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INTRODUCTION |
Several lines of evidence have established that DNA methylation
patterns are tissue specific and are generated during development by
rounds of de novo methylation and demethylation events
(1-4). Two modes of demethylation have been documented: site-specific demethylation that coincides in many instances with the onset of gene
expression of specific genes (5, 6) and a general genome wide global
demethylation that occurs during early development in vivo
(7, 8), during cellular differentiation (9-11), and in cancer cells
(12). The global demethylation is consistent with the hypothesis that a
general demethylase activity that is activated at specific points
in development or oncogenesis exists (13). However, until recently the
identity of the enzymes responsible for demethylation has been a
mystery. It has been generally assumed that an activity that can
transform a methylated cytosine to a cytosine by direct removal of a
methyl group does not exist. Therefore, it has been proposed previously
that demethylation involves either a glycosylase and repair activity
(10, 13, 14) or a nucleotide excision and replacement activity (15).
Whereas a demethylation process that involves a sequence of
glycosylase, nuclease, repair, and ligase enzymatic activities could
possibly be involved in site specific demethylation, it is hard to
understand how it can catalyze genome wide global demethylation. Global
nicking and repair would have a serious impact on the integrity of the
genome. What enzymatic activity is responsible for global demethylation?
We have recently identified and cloned from human cancer cells a
bona fide DNA demethylase that catalyzes the hydrolytic
removal of methyl residues from methyl cytosine in DNA (16). DNA
demethylase demethylates both fully methylated and hemimethylated DNA,
shows dinucleotide specificity, and can demethylate mCpG sites in
different sequence contexts (16). Since this enzyme can remove methyl groups from DNA without damaging the DNA, it is a candidate to be
involved in global hypomethylation. One essential property of an enzyme
that removes methylation from wide regions of the genome could be processivity.
In this paper we have used sodium bisulfite DNA mapping to determine
whether purified demethylase demethylates DNA in a processive or
distributive manner in an isolated system. Bisulfite treatment that is
followed by PCR amplification, cloning, and sequencing of individual
molecules of DNA allows one to determine the state of methylation of a
single DNA molecule at a time, at single base resolution (17). This
allows for the analysis of an interaction of an enzyme with a single
substrate molecule. We test here whether methylated murine and
bacterial gene sequences are processively or distributively
demethylated by purified demethylase and whether this process is
affected by the properties of the methylated sequence.
The processive nature of DNA demethylase in vitro
demonstrated in this manuscript provides a model to explain the
mechanism involved in genome wide hypomethylation.
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MATERIALS AND METHODS |
Purification of Demethylase--
Nuclear extracts were prepared
from A549 (ATCC: CCL 185) cultures at near confluence as described
previously (18). A freshly prepared nuclear extract (1 ml, 6 mg) was
diluted to a conductivity equivalent to 0.2 M NaCl in
buffer L (10 mM Tris-HCl, pH 7.5, 10 mM
MgCl2) and applied onto a DEAE-Sephadex A-50 column
(Amersham Pharmacia Biotech) (2.0 × 1 cm) that was
pre-equilibrated with buffer L at a flow rate of 1 ml/min. Following a
15-ml wash with buffer L, proteins were eluted with 5 ml of a linear
gradient of NaCl (0.2-5.0 M). 0.5-ml fractions were
collected and assayed for demethylase activity. Demethylase eluted
between 4.9 and 5.0 M NaCl. Active DEAE-Sephadex column
fractions were pooled, adjusted to 0.2 M NaCl by dilution,
and loaded onto an SP-Sepharose column (Amersham Pharmacia Biotech)
(2.0 × 1 cm). Following washing of the column as described above,
the proteins were eluted with 5 ml of a linear NaCl gradient (0.2-5.0
M). 0.5-ml fractions were collected and assayed for
demethylase activity. Demethylase activity eluted around 5.0 M NaCl. Active fractions were pooled, adjusted to 0.2 M NaCl by dilution, and applied onto a Q-Sepharose
(Amersham Pharmacia Biotech) column (2.0 × 1 cm), and proteins
were eluted as described above. The demethylase activity eluted around
4.8-5.0 M NaCl. The pooled fractions of Q-Sepharose column
were loaded onto a 2.0 × 2.0 cm DEAE-Sephacel column (Amersham
Pharmacia Biotech) and eluted with 10 ml of buffer L. The activity was
detected at fraction 4, which is very near the void volume. Demethylase
is purified 500,000-fold using this protocol.
Demethylase activity was assayed by measuring the conversion of
methyl-dCMP (mdCMP)1 in a
poly(mdC-[32P]dG)n double-stranded DNA stranded
to dCMP as described previously (18). One unit of demethylase is
defined as the activity required to transform 1 pmol of mdCMP to dCMP
in 1 h at 37 °C (16).
In Vitro Methylation of Substrates--
PMetCAT+ plasmid or
pBluescript SK(+) plasmid were methylated in vitro by
incubating 5 µg of plasmid DNA with 8 units of SssI CpG
DNA methyltransferase (19) (New England Biolabs Inc.) in a buffer
recommended by the manufacturer containing 320 µM
S-adenosylmethionine, at 37 °C for 2 h. After
repeating this procedure five times, full protection from
HpaII digestion was observed.
Demethylase Reaction--
400 ng of methylated pMetCAT+ or
pBluescript SK plasmids were incubated with 1.4 units of
DEAE-Sephadex-purified dMTase in buffer containing 20 µg of RNase A,
for 10 s to 2 h. The reaction was stopped and purified by
phenol/chloroform extraction and ethanol precipitation and subjected to
bisulfite mapping.
Bisulfite Mapping--
Bisulfite mapping was performed as
described previously with minor modifications (17). Fifty nanograms of
sodium bisulfite-treated DNA samples were subjected to PCR
amplification using the first set of primers described below. PCR
products were used as templates for subsequent PCR reactions utilizing
nested primers. The PCR products of the second reaction were then
subcloned using the Invitrogen TA cloning kit (we followed the
manufacturer's protocol), and the clones were sequenced using the T7
sequencing kit (Amersham Pharmacia Biotech) (we followed the
manufacturer's protocol, procedure C). The primers used for the DNA
MeTase genomic region (Gen BankTM accession number
M84387) were: MET5'1, 5'-ggattttggtttatagtattgt-3'; MET5' (nested),
5'-ggaattttaggtttttatatgtt-3'; MET3'1, 5'-ctcttcataaactaaatattataa-3'; and MET3' (nested), 5'-tccaaaactcaacataaaaaaat-3' (245-265). The primers used for the bacterial DNA chloramphenicol acetyltransferase (CAT) genomic region (GenBankTM accession number U65077)
were: CAT5'1, 5'-ttgtttaatgtatttataattacat-3'; CAT5'(nested),
5'taaagaaaaataagtataagtttta-3'; CAT3'1, 5'-ctcacccaaaaattaactaaaa-3'; CAT3' (nested), 5'tttaaaaaaataaaccaaattttca-3'. Eighteen control clones
were sequenced using methyltransferase primers (0 s), 14 clones at
10 s and 30 s and 12 clones at 2 h. Six control clones were sequenced using CAT primers (0 s), 5 clones at 30 s and 8 clones at 2 h.
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RESULTS |
Two Possible Modes of Demethylation--
Enzymes interacting with
DNA can act either processively or distributively. A processive
mechanism implies that once the demethylase lands on, or centers itself
on, a DNA molecule, it slides along the DNA molecule and demethylates a
stretch of CpGs in cis as it progresses along the DNA strand
(see Fig. 1 for model). The alternative
distributive mechanism implies that each demethylation event requires
an independent interaction of the enzyme with the DNA. The enzyme is
dislodged from the DNA after demethylation of each CpG site and will
then land on any accessible methylated CpG site on any molecule of DNA
in the reaction mixture in no specific order. These two models can be
differentiated if one can monitor the intermediate stages of a
demethylation reaction of a sequence of methylated CG sites on
individual molecules of DNA. A distributive mechanism predicts that the
demethylated sites will be interspersed within methylated sites and
will be randomly distributed in the different DNA molecules (Fig.
1A). On other hand, if demethylation is progressive, two
classes of molecules will be identified at an intermediate stage,
molecules that bear stretches of nonmethylated sites and others that
are fully methylated (Fig. 1B).

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Fig. 1.
Two alternative models for DNA demethylation
by demethylase. Two molecules of DNA bearing methylated CG sites
(filled circles) are shown. Demethylated CG sites are
indicated as open circles. The distributive model
(A) predicts that demethylase (represented by shaded
ovals) interacts with each site independently. The enzyme is
dislodged from the DNA after demethylation of each site. The last two
lines show the predicted pattern of methylation of the two DNA
molecules according to this model. In the processive model
(B), the demethylase enzyme lands on a molecule of DNA and
proceeds to demethylate the molecule in cis. The
arrows indicate the direction of the enzyme. The last two
lines illustrate the pattern of methylation of the DNA molecules, if
the enzyme works in a processive manner and the reaction is terminated
before completion.
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Restriction Enzyme Analysis of the Pattern of Demethylation in
Vitro of a CpG-methylated Plasmid--
pBluescript(+) plasmid was
fully methylated in vitro and incubated at 37 °C with 1.4 units of demethylase for either 25 min or 3 h. Following
demethylation, the plasmid was subjected to digestion with
HpaII, which cleaves the sequence CCGG when the internal C
is not methylated. As shown in Fig. 2,
demethylation of the plasmid is completed after 3 h, as indicated
by the complete digestion of the plasmid with HpaII and the
appearance of all the predicted HpaII fragments. At 25 min
the plasmid is incompletely demethylated as indicated by the presence
of plasmid DNA that is resistant to HpaII cleavage. However,
the pattern of cleavage by HpaII is consistent with a
processive mechanism of demethylation. If demethylation is
distributive, the demethylated CpGs should be randomly spread,
resulting in a gradient of sizes of HpaII partial fragments.
However, the results in Fig. 2 show two classes of bands, fully
digested HpaII fragments and plasmids that are completely
resistant to HpaII.

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Fig. 2.
Restriction enzyme analysis of in
vitro demethylation of a methylated SK plasmid. Fully
methylated pBluescript plasmid (with MSssI, which methylates
all CG sequences in a plasmid) was incubated with either buffer L
(lane 2) or demethylase for 25 min (lane 3) or
180 min (lane 4), digested with HpaII restriction
enzyme, fractionated on a 2% agarose gel, and was then subjected to
Southern blot transfer and hybridization with a 32P-labeled
pBluescript SK DNA. Lane 1 contains undigested SK plasmid.
The bands corresponding to undigested intact plasmid and the bands
corresponding with the expected HpaII fragments within
pBluescript are indicated.
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Bisulfite Mapping of in Vitro Demethylated Mouse dnmt1
Sequences--
Whereas the restriction enzyme pattern of digestion
shown above suggests a processive mechanism of demethylation, it only allows us to look at a population of plasmids at a given time. To fully
demonstrate a processive mechanism, one has to be able to follow the
fate of methylated CG sites on a single molecule of DNA. Bisulfite
mapping, which is followed by PCR amplification, cloning, and
sequencing of individual clones allows one to determine the state of
methylation of a single DNA molecule at a time, at single base
resolution (17). Fully methylated pMetCAT+ plasmid DNA, which has been
described previously (18) (Fig.
3A and
4A, zero time) bears a genomic
fragment of the dnmt1 gene and a bacterial chloramphenicol
acetyltransferase gene on the same DNA molecule. Thus, it allows us to
follow the demethylation of a stretch of CpG sites located in a low
density CG region, which is characteristic of many vertebrate genes
(dnmt1) as well as a dense CG region in the bacterial CAT
(Fig. 4). In vitro methylated pMetCAT was incubated with
purified demethylase for different time intervals as indicated in Fig.
3 and Fig. 4. The demethylated DNA was treated with sodium bisulfite
(which results in the conversion of nonmethylated cytosines to
thymidine, whereas methylated cytosines are protected), amplified by
PCR using appropriate primers for the indicated region of
dnmt1, and subcloned. Subclones, each of which represents a single molecule of DNA treated with demethylase for a specific amount
of time, were sequenced.

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Fig. 3.
Bisulfite mapping of the dnmt1
region in methylated pMetCAT+ plasmid at different time intervals
following initiation of demethylation in vitro.
A, physical map of the regulatory genomic region residing
upstream of exon 2 of mouse dnmt1 included in the pMetCAT
plasmid (18) (exons are indicated by filled boxes, and the
intronic sequence is indicated by a line). A blowup of the
region amplified following bisulfite treatment is shown under the
physical map; the different CpG sites in the fragment are presented as
ovals. The percentage of methylated cytosines per site in
12-18 clones analyzed by bisulfite mapping per time interval
(indicated by the different lines under the physical map) were
determined and are presented as different shadings of the circles
representing each of the sites (heavily methylated sites (>75%) are
indicated by filled circles, partially methylated sites
(50-75%) are indicated by a hatched circle, partially
methylated sites (25-50%) are indicated by a gray circle,
and nonmethylated (<25%) sites are indicated by an open
circle). B, the methylation pattern of individual
clones of amplified dnmt1 region from bisulfite-treated
methylated pMetCAT plasmids that were incubated for different time
intervals (10 s, 30 s, and 2 h). Each line represents one
clone. Filled circles refer to methylated CG dinucleotides,
and empty circles indicate demethylated CG dinucleotides.
"n" indicates the number of clones analyzed for each
time interval. C, a representative sequencing gels of
bisulfite treated methylated pMetCAT DNA is presented per condition.
Lollipops indicate the specific CpG sites by their position.
The numbering is according to GenBankTM accession number
M84387. Filled lollipops indicate fully methylated sites,
and empty lollipops indicate nonmethylated sites.
"n" indicates the number of clones with identical
methylation patterns.
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Fig. 4.
Bisulfite mapping of the CAT region in
methylated pMetCAT+ plasmid at different time intervals following
initiation of demethylation in vitro.
A, physical map of the bacterial CAT region in pMetCAT. A
blowup of the region amplified is shown under the physical map; the
different CpG sites in the fragment are presented as ovals.
The percentage of methylated cytosines per site for all the clones
analyzed per time interval (indicated by different lines under the
physical map) were determined and are presented as different shadings
of the circles representing each of the sites as indicated (see legend
to Fig. 3A). B, the methylation pattern for a
sample of individual clones of amplified CAT region from
bisulfite-treated methylated pMetCAT plasmids that were incubated for
different time intervals (10 s, 30 s, and 2 h) are displayed.
Each line with circles represents one clone
mapped within the methyltransferase enhancer region. Filled
circles refer to methylated CG dinucleotides, and empty
circles indicate demethylated CG dinucleotides. "n"
refers to the number of clones analyzed for each time interval.
C, a representative sequencing gel of bisulfite treated DNA
is presented per condition. Lollipops indicate the specific
CpG sites by their position. The numbering is according to
GenBankTM accession number U65077. Filled
lollipops indicate fully methylated sites, and empty
lollipops indicate nonmethylated sites. The two sequencing gels on
the left-hand side present nucleotides in the order of ATCG,
whereas the two gels on the right-hand side present the
nucleotides in the order of TAGC. "n" indicates the
number of clones with identical methylation patterns.
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We first calculated the average methylation at each site in a
representative population of in vitro demethylated pMetCAT
plasmids. This allowed us to monitor the general progression of the
demethylation reaction and to identify sites that exhibited either
specific resistance or sensitivity to demethylation. Fig. 3A
shows the map of the CG sites in the dnmt1 region and the
average percentage methylation of each site in a representative
population. The 265-bp region amplified contains 5 CG sites or 1 CG
site in 50. At 10 s 11 out of 14 clones observed remain fully
methylated (78%); however, methylation decreases with increased time.
At 30 s, 8 clones remain fully methylated out of 14 (57%), and
all 12 clones are fully demethylated (0% methylation) at 2 h. As
appears from this experiment, demethylation of all the CGs in the
dnmt1 region proceeds at the same rate.
Since each CG site has the same probability of being demethylated
at interim time points of the reaction, the pattern of methylation of
individual molecules of DNA should be a consequence of either the
distributive or processive property of the enzyme. As observed in Fig.
3C, one quarter of the molecules are fully demethylated, whereas the other group of molecules is completely methylated at
30 s. Even at 10 s we did not see a demethylated CpG, which is bilaterally flanked with methylated CpGs. This pattern is consistent with a processive mechanism. This assay was repeated, and the results
were confirmed by looking at a bacterial sequence with a higher CpG
density, within the same plasmid.
In Vitro Demethylation of a Dense CG Region Residing within the
CAT--
Bisulfite mapping of the state of methylation of a second
region in the same pMetCAT+ plasmid allowed us to confirm our initial observations regarding the processivity of the reaction and to determine whether demethylation is dependent on the properties of the
sequence. We amplified a 250-base pair sequence within the bacterial
CAT gene, with a CpG density of 5.2% (13 CpGs within a region of 250 bp), which is more than two times times greater than the density of the
dnmt1 region. Fig. 4A shows a physical map of the
CAT gene and the location of the CpG sites that were mapped. The
average methylation of the CG sites at different time intervals through
the demethylation reaction was calculated, and it demonstrates that the
demethylation of the CAT region which is co-linear with the
dnmt1 region is slower (Fig. 4A). This is consistent with the slower demethylation of the SK plasmid, which is
also CG-rich relative to the dnmt1 region (Fig. 3). At zero time, all 13 CpGs are fully methylated and remain so at 30 s, unlike the pattern seen in the dnmt1 region (Fig.
3A). Even at 2 h, the demethylation reaction is not
complete, 2 clones out of 8 are still methylated. When the state of
methylation of independent molecules is examined (Fig. 4B),
the distribution of demethylated sites is not random among the
different plasmid molecules. Some molecules are fully methylated
whereas others bear a stretch of demethylated CGs. This again is
consistent with a processive mechanism. Once demethylation is
initiated in this region it progresses in cis.
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DISCUSSION |
Global changes in demethylation require enzymatic activities that
can transform methylated cytosines, which are spread over large
segments of DNA to nonmethylated cytosines. One attractive explanation
might be that the demethylase enzyme is a processive enzyme. The
mechanisms responsible for demethylation have been unknown, since
the identity of the enzymatic activities responsible for demethylation
was a mystery. Our identification of a bona fide demethylase
(16) allows us to address the mechanisms of demethylation for the first
time. In this manuscript we use the bisulfite mapping method (17),
which provides a unique opportunity to dissect the interaction of an
enzyme with a single substrate molecule at a time. Using this method,
we demonstrate that the demethylase is a processive enzyme. We suggest
that the critical step in demethylation is the interaction of the
enzyme with the substrate. Once demethylation is initiated, it will
proceed uninterrupted for at least 250 bp at a high rate, since no
partial demethylation of the dnmt1 region or the CAT region
is detected even after 30 s at single molecule resolution.
The presence of regions exhibiting different density of methylated CGs
on the same molecule of DNA allowed us to determine whether these
differences will affect demethylation and its processivity. It is
obvious that the processivity of the demethylase does not extend
to the CAT region from the dnmt1 region, since the CAT region (Fig. 4B), which is co-linear with the
dnmt1 region (Fig. 3B), is demethylated more
slowly. This is consistent with the demethylase proceeding to a certain
distance before being dislodged. However, even within the CAT region,
once demethylation is initiated, it proceeds in cis, since
we have not identified molecules that are partially methylated in this
region. The difference in the demethylation of the CAT and
dnmt1 regions must reflect a lag in initiation of
demethylation in these two different regions. These data are consistent
with the interaction of demethylase with the substrate as being the
rate-limiting step. We suggest that the interaction of demethylase
with DNA can vary based on the properties of the region; some regions
such as the dnmt1 sequence on pMetCAT show a high propensity
to interact with the enzyme, whereas others such as the CAT regions
display a low affinity. Once the demethylase lands on a specific
region, it migrates processively to a certain distance until it
recognizes a stop signal or dislodges from the DNA.
It has been proposed previously that demethylation in vivo
proceeds in cis from centers of demethylation (20),
resulting in demethylation of certain stretches of the genome. The data presented here are consistent with this model.
Whereas additional experiments are required to establish that
demethylation in vivo progresses processively from centers
of methylation as suggested here, the data presented here demonstrate that the demethylase is a processive enzyme and is consistent with this model.