From the NICHD, National Institutes of Health, Bethesda, Maryland 20814
Received for publication, December 5, 2000, and in revised form, February 20, 2001
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ABSTRACT |
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CpG methylation is maintained in daughter
chromatids by the action of DNA methyltransferase at the replication
fork. An opportunity exists for transcription factors at replication
forks to bind their cognate sequences and thereby prevent remethylation
by DNA methyltransferase. To test this hypothesis, we injected a
linearized, methylated, and partially single-stranded reporter plasmid
into the nuclei of Xenopus oocytes and followed changes in
the transcriptional activity after DNA replication. We find that
dependent on Gal4-VP16, the action of DNA methyltransferase, and
replication-coupled chromatin assembly DNA replication provides a
window of time in which regulatory factors can activate or repress gene
activity. Demethylation in the promoter region near the GAL4 binding
sites of the newly synthesized DNA did not occur even though the Gal4
binding sites were occupied and transcription was activated. We
conclude that "passive" demethylation at the replication fork is
not simply dependent on the presence of DNA binding transcriptional activators.
CpG methylation is causal for transcriptional silencing,
imprinting, and X inactivation (1-4). CpG island hypermethylation at
promoter elements of tumor suppressor genes correlates with their
repression (5-8). The regulatory mechanisms by which DNA methylation
interferes with gene activity have begun to be unraveled (2). Large
repressor complexes containing methyl-CpG-binding proteins and histone
deacetylases have been characterized (9-11), directly linking CpG
methylation to transcriptional repression. To achieve the precise
epigenetic control of gene expression, the mechanisms of maintenance
methylation, de novo methylation, and demethylation must be
strictly regulated.
Several mechanisms have been proposed to keep a promoter in a CpG
island methylation-free state (reviewed in Ref. 12). Active demethylation occurs during early mouse development (13). However, the
existence of proteins exerting demethylase activity has been controversial and remains obscure (Ref. 14 and references therein). Alternatively, DNA replication might be involved in controlling the
methylation state of promoters (15). Replicative processes might
provide a window of opportunity for transcription factors to bind to
hemimethylated DNA and thereby prevent remethylation and subsequent
gene repression. Transcription factor binding might interfere with the
remethylating activity of DNA methyltransferases at the replication
fork (16, 17).
Transcription factor binding during the replication of DNA leads to
subsequent demethylation concomitant with DNA replication (18). In
Xenopus eggs it has been shown that replication, together with factor binding but not ongoing transcription, results in partial
locus-specific demethylation (15). Alternatively, nonmethylated CpG
islands may result from a transcriptionally active gene, the sequences
of which also function as an origin of DNA replication at a very early
developmental stage (19).
All enzymatic activities and co-factors necessary for efficient DNA
replication are present in the Xenopus oocyte. Although oocytes do not initiate DNA replication on double-stranded DNA, they
can convert single-stranded DNA into double-stranded DNA and support
the concomitant assembly of chromatin. Previous studies using
single-stranded unmethylated DNA showed the relief of nucleosomal repression by transcriptional activators and that the assembly of
transcription complexes and replication-coupled chromatin assembly are
highly competitive processes (20, 21). Here we examine whether
transcription factors compete with the DNA methylation maintenance
machinery on CpG-methylated templates directly at the replication fork
and whether this competition can determine the transcriptional fate of
a reporter gene. Our approach is first to in vitro methylate
linearized template DNA and subsequently to use the enzyme exonuclease
III to generate unidirectional deletions. The modified DNA is then
microinjected into frog oocytes. Using this partially single-stranded
methylated template, we find that the stable binding of transcriptional
activators or repressors during the synthesis of the second DNA strand
programs the gene to remain either active or repressed. This
programming is, under our experimental conditions, independent of
complete demethylation near the binding site. Hence, factor binding
during the time window of second strand synthesis might tag the sites
for complete demethylation once replication is completed.
Plasmids--
The construct pGEMH2BLuc used in this study is
derived from the vector PGEM-3Zf(+) (Promega). The plasmid was cut with
EcoRI and HindIII and ligated with an
EcoRI/HindIII fragment from the plasmid pE4
containing five Gal4 binding sites and the adenovirus E4
promoter (kindly provided by Mike Carey, University of
California, Los Angeles). The minimal adenovirus E4 promoter was
replaced with the TaqI/MluI fragment of the
histone H2B promoter. The luciferase gene sequence ranging from +1 to
+200 was polymerase chain reaction-amplified using specific primers
containing BamHI and SmaI restriction sites and
was subsequently cloned into the SmaI/BamHI
linearized vector. Expression plasmids encoding fusion proteins of
MeCP2 and Gal4-VP16 used in this study are described in Ref.
9.
Exonuclease III Digestion--
Templates for second strand
synthesis in vitro and in vivo were generated
using exonuclease III (exo
III)1 (Promega), which
produces unidirectional deletions starting at the 3' end. To protect
one end from digestion, the plasmid pGEMH2Bluc was cut with
KpnI and SmaI. Deletions were produced starting
from the SmaI end and heading into the regulatory sites of
the H2B promoter and upstream Gal4 sites. 10 µg of the linear
fragment were incubated with 500 units of exonuclease III at 37 °C
for 10 min in a buffer containing 66 mM Tris/Cl, pH 8.0, and 0.66 mM MgCl2. To stop the reaction,
samples were heat-inactivated by incubation at 65 °C for 20 min. The
lengths of the deletions were determined by the treatment of aliquots
from the reactions with mung bean nuclease to cleave single-stranded
DNA overhangs and by comparing them with the size of the wild-type
unmodified template. After purification of the modified DNA by standard
methods, the fragments were either microinjected into oocytes or
subjected to second strand synthesis in a cell-free egg extract derived from Xenopus laevis.
In Vitro Methylation of DNA--
All CpG sites in the plasmid
PGEMH2BLuc were methylated in vitro using SssI
methylase following the instructions of the manufacturer (New England
Biolabs). After phenol-chloroform extraction, the DNA was precipitated.
The efficiency and amount of methylation were checked by restriction
analysis with the methylation-sensitive restriction endonuclease
HpaII.
Preparation of X. laevis Low Spin Supernatant Extract and Second
Strand Synthesis--
Female frogs were primed with 200 IU of human
choriogonadotropin, and eggs were dejellied and crushed in a buffer
containing 50 mM Tris/Cl, pH 7.5, 50 mM KCl,
2.5 mM MgCl2, and 2 mM
2-mercaptoethanol as described (21). 5 µl of egg extract (50 µg of
protein), 500 µM each dCTP, dTTP, and dGTP, and 0.5 µCi
of [32P]dATP were mixed with 500 ng of exonuclease
III-digested template, and the reaction was allowed to proceed for
2 h at room temperature. The buffer system used
contained 10 nM Microinjection into X. laevis Oocytes and Primer
Extension--
Oocytes preparation and DNA and RNA microinjections
were performed as described (22). In vitro transcribed
mRNA (2-5 ng) encoding the Gal4-hybrid protein was injected into
the cytoplasm, and 6-8 h later 1 ng of DNA template was injected into
each oocyte nucleus. Total RNA of the injected samples was
precipitated, and the pellets were washed with 70% ethanol and
dissolved in 20 µl of diethyl pyrocarbonate-treated water. The
transcription was monitored by primer extension as described (22). An
oligonucleotide complementary to the luciferase gene from +126 to +154
was used for primer extension analyses
(5'-ATGTTCACCTCGATATGTGCATCTGTAAA-3').
DNase I-hypersensitive Site Analysis--
Oocytes (20/sample)
were injected with mRNA and template DNA as described above and
homogenized in DNase I digestion buffer (10 mM HEPES, pH
8.0, 50 mM KCl, 5 mM MgCl2, 1 mM dithiothreitol, 0.1% Nonidet P-40, and 5% glycerol).
The oocyte suspension was divided into four fractions and subsequently
digested with 10-100 units of DNase I (Life Technologies, Inc.) at
room temperature for 1 min and was stopped with 1 volume of 1% SDS/20
mM EDTA. The samples were then treated with 3 µl of DNase
I-free RNase (Roche Molecular Biochemicals) for 30 min at
37 °C, and proteinase K was added for 2 h at 55 °C. Proteins
were extracted with 1 volume of phenol-chloroform, and the clear
supernatants were precipitated with isopropanol and washed with 70%
ethanol. The DNA pellets were dissolved in 20 µl of TE buffer (50 mM
Tris/HCl, 1 mM EDTA, pH 7.6) and subjected to restriction
digestion with 20 units of KpnI/sample. Digested DNA samples
were analyzed by electrophoresis using 2% agarose gels, blotted onto
Hybond-N membranes, and hybridized with a random primed probe
comprising the KpnI/HindIII fragment of the
subcloned Gal4-H2B-Luciferase insert.
Micrococcal Nuclease Treatment of Oocytes--
Injected oocytes
(20/sample) were homogenized in MNase buffer (DNase I buffer + 3 mM CaCl2). The oocyte extract was divided into
four fractions and subsequently digested with increasing amounts (1-10
units) of MNase (Worthington) at room temperature for 15 min. Reactions
were stopped with 1 volume of a buffer containing 1% SDS/20
mM EDTA. DNA was prepared according to standard methods and
analyzed by electrophoresis, blotted, and hybridized as described above.
Establishing a System Suitable for Analysis of the Transcription of
Replicating Methylated DNA--
To examine whether transacting factor
binding interferes with the process of DNA remethylation during the
time course of replicative processes, we used a single-stranded
methylated H2B minimal promoter fused to five upstream Gal4 binding
sites to assay for second strand synthesis and transcription (Fig.
1A). Because it is presently not possible to methylate cytosines efficiently within CpG sequences of
single-stranded DNA using a bacterially derived methylase, we
methylated double-stranded DNA and then generated heteroduplex DNA
using exonuclease III, which creates unidirectional deletions starting
from the 3' end of the template. After linearization of the plasmid DNA
with SmaI and KpnI and subsequent treatment with
exonuclease III, we were able to generate deletions ranging from 400 to
3000 base pairs (Fig. 1B). Using a low spin supernatant derived from X. laevis eggs (containing the functional
replication machinery) we were able to demonstrate incorporation of
radioactive nucleotides into newly synthesized DNA (Fig.
2, A and B).
Cleavage with HpaII and comparison of methylated and
nonmethylated templates shows that there is no major change in the
methylation status of all HpaII sites in the H2B promoter
after DNA synthesis (Fig. 2, C and D,
lanes 4-6). Aphidicolin sensitivity
indicates that DNA polymerase The Efficient Transcription of Linearized Methylated Templates
Depends on a Transcriptional Activator--
As described previously,
DNA topology plays a significant role in transcription and template
linearization and has been reported to exert a dramatic decrease in
transcription efficiency (23, 24). The exact mechanism of this
effect is not known and may be explained by a decreased affinity of
transacting factors for their cognate sequences. Transcription of
microinjected linearized DNA into frog oocytes is restored by
co-injection of strong activators like Gal4-VP16 (Fig.
3A, lanes 8,
9, 12, 13, 16, and
17). In contrast, when we use a supercoiled circular
template, the stimulation of transcription caused by the activator is
less pronounced (Fig. 3A, compare lanes 2 and 3 with lanes 4 and 5). Template
methylation abolishes transcription completely in the absence of the
activator (Fig. 3A, lanes 6 and 7)
(25). Gal4-VP16 is capable of binding to methylated DNA when present
before chromatin assembly rendering the microinjected templates
transcriptionally active (Fig. 3A, lanes 8,
9, 12, 13, 16, and
17). In addition, microinjected linear template DNA is
sufficiently stable in the oocyte to allow efficient packaging into
nucleosomal arrays during an incubation of 12 h after injection
(Fig. 3B).
Gal4-VP16 Can Overcome Methylation-dependent Repression
of Transcription by Binding during Second Strand Synthesis--
The
key question to be addressed in our assays is whether binding of a
transcriptional activator to its cognate sequence can overcome
transcriptional repression caused by cytosine methylation concomitant
with replication-coupled chromatin assembly during the step of DNA
synthesis. We microinjected the RNA for Gal4-VP16 6 h prior to the
injection of template DNA to enable the activator to be present during
the synthesis of the new strand and replication-coupled positioning of
the nucleosomes. The presence of Gal4-VP16 renders both the methylated
double-stranded control template and the partially single-stranded
methylated template transcriptionally active (Fig. 4A, lanes 7 and
9). Transcription depends on the activation domain of VP16
(Fig. 4C, compare lanes 2 and 3 with
lanes 6 and 7). We next addressed the effects of
the kinetics of chromatin assembly on transcription under these
conditions. Double-stranded DNA is slowly packaged into chromatin,
whereas single-stranded DNA is rapidly assembled into nucleosomes
because of a replication-coupled mechanism (20, 21). To test the
dependence of chromatinization in our system, RNA encoding Gal4-VP16
and CpG-methylated DNA were injected at different time points (as shown
in the flow diagram in Fig.
5A). When present before
chromatin assembly, Gal4-VP16 leads to transcriptional activation of
both the double-stranded and exo III-treated template (Fig.
5B, lanes 1-4). But when the activator
RNA is injected during or after replication-coupled chromatin assembly,
the activating potential of Gal4-VP16 is decreased in the case of exo
III-treated DNA (Fig. 5B, lanes 7 and
8 and lanes 11 and 12). As shown in a
DNase I hypersensitivity assay (Fig. 5D), the accessibility
of Gal4-VP16 to its binding sites (and therefore the transactivating
potential) decreases once rapid chromatinization after the injection of
exo III-treated DNA occurs. DNase I-hypersensitive sites around the H2B
promoter are lost when activator RNA is injected 2 h later than
the partially single-stranded template (Fig. 5D, lanes
7 and 8 and lanes 11 and 12).
The Transcriptional State of a Gene Can Be Programmed during
Replication of Methylated DNA--
To determine whether second strand
synthesis of methylated DNA provides a "window of opportunity" to
assemble either an active or a repressive transcriptional state, we
microinjected RNA encoding the MeCP2 repression domain, fused to a
Gal4-DNA binding domain, into oocytes before injection of the
nuclease-modified template DNA. MeCP2 has been reported to repress
transcription in vivo by recruiting histone deacetylases (9,
10). Incubation of the injected oocytes with increasing amounts of
trichostatin A, a potent inhibitor of histone deacetylases,
relieved the transcriptional inhibition because of endogenous
histone-deacetylase repressor complexes recruited to methylated DNA as
compared with injection of non-trichostatin A-treated oocytes (Fig.
6, compare lane 2 with
lanes 3-6). However, in the presence of the
hybrid MeCP2-Gal4, this alleviation of repression by trichostatin A is
less pronounced (lanes 8-11). One explanation for
this finding is the specific targeting of abundant protein repressor
complexes to Gal4 binding sites upstream of the histone H2B promoter.
The process of replication or second strand synthesis is therefore a
time window for commitment of the transcriptional status toward
repression.
Gal4-VP16 Expression Does Not Lead to Demethylation during Second
Strand Synthesis--
To determine whether binding of Gal4-VP16
precluded remethylation of newly synthesized templates, we performed
restriction analyses to study the methylation status of microinjected
DNA after the microinjection of either double-stranded or exonuclease III-treated single-stranded templates (Fig.
7). It is apparent that the bulk of
template treated with exonuclease is size-reduced (Fig. 7A,
compare lanes 1 and 3). In contrast, when
injected into oocytes followed by purification and subsequent
restriction, the full-length HindIII fragment appears,
indicating the resynthesis of the second DNA strand (Fig.
7B, lanes 4, 10, and 12).
The sensitivity of HpaII against restriction cleavage is
restored in the case of the DNA that is injected in single-stranded
methylated form (compare lanes 6 and 12). This
indicates that no complete demethylation of both strands occurs either
in the absence (Fig. 7B) or presence (Fig. 7C) of
functional Gal4-VP16. On the contrary, no apparent de novo
methylation is observed for the injected nonmethylated template (Fig.
7B, lanes 4-6), from which
we conclude that demethylation is not induced by transcription factor
binding near the HpaII sites within the H2B promoter in the
frog oocyte during ongoing synthesis of the second strand
under our experimental conditions.
There are two major findings made in this study. First,
transcriptional activators or repressors are capable of programming the
transcriptional state in the context of methylated CpG dinucleotides within regulatory sites during DNA synthesis. Second, upon binding of
transactivators, no demethylation events were observed on newly synthesized CpG-methylated DNA, indicating that the target sites for
DNMTs are accessible even in the presence of site-specific DNA-binding proteins.
DNA Methylation, Methyl CpG-dependent Gene Silencing,
and the Replication Machinery Are Associated--
Components of the
DNA methylation machinery such as DNMT1 associate with replication foci
(26). DNMT1 forms a complex with proliferating cell nuclear
antigen in vivo (27). In addition, the inhibition of
DNMT1 diminishes DNA replication, and DNMT1 interacts with histone
deacetylases and the transcriptional repressor DMAP1 at
replication foci (28, 29). It has been proposed that remethylation of
newly synthesized CpG islands is directly coupled to the migrating
replication fork. After DNA methylation and once chromatin is
assembled, methyl-CpG-binding proteins could recruit histone
deacetylases (9, 10). Methyl-CpG-binding proteins like
methyl-CpG-binding protein-2 and -3 have been reported to bind
hemimethylated DNA at the replication fork in late S phase (30). DNA
methylation at specific cytosine residues might also serve as a signal
recognized by the replication machinery, and this may lead to
initiation of replication at more specific genomic sites. This
mechanism may be used only by a subset of origins because not every
origin of replication appears to have methylated CpG sequences
(31).
DNA Demethylation during Replication--
Little is known
concerning factors that are involved in regulation of the cellular
methylation pattern. DNA methylation at the specific dinucleotide
sequence "CpG" plays a key regulatory role for the transcriptional
activity for some loci and potentially for other nuclear processes like
replication, DNA repair, and recombination (31, 32, 33). In addition,
aberrant methylation of CpG islands in the vicinity of active promoters
correlates strongly with the escape from normal cell cycle regulation
and therefore oncogenesis (34). Under normal physiological conditions, these CpG islands are methylation-free except for sequences located on
the inactive X chromosome where CpG methylation is part of the cellular
dosage compensation mechanism in females (reviewed in Ref. 35).
Progress has been made in elucidating the underlying mechanisms for
specification and maintenance of an unmethylated CpG island. Protein-DNA interactions at specific CpG dinucleotides are crucial for
defining the unmethylated state. In those studies the oriP region of
the Epstein-Barr virus was used to demonstrate a site-specific demethylation once the specific factor Epstein-Barr nuclear antigen-1 was bound followed by subsequent replication (18).
DNA demethylation was shown to be a two-step process requiring
transacting factor association and replication of a specific sequence.
Other transcription factors were shown to operate in an analogous
manner in other systems. It has been demonstrated for X. laevis embryos that a massive demethylation occurs at specific regulatory sites of reporter genes after the mid-blastula transition (15). These observations lead to the hypothesis that the replicative processes concomitant with the binding of transcription factors play a
role in keeping a CpG sequence free of methylation. An alternative
hypothesis is that active demethylating proteins are involved in
establishing the demethylated state. The existence of such demethylases
has been controversial (10, 11, 36). At present, to our knowledge none
of the studies described attempted to address the question of how the
transcriptional state of a gene is affected once the regulatory site
for a transcription factor has been methylated and DNA synthesis of the
second strand is ongoing. Two possible scenarios can be envisaged. (i)
Binding of the transcriptional regulator and the remethylating activity like DNMT1 would compete for their target sites, depending on the
affinity toward the particular site, and the influence of remethylation
may dominate over transcription factor binding, resulting in
transcriptional repression. (ii) Another possibility is that the
transacting factor would dominate over remethylating activities and
therefore program the transcriptional state, allowing rapid initiation
of transcription.
It is technically challenging to set up an experimental system
that allows the investigation of newly synthesized methylated DNA and
its effect on gene transcription. Because of the low activity of
bacterial CpG methylases on single-stranded DNA, in vitro
CpG methylation of single-stranded circular DNA is not efficient. To
circumvent this problem we linearized plasmid DNA harboring regulatory
sequences and then in vitro methylated the template DNA with
the methylase SssI. To generate a partially single-stranded methylated template, we digested the linear DNA with exonuclease III,
giving rise to unidirectional deletions of one strand as a function of
time. The resulting heteroduplex DNA was used as a template in oocyte
microinjection studies. From initial studies using oocyte
microinjections, we concluded that linearized DNA is sufficiently
stable in the oocyte after a 12-h incubation and is assembled into
nucleosomes (see Fig. 3B). However, as reported by others,
linearization of supercoiled plasmid DNA leads to a decrease in
transcriptional efficiency (23, 24). Our studies confirmed these
previous findings, but it became clear that binding of a strong
transcriptional activator such as Gal4-VP16 is capable of overcoming
the negative influence of template linearization on transcription. We
tested other systems such as the thyroid hormone
receptor-
Using single-stranded circular DNA as a template for
transcription, earlier studies have demonstrated that transcription
complex formation and nucleosome assembly, involving all four core
histones, are competitive processes (21). Via its interaction with the replication protein-proliferating cell nuclear antigen, the chromatin assembly factor-1 directs chromatin assembly of newly replicated DNA
toward repression (37, 38). Furthermore, it has been proposed that
heavily methylated DNA-like repetitive sequences and satellite DNA
replicate late in S phase and that transcription of these sequences is
down-regulated. It is tempting to speculate that in order to render
methylated genes transcriptionally active replication processes should
provide a time window allowing genes to be programmed by functional
transcription complexes. From our studies it became clear that the
binding of transcriptional activators or repressors during the
formation of the second strand and targeting them to a specific
regulatory site are sufficient to direct the transcriptional status of
a methylated promoter, perhaps by directly interfering with the
chromatin assembly factor-1 chromatin assembly pathway.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glycerophosphate, 3 mM creatine phosphate, 5 mM MgCl2, 3 mM ATP, and 10 ng creatine kinase. The reactions were
stopped with proteinase K and incubation for 1 h at 37 °C. DNA
fragments were further purified using the Rapid PCR Purification system
according to the manufacturer's instructions (Life Technologies,
Inc.).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
is responsible for the polymerizing
activity (Fig. 2, C and D, lanes
10-12). We conclude that in vitro methylated and further modified templates are subjected to second strand synthesis
carried out by DNA polymerase
-primase.
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Fig. 1.
Constructs used in the present study and the
strategy for generating single-stranded DNA. A, plasmid
DNA used in this study was generated as described under "Experimental
Procedures." 5× Gal4 binding sites were cloned in front of a
complete histone H2B promoter and a partial luciferase sequence. After
linearization with KpnI and SmaI, one strand was
deleted unidirectionally with exo III. B, the length of
deleted DNA under our experimental conditions varies between 1 and
2.5 kilobase pairs (kB) as determined by mung bean
nuclease treatment and subsequent religation of samples. Lanes
2-6 represent time points for exo III digestion for 3 (lanes 2 and 3), 5 (lanes 4 and
5), and 10 min (lane 6). Lane 1 represents no digestion. bp, base pairs.
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Fig. 2.
Exo III-modified DNA is subjected to second
strand synthesis carried out by DNA polymerase
present in X. laevis egg
extract. A, agarose gel showing the time course of
incorporation of radioactive nucleotides into the newly synthesized
strand after 15 (lane 1), 30 (lane 2), 45 (lane 3), 60 (lane 4), 90 (lane 5),
and 120 min (lane 6). RP denotes the position of
the full-length replication product according to a replicated
single-stranded circular marker DNA, and RI reflects the
position of smaller products of DNA synthesis. B,
quantitation of the results shown in A. C
and D, a restriction analysis of newly synthesized DNA.
Replication products generated after 45 min of incubation in egg
extract were purified and digested with a subset of restriction enzymes
as indicated. bp, base pairs. C shows the results
for nonmethylated DNA, and D shows the results obtained when
methylated DNA is used. A. U., arbitrary units.
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Fig. 3.
Template linearization leads to loss of
transcriptional efficiency in the absence of transcriptional activators
like Gal4-VP16. A, 20 oocytes for each lane were
microinjected with 2 and 5 ng of RNA encoding the activator Gal4-VP16
6 h prior to DNA template injection (1 ng). After overnight
incubation at 18 °C, transcription was assayed via primer extension
as described under "Experimental Procedures." H2BLuc
denotes the position of the test RNA, and H4 denotes the
position of the endogenous H4 RNA as an RNA recovery control.
Nonmethylated was compared with methylated DNA, and supercoiled
(sc) was compared with linearized (lin) DNA.
B, linearized template DNA is effectively packaged into
nucleosomal arrays in the oocyte. 12 h after injection half of the
oocytes used for the transcription experiments were crushed in
micrococcal nuclease buffer and subsequently treated with increasing
amounts of micrococcal nuclease (Worthington). The reactions were
stopped, and DNA was prepared according to standard procedures and
blotted onto Hybond-N membranes after electrophoresis. The membranes
were subsequently hybridized against a randomly labeled fragment
comprising the inserted DNA used in this study.
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Fig. 4.
Gal4-VP16 renders injected templates active
when present before chromatin assembly regardless of whether
double-stranded, single-stranded exoIII-treated, nonmethylated, or
methylated DNA is used. A, for each lane, 20 oocytes were injected with 5 ng of Gal4-VP16 RNA 6 h prior to DNA
template injection (1 ng), and transcription was assayed by primer
extension. A quantification of the results is shown in B. A.U., arbitrary units. C, 5 ng of RNA encoding
the Gal4-DNA binding domain (Gal4DBD) alone was injected
into 20 oocytes/lane 6 h prior to DNA injection, and transcription
was assayed as in A. H2BLuc denotes the
position of the test RNA, and H4 denotes the position of the
endogenous H4 RNA as an RNA recovery control.
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Fig. 5.
Rapid chromatin assembly on the partially
single-stranded methylated template leads to transcriptional
repression. A, flow diagram showing the time course of
RNA and DNA injection experiments. B, oocytes were
microinjected with 2 and 5 ng of Gal4-VP16 RNA prior to template DNA
(panel I, pre), at the same time with the DNA
template (panel II, t=0), or 2 h after the
DNA (panel III, post 2 h). Double-stranded
was compared with exo III-treated DNA (lanes 1,
2, 5, 6, 9, and
10 versus lanes 3, 4,
7, 8, 11, and 12). A
quantification of the results is shown in C. A.U., arbitrary units. D, DNase I-hypersensitive
site analysis for the conditions shown in B. After an
incubation of 12 h the oocytes were crushed in DNase I buffer and
subsequently treated with increasing amounts of DNase I (Promega). The
reactions were stopped, and DNA was purified and digested with
KpnI to counteract any possible religations that may have
occurred in the oocyte. After electrophoresis DNA fragments were
blotted onto a Hybond-N membrane and hybridized against a randomly
labeled probe hybridizing from the beginning of the luciferase sequence
to the Gal4 binding sites. Arrows denote the sites located
in the promoter region of the template, which seem to be hypersensitive
against DNase I treatment. H2BLuc denotes the position of
the test RNA, and H4 denotes the position of the endogenous
H4 RNA as an RNA recovery control.
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Fig. 6.
Targeting of the MeCP2 repression domain
during second strand synthesis leads to transcriptional repression of
injected constructs. A, after microinjection of 5 ng of
repressor RNA (MeCP2 repression domain fused to the Gal4 DNA binding
domain; Gal4MRD) 6 h prior to nuclear DNA injections, the oocytes
were incubated in MBSH buffer containing increasing amounts of
trichostatin A (lanes 3-6 and lanes
8-11). Results were quantitated in B. TSA, trichostatin A. H2BLuc denotes the
position of the test RNA, and H4 denotes the position of the
endogenous H4 RNA as an RNA recovery control. A.U.,
arbitrary units.
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Fig. 7.
Second strand synthesis does not lead to
demethylation in either the absence or presence of Gal4-VP16.
A, Southern blot showing the DNA fragment pattern before
oocyte injection and after restriction digest with the enzymes
indicated (nonmethylated DNA). The bulk of DNA in lane 3 depicts the population of exonuclease III-deleted DNA. The
arrow denotes the 600-base pair HindIII fragment.
B, second strand synthesis in the oocyte leads to an
unchanged methylation status in the vicinity of the Gal4 sites and the
H2B promoter in the absence of Gal4-VP16. C, Gal4-VP16
expression in the oocyte has no influence on the methylation pattern
shown in B.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-promoter and found that transcriptional efficiency is completely lost after linearization (data not shown).
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ACKNOWLEDGEMENTS |
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We thank Drs. Trevor Collingwood and Fyodor Urnov for help regarding advice for the oocyte microinjection technique. We are also grateful to Drs. Mike Carey and Geert-Jan Veenstra for providing plasmid DNAs used for generating the constructs described in this study. We are further grateful to Timur Yusufzai and Dr. Melvin L. DePamphilis for critical reading of the manuscript before publication.
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FOOTNOTES |
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* 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 grant from the Deutsche Forschungsgemeinschaft. To
whom correspondence should be addressed: NICHD, National Institutes of
Health, Bldg. 6, Rm. 3A17, Bethesda, MD 20814. Tel.: 301-496-1208; Fax:
301-480-9354; E-mail: stunkelw@mail.nih.gov.
§ Supported by a grant from the European Molecular Biology Organization. Present address: CNRS, UPR 9079, Villejuif, France.
¶ Present address: Sangamo Biosciences, Inc., Point Richmond Tech Center, 501 Canal Blvd., Ste. A100, Richmond, CA 94804.
Published, JBC Papers in Press, March 6, 2001, DOI 10.1074/jbc.M010967200
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ABBREVIATIONS |
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The abbreviations used are: exo III, exonuclease III; DNMT, DNA methyltransferase.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Feil, R., and Kelsey, G. (1997) Am. J. Hum. Genet. 61, 1213-1219[CrossRef][Medline] [Order article via Infotrieve] |
2. | Bird, A. P., and Wolffe, A. P. (1999) Cell 99, 451-454[Medline] [Order article via Infotrieve] |
3. |
Srivastava, M.,
Hsieh, S.,
Grinberg, A.,
Williams-Simons, L.,
Huang, S. P.,
and Pfeifer, K.
(2000)
Genes Dev.
14,
1186-1195 |
4. | Wolffe, A. P. (2000) Curr. Biol. 10, R463-R465[CrossRef][Medline] [Order article via Infotrieve] |
5. | Kundu, T. K., and Rao, M. R. (1999) J. Biochem. (Tokyo) 125, 217-222[Abstract] |
6. | Costello, J. F., Fruhwald, M. C., Smiraglia, D. J., Rush, L. J., Robertson, G. P., Gao, X., Wright, F. A., Feramisco, J. D., Peltomaki, P., Lang, J. C., Schuller, D. E., Yu, L., Bloomfield, C. D., Caligiuri, M. A., Yates, A., Nishik, W. R., Su Huang, H, Petrelli, N. J., Zhang, X., O'Dorisio, M. S., Held, W. A., Cavenee, W. K., and Plass, C. (2000) Nat. Genet. 24, 132-138W[CrossRef][Medline] [Order article via Infotrieve]. R. |
7. |
Muller, C.,
Readhead, C.,
Diederichs, S.,
Idos, G.,
Yang, R.,
Tidow, N.,
Serve, H.,
Berdel, W. E.,
and Koeffler, H. P.
(2000)
Mol. Cell. Biol.
20,
3316-3329 |
8. | Song, S. H., Jong, H. S., Choi, H. H., Kang, S. H., Ryu, M. H., Kim, N. K., Kim, W. H., and Bang, Y. J. (2000) Int. J. Cancer 87, 236-240[CrossRef][Medline] [Order article via Infotrieve] |
9. | Jones, P. L., Veenstra, G. J., Wade, P. A., Vermaak, D., Kass, S. U., Landsberger, N., Strouboulis, J., and Wolffe, A. P. (1998) Nat. Genet. 19, 187-191[CrossRef][Medline] [Order article via Infotrieve] |
10. | Nan, X., Ng, H. H., Johnson, C. A., Laherty, C. D., Turner, B. M., Eisenmann, R. N., and Bird, A. (1998) Nature 393, 386-389[CrossRef][Medline] [Order article via Infotrieve] |
11. | Wade, P. A., Gregonne, A., Jones, P. L., Ballestar, E., Aubry, F., and Wolffe, A. P. (1999) Nat. Genet. 23, 62-66[Medline] [Order article via Infotrieve] |
12. | Hsieh, C. L. (2000) Curr. Opin. Genet. Dev. 10, 224-228[CrossRef][Medline] [Order article via Infotrieve] |
13. | Oswald, J., Engemann, S., Lane, N., Mayer, W., Olek, A., Fundele, R., Dean, W., Reik, W., and Walter, J. (2000) Curr. Biol. 10, 475-478[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Wolffe, A. P.,
Jones, P. L.,
and Wade, P. A.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
5894-5896 |
15. |
Matsuo, K.,
Silke, J.,
Georgiev, O.,
Marti, P.,
Giovannini, N.,
and Rungger, D.
(1998)
EMBO J.
17,
1446-1453 |
16. | Kirillov, A., Kistler, B., Mostoslawsky, R., Cedar, H., Wirth, T., and Bergman, Y. (1996) Nat. Genet. 13, 435-441[Medline] [Order article via Infotrieve] |
17. |
Lin, I. G.,
Tomzynski, T. J.,
Ou, Q.,
and Hsieh, C. L.
(2000)
Mol. Cell. Biol.
20,
2343-2349 |
18. |
Hsieh, C. L.
(1999)
Mol. Cell. Biol.
19,
46-56 |
19. | Antequera, F., and Bird, A. (1999) Curr. Biol. 9, R661-R667[CrossRef][Medline] [Order article via Infotrieve] |
20. | Almouzni, G., Mechali, M., and Wolffe, A. P. (1990) EMBO J. 9, 573-582[Abstract] |
21. | Almouzni, G., and Wolffe, A. P. (1993) Genes Dev. 17, 2033-2047 |
22. |
Li, Q.,
Imhof, A.,
Collingwood, T. N.,
Urnov, F. D.,
and Wolffe, A. P.
(1999)
EMBO J.
18,
5634-5652 |
23. | Harland, R. M., Weintraub, H., and McKnight, S. L. (1983) Nature 302, 38-43[Medline] [Order article via Infotrieve] |
24. | Wang, J. C., and Lynch, A. S. (1993) Curr. Opin. Genet. Dev. 3, 764-768[Medline] [Order article via Infotrieve] |
25. | Kass, S. U., and Wolffe, A. P. (1997) Curr. Biol. 7, 157-165[Medline] [Order article via Infotrieve] |
26. | Leonhardt, H., Rahn, H. P., and Cardoso, M. C. (1998) J. Cell. Biochem. Suppl. 30, 243-249 |
27. |
Chuang, L. S.,
Ian, H. I.,
Koh, T. W.,
Ng, H. H.,
Xu, G.,
and Li, B. F.
(1997)
Science
277,
1996-2000 |
28. |
Knox, J. D.,
Araujo, F. D.,
Bigey, P.,
Slack, A. D.,
Price, G. B.,
Zannis-Hadjopoulos, M.,
and Ssyf, M.
(2000)
J. Biol. Chem.
275,
17986-17990 |
29. | Rountree, M. R., Bachman, K. E., and Baylin, S. B. (2000) Nat. Genet. 25, 269-277[CrossRef][Medline] [Order article via Infotrieve] |
30. |
Tatematsu, K. I.,
Yamazaki, T.,
and Ishikawa, F.
(2000)
Genes Cells
5,
677-688 |
31. |
Rein, T.,
Kobayashi, T.,
Malott, M.,
Leffak, M.,
and DePamphilis, M. L.
(1999)
J. Biol. Chem.
274,
25792-25800 |
32. | Hare, J. T., and Taylor, J. H. (1989) Cell Biophys. 15, 29-40[Medline] [Order article via Infotrieve] |
33. | Hsieh, C. L., and Lieber, M. R. (1992) EMBO J. 11, 315-325[Abstract] |
34. |
Volkers, N.
(2000)
J. Natl. Cancer Inst.
92,
789-790 |
35. | Richardson, B., and Yung, R. (1999) J. Lab. Clin. Med. 134, 333-340[Medline] [Order article via Infotrieve] |
36. | Bhattacharya, S. K., Ramchandani, S., Cervoni, N., and Szyf, M. (1999) Nature 18, 579-583 |
37. | Shibahara, K., and Stillman, B. (1999) Cell 96, 575-585[Medline] [Order article via Infotrieve] |
38. |
Moggs, J. G.,
Grandi, P.,
Quivy, J. P.,
Jonsson, Z. O.,
Hubscher, U.,
Becker, P. B.,
and Almouzni, G.
(2000)
Mol. Cell. Biol.
20,
1206-1218 |