 binding region of DNA MeTase 5` upstream sequences, we incubated a 648 bp fragment containing these sequences with c-Jun protein and subjected the bound products to a DNase I footprint analysis as described in materials and methods (Fig. 4B). Two potential AP-1 sites (TGACTCA (-1744, -1738, and TGACTGA(-1650, -1644)) were protected from DNase cleavage as indicated in Fig. 4B. These experiments have therefore established that the AP-1 sites in the 5` of the DNA MeTase gene are functional AP-1 sites in vitro.
Figure 5:
Transcription of the endogenous DNA MeTase
gene is induced by forced expression of exogenous Ha-ras. P19
cells were stably transfected with either (10 µg) pZEM or (10
µg) pZEM Ha-ras encoding the Ha-ras oncogene
which contains activation mutations in codon 12 (Gly to Arg) and 59
(Ala to Thr) and (1 µg) pUCSVneo as a selectable marker. G418 (0.5
mg/ml) resistant colonies were selected and propagated. Preliminary
Southern blot analysis using a P-labeled Ha-ras probe (0.7 kb HindIII-PstI fragment) has been
performed to identify transfectants and three randomly picked positive
clones per each transfected plasmid were used for our further studies. A, Northern blot analysis using a ras probe. The ras transfected panels were exposed for a short time to enable
their presentation alongside with the nontransfected controls. The
viral ras directs a 2-kb transcript that is easily
distinguishable from the shorter endogenous ras messages
(1-1.4 kb). As observed in the figure (left panel) the ras transfectants express large amounts of ras that
is probably initiated at multiple sites in addition to the viral
initiation site. The filter was stripped from the radioactivity and
rehybridized with an 18 S rRNA-specific
P-labeled
oligonucleotide (45) (bottom panel). B,
P-labeled RNA (1
10
dpm) transcribed
in nuclei prepared from ras-transfected P19 cells (lanes
11 and 17) and pZEM control (lane 1) was
hybridized with filter immobilized SKmet5` (10) containing a
DNA MeTase genomic fragment encoding the 5` transcribed
regions of the gene and SK plasmid as a negative control. C,
RNase protection assay of DNA MeTase mRNA. RNA prepared from
three P19 pZEM
transfectants (lanes 6, 11, and 17) and pZEM control transfectants was
subjected to an RNase protection assay using a 700-bp riboprobe (probe
A) (10) encoding the DNA MeTase genomic sequence from
-0.39 to +318. The major bands representing the two major
initiation sites are indicated (92 and 90). The first
exon will give a 99-bp protected fragment. Additional minor initiation
sites are markedly represented in the two ras transfectants
that express significant levels of DNA MeTase. The experiment has been
repeated twice with similar results. D, quantification of
transcription rate and steady state mRNA levels in Ras transfectants versus controls. 1) Run on assays: a run on assay similar to
the one presented in B was performed on nuclei prepared from
three independent ras transfectants (6, 11, 17) and three control transfectants
pZEM (lanes 1, 2, and 3). The intensity of the signal obtained
following hybridization of the labeled RNA with SKmet5` was determined
by densitometry (Scanalytics Masterscan) and the average for the three
lines is presented with the S.D. 2) mRNA- quantification of the signal
obtained in the autoradiogram presented in C at the 92-, 90-, and 99-bp
fragments hybridizing with DNA MeTase mRNA. The average for the three ras-transfected and the three pZEM-transfected lines is
presented with the S.D.
This report addresses the hypothesis that the activity of DNA
MeTase is regulated by nodal cellular signaling pathways. While this
fact by itself is not sufficient to demonstrate that regulation of DNA MeTase is an important cellular control point, it is
consistent with that hypothesis(23, 39) . We
demonstrate in this study that when Ras-Jun signaling pathway is
activated by either expressing high levels of exogenous Ras or Jun, the
DNA MeTase promoter is significantly induced ( Fig. 1and Fig. 3), that this activation is mediated by
AP-1 recognition sequences in the 5` region of the gene
(-175-1640) and that it is inhibited by the glucocorticoid
receptor in a ligand dependent manner (Fig. 2). This inhibition
is most probably mediated by protein-protein interactions with AP-1 as
has been shown for other AP-1 induced promoters since the DNA MeTase 5` region does not contain a full glucocorticoid
recognition site(10) . Forced expression of an exogenous
Ha-ras induces transcription and an increase in the steady
state levels of endogenous DNA MeTase mRNA (Fig. 5). Induction
of Jun appears to be associated with induction of differentiation in
P19 cells(40) . Since Ras has multiple effects on P19 cells it
is hard to assess the relative effect of increased DNA MeTase activity
on DNA methylation patterns. Our ras transfectants induce high
levels of demethylase activity which complicates the
analysis of the effects that induction of DNA MeTase has on the pattern
of methylation. Whereas other cell systems should be sought to study
the effects of an increase in Ras activity on DNA methylation, P19
cells are an excellent model to dissect AP-1 and glucocorticoid
responsiveness of the exogenous promoter because they do not express
high levels of c-jun(41) . Using other cell systems
to study the effects induction of Ras and Jun might have on DNA
methylation, we have recently shown that inhibition of the Ras-Jun
pathway in an adrenocortical tumor cell line Y1 results in inhibition
of DNA methylation activity and reduction in methylation levels of CpG
dinucleotides. (
)What is the significance of regulation of
DNA MeTase by Ras and Jun? One documented instance when DNA MeTase is markedly up-regulated is cancer; many cancer cell
lines and colon cancer cells in vivo express significantly
higher levels of DNA MeTase(11, 12) . In this
report we suggest a possible molecular route through which oncogenic
pathways can lead to induction of DNA MeTase. DNA MeTase exhibits very low constitutive levels of expression ( Fig. 1and Fig. 3). This basal level of expression is
responsive to the proliferative state of the cell by a
posttranscriptional up-regulation as we have previously
shown(24) . However, in addition to this basal control it is
possible that cellular signaling systems such as the one induced by Ras
can induce high activities of DNA MeTase in a programmed
manner at distinct sites and times in development. When these signaling
pathways are aberrantly up-regulated as happens in many cancer cells,
DNA MeTase promoter is induced, possibly resulting in
elevation of DNA MeTase (11, 12) and the
hypermethylation of specific genomic regions in cancer
cells(17) . While it is clear that factors other than the level
of activity of DNA MeTase must play a role in shaping the methylation
pattern of the genome(43) , the level of DNA MeTase activity is
most probably an important factor. It is possible however that in
addition to AP-1, other pathways control DNA MeTase gene
expression. What are the changes in DNA methylation that are critical
for cellular transformation? Several mechanisms have previously been
suggested such as silencing of tumor suppressor genes (13, 16) , an increase in spontaneous mutations
resulting from deamination of methylated cytosines (44) or
altered regulation of replication(23) . In contrast to the
induction of cellular proliferation by Ras in many cell types, Ras can
induce the differentiation of PC12 cells(42) . Induction of DNA
MeTase might play an important role in these processes as well.
Although additional experiments are required to determine whether DNA
MeTase plays a critical role in the transformation process as a
downstream effector of Ras, the results presented in this report
establish a molecular link between cellular signaling pathways and the
machinery responsible for controlling the pattern of modification of
the genome.