From the
We demonstrate that expression of v-Ha-ras in mouse
embryonal P19 cells results in genome-wide demethylation. Analysis of
the pattern of methylation of specific genes reveals that different
types of genes are demethylated in the ras transfectants:
skeletal muscle specific genes, a gene specifically expressed in the
adrenal cortex (c21), ubiquitous genes, and exogenously
introduced sequences. Transient transfection and in vitro demethylation assays reveal that the ras transfectants
express high levels of a general DNA demethylation activity. This
demonstrates that the general DNA demethylation activity in mouse
embryonal cells is controlled by an important cellular signal
transducer and that DNA demethylase is a potential downstream effector
of Ras.
DNA methylation patterns are a hallmark of vertebrate genomes.
The cytosine moieties residing in the dinucleotide sequence CpG are
generally methylated in vertebrate genomes; however, some CpGs are not
methylated. The distribution of nonmethylated cytosines is tissue- and
site-specific, resulting in a pattern of
methylation
(1, 2) . It is clear that the pattern of
methylation is established by a sequence of methylation and
demethylation events
(3, 4) . Although it was originally
suggested that DNA demethylation is accomplished by a passive loss of
methyl groups during replication
(5) , it is now clear that an
active process of demethylation occurs in embryonal cells
(6) ,
in differentiating cell lines
(7, 8, 9) , and in
response to estrogen treatment
(10) . Whereas site-specific
hypomethylation occurs during development, it is unclear whether the
demethylation machinery per se is site-specific or whether
site specificity is determined by other signals. A critical question
for understanding the biological role of demethylation is whether it is
an indirect result of other site-specific events, such as
transcriptional activation, or whether mammalian cells bear a
demethylation activity that is independently programmed with
development. If demethylation is a programmed event, then the question
is whether it is regulated by central cellular control points.
We
have recently shown that mouse embryonal teratocarcinoma P19 cells
express a general demethylase activity. This activity was partially
purified and was shown to demethylate DNA in the absence of exogenous
dCTP, suggesting that it does not act via an excision repair
mechanism.
The c21 gene is heavily methylated in P19 cells
and the pZEM transfectants, showing a high molecular band at 9
kb
We analyzed the state of methylation of a gene that is
specifically expressed in myogenic lineages. The acetylcholine receptor
thy-1 is expressed in P19 cells
(24) but not in a
number of other cell types. The HpaII sites located in a CpG
island upstream from the transcription initiation site of
thy-1 are hypomethylated in P19 pZEM transfectants as well as
P19 cells as demonstrated by the presence of the 0.6-kb HpaII
fragment. One of these HpaII sites (site 2) undergoes de
novo methylation in the ras 6 and 11 transfectants as
indicated by the relative intensification of the 0.8-kb partially
methylated HpaII fragment (Fig. 2C; see also
the results of a densitometric analysis presented in
Fig. 2D). These results imply that even under conditions
where general genomic demethylation is taking place, some sites will
remain selectively methylated or even undergo de novo methylation.
The DNA methyltransferase gene is expressed in all
proliferating cells and in P19 cells
(19) . The 5` region of the
DNA methyltransferase bears a HpaII site, which is located in
proximity to a cluster of AP-1 sites 1.7 kb upstream of the
transcription initiation site (physical map in Fig. 2E).
Demethylation of this site will result in a 1.3-kb fragment following
HpaII and HindIII digestion. The majority of P19
cells is methylated at this HpaII site as indicated by the
presence of the hypermethylated 4-kb HindIII fragment in all
three pZEM transfectants. A large fraction of the population of ras transfectants is hypomethylated at this site as indicated by the
relative diminution of the 4-kb fragment and the increase in the
relative intensity of the unmethylated fragment.
We determined the
state of methylation of the 3` region of human growth hormone (HGH)
cDNA included in the pZEM and pZEMras construct. Both constructs
include the same promoter and 3` sequences and should express similar
transcriptional activity (both constructs are expressed in nonembryonal
cells, data not shown). Whereas the transfected pZEM is heavily
methylated in the control pZEM transfectants, the HGH 3` region is
markedly hypomethylated in all ras transfectants
(Fig. 2F) as indicated by the presence of the expected
HpaII low molecular fragments at 0.7, 0.5, and 0.3 kb. Some
low level of de novo methylation is observed in the ras transfectants (Fig. 2F, compare the MspI
and HpaII lanes; the partially methylated fragments are
indicated by asterisks). This marked difference between P19
and ras transfectants suggests that de novo methylation is inhibited by v-Ha-Ras. There are three possible
explanations for the inhibition of de novo methylation in
ras transfectants: Ras might inhibit de novo methylation per se; maintenance methylation might be
inhibited; and de novo methylation might be intact, but
demethylation activity is induced in ras transfectants.
Similarly, the genome-wide demethylation observed in ras transfectants can be explained by inhibition of maintenance
methylation, de novo methylation, or activation of
demethylation. Alternatively, genome-wide demethylation might be caused
by wide changes in chromatin structure altering the accessibility of
DNA to the methylation machinery.
The plasmid was methylated in vitro with
SssI methylase
(27) , which methylates all CpG sites,
and introduced into P19 cells, pZEM, and PZEMras transfectants by
DNA-mediated gene transfer. Each experiment was repeated twice, and
each transfection was done in triplicate. The autoradiogram in
Fig. 3
(A and B) presents one representative
experiment for each condition. DNA was prepared from nuclei purified
from the different transfectants 48 h after transfection and was
subjected to either HpaII or MspI digestion.
Following agarose gel fractionation and Southern blot transfer, the
filters were sequentially hybridized with either a CAT probe
(Fig. 3A) or a methyltransferase 5` probe
(Fig. 2B). The presence of mRNA encoding v-Ha-Ras in the
ras transfectants was verified by subjecting RNA made from the
cytosolic fraction to Northern blot analysis and hybridization with a
ras probe (Fig. 3C).
To determine that loss
of methylation of the transiently transfected plasmids does not result
from replication in the absence of methylation, DNA prepared from the
transient transfectants was subjected to digestion with DpnI.
DpnI cleaves DNA only when its GATC sites are methylated on
both strands. Because bacterial cells but not mammalian cells bear the
methylase required for methylating GATC sites, sensitivity to
DpnI implies that the plasmid did not replicate in the
mammalian cell. As observed in Fig. 3D, the transiently
transfected plasmid maintained its sensitivity to DpnI
digestion. Especially instructive is an engineered site in the plasmid
(Fig. 3D, bottom panel, for localization and
description) where the DpnI cleavage site is located 3 base
pairs apart from the HpaII cleavage site. Whereas >95% of
the molecules are demethylated at this site in the ras transfectants as indicated by the sensitivity to HpaII
cleavage (Fig. 3B, the 2.3-kb HpaII fragment),
>95% of the transfected molecules are still sensitive to
DpnI cleavage of this site (Fig. 3D, see
bottom panel for location of the site), implying that the
adenine residue is still methylated on both strands. This excludes
replication and repair as a mechanism for demethylation. This does not
exclude, however, very short patch excision repair (less than 3 bases)
as a possible mechanism, as has been previously suggested (28).
The
autoradiograms were scanned, and the fraction of molecules that were
fully demethylated at each point was determined
(Fig. 3E). The transfection efficiency was similar in
the different transfectants as determined by hybridization with a
plasmid probe and normalizing to the level of nuclear DNA at each point
(data not shown). As observed in Fig. 3, the levels of
demethylation of both the exogenous Met 5` region and the bacterial CAT
sequences are increased severalfold (10-fold in ras 6 to
100-fold in the ras 11 transfectant) over the rate of
demethylation observed in control lines. The fact that demethylation of
murine, bacterial, and herpesvirus sequences is observed is consistent
with the hypothesis that v-Ha-ras expression induces the
expression of a general demethylation activity.
Although it is well established that demethylation of
specific sites in DNA is associated with
development
(1, 2, 3, 4) , it is unclear
whether site-specific demethylation events that have been extensively
documented are side effects of readjustments of chromatin structure or
whether they reflect an independent cellular process. In this report we
show that genome-wide demethylation events could be programmed by an
important cellular signaling pathway and that it can occur independent
of the full manifestations of either differentiation or cellular
transformation. The fact that ras transfectants demethylate
transiently transfected exogenous DNA encoding a number of different
sequences is consistent with the hypothesis that ras induces a
general demethylation activity. These conclusions are further supported
by the observation that nuclear extracts prepared from ras
transfectants bear a measurable CpG demethylating activity. The
chemical nature of the demethylation reaction is still unclear and will
be determined in future experiments.
Is this demethylation related
to site-specific demethylation of CpG islands that occurs in embryonal
cells? How can induction of a general demethylation activity lead to a
specific pattern of methylation? We do not have the data to answer this
question, but it has been previously hypothesized that the final
demethylation pattern will reflect an interplay between a general
demethylase(s) and local site-specific signals
(29) . Another
interesting observation is that expression of ras results in
some limited de novo methylation of specific sites
(Fig. 2C). The fact that both methylation and
demethylation can be induced by Ras might be relevant to the question
of how genome-wide hypomethylation
(30) and hyperactivation of
DNA methyltransferase can coexist
(31, 32) in cancer
cells as has been previously suggested
(29) . The fact that
general demethylation could be induced by an important cellular signal
transducer raises the interesting possibility that the control of the
availability of a demethylase might be an important site through which
the state of methylation of the genome might be regulated.
We thank Dr. Olson for the myogenin cDNA and Dr.
Gardner for the acetylcholine receptor (
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
To address the question of whether
this activity is regulated by cellular signaling pathways, we
introduced exogenous v-Ha-ras into P19 cells by DNA-mediated
gene transfer. The involvement of signaling pathways triggered by Ras
in differentiation of P19 cells has been studied before. Whereas both
Src
(11) and Jun, a downstream effector of Ras
(12) , can
induce the differentiation of P19 cells, forced expression of oncogenic
Ras does not induce or inhibit differentiation of P19
cells
(13) . Ras might still induce some events that are part of
the differentiation program. General genome-wide demethylation is one
of the events associated with induction of differentiation of embryonal
carcinoma cells
(14) . The fact that Ras does not induce full
differentiation or cellular transformation of P19 cells will enable one
to differentiate between events that are consequences of
differentiation and those that are specifically triggered by Ras.
Stable Transfection and Analysis of Genomic DNA
Methylation
The pZEMras expression vector bearing a 2.3-kb
BamHI-EcoRI fragment encoding v-Ha-ras from
the murine mammary tumor virus v-Ha-ras plasmid
(15) or
the pZEM control vector
(16) was cointroduced into P19 cells
with pUCSVneo (1 µg) as a selectable marker by DNA-mediated gene
transfer using the calcium phosphate protocol
(17) .
G418-resistant cells were cloned and propagated in selective medium and
analyzed for expression of the transfected DNA by Southern and Northern
blot analysis
(17) . DNA was extracted from three P19 PZEM
control transfectants (1, 2, and 3) and three P19 lines transfected
with pZEMras and subjected to a nearest neighbor analysis as described
previously
(8) . 2 µg of DNA were incubated at 37 °C for
15 min with 0.1 unit of DNase and 2.5 µl of
[-
P]dGTP (3000 Ci/mmol from Amersham). 2
units of Kornberg DNA polymerase (Boehringer Mannheim) were then added,
and the reaction was incubated for an additional 25 min at 30 °C.
50 µl of water were then added to the reaction mixture, and the
unincorporated nucleotides were removed by spinning through a Microspin
S-300 HR column (Pharmacia Biotech Inc.). The labeled DNA (20 µl)
was digested with 70 µg of micrococcal nuclease (Pharmacia) in the
manufacturer's recommended buffer for 10 h at 37 °C. Equal
amounts of radioactivity were loaded on TLC phosphocellulose plates
(Merck), and the 3`-mononucleotides were separated by chromatography in
one dimension (isobutyric acid:H
O:NH
OH in the
ratio 66:33:1). The chromatograms were exposed to XAR film (Eastman
Kodak), and the autoradiograms were scanned by scanning laser
densitometry (Scanalytics one-dimensional analysis). Spots
corresponding to cytosine and 5-methylcytosine-dimensional were
quantified, and the percentage of nonmethylated CpG dinucleotide
sequences was calculated. To study demethylation of specific genes,
genomic DNA (10 µg) was extracted from three pZEM (1, 2, and 3) and
three pZEMras (6, 11, and 17) transfectants and subjected to digestion
with HindIII (Boehringer Mannheim, 25 units) followed by
digestion with either 25 units of MspI(M), which is
insensitive to CC
GG methylation, or HpaII(H),
which is sensitive to methylation, for 8 h at 37 °C. At least three
different DNA preparations isolated from three independent passages
were assayed per transfectant to exclude variability between different
DNA samples and verify the stability of DNA methylation patterns. A
representative experiment is presented.
Transient Transfection and Nuclear DNA
Extraction
pMetCAT+ plasmid was methylated in vitro by incubating 20 µg of plasmid with 10 units of SssI
CpG DNA methyltransferase
(27) (New England Biolabs Inc.) in a
buffer recommended by the manufacturer. To verify that the plasmids
were fully methylated, we repeated the methylation reaction (after full
protection from HpaII had been observed) for two additional
times. Methylated pMetCAT+ DNA (1-10 µg) (physical map
in Fig. 3A, bottom panel) was introduced into
P19 cells (8 10
/well in a six-well tissue culture
dish (Nunc)) by DNA-mediated gene transfer using Lipofectin (Life
Technologies, Inc.) according to the manufacturer's
recommendations. We used different concentrations of DNA/transfection
mix to exclude differences in transfected plasmid concentration as an
explanation for the observed differences in the level of DNA
demethylation (1-10 µg). To avoid contamination of the
transfected DNA with non-nuclear plasmid DNA, we first isolated the
nuclei. Cells were dislodged by scraping in phosphate-buffered saline
buffer, nuclei were isolated as described previously, and DNA was
extracted
(18) .
Figure 3:
Demethylation of transiently transfected
pMetCAT+ plasmid in P19 pZEM and pZEMras transfectants. P19,
PZEM1, and PZEMras 6 and 11 transfectants were transiently transfected
with methylated pMetCAT plasmid as described under ``Materials and
Methods.'' 10 µl of nuclear DNA prepared from the transiently
transfected cells (1 and 3 µg of plasmid/transfectant) were
subjected to HpaII and MspI digestion, Southern blot
transfer, and hybridization to pOCAT (A) or Met 5` probes
(B). Nontransfected DNA is fully methylated as indicated in
the first pair of lanes. The physical map of the transfected
pMetCAT+ plasmid is presented in the bottom panel of
A. The CAT sequence is hatched, the herpesvirus
tk gene is shaded, the DNA methyltransferase 5`
region is indicated as a line, the exons are filled
boxes, and the AP-1 sites are shaded ovals.
HpaII sites (H), the BamHI site
(BHI), and the HindIII site (HIII) are
shown. The expected HpaII fragments are indicated under the
physical map. C, Northern blot analysis. RNA was prepared from
the cytosolic fraction made from pZEM (lines 1, 2, and 3) and pZEMras
transfectants and subjected to Northern blot analysis and hybridization
with a P-labeled ras probe (0.7-kb
HindIII-PstI fragment (15)) according to standard
procedures (17). The viral ras directs a 2-kb transcript that
is easily distinguishable from the shorter endogenous ras
messages (1-1.4 kb). D, DpnI cleavage of
transiently transfected pMetCAT DNA. SssI-methylated
(M) and nonmethylated (NM) pMetCAT DNA and DNA
prepared from control P19 and pZEM and pZEMras transfectants
transiently transfected with pMetCAT (3 µg) were subjected to
digestion with DpnI (Boehringer Mannheim), Southern blotting,
and hybridization with a POCAT probe (indicated in the bottom
panel). The bottom panel is a physical map of the pMetCAT
construct; the DpnI sites analyzed by the POCAT probe are
indicated (D). The first DpnI in the CAT region of
the plasmid is designed to be located 3 base pairs apart from a
HpaII site. The sequence of the combined site is presented
relative to the physical map. The arrows indicate the
methylated C or A, and the HpaII and DpnI recognition
sites are underlined. E, an autoradiogram like the
one shown in A was scanned (Masterscan Scanalytics), and the
intensity of hybridization to pOCAT of fragments corresponding to
nonmethylated HpaII sites and methylated HpaII sites
was determined. The ratio of nonmethylated to methylated molecules was
then determined. The first bar (shaded) in each set
indicates cells transfected with 1 µg of plasmid, and the
second bar (hatched) indicates transfectants
initially transfected with 3 µg of DNA.
In Vitro Demethylation Assay
A
P-labeled, fully methylated dCpG substrate was prepared as
follows. 100 ng of a double-stranded (m
dC-dG) oligodimer
(Pharmacia) were denatured by boiling, which was followed by partial
annealing at room temperature. The complementary strand was extended
with the Klenow fragment (Boehringer Mannheim) using m
dCTP
(Boehringer Mannheim) and [
-
P]dGTP (100
µCi, 3000 Ci/mmol), and the unincorporated nucleotides were removed
by chromatography through a NAP-5 column (Pharmacia). Nuclear extracts
were prepared from logarithmic cultures of pZEM (lines 1, 2, and 3) and
pZEMras transfectants (lines 6, 11, and 17) as described previously and
incubated with 1 ng of fully methylated CpG substrate for 30 min at 37
°C in a buffer containing 10 mM Tris-HCl, pH 7.6, 25%
glycerol (v/v), and 5 mM EDTA. The reacted DNA was purified by
phenol/chloroform extraction and subjected to micrococcal nuclease
digestion to 3` mononucleotides, TLC (using polyethyleneimine thin
layer chromatography, Brinkman), and autoradiography as described
above.
Exogenous Expression of Ras Induces Genomic DNA
Demethylation
P19 cells were cotransfected with pUCSVneo and
either pZEMras
(15) or pZEM
(16) as a control.
G418-resistant colonies were isolated, and cells bearing the pZEM and
pZEMras plasmids were identified by Northern and Southern blot analyses
(data not shown). (Fig. 2F is a Southern blot
demonstrating the presence of the pZEM and pZEMras constructs, and the
Northern blot in Fig. 3indicates the presence of the
v-Ha-ras message in the selected transfectants.) Three
transfectants/plasmid were randomly selected for our studies. The
general level of CpG methylation in the ras transfectants and
controls was determined by nearest neighbor analysis
(8) . The
results presented in Fig. 1show a slight reduction in total DNA
methylation in the ras transfectants. The DNA
methyltransferase promoter bears AP-1 sites
(19) and has been
shown by us to be inducible with Ras or Jun
(20) . The general
DNA methylation level might therefore reflect a balance of methylation
and demethylation events induced by Ras.
Figure 2:
The state of methylation of specific genes
in ras transfected and control pZEM transfectants. DNA
prepared from three independent pZEM and pZEMras transfectants was
loaded in the lanes presented in the figure in the following
order from left to right: pZEM 1, 2, and 3 and
pZEMras 6, 11, and 17. The digested DNA was size-fractionated on a 1.5%
agarose gel, Southern blot transferred onto Hybond-N+ in 0.4
M NaOH, and hybridized with the respective
P-labeled DNA probe according to standard protocols (17).
The filters were stripped of radioactivity by boiling the filter for 15
min in 0.2
SSC, 1% sodium dodecyl sulfate buffer and
rehybridized with other probes. A, c21 3.8. A 3.8-kb
BamHI fragment containing the coding portions of the c21 gene was used as a probe. The physical map of the gene is
presented in the bottom panel. The locations of HpaII
sites are indicated as H. The HpaII fragments that
are expected if the CCGG sites are not methylated are indicated
relative to the physical map. The partial 1.9-kb fragment observed in
the ras transfectants is indicated (1.9*). The
locations of the exons are indicated as closed boxes, and the
transcription initiation site is indicated by an arrow. The
probe used is shown as a thickline relative to the
physical map. B, acetylcholine receptor
subunit
promoter. The 1-kb HindIII (HIII) fragment 5` region
probe (22) is indicated as a thick line in the bottom
panel relative to the location of the HpaII sites
(H). The expected HpaII fragments from a
nonmethylated versus a methylated gene are indicated under the
physical map. C, thy-1. The probe used is a
HpaII-XbaI 214-base pair fragment located upstream
from the first exon as shown in the bottom panel relative to a
physical map of the region. The expected HpaII (H)
fragments are indicated. The partially methylated fragment is indicated
as an asterisk relative to the autoradiogram. The internal
site shows some residual resistance to MspI; we have observed
this before (25) with multiple DNA preparations. D,
quantification of de novo methylation of the thy-1HpaII site in ras transfectants. The
autoradiogram shown in E was scanned using Masterscan
(Scanalytics one-dimensional analysis), and the ratio of the methylated
0.8-kb HpaII fragment to the nonmethylated 0.6-kb fragment was
determined. The bars for pZEM 1, 2, and 3 and Ras 6, 11, and
17 are presented (from left to right). E, 5`
region of DNA methyltransferase. The physical map of the DNA
methyltransferase 5` region is indicated. Filled boxes are
exons, the arrows indicate the transcription initiation sites,
and the shadedovals indicate the location of the
AP-1 recognition sequences. The probe is a 1.3-kb BamHI
(BHI)-HindIII (HIII) fragment indicated as a
thick line, and the HindIII-HpaII fragments
expected for a methylated or a nonmethylated HpaII
(H) site are indicated relative to the physical map.
F, 3` region of HGH. The state of methylation of the
transfected plasmid was studied using a 0.77-kb probe encoding the 3`
HGH sequence included in the expression vector as indicated (thick
line) in the bottompanel. The physical map and
the expected HpaII (H) fragments (as well as the
observed partial HpaII fragments, which are indicated as
asterisks next to the autoradiogram, for the pZEMras) for both
the pZEM and pZEMras plasmids are shown. Metallothionein sequences are
lightly shaded, v-Ha-ras sequences are darkly
shaded, and HGH sequences are
hatched.
Figure 1:
State of
genomic methylation of P19 pZEM and P19 ras transfectants. The
state of methylation of genomic DNA of P19 pZEM and P19 ras transfectants was determined as described under ``Materials
and Methods.'' Results are presented as the averages of three
independent transfectants/group ± S.D. The experiment was
repeated three times. The statistical significance of the difference
between the pZEM and pZEM ras groups was determined by a t test (p < 0.01).
State of Methylation of Specific Genes
To
determine whether Ras induces demethylation or hypermethylation in P19
cells and whether these changes are general or targeted at a certain
class of genes, we analyzed the state of methylation of specific genes
using MspI/HpaII restriction enzyme analysis,
Southern blotting, and hybridization with specific probes. Five classes
of genes were selected: (a) a gene that is expressed in
skeletal muscles (skeletal muscles are one of the prominent cell types
into which P19 cells can differentiate
(21) ), the acetylcholine
receptor chain
(22) ; (b) a gene that is
specifically expressed in the adrenal cortex but is normally heavily
methylated and repressed in P19 cells, steroid 21-hydroxylase
(c21)
(23) ; (c) a gene that is specifically
expressed in P19 cells, thy-1 (24); (d) a gene that
is ubiquitously expressed in all dividing tissues, the 5` region of the
DNA methyltransferase
(19) ; and (e) the 3` region of
the human growth hormone gene, which was exogenously introduced into
P19 cells
(16) (exogenously transfected DNA is methylated de
novo in mouse embryonal cells
(25) ). DNA was prepared from
exponentially growing cells, digested with HindIII and either
MspI or HpaII, and sequentially hybridized with the
different gene-specific probes indicated in Fig. 2. The pattern
of methylation observed per each transfectant and the P19 controls were
stable (data not shown), suggesting that, once forced expression of Ras
triggers a change in DNA methylation, the new state of methylation is
maintained.
(
)
following HpaII digestion, whereas
the MspI pattern reflects the expected fragments at 1, 0.9,
0.8, 0.7, 0.4, and 0.15-0.2 kb (Fig. 2A). All
three ras transfectants show a diminution of the 9-kb, high
molecular weight fragment following HpaII digestion and a
distinct partially methylated HpaII fragment migrating as a
1.9-kb fragment (see physical map in Fig. 2A for
possible origin of this fragment). In addition to the site-specific
demethylation at the sites flanking the 1.9-kb partial HpaII
fragment, some of the fully hypomethylated low molecular fragments are
now observed at 0.8, 0.7, 0.4, and 0.15 kb. The demethylation of
c21, which is not expressed in P19 cells (data not shown),
suggests that this demethylation is not an effect of induction of gene
expression.
chain 5` region 1.3-kb HindIII fragment bears one CCGG
site (physical map in Fig. 2B). The majority of the
population of pZEM control transfectants is methylated at this site as
indicated by the relative abundance of the 1-kb fully methylated
HindIII fragment. In the ras transfectants, most of
these sites are demethylated as implied by the presence of the 0.3- and
0.7-kb HpaII fragments (Fig. 2B). The myoD and myogenin genes are also heavily methylated in pZEM controls
and demethylated in ras transfectants (data not shown).
Transient Transfection Demethylation Assay
To
address the question of whether Ras induces a general demethylation
activity in P19 cells, we utilized a transient demethylation assay. We
have previously shown that P19 cells cannot methylate transiently
introduced exogenous DNA de novo, but they possess an activity
that demethylates in vitro methylated plasmid DNA. Therefore, a transient transfection demethylation assay can
measure the level of demethylation activity in the cell without
confounding de novo methylation. We utilized the previously
described pMetCAT+ plasmid as a substrate
(19) . This
plasmid bears the 2-kb DNA methyltransferase 5` region inserted in
front of the bacterial CAT gene from pOCAT and a viral herpes tk gene
(26) . The HpaII site residing 5` of the DNA
methyltransferase fragment (-1931) (Fig. 3A,
physical map) is methylated in P19 cells and demethylated in ras transfectants (Fig. 2E). The cat portion
of the plasmid is a bacterial sequence that does not have a homologue
in the P19 genome and could be used to measure nonspecific
demethylation.
In Vitro Demethylation Assays
To further determine
whether Ras induces a demethylation activity that is independent of DNA
replication, we determined whether nuclear extracts prepared from
ras transfectants bear a higher demethylase activity than
extracts prepared from control P19 cells. We synthesized a fully
methylated P-labeled double-stranded
poly-m
dCpG oligonucleotide in vitro using
m
dCTP and [
-
P]dGTP as described
in the legend to Fig. 4. 1 ng of the methylated substrate was
incubated with 10 µg of 0.4 M NaCl nuclear extracts
prepared from three pZEM and three pZEMras transfectants for 30 min at
37 °C in the absence of exogenous dNTPs. The reacted DNA was
digested to 3` mononucleotides with micrococcal nuclease and subjected
to thin layer chromatography to separate methylated and nonmethylated
3`
P-labeled dCMPs. The autoradiograms were scanned, and
the relative intensity of demethylated dCMP was determined. As observed
in Fig. 4, the extracts prepared from ras P19
transfectants bear a measurable level of an active demethylase, whereas
this activity is barely detectable under these conditions in the
control transfectants.
Figure 4:
In vitro demethylation assay.
P-Labeled, fully methylated dCpG substrate was incubated
(for 0.5 h) with nuclear extracts (10 µg) prepared from P19pZEM and
pZEMras transfectants. The relative abundance of m
dCMP and
dCMP was determined after subjecting the reacted DNA to micrococcal
nuclease digestion and chromatographic separation as described under
``Materials and Methods.'' The top panel shows a
representative chromatogram. The autoradiograms were scanned, spots
corresponding to dCMP and m
dCMP were quantified, and the
percentage of nonmethylated dCMP was calculated. The bottom panel presents results obtained for extracts prepared from three
different transfectant lines/plasmid.
subunit) promoter probe.
The excellent technical help of Anik Boudreau is sincerely
acknowledged. We thank Dr. Gary Tanigawa for critical reading of the
manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.