A SNF2-like protein facilitates dynamic control of DNA methylation.

Abstract

DRD1 is a SNF2-like protein previously identified in a screen for mutants defective in RNA-directed DNA methylation of a seed promoter in Arabidopsis. Although the initial study established a role for DRD1 in RNA-directed DNA methylation, it did not address whether DRD1 is needed for de novo or maintenance methylation, or whether it is required for methylation of other target sequences. We show here that DRD1 is essential for RNA-directed de novo methylation and acts on different target promoters. In addition, an unanticipated role for DRD1 in erasure of CG methylation was shown when investigating maintenance methylation after segregating away the silencing trigger. DRD1 is unique among known SNF2-like proteins in facilitating not only de novo methylation of target sequences in response to RNA signals, but also loss of methylation when the silencing inducer is withdrawn. The opposing roles of DRD1 could contribute to the dynamic regulation of DNA methylation.

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ASNF2-likeproteinfacilitatesdynamiccontrolofDNA
methylation
Tatsuo Kanno1*, Werner Aufsatz1*, Estelle Jaligot1, Michael Florian Mette2, Marjori Matzke1+
& Antonius J.M. Matzke1
1Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, UZA2, Pharmazie Zentrum, Vienna, Austria,
and2IPK Gatersleben, Gatersleben, Germany
DRD1 is a SNF2-like protein previously identified in a screen for
mutants defective in RNA-directed DNA methylation of a seed
promoter in Arabidopsis. Although the initial study established a
role for DRD1 in RNA-directed DNA methylation, it did not
address whether DRD1 is needed for de novo or maintenance
methylation, or whether it is required for methylation of other
target sequences. We show here that DRD1 is essential for RNA-
directed de novo methylation and acts on different target
promoters. In addition, an unanticipated role for DRD1 in
erasure of CG methylation was shown when investigating
maintenance methylation after segregating away the silencing
trigger. DRD1 is unique among known SNF2-like proteins in
facilitating not only de novo methylation of target sequences in
response to RNA signals, but also loss of methylation when the
silencing inducer is withdrawn. The opposing roles of DRD1
could contribute to the dynamic regulation of DNA methylation.
Keywords: demethylation; de novo methylation; maintenance
methylation; RNA-directed DNA methylation; SNF2-like protein
EMBO reports advance online publication 10 June 2005;
doi:10.1038/sj.embor.7400446
INTRODUCTION
RNA-directed DNA methylation has been documented in plants
(Mathieu & Bender, 2004) and in human cells (Kawasaki & Taira,
2004; Morris et al, 2004). Genetic analyses in Arabidopsis have
identified the DNA methyltransferases that catalyse de novo
cytosine (C) methylation in response to RNA signals (Cao et al,
2003; Aufsatz et al, 2004) and maintain symmetrical C(N)G
methylation (N is A, T or C) in cooperation with histone-modifying
enzymes (Jones et al, 2001; Aufsatz et al, 2002a; Jackson et al,
2002; Malagnac et al, 2002). A plant-specific SNF2-like putative
chromatin-remodelling protein, DRD1, was identified in a screen
for mutants impaired in RNA-directed DNA methylation and
silencing of the seed-specific a0promoter (Kanno et al, 2004).
From the initial genetic screen, it was unclear whether drd1
mutants are deficient in de novo methylation or in maintenance
methylation. In the case of RNA-directed DNA methylation, de
novo methylation leads to the modification of Cs in all sequence
contexts within the region of RNA–DNA sequence identity,
whereas maintenance methylation refers to perpetuation of
symmetrical C(N)G methylation during DNA replication in the
absence of the RNA trigger (Matzke et al, 2004). To further
understand the role of DRD1 in RNA-directed DNA methylation,
we examined separately the effects of a drd1 mutation on
RNA-directeddenovomethylation
methylation. We report here that DRD1 influences both of these
processes in a manner that facilitates the dynamic regulation of
DNA methylation.
andonmaintenance
RESULTS AND DISCUSSION
Transgenic systems to study RNA-mediated silencing and methy-
lation of the seed-specific a0promoter (Kanno et al, 2004) and the
constitutive nopaline synthase (NOS) promoter (Aufsatz et al,
2002b) have been described previously. These systems rely on a
double-stranded RNA—encoded at a silencer H complex (hygro-
mycin resistance)—that is homologous to the respective target
promoter sequence. The double-stranded RNAs are processed to
short RNAs, 21–24 nucleotides in length, which are thought to
trigger de novo methylation and silencing of the homologous
target promoter at an unlinked target K complex (kanamycin
resistance; supplementary Fig 1 online). In the absence of the
silencer, the respective target promoter is active and unmethylated
(Fig 1A: left, a0promoter; right, NOS promoter), whereas it is
repressed and methylated in the presence of the silencer (Fig 1B:
left, a0promoter; right, NOS promoter).
In drd1 mutants, non-CG methylation of the target a0promoter
is greatly reduced, as evidenced by complete digestion with
methylation-sensitive restriction enzymes abbreviated F, Sc, Ps,
Pa, E and Ba (Fig 1C, left: data are for third-generation drd1-6
plants). By contrast, substantial CG methylation is retained,
Received 31 January 2005; revised 4 May 2005; accepted 5 May 2005; published
online 10 June 2005
*These authors contributed equally to this work
+Corresponding author. Tel: þ43 1 4277 29740; Fax: þ43 1 4277 29749;
E-mail: marjori.matzke@gmi.oeaw.ac.at
1Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences,
UZA2, Pharmazie Zentrum, Althanstrasse 14/2D-541, 1090 Vienna, Austria
2Institute of Plant Genetics and Crop Plant Research (IPK), Correnstrasse 3,
D-06466 Gatersleben, Germany
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as indicated by the persistent double band generated by the
restriction enzyme abbreviated H (Fig 1C, left). Similar findings for
drd1-1 led to the suggestion that DRD1 is important primarily for
non-CG methylation (Kanno et al, 2004).
To analyse whether DRD1 is needed for RNA-directed de novo
methylation of target sequences, crosses were made to generate
F1 plants in which a naive target K complex was combined with
the silencer H complex in either wild-type (D/D; Fig 2D) or
homozygous drd1-6 (d/d) plants (Fig 2E). Methylation of the target
promoter was then examined in the resulting F1 progeny.
In wild-type F1 plants, the target a0promoter showed increased
methylation in CGs and in non-CGs after introducing the silencer
H complex (Fig 1D, left). The level of methylation observed in
wild-type F1 plants approximated that seen in plants in which the
target complex and silencer complex had been together in the
same genome for several generations (Fig 1B, left). The similar
methylation patterns indicate that the maximum attainable level of
RNA-directed methylation of the target a0promoter is essentially
reached in the first generation containing both transgene
complexes. By contrast, the target a0promoter did not acquire
detectable methylation after being combined with the silencer H
complex in homozygous drd1-6 plants (Fig 1E, left), which
showed a hybridization pattern identical to that of the unmethy-
lated target a0promoter (Fig 1A, left). The lack of methylation was
not because of inadequate production of RNA signals, as indicated
by the continued presence of a0promoter short RNAs in drd1-6
plants (Fig 3A).
The target NOS promoter also becomes methylated de novo
at CGs and non-CGs in wild-type plants after introducing the
respective silencer H construct (Fig 1D, right; Aufsatz et al, 2004),
but it failed to acquire measurable methylation in the drd1-6
mutant (Fig 1E, right). Bisulphite sequencing confirmed that no
detectable methylation was induced at the target NOS promoter in
drd1-6 mutants, whereas methylation was observed at Cs in all
sequence contexts within the region of RNA–DNA sequence
identity in wild-type plants (supplementary Fig 2 online). NOS
promoter short RNAs were detected in the F1 plants (Fig 3B),
indicating that RNA signals were available but unable to induce
methylation in the drd1-6 mutants. In contrast to the a0promoter,
the level of NOS promoter methylation was less in F1 progeny
than in plants in which the target and silencer had been
together for several generations (compare Fig 1D, right, with
Fig 1B, right). Despite this difference, the results show that
DRD1 is indispensable for RNA-directed de novo methylation
of two distinct promoters.
The efficiency of maintenance methylation was examined in
wild-type and drd1-6 plants after crossing out the respective
silencer H complexes to remove the source of the RNA signals. In
wild-type F2 progeny descended from DRD1 wild-type parents
(Fig 2F), the target a0promoter lost both CG and non-CG
methylation after segregating away the silencer complex (Fig 4F,
left), resulting in a hybridization pattern that is indistinguishable
from the unmethylated a0promoter (Fig 1A, left). An identical
pattern of methylation (Fig 4G, left) was observed in wild-type F2
progeny descended from the drd1-6 mutant (Fig 2G). Bisulphite
sequencing confirmed the loss of non-CG methylation and
showed only some residual methylation at two CG dinucleotides
(supplementary Fig 3A online). Thus, in the a0promoter silencing
system, almost all methylation is lost in wild-type progeny when
the source of the RNA signal is withdrawn. Unexpectedly,
however, in drd1-6 progeny lacking the silencer H complex
(Fig 2H,I), substantial CG methylation was detected, even though
non-CG methylation was lost. Retention of CG methylation is
again exemplified by the persistent double band generated by the
restriction enzyme abbreviated H. This double band was observed
in drd1-6 progeny that are homozygous (Fig 4H, left) and
hemizygous (Fig 4I, left) for the target K complex, indicating no
dependence on dosage of the target promoter. Bisulphite
sequencing confirmed the enhanced maintenance of CG methyla-
tion in the drd1-6 mutant (supplementary Fig 3B online) relative to
wild-type plants (supplementary Fig 3A online).
K/K;H/H;D/D
K/K;H/H;d/d
K/ – ;H/ – ;d/d
K/ – ;H/ – ;D/D


α′ promoter
–H F Sc
NOS promoter
PS––BNPs Pa E Ba
K/K; – / – ;D/D
NA
?
?
?
?
??
?
?
?
A
B
C
D
E
Fig 1|Analysis of de novo methylation. Methylation of the target a0
promoter was analysed using restriction enzymes diagnostic for non-CG
methylation (F, Sc, Ps, Pa, E, Ba) or CG methylation (H). Methylation of
the nopaline synthase (NOS) promoter was analysed using restriction
enzymes diagnostic for non-CG methylation (N), CG methylation (P, B)
or both (S). The respective methylation-sensitive enzymes were added
after a standard digest with non-methylation-sensitive restriction
enzymes (‘?’ lanes). The position of the methylated fragment is denoted
by the dots to the left of each blot. Shifts to the smaller fragment(s)
indicate no methylation at the site(s) tested. Maps of the promoters
showing positions of restriction enzyme sites and the probes used for
hybridization are depicted in supplementary Fig 1 online. Genotypes of
the plants analysed are shown to the right; dashes indicate a hemizygous
transgene locus. Bold letters to the left represent boxed genotypes shown
in the breeding schemes in Fig 2. Abbreviations: K, target complex; H,
silencer complex; d, drd1-6 mutant; D, wild type; NA, not applicable.
Abbreviations of enzymes and their recognition sequences (sensitivity to
C methylation indicated by the superscript ‘m’): a0promoter: B, BstUI
(mCGmCG); Ba, BamHI (GGATmCmC); E, EcoT22I (ATGmCAT);
F, Fnu4HI (GmCmNGmC, if N is C); H, HpyCH4IV (AmCGT);
NOS promoter: N, NheI (GCTAGmC); P, Psp1406I (AAmCGTT);
Pa, PagI (TmCATGA); Ps, PstI (mCTGmCAG); S, SacII (mCmCGmCGG);
Sc, ScrF1 (CmCmNGG (if N is C).
SNF2-like protein and DNA methylation dynamics
T. Kanno et al
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A comparable result was obtained when analysing mainte-
nance methylation in the NOS promoter system. In contrast to the
a0promoter, the target NOS promoter retains significant methyla-
tion in the absence of the silencer H complex in wild-type plants:
although non-CG methylation seems to be lost (exemplified by
complete digestion with the enzyme abbreviated N (NheI)),
considerable CG methylation remains, as shown by only partial
digestion with restriction enzymes abbreviated P (Psp1406I) and
B (BstUI) (Fig 4F, right). Similarly to the a0promoter, however,
the NOS promoter shows increased CG methylation in drd1-6
progeny. This is evidenced by poorer digestion with enzymes P
and B in drd1-6 mutants (Fig 4H,I, right) than in wild-type plants
(Fig 4F,G, right). By contrast, cleavage by enzyme N, which is
diagnostic of non-CG methylation, is equivalent in both drd1-6
mutants and wild-type plants (Fig 4F–I, right). Collectively, these
findings show a previously unsuspected role for DRD1 in the
complete erasure of CG methylation after segregating away the
silencer H complex that encodes the RNA trigger.
The results of the experiments reported here, which have
examined separately de novo and maintenance methylation,
necessitate a revision of the original proposal that DRD1 is
primarily important for non-CG methylation (Kanno et al, 2004).
DRD1 is required for RNA-directed de novo methylation of Cs in
all sequence contexts, including methylation of CG dinucleotides
(Fig 5). However, DRD1 is also necessary for efficient loss of
methylation, particularly CG methylation, once the source of the
RNA signal is removed. The latter requirement is more evident for
the target a0promoter, which loses almost all CG methylation in
wild-type plants after segregating away the silencer complex but
shows elevated CG methylation in the absence of the silencer in
the drd1-6 mutant. Therefore, the methylation pattern observed in
third-generation drd1 plants can be explained as an additive
defect caused by inadequate erasure of CG methylation coupled
to failed de novo methylation, which affects predominantly
non-CG nucleotide groups (Fig 5). Whether the remaining CG
methylation in drd1 mutants is due to more robust maintenance
or to a deficiency in either passive loss or active removal of CG
methylation is not known. Passive loss occurs when there is a
failure to maintain methylation during successive rounds of DNA
replication. By contrast, active demethylation requires a demethy-
lase activity and can take place in nondividing cells (Santos et al,
2002). In animal systems, DNA glycosylases, which are normally
involved in base excision repair, have been reported to be
involved in active demethylation of CG dinucleotides (Kress et al,
2001). In Arabidopsis, two large proteins that contain DNA
glycosylase domains, DEMETER (Xiao et al, 2003; Kinoshita et al,
2004) and REPRESSOR OF SILENCING 1 (Gong et al, 2002), have
been implicated in the removal of CG methylation and the re-
activation of silenced genes. Additional work is required to clarify
how DRD1 contributes to the loss of CG methylation.
Although DRD1 seems to be a common requirement for RNA-
directed DNA methylation, the two target promoters tested here
differ in the rate of their response to the introduction or
withdrawal of RNA signals in wild-type plants. The target a0
promoter is very responsive, acquiring a full level of methylation
in the first generation that combines the target and silencer
complexes and losing most methylation in the first generation after
crossing out the silencer complex. By contrast, the target NOS
promoter does not attain the full level of RNA-directed methyla-
tion in the F1 generation and retains substantial CG methylation
De novo set-up
K/ – ; – / – ;d/D – / – ;H/ – ;d/D
X
– / – ; – / – ;d/d K/K; – / – ;D/D
– / –;H/H;D/D
K/ – ;H/ – ;d/d K/ –;H/ –;D/D
K/ –; H/ –;D/D K/ –; H/ –;d/D
K/K; – / – ;d/d
K/ – ; – / – ;d/d
K/K; – / – ;D/D
K/K; – / – ;D/D
– / – ; – / – ;d/D K/K; H/H;D/D
X
or
Col-0
X
K/K; H/H;D/D
X
Col-0
K/K; H/H;d/d
Maintenance set-up
XX
A
ED
BC
I
FH
G
Fig 2|Breeding schemes. De novo methylation analysis: wild-type plants that were homozygous for either the target K complex (K/K;?/?;D/D) or the
silencer H complex (?/?;H/H;D/D) were crossed with a transgene-free, homozygous drd1-6 mutant (?/?;?/?;d/d), producing respective heterozygous
drd1-6 progeny (K/?;?/?;d/D and ?/?;H/?;d/D). Heterozygous drd1-6 plants containing either the target K complex or silencer H complex were
crossed and F1 progeny that were either drd1-6 mutant (K/?;H/?;d/d) or wild type (K/?;H/?;D/D) were identified by genotyping. Maintenance
methylation analysis: a0promoter system: segregating the target K complex from the silencer H complex in wild-type (K/K;H/H;D/D) or homozygous
drd1-6 plants (K/K;H/H;d/d) required an initial backcross to untransformed wild-type plants (Col-0). This produced progeny that were hemizygous for
the K and H complexes in either wild-type (K/?;H/?;D/D) or heterozygous drd1-6 (K/?;H/?;d/D) backgrounds. The nopaline synthase (NOS) promoter
system: the K/K;H/H;D/D plants were likewise crossed to Col-0 to produce wild-type progeny (K/?;H/?;D/D). To generate the K/?;H/?;d/D genotype,
wild-type plants that were homozygous for the respective target K complex and silencer H complex (K/K;H/H;D/D) were crossed with a transgene-free,
heterozygous drd1-6 mutant (?/?;?/?;d/D). For both silencing systems, wild-type plants (K/?;H/?;D/D) were selfed and the resulting F2 progeny
genotyped to identify those containing K and lacking H (K/K;?/?;D/D). The heterozygous drd1-6 plants (K/?;H/?;d/D) were also selfed and the
resulting F2 progeny were genotyped to identify those containing K and lacking H in wild-type (K/K;?/?;D/D) and homozygous drd1-6 mutant
(K/(K);?/?;d/d) backgrounds. The boxed and lettered genotypes correspond to the lettered blots in Figs 1,4.
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T. Kanno et al
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after removing the source of the trigger RNA. The reasons for these
differences in response time are not known. In both systems, the
respective short RNAs seem to be present in comparable amounts.
Variations in the sequence composition, structure or chromatin
condensation state of the two target complexes might alter their
sensitivity to RNA signals (Matzke et al, 2004). For example, there
are several copies of the NOS promoter at the corresponding
target K complex (Aufsatz et al, 2002b), whereas only a single
copy of the a0promoter is present at the corresponding target
K complex (Kanno et al, 2004). The higher density of CG
dinucleotides in the NOS promoter (supplementary Fig 2 online)
may contribute to more efficient maintenance of CG methylation
at this promoter. By contrast, the a0
comparatively deficient in CG dinucleotides (supplementary Fig
3 online), is not repressed by CG methylation but instead seems to
be silenced by non-CG methylation (Kanno et al, 2004). As shown
here, this non-CG methylation must be established de novo in
each generation by a DRD1-dependent, RNA-directed pathway.
The fact that the a0promoter is silenced by non-CG methylation
probably allowed us to identify the drd1 mutant in the
corresponding genetic screen. By contrast, proteins required for
CG methylation, the DNA methyltransferase MET1 (Aufsatz et al,
2002a) and histone deacetylase 6 (Aufsatz et al, 2002b), were
promoter, which is
identified in a genetic screen carried out on the NOS promoter
silencing system.
Several other SNF2-like proteins, including DDM1 (Jeddeloh
et al, 1999), Lsh (Dennis et al, 2001) and ATRX (Gibbons et al,
2000), can regulate DNA methylation. However, DRD1 is the
only one of these factors that has been implicated both in RNA-
directed de novo methylation of cytosines in all sequence contexts
and in erasure of CG methylation. The drd1 alleles identified in
the original genetic screen contain mutations in functionally
implicated regions of the SWI2/SNF2 ATPase domain (Kanno
et al, 2004), suggesting that DRD1 functions as a chromatin-
remodelling protein to disrupt histone–DNA contacts and/or
displace nucleosomes. One possibility is that DRD1 is specialized
to allow RNA signals to access homologous target DNA in a
chromatin context. Depending on the availability of RNA signals
and various DNA-modifying enzymes in different cell types,
DRD1 activity could facilitate RNA-guided de novo methylation
catalysed by DNA methyltransferases or demethylation of CG
dinucleotides catalysed by DNA glycosylases. In this model,
DRD1 is a pivotal factor in the dynamic regulation of promoter
activity, which might contribute to developmental plasticity,
epigenetic reprogramming or adaptation of plants to the environ-
ment. Future research will have to focus on determining the full
scope of DRD1 activity by using genomics approaches to identify
endogenous targets in the genome.
Speculation
In view of the proposed role of DRD1 in opening chromatin to
RNA signals to create a substrate for de novo methylation, one can
ask whether loss of CG methylation, which also requires DRD1,
occurs at DNA sequences targeted by short RNAs (Fig 5, short
K/K;H/H;D/D
K/K;H/H;D/D
21 nt
21 nt
K/K;H/H;d/d K/K; – / – ;D/D
K/K; – / – ;D/D
ND
K/ – ;H/ – ;d/d K/ – ;H/ – ;D/D
A
B
Fig 3|Short RNA analysis. (A) Northern blot analysis of a0promoter
short RNAs in wild-type plants containing only the target complex
(K/K;?/?;D/D) or both the target and silencer complexes (K/K;H/H;D/D),
or in the drd1-6 mutant (K/K;H/H;d/d). (B) Northern blot analysis of
nopaline synthase (NOS) promoter short RNAs in F1 progeny from the
‘de novo’ cross (Fig 2) that were homozygous for the drd1-6 mutation
(K/?;H/?;d/d) or wild type (K/?;H/?;D/D), and in wild-type plants that
were either homozygous for the target K complex only (K/K;?/?;D/D)
or double homozygous for the target K complex and silencer H complex
(K/K;H/H;D/D). In wild-type plants, NOS promoter short RNAs are
approximately twice as abundant in homozygous H/H as in hemizygous
H/? plants. The quantities of short RNAs are increased in drd1-6
mutants, perhaps because they are not used and turned over efficiently
in these plants. Ethidium bromide staining of the principal RNA species
in the samples is shown as a loading control. ND indicates that a0
promoter short RNAs were not tested in the genotype K/?;H/?;D/D.
α′ promoter
F Sc
K/K; – / – ;D/D
K/(K); – / – ;D/D
K/K; – / – ;d/d
K/ – ; – / – ;d/d
–– Ps Pa
F
G
H
I
–P
•
•
•

•
NOS promoter
S BNH E Ba
•
•
•
•
Fig 4|Maintenance methylation analysis. Methylation of the target
promoter at the respective K complex (a0promoter (left) and nopaline
synthase (NOS) promoter (right)) after segregating away the respective
silencer H complex was studied using methylation-sensitive restriction
enzymes as described in the legend to Fig 1. The genotypes of the plants
analysed are shown to the right. The bold letters to the left represent the
boxed genotypes shown in the breeding scheme in Fig 2. ‘K/(K)’ denotes
both homozygous K/K and hemizygous K/? plants. The plants shown in
(G,H,I) are siblings.
SNF2-like protein and DNA methylation dynamics
T. Kanno et al
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RNAs in parentheses). Although the silencing complex that
encodes the trigger RNA was segregated away in plants used to
assess maintenance methylation, we cannot exclude the possibility
that parental RNA signals are carried over into cells of the next
generation, providing a short time period early in development
when RNA-guided demethylation could occur. For example,
DEMETER acts only in the central cell of the female gametophyte
to remove CG methylation (Xiao et al, 2003; Kinoshita et al,
2004). Thus, whether RNA-directed de novo methylation or
RNA-guided demethylation occurs would be governed by the
differentialcell-type-specific
methyltransferases or DNA glycosylases, respectively.
availabilityofeitherDNA
METHODS
Plant material and genotyping. All experiments were performed
on drd1-6 mutant (Kanno et al, 2004) or wild-type Arabidopsis
thaliana (ecotype Columbia-0). The genotypes of plants used for
molecular analyses were determined by PCR-based screens, as
described in the supplementary information online.
DNA methylation analysis. DNA was isolated from genotyped
plants and analysed for cytosine methylation, using methods
described previously (Aufsatz et al, 2002a,b, 2004; Kanno et al,
2004). Southern data results were reproduced for at least two
plants of each genotype. For the drd1-6 mutant shown in Fig 1C
(left), DNA was isolated from plants of the M3 generation
(genotype K/K;H/H;d/d).
Short RNA analysis. NOS promoter and a0promoter short
RNAs were isolated and analysed by northern blotting, as
described previously (Aufsatz et al, 2002a,b, 2004; Kanno
et al, 2004).
Supplementary information is available at EMBO reports online
(http://www.emboreports.org).
ACKNOWLEDGEMENTS
We acknowledge financial support from the Austrian Fonds zur
Fo ¨rderung der wissenschaftlichen Forschung (Grant P-15611-B07) and
the European Union (contract no. HPRN-CT-2002-00257).
REFERENCES
Aufsatz W, Mette MF, van der Winden J, Matzke M, Matzke AJM (2002a)
HDA6, a putative histone deacetylase needed to enhance DNA
methylation induced by double stranded RNA. EMBO J 21:
6832–6841
Aufsatz W, Mette MF, van der Winden J, Matzke AJM, Matzke M (2002b)
RNA-directed DNA methylation in Arabidopsis. Proc Natl Acad Sci USA
99: 16499–16506
Aufsatz W, Mette MF, Matzke AJM, Matzke M (2004) The role of MET1 in
RNA-directed de novo and maintenance methylation of CG
dinucleotides. Plant Mol Biol 54: 793–804
Cao X, Aufsatz W, Zilberman D, Mette MF, Huang MS, Matzke M, Jacobsen
SE (2003) Role of the DRM and CMT3 methyltransferases in RNA-
directed DNA methylation. Curr Biol 13: 2212–2217
Dennis K, Fan T, Geiman T, Yan Q, Muegge K (2001) Lsh, a member of the
SNF2 family, is required for genome-wide methylation. Genes Dev 15:
2940–2944
Gibbons RJ, McDowell TL, Raman S, O’Rourke DM, Garrick D, Ayyub H,
Higgs DR (2000) Mutations in ATRX, encoding a SWI/SNF-like protein,
cause diverse changes in the pattern of DNA methylation. Nat Genet 24:
368–371
Gong Z, Morales-Ruiz T, Ariza RR, Rolda ´n-Arjona T, David L, Zhu J-K (2002)
ROS1, a repressor of transcriptional gene silencing in Arabidopsis,
encodes a DNA glycosylase/lyase. Cell 111: 803–814
Jackson JP, Lindroth A, Cao X, Jacobsen SE (2002) Control of CpNpG
methylation by the KRYPTONITE histone H3 methyltransferase. Nature
416: 556–560
Jeddeloh JA, Stokes TL, Richards EJ (1999) Maintenance of genomic
methylation requires a SWI2/SNF2-like protein. Nat Genet 22:
94–97
Jones L, Ratcliff F, Baulcombe DC (2001) RNA-directed transcriptional gene
silencing in plants can be inherited independently of the RNA trigger and
requires Met1 for maintenance. Curr Biol 11: 747–757
CG CNG CNN
CG CNG CNN
DRD1
CG CNG CNN
DRD1
CG CG CNG CNN
drd1
De novo (+ RNA)
Maintenance (– RNA)
α′ promoter
) ?
(
Fig 5|Dual role of DRD1 in the dynamic regulation of RNA-directed DNA methylation. DRD1 is required for RNA-directed de novo methylation of Cs
in all sequence contexts for the target promoters tested here. DRD1 is also needed for the complete erasure of methylation after segregation of the
source of the RNA signal, as indicated by enhanced retention of CG methylation in drd1 mutants. This is particularly clear for the target a0promoter,
which normally loses most methylation after withdrawing the silencing trigger. Methylation is indicated by filled circles, the target promoter by the red
line and short RNAs by red dashed lines.
SNF2-like protein and DNA methylation dynamics
T. Kanno et al
&2005EUROPEAN MOLECULAR BIOLOGY ORGANIZATION EMBO reports VOL 6 | NO 7 | 2005
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