Special Focus Issue – Review
DNA Methylation in Plants: Relationship to Small
RNAs and Histone Modifications, and Functions
in Transposon Inactivation
Hidetoshi Saze1,2,7, Kazuo Tsugane3,4,7, Tatsuo Kanno1,5,7 and Taisuke Nishimura1,6,*
1
PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama, 332-0012 Japan
Plant Epigenetics Unit, Okinawa Institute of Science and Technology, 1919-1 Tancha, Onna-son, Okinawa, 904-0495 Japan
3
National Institute for Basic Biology, Okazaki, 444-8585 Japan
4
Graduate University for Advanced Studies (Sokendai), Okazaki, 444-8585 Japan
5
Plant Genome Engineering Research Unit, Agrogenomics Research Center, National Institute of Agrobiological Sciences,
Kannondai 2-1-2, Tsukuba, Ibaraki, 305-8602 Japan
6
Laboratory of Plant Cell Function, Bioscience and Biotechnology Center, Nagoya University, Furoucho, Chikusa-ku, Nagoya,
464-8601 Japan
7
These authors contributed equally to this work
*Corresponding author: E-mail, [email protected]; Fax, +81-52-789-5214
(Received October 21, 2011; Accepted January 20, 2012)
2
DNA methylation is a type of epigenetic marking that
strongly influences chromatin structure and gene expression
in plants and mammals. Over the past decade, DNA methylation has been intensively investigated in order to elucidate
its control mechanisms. These studies have shown that small
RNAs are involved in the induction of DNA methylation,
that there is a relationship between DNA methylation and
histone methylation, and that the base excision repair pathway has an important role in DNA demethylation. Some
aspects of DNA methylation have also been shown to be
shared with mammals, suggesting that the regulatory pathways are, in part at least, evolutionarily conserved.
Considerable progress has been made in elucidating the
mechanisms that control DNA methylation; however,
many aspects of the mechanisms that read the information
encoded by DNA methylation and mediate this into downstream regulation remain uncertain, although some candidate proteins have been identified. DNA methylation has a
vital role in the inactivation of transposons, suggesting that
DNA methylation is a key factor in the evolution and adaptation of plants.
Keywords: DNA demethylation DNA methylation Histone modification MBD RdDM Transposon.
Editor-in-Chief’s choice
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Abbreviations: BER, DNA base excision repair; dsRNA, doublestranded RNA; H3K36, histone H3 lysine 36; H3K4, histone H3
lysine 4; H3K9, histone H3 lysine 9; LINE, long-interspersed
nuclear element; LTR, long terminal repeat; MBD, methylCpG-binding domain; MBP, methyl-CpG-binding protein;
PcG, plolycomb group protein; PHD, plant homeodomain;
piRNA, PIWI-interacting RNA; Pol, DNA-dependent RNA
polymerase; RdDM, RNA-directed DNA methylation; RNAi,
RNA interference; SINE, short-interspersed element; siRNA,
small interfering RNA; SRA, SET and RING finger-associated;
TBP, TATA-binding protein.
Introduction
Gene expression in eukaryotes is highly influenced by chromatin structure. For example, gene expression is inactivated in
compact chromatin which consists of highly packed nucleosomes (heterochromatin) but is active in less condensed
chromatin (euchromatin). Although the mechanism(s) that
defines chromatin status is still elusive, some of the chemical
modifications of DNA and histone proteins, known as epigenetic marks, that may influence chromatin structure have been
identified. Methylation of the fifth carbon of cytosine residues is
one of the most extensively studied epigenetic modifications
in both plants and mammals. While cytosine methylation is
mostly limited to CG dinucleotide sequence contexts in mammalian genomes, except for embryonic stem cells in which 25%
of cytosine methylations are found in a non-CG context (Lister
et al. 2009), plants additionally harbor methylated cytosines in
CHG and CHH sequence contexts (H = A, C or T) throughout
their genomes (Cokus et al. 2008). DNA methylation is relatively stable and can be maintained during DNA replication.
This stability is also important for plant defenses against transposons (mobile elements) that can induce mutations and
chromosome instability. The proper regulation of DNA methylation is crucial for mammalian development, and abnormalities in the patterns of methylation are closely associated with
Plant Cell Physiol. 53(5): 766–784 (2012) doi:10.1093/pcp/pcs008, available FREE online at www.pcp.oxfordjournals.org
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Plant Cell Physiol. 53(5): 766–784 (2012) doi:10.1093/pcp/pcs008 ! The Author 2012.
DNA methylation in plants
human diseases (Bird 2002, Robertson 2005). DNA methylation
also plays pivotal roles in the development and environmental
responses of plants. Recent studies have suggested that dominance relationships in self-incompatibility, sex determination
and shoot regeneration involve DNA methylation for their
proper regulation, as well as the genomic imprinting described
by Ikeda et al. in this issue (Shiba et al. 2006, Martin et al. 2009,
Li et al. 2011). In addition, the dynamics of the DNA methylation pattern associated with developmental processes have
been unveiled through studies of gametophyte development
(see Dickinson and Gutierrez-Marcos in this issue). On the
other hand, a decrease in the DNA methylation level influences
the rates of infection by bacterial pathogens (Pavet et al. 2006).
In this issue, Mittelesten-Scheid and Pecinka have reviewed
epigenetic regulation including DNA methylation under stress
adaptation. In the last decade, the mechanisms that regulate
DNA methylation have been gradually elucidated in plants.
Many of the advances in this field have derived from studies
using Arabidopsis mutants, which have enabled identification
of many factors involved in DNA methylation and have demonstrated that some mechanisms are conserved and shared with
mammals. In this comprehensive review we briefly describe
the achievements of these studies and outline the regulatory
mechanisms of DNA methylation. In addition, we will discuss
the current understanding of the roles of DNA methylation in
transposon regulation.
DNA Methyltransferases in Plants
In Arabidopsis, two maintenance type and one de novo
type of DNA methyltransferase have been identified. DNA
METHYLTRANSFERASE 1 (MET1), a homolog of the mammalian Dnmt1 methyltransferase, catalyzes methylation at a CG
dinucleotide site, whereas CHROMOMETHYLASE 3 (CMT3),
a plant-specific DNA methyltransferase, catalyzes methylation
at CHG sites. Both CG and CHG sites are symmetric sequences,
therefore, the methylation patterns at these sites can be
transmitted to and maintained in the sister strand during
DNA replication by the activities of MET1 and CMT3, respectively. Asymmetric CHH methylation is maintained by de novo
methylation that is catalyzed by CMT3 and DOMAINS
REARRANGED METHYLTRANSFERASE 2 (DRM2), an ortholog
of the mammalian Dnmt3a/b de novo methyltransferase
(Cao et al. 2003, Chan et al. 2005). A recent study showed
that DRM3, a catalytically mutated DNA methyltransferase
paralog, also plays an important role in establishing de novo
cytosine methylation in all sequence contexts in the process
of RNA-directed DNA methylation (RdDM) by stimulating
the activity of DRM2 (Henderson et al. 2010). In mammals,
it has been shown that the interaction between Dnmt3a and
the catalytically mutated DNA methyltransferase Dnmt3L is
required for establishing de novo DNA methylation during
germline development (Jia et al. 2007). Although phylogenetic
analyses indicate that DRM2/DRM3 and Dnmt3a/Dnmt3L
evolved independently in plants and animals, it seems likely
that both organisms acquired a similar mechanism for de
novo DNA methylation. It would be important to investigate
the relationship between DRM2 and DRM3 biochemically for
an in-depth understanding of the mechanism of de novo DNA
methylation in plants.
RNA-Directed DNA Methylation
Atypical DNA-dependent RNA polymerases
RdDM was first described by Wassenegger et al. (1994) and
refers to an RNA interference (RNAi)-based chromatin modification process in which 24 nucleotide (nt) small interfering
RNAs (siRNAs) induce de novo cytosine methylation in all sequence contexts (CG, CHG and CHH) in DNA regions with
sequence complementarity to the siRNAs (Law and Jacobsen
2010, Haag and Pikaard 2011, X. J. He et al. 2011, Kanno and
Habu 2011). In mammals, 25–30 nt PIWI-interacting RNA
(piRNA) is required for the control of transposable elements
via DNA methylation during male gametogenesis (Thomson
and Lin 2009). In Arabidopsis, RdDM is involved in several biological processes, such as biotic/abiotic stress responses and
plant development, in addition to maintaining the inert state
of transposons/repetitive sequences (Law and Jacobsen 2010,
Haag and Pikaard 2011, X. J. He et al. 2011, Kanno and Habu
2011). The molecular mechanism for RdDM involves two steps:
first, biogenesis of the 24 nt siRNAs; and, secondly, the conversion of siRNA signals into de novo DNA methylation at the
target DNA regions. Each step requires a plant-specific
DNA-dependent RNA polymerase, abbreviated as Pol IV and
a Pol V, respectively.
Phylogenetic analyses of NRPD1 and NRPE1, the largest subunits of Pol IV and Pol V, respectively, showed that these atypical plant-specific polymerases evolved from Pol II for their
specialized function in the RdDM pathway. In agreement
with this conclusion, three mass spectrometric analyses have
reported that both Pol IV and Pol V complexes consist of 12
subunits and that half of these subunits (the sixth, and 8th–
12th subunits) are identical to those of Pol II (Huang et al. 2009,
Ream et al. 2009, Law et al. 2011). The subunit compositions of
each polymerase are summarized in Table 1. The second largest
subunit, NRPD2/NRPE2, and the fourth subunit, NRPD4/
NRPE4, are identical in Pol IV and Pol V but are different
from their Pol II counterparts. There are two highly homologous
proteins in the third subunit in Arabidopsis: Pol II harbors
NRPB3/NRPD3A/NRPE3A, while Pol IV and Pol V can harbor
either NRPB3/NRPD3A/NRPE3A or NRPD3B/NRPE3B. In the
fifth subunit, Pol II contains NRPB5/NRPD5 and Pol V has
NRPE5, while Pol IV has either NRPB5/NRPD5 or NRPD5B/
NRPE5B, a homolog of NRPE5 (Huang et al. 2009, Law et al.
2011). With respect to the seventh subunit, the Arabidopsis
genome contains NRPB7 of Pol II and three other genes,
NRPD7A, NRPD7B/NRPE7B and NRPB7-like. To date, NRPD7A
has only been identified in Pol IV, while NRPD7B/NRPE7B has
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H. Saze et al.
Table 1 The subunit compositions for Pol II, Pol IV and Pol V in Arabidopsis
Subunit
1
2
3
4
5
6
7
8
9
10
11
12
Protein
NRPB1
NRPD1
NRPE1
NRPB2
NRPD2/NRPE2
NRPB3/NRPD3A/NRPE3A
NRPD3B/NRPE3B
NRPB4
NRPD4/NRPE4
NRPB5/NRPD5A
NRPD5B/NRPE5B
NRPE5
NRPB6A/NRPD6A/NRPE6A
NRPB6B/NRPD6B/NRPE6B
NRPB7
NRPD7A
NRPD7B/NRPE7B
NRPB8A/NRPE8A
NRPB8B/NRPD8B/NRPE8B
NRPB9A/NRPD9A/NRPE9A
NRPB9B/NRPD9B/NRPE9B
NRPB10/NRPD10/NRPE10
NRPB11/NRPD11/NRPE11
NRPB12/NRPD12/NRPE12
AGI number
At4g35800
At1g63020
At2g40030
At4g21710
At3g23780
At2g15430
At2g15400
At5g09920
At4g15950
At3g22320
At2g41340
At3g57080
At5g51940
At2g04630
At5g59180
At3g22900
At4g14660
At1g54250
At3g59600
At3g16980
At4g16265
At1g11475
At3g52090
At5g41010
Pol II
Pol IV
Pol V
Summary of the mass spectrometric analysis data from Pikaard’s (Ream et al. 2009), Baulcombe’s (Huang et al. 2009) and Jacobsen’s laboratories (Law et al. 2011)
on the subunit compositions of Pol II, Pol IV and Pol V in Arabidopsis. The confirmed subunit of each polymerase complex is indicated by an orange box. The light
green boxes indicate the subunit that could not be distinguished in related proteins due to the absence of unique peptides.
been found in a subset of Pol IV and Pol V (Ream et al. 2009,
Law et al. 2011). It remains unclear how the particular subunit
compositions of Pol IV/Pol V affect the specific functions of
each polymerase, and resolving this will be an important issue
for future studies.
Small RNA biogenesis in RdDM
Although the RNA polymerase activity of Pol IV and its transcripts have not been confirmed either in vivo or in vitro, Pol
IV is essential for production of siRNAs. Thus, Pol IV is considered to be an active polymerase for transcription of the target
region of RdDM (Fig. 1A; Herr et al. 2005, Pontes et al. 2006,
Zhang et al. 2007a). As a consequence of a physical interaction
between Pol IV and RNA-DEPENDENT RNA POLYMERASE
2 (RDR2) (Law et al. 2011), the Pol IV transcript appears
to convert immediately into double-stranded RNA (dsRNA).
CLASSY 1 (CLSY1), the SWI2/SNF2-like chromatin remodeling
factor, may have a function at some point in the early
stages of siRNA biogenesis (Smith et al. 2007). This dsRNA
is then chopped into 24 nt siRNAs by DICER-LIKE 3 (DCL3)
(Xie et al. 2004); in turn, these siRNAs undergo a ‘maturation’
process mediated by HUA ENHANCER 1 (HEN1) in which a
methyl group is added to their 30 end (Yu et al. 2005). The
mature siRNAs are loaded onto ARGONAUTE 4 (AGO4) to
form a silencing effector complex. A recent study identified
768
SAWADEE HOMEODOMAIN HOMOLOG 1 (SHH1), which
physically interacts with NRPD1, as a novel component of
siRNA biogenesis. Although the biochemical function of the
protein in siRNA biogenesis is still unclear, the authors proposed that it might work as a transcription factor for recruiting
Pol IV to the target DNA region. This proposal was based on
the potential DNA binding ability of SHH1, which is characterized by a cryptic homeodomain and a SWADEE domain (Law
et al. 2011).
Molecular mechanism of de novo DNA
methylation in RdDM
Forward and reverse genetic approaches have identified a large
number of proteins that are associated with the establishment
of de novo methylation at sites homologous to the siRNAs.
These proteins include the subunits of Pol V (Kanno et al.
2005, Onodera et al. 2005, Pontier et al. 2005, He et al. 2009a,
Lahmy et al. 2009), SUPPRESSOR OF TY INSERTION 5-LIKE
(SPT5L)/KOW DOMAIN-CONTAINING TRANSCRIPTION
FACTOR 1 (KTF1) (Bies-Etheve et al. 2009, He et al. 2009a,
Huang et al. 2009), DEFECTIVE IN RNA-DIRECTED DNA
METHYLATION 1 (DRD1) (Kanno et al. 2004), DEFECTIVE IN
MERISTEM SILENCING 3 (DMS3)/INVOLVED IN DE NOVO 1
(IDN1) (Kanno et al. 2008, Ausin et al. 2009), RNA-DIRECTED
DNA METHYLATION 1 (RDM1) (Gao et al. 2010), DEFECTIVE
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DNA methylation in plants
A
DMS4
? SHH
Pol VI
RDR2
Pol IV transcript
CLSY
dsRNA
DCL3
siRNA
HEN1
Methylated
siRNA
Silencing effector complex
AGO4
B
IDN2
DMS3
?
?
Pol V transcript
DMS4
KTF1
DMS3
DRD1
1
RDM1
DRM2
Pol V
RDM
AGO4
?
SUVH
2/9
?
DRM3
Fig. 1 Hypothetical model for the RNA-directed DNA methylation. (A) As a result of the physical interaction between Pol IV and
RNA-DEPENDENT RNA POLYMERASE 2 (RDR2), it seems likely that the Pol IV transcripts are copied into double-stranded RNA (dsRNA)
immediately after transcription. SAWADEE HOMEODOMAIN HOMOLOG 1 (SHH1) may interact directly with the target DNA region to
facilitate the transcription of RNA polymerase IV (Pol IV). DEFECTIVE IN MERISTEM SILENCING 4 (DMS4)/RNA-DIRECTED DNA
METHYLATION4 (RDM4) might be required for directing the Pol IV complex to the target region. The putative chromatin remodeling
factor CLASSY 1 (CLSY1) is likely to be involved in (an) early step(s) of the RdDM pathway. The dsRNA is chopped into 24 nucleotide small
interfering RNAs (siRNAs) by DICER-LIKE 3 (DCL3); subsequently, HUA ENHANCER1 (HEN1) adds a methyl group (purple circle) to the 30 ends
of the siRNA. One or other strand of the methylated siRNA is then loaded onto ARGONAUTE 4 (AGO4) to form the silencing effector complex.
(B) Pol V can transcribe non-coding RNA from the RdDM target region, and this non-coding RNA is thought to act as a scaffold RNA to recruit
the silencing effector complex, presumably via siRNA–non-coding RNA sequence complementarity. INVOLVED IN DE NOVO 2 (IDN2)/
RNA-DIRECTED DNA METHYLATION 12 (RDM12) and DEFECTIVE IN MERISTEM SILENCING 3 (DMS3)/INVOLVED IN DE NOVO 1 (IDN1)
might be required to stabilize this siRNA–non-coding RNA base pairing. In addition, the possible interaction of Pol V, AGO4 and SUPPRESSOR
OF TY INSERTION 5-LIKE (SPT5L)/KOW DOMAIN-CONTAINING TRANSCRIPTION FACTOR 1 (KTF1), which can also bind to RNA, may
contribute to recruit DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) to the target locus. Since RNA-DIRECTED DNA
METHYLATION 1 (RDM1) can interact with both AGO4 and DRM2 in addition to its ability to bind to methylated DNA, this protein may
anchor DRM2 at the DNA region, where it is methylated by the RdDM machinery. DRM3 and SUVH2/SUVH9 may require for facilitating de novo
DNA methylation mediated by DRM2. DMS3 and RDM1 together with DEFECTIVE IN RNA-DIRECTED DNA METHYLATION 1 (DRD1) are also
found in the DDR complex that is thought to facilitate Pol V transcription. DMS4 might assist in the recruitment of Pol V to the target region.
Methylated cytosines are indicated by yellow dots.
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H. Saze et al.
IN MERISTEM SILENCING 4 (DMS4)/RNA-DIRECTED DNA
METHYLATION 4 (RDM4) (He et al. 2009b, Kanno et al.
2010), INVOLVED IN DE NOVO 2 (IDN2)/RNA-DIRECTED
DNA METHYLATION 12 (RDM12) (Ausin et al. 2009, Zheng
et al. 2010), SUVH2 or SUVH9 (Johnson et al. 2008), DRM2 and
DRM3 (Cao and Jacobsen 2002, Henderson et al. 2010,
Naumann et al. 2011) (Fig. 1B).
Pol V-dependent non-coding RNAs that have 50 modification such as triphosphate or a 7meG cap structure, but lack a
poly(A) tail accumulate at several RdDM target loci; AGO4 can
interact with the C-terminal domain of Pol V via the WG/GW
motif, suggesting that the function of Pol V is to serve as a
scaffold RNA and/or to recruit the siRNA–AGO4 complex to
the methylation target region by siRNA–Pol V transcript sequence complementarity (El-Shami et al. 2007, Wierzbicki et al.
2008, Wierzbicki et al. 2009). A recent study showed that
non-coding RNAs are transcribed by Pol II for a subset of
RdDM target loci, and suggested that they are required to recruit Pol IV and/or Pol V to the target region (Zheng et al.
2009). The identities of the factors that provide locus specificity
among the RdDM targets have yet to be determined. SPT5L/
KTF1, a homolog of the yeast transcription elongation factor
Spt5, binds to non-coding RNA and also AGO4 via the WG/GW
motif in its C-terminus. SPT5L/KTF1 may contribute to the
stabilization of the association between the siRNA–AGO4 complex and scaffold RNA (Bies-Etheve et al. 2009, He et al. 2009a).
IDN2/RDM12 shares some homology with SUPPRESSOR
OF GENE SILENCING 3 (SGS3), which is involved in posttranscriptional gene silencing. IDN2/RDM12 possibly interacts
with dsRNA via its XS domain; the levels of accumulation of
siRNAs are largely unaffected in the idn2 mutant (Ausin et al.
2009), suggesting that IDN2/RDM12 has a downstream role in
the siRNA biogenesis pathway. DMS3/IDN1, which has a hinge
domain region similar to the structural maintenance of
chromosome (SMC) protein, is expected to form a homodimer
and may function as a clamp to stabilize homologous nucleic
acids (Kanno et al. 2008, Ausin et al. 2009). IDN2/RDM12 and
DMS3/IDN1 might contribute to the stabilization of base pairing between siRNA and scaffold RNA. RDM1 can interact with
both AGO4 and DRM2, and can possibly bind to methylated
single-stranded DNA through its unique fold that has a
pocket-like structure (Gao et al. 2010). These characteristics
of RDM1 suggest that it might work as a bridge between a
silencing effector complex and a methylated/methylating
target DNA region to recruit DRM2 together with DRM3.
Johnson et al. (2008) reported that SUVH2 or SUVH9
which have SET and SRA (SET and RING finger-associated)
domains are required for DRM2-dependent DNA methylation
in a locus-specific manner. Although some SET domain proteins mediate histone methylation (see the following section
‘Histone methylation and DNA methylation’), no change in
the histone methylation pattern was observed in heterochrmatin in suvh2 and suvh9 mutants. On the other hand, it has
been demonstrated that the SRA domain, which is considered as a methylcytosine-binding domain (see the section
770
‘The downstream mechanisms of DNA methylation’), of
SUVH2 and SUVH9 can bind to methylated cytosines in the
CG context and to methylated cytosines in the CHH context in vitro. A model where binding of SUVH2 or SUVH9 to
methylated cytosine may be required to recruit or retain DRM2
at the RdDM target locus has been proposed (Johnson et al.
2008).
DRD1, a SWI2/SNF2-like chromatin remodeling factor, is
required for the accumulation of non-coding RNAs transcribed
by both Pol V and Pol II (Zheng et al. 2009), and was found to
form a protein complex, DDR, with DMS3/IDN1 and RDM1
(Law et al. 2010). The proposed function of the DDR complex
is to recruit Pol V to the templates and/or to activate Pol V for
the transcription of non-coding RNAs. Notably, it has been
shown that DMS3 is not essential for production of Pol
II-dependent non-coding RNA but that DRD1 is indispensable
(Zheng et al. 2009), indicating that the DDR complex is present
only in some RdDM target loci. DMS4/RDM4 has homology to
the yeast protein Interacts With RNA polymerase II 1 (IWR1), a
dissociable factor of the core transcription machinery of Pol II
(He et al. 2009b, Kanno et al. 2010). The Arabidopsis mutant
dms4/rdm4 shows pleiotropic morphological defects in addition to reactivation of RdDM targets. Many thousands of Pol
II transcripts show altered levels of accumulation in the dms4/
rdm4 mutant, and there is evidence that DMS4/RDM4 can
physically interact with Pol II, Pol IV and Pol V in vivo (He
et al. 2009b, Kanno et al. 2010, Law et al. 2011). Recently, two
different biological functions for yeast IWR1 were identified:
first, IWR1 is required for importing the Pol II complex from
the cytoplasm into the nucleus (Czeko et al. 2011); and, secondly, IWR1 is important for recruitment of the TATA-binding
protein (TBP) to loci transcribed by Pol I, Pol II and Pol III
(Esberg et al. 2011). By analogy with the yeast data, DMS4/
RDM4 may bind to Pol II, Pol IV and Pol V to aid import of
the polymerases into the nucleus or to facilitate polymerase
activity at the RdDM target loci.
Histone Methylation and DNA Methylation
Histone methylation and DNA methylation in
eukaryotic organisms other than plants
In addition to the RNAi-based mechanisms overviewed in the
previous sections, covalent modifications of histone proteins
play an important role in the establishment and maintenance
of DNA methylation. In particular, methylation of histone H3
lysine 9 (H3K9) is essential for the formation of the heterochromatic state (Grewal and Jia 2007). Although some species,
including the eukaryotic model organisms Drosophila melanogaster, Caenorhabditis elegans and Schizosaccharomyces pombe,
have lost the capacity for cytosine methylation at some stage of
evolution (Zemach et al. 2010), they have retained the H3K9
methylation and RNAi systems. In the filamentous fungus
Neurospora crassa, DNA methylation is solely dependent on
H3K9 methylation mediated by the histone methyltransferase
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DNA methylation in plants
Dim-5, and RNAi has little or no effective role in DNA
methylation (Tamaru and Selker 2001, Freitag et al. 2004).
In mammals, H3K9 methylation is mediated by SET domain
proteins including G9a and SUV39H1 (Peters et al. 2001,
Tachibana et al. 2002). H3K9 methylation is recognized by
the chromodomain of Heterochromatin Protein 1 (HP1),
which further recruits Dnmt1 (Fuks et al. 2003a), indicating a
link between DNA methylation and H3K9 methylation. Indeed,
loss of H3K9 methyltransferase function results in the reduction
of DNA methylation in repetitive major satellite DNA, or
at promoters of endogenous genes (Lehnertz et al. 2003,
Tachibana et al. 2008). It is well established that the H3K9
methyltransferase Clr4 of the fission yeast S. pombe is recruited
by an siRNA-containing complex called RITS (Grewal and Jia
2007), whereas the role of siRNAs in recruitment of the H3K9
methyltransferases to target loci in other organisms is still not
well understood. Recent studies have demonstrated that
chromatin-modifying enzymes, such as the polycomb group
proteins (PcG) and G9a in mammals, can bind to long
non-coding RNAs that recruit modifiers to target loci and
induce transcriptional repression (Nagano et al. 2008, Pandey
et al. 2008). It has been reported that a cis-acting non-coding
RNA functions similarly in Arabidopsis to recruit PcG to target
loci (Heo and Sung 2011), suggesting that it is possible that
targeting mechanisms analogous to those of animal species
are also present in plants.
Histone modifications and DNA methylation
in plants
In plants, the histone modification status influences DNA
methylation. As in other organisms, H3K9 methylation in
plants is mediated by SET domain proteins, which are essential
for transposon silencing and proper plant development
(Jackson et al. 2002, Malagnac et al. 2002, Ding et al. 2007). In
plant genomes, H3K9 methylation is enriched at repeat sequences and is associated with both DNA methylation and
small RNAs, which are essential for the formation of constitutive heterochromatin (Bernatavichute et al. 2008, Lister et al.
2008, Zhou et al. 2010). In Arabidopsis, 15 protein homologs of
the H3K9 methyltransferase SU(VAR)39 have been identified
(Baumbusch et al. 2001). Among these, KRYPTONITE/
SU(VAR) HOMOLOG 4 (KYP/SUVH4) primarily methylates
H3K9 at repeat sequences in a redundant fashion with the
homologs SUVH5 and SUVH6 (Jackson et al. 2002, Malagnac
et al. 2002, Ebbs and Bender 2006, Bernatavichute et al. 2008).
KYP/SUVH4 catalyzes mono- and dimethylation of K9 of H3
(Jackson et al. 2004). On the other hand, a recent study showed
that the SET domain protein SUVR4 can convert monomethylated H3K9 to the trimethylated state especially in the presence
of ubiquitin, and this activity affects transcriptional silencing of
transposons (Veiseth et al. 2011).
Despite the conservation of SU(VAR)39-like histone methyltransferases and H3K9 methylation in plants, the readout
mechanism for the H3K9 methylation signal on chromatin
and its effect on DNA methylation seem to be different
between animals and plants. For example, there is only
one homolog of HP1 in Arabidopsis, named LHP1/TFL2,
and it binds to methylated H3K27 rather than methylated
H3K9 (Zhang et al. 2007b). In plants, H3K9 methylation is
recognized by a plant-specific DNA methyltransferase named
CHROMOMETHYLASE 3 (CMT3) (Lindroth et al. 2004). CMT3
mediates DNA methylation at non-CG sites, with a preference
for CHG sites (Bartee et al. 2001, Lindroth et al. 2001). The
SUVH family (SUVH1–SUVH10) in Arabidopsis has an SRA
domain in the N-terminus, which preferentially binds to methylated cytosine by flipping out 5-methylcytosine from the
DNA strand (Arita et al. 2008, Rajakumara et al. 2011). Thus,
the histone H3K9 methyltransferases are recruited by DNA
methylation via the SRA domain, and probably add H3K9
methylation marks to these loci. Sites of H3K9 methylation
can also bind to the chromodomain encoded by CMT3, thus
forming a self-reinforcing loop of repressive epigenetic marks at
heterochromatic loci (Johnson et al. 2007) (Fig. 2).
As mentioned above, the mechanisms that establish H3K9
methylation/CHG methylation at target loci are still not well
understood. Although the RNAi-based pathway might act upstream of H3K9 methylation as in S. pombe, several lines of
evidence suggest that the two pathways act independently in
plants. For example, inactivation of both the RdDM and the
H3K9 methylation pathways in Arabidopsis results in pleiotropic developmental defects that are not observed in mutants
of either pathway (Chan et al. 2006). The developmental defects are due to the ectopic expression of an F-box gene named
SUPPRESSOR OF drm1 drm2 cmt3 (SDC) (Henderson and
Jacobsen 2008). In wild-type somatic cells, SDC is transcriptionally silenced due to dense DNA methylation at seven tandem
repeats in the promoter; this methylation is redundantly maintained by the RdDM and the H3K9 methylation pathways.
Additional evidence for the independence of H3K9 methylation
from RdDM comes from the fact that increases in H3K9
KYP
IBM1
K9
M
K9
CMT3
H3K9 methylation
M
M
K9
non-CG
DNA methylation
Genes
K9
KYP
Repeats
Fig. 2 Active demethylation of H3K9 methylation at genic regions by
IBM1. Histone H3K9 methyltransferase KRYPTONITE (KYP) can target
both genes and repeats. H3K9 methylation can further recruit the
non-CG methyltransferase CHROMOMETHYLASE 3 (CMT3) at repeats, whereas IBM1 prevents accumulation of the H3K9 methylation
at genic regions.
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methylation can occur in the absence of RdDM factors.
In Arabidopsis, H3K9 methylation is antagonized by a
jumonji-C type histone demethylase INCREASED BONSAI
METHYLATION 1 (IBM1) (Saze et al. 2008, Miura et al. 2009,
Inagaki et al. 2010). In the ibm1 mutant, >4,000 actively transcribed genes become hypermethylated, particularly in CHG
contexts, which concomitantly accumulate H3K9 methylation
in their gene body (Miura et al. 2009, Inagaki et al. 2010).
Hypermethylation is nearly completely suppressed by mutation
of cmt3 or kyp (Saze et al. 2008, Inagaki et al. 2010), implying
that H3K9 methylation/CHG methylation might occur more
frequently in euchromatic regions than previously thought,
although this methylation is eventually masked due to its
active removal by IBM1 (Fig. 2). Importantly, ibm1-induced
DNA hypermethylation does not depend on the RdDM pathway, since mutations of factors in the RdDM pathway, including
the de novo methyltransferase DRM2, cannot suppress the
effect of the mutation (Inagaki et al. 2010). The accumulation
of CHG methylation peaks mainly in the middle of transcribed
genes suggests a link between H3K9 methylation and transcription (Miura et al. 2009).
In addition to H3K9 methylation and histone methyltransferases, other histone modifications and chromatin modifiers
can influence the maintenance of DNA methylation. In particular, pathways involved in the removal of active epigenetic
marks from heterochromatin are important for maintenance
of the repressive state. One example is monoubiquitination of
histone H2B (H2Bub), which is associated with transcriptional
activation, and is required for further deposition of active histone marks such as H3K4 and H3K36 methylation (Schmitz
et al. 2009) (see Tamada et al. in this issue). In Arabidopsis,
loss of ubiquitin protease UBP26 function, which can remove
monoubiquitination at Lys143 of H2B, results in the reduction
of DNA methylation and an accumulation of H3K4 methylation
at silenced transgenes and transposons (Sridhar et al. 2007). In
addition, direct removal of H3K4 methylation by distinct classes
of histone demethylase is also required for the maintenance of
DNA methylation. For example, defects in Arabidopsis H3K4
demethylase homologs of Lysine Specific Demethylase 1 (LSD1)
cause the accumulation of H3K4 methylation and a loss of DNA
methylation at the promoter of the FWA gene (Jiang et al.
2007). Similarly, jumonji-C domain-containing protein JMJ14
probably maintains non-CG methylation through removal of
H3K4 trimethylation downstream of the RdDM pathway
(Deleris et al. 2010, Searle et al. 2010). Hence, histone modifications are balanced and coordinated by positive and negative
regulators, which can define the functional chromatin domains
along the chromosomes (Roudier et al. 2011).
DNA Demethylation
Genome-wide and locus-specific DNA demethylation are important processes during development and in environmental
responses in mammals (Zhu 2009, Wu and Zhang 2010).
772
In plants, allele-specific activation of imprinted genes is
known to be regulated by DNA demethylation (see the
review by Ikeda et al. in this issue). DNA demethylation can
be achieved by both active and passive mechanisms. Active
mechanisms remove methylation marks from DNA, whereas
passive demethylation results from the inhibition of maintenance activity for DNA methylation during DNA replication.
One such passive mechanism is the repression of MET1 expression during female gametogenesis that results in a decrease in
the DNA methylation level in some imprinted genes (Jullien
et al. 2008). Certain demethylation processes in plants and
mammals occur rapidly and thus cannot be achieved by a passive mechanism that would need several rounds of DNA replications (Zhu 2009, Wu and Zhang 2010).
The identities of active mechanisms have been investigated
and models for their behavior have been suggested for both
plants and animals. The ROS1, DME, DML2 and DML3 proteins
of Arabidopsis, which belong to a small family of DNA glycosylases involved in DNA base excision repair (BER), may be the
main component of an active demethylation mechanism. The
proteins are bifunctional enzymes and have an apyrimidinic
lyase activity for cleaving phosphodiester bonds in the DNA
backbone at abasic sites (Agius et al. 2006, Gehring et al. 2006,
Morales-Ruiz et al. 2006, Penterman et al. 2007). The proteins
can also specifically recognize 5-methylcytosine residues and,
in vitro, attack the glycosidic bond to remove the methylcytosine base (Agius et al. 2006, Gehring et al. 2006, Morales-Ruiz
et al. 2006, Penterman et al. 2007). The unmethylated cytosine
may be inserted in a reaction mediated by an unknown DNA
polymerase and DNA ligase in a manner similar to BER. Ectopic
hypermethylation at specific loci was observed in mutants of
these DNA glycosylase genes, which supports the hypothesis
that they are active DNA demethylase enzymes (Choi et al.
2002, Gong et al. 2002, Penterman et al. 2007, Zhu et al. 2007).
Although no orthologs of DNA glycosylases of the ROS1/
DME family have yet been identified in animals, nevertheless
it is likely that BER is an important component of their mechanisms for DNA demethylation. Chick thymine DNA glycosylase and chick and human methyl-CpG-binding domain
protein 4 (MBD4) have a DNA glycosylase activity for
5-methylcytosine, but it is much lower than the activity of
thymine glycosylase for T/G mismatch repair (Jost et al. 1995,
Zhu et al. 2000a, Zhu et al. 2000b). Therefore, deamination of
5-methylcytosine to thymine has been suggested to be an important step in the DNA demethylation process in animals
(Kangaspeska et al. 2008, Métivier et al. 2008). In addition to
deamination, oxidation of 5-methylcytosine may play a role in
demethylation. TET1 oxidase converts 5-methylcytosine to
5-hydroxymethylcytosine, which can be a substrate for deamination enzymes, and the resultant hydroxymethyluracil may be
replaced by methylcytosine through the threonine dehydrogenase and BER pathways (Kriaucionis and Heintz, 2009,
Tahiliani et al. 2009, Cortellino et al. 2011, Guo et al. 2011).
Recently it was reported that TET1 oxidase catalyzes iterative
oxidation to yield 5-formylcytosine and, subsequently,
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DNA methylation in plants
5-carboxylcytosine, which can be a substrate for thymine
DNA glycosylase and/or other unknown decarboxylases
(Y.-F. He et al. 2011, Ito et al. 2011). Hydroxymethylcytosine,
however, cannot be recognized by maintenance mechanisms
for DNA methylation, suggesting a role for passive DNA
demethylation (Valinluck and Sowers 2007). In plants, TET1
and 5-hydroxycytosine have not been reported to date. Their
absence may be associated with the evolution of DNA glycosylases that catalyze 5-methylcytosine directly.
Currently, little is known about the targeting mechanism
that regulates DNA demethylation in plants and animals.
However, in Arabidopsis, mutations in the DNA demethylation
pathway have been shown to change DNA methylation levels in
a small set of genes, indicating the existence of a targeting
mechanism for the DNA demethylation machinery that leads
to demethylation at specific loci (Choi et al. 2002, Gong et al.
2002, Penterman et al. 2007, Zhu et al. 2007). Arabidopsis ROS3
was isolated as one of the factors involved in the ROS1
demethylation pathway (Zheng et al. 2008). The ros3 mutant
has a similar phenotype to that of ros1, and the ROS3 protein
has been shown to co-localize with the ROS1 protein in nucleoplasmic foci and in the nucleolus. ROS3 has an RNA recognition
motif which can bind small, single-stranded RNAs with specific
sequences. It will be interesting to determine whether the small
RNAs are associated with the DNA demethylation machinery
and are involved in targeting demethylation to specific loci, just
as RdDM does.
The Downstream Mechanisms of
DNA Methylation
How are signals from DNA methylation mediated into downstream actions, such as changes in chromatin structure and
regulation of gene expression? This is one of the most intriguing
questions with respect to understanding the roles of DNA
methylation. Although our understanding of the regulation of
DNA methylation has increased (see the above sections), the
downstream effects of DNA methylation remain less clear.
Several models have been suggested for mechanisms that
might read the information encoded by DNA methylation
(Joulie et al. 2010). One possible mechanism involves the alteration of chromatin structure. DNA methylation modifies
the flexibility of DNA, which may then influence nucleosome
positioning (Segal and Widom 2009). Changes in nucleosome
positioning can influence the accessibility of the DNA by the
various factors involved in transcriptional regulation. A second
possibility suggests a more direct effect in which transcription
factors are prevented from binding to their target sequences. In
mammals, inhibition of the binding of CTCF, a chromatin insulator, by DNA methylation is crucial for the regulation of
imprinted genes (Bell and Felsenfield 2000, Hark et al. 2000).
Additionally, the binding activities of various transcription factors, such as CREB or E2F, to the DNA sequences can be blocked
by DNA methylation (Iguchi-Ariga and Schaffner 1989,
Campanero et al. 2000). The plant homologs of these transcription factors are also prevented from binding to their targets
by DNA methylation (Inamdar et al. 1991, Scebba et al. 2003),
although the physiological roles of this inhibition by DNA
methylation are unclear.
The third and most studied model proposes that methylCpG-binding proteins (MBPs) recognize methylated DNA and
mediate downstream signals (Clouaire and Stancheva 2008,
Sasai and Defosez 2009). MBPs are classified into three families
by the differences in their motifs for recognizing DNA methylation. The first identified motif was the MBD (Lewis et al. 1992,
Hendrich and Bird 1998). Mammalian MBD proteins are known
to be involved in the protein complexes associated with histone
deacetylase, methyltransferase and/or DNA methyltransferase
activities that function for transcriptional repression (Nan et al.
1997, Nan et al. 1998, Jones et al. 1998, Ng et al. 1999, Zhang
et al. 1999, Ng et al. 2000, Fuks et al. 2003b, Fujita et al. 2003,
Kimura et al. 2003, Sarraf and Stancheva 2004, Kondo et al.
2005, Nan et al. 2007), which is consistent with the silencing
activity of DNA methylation in the promoter region. MBD
proteins have also been found in plants. The Arabidopsis
genome has 13 genes encoding proteins with an MBD motif
(Zemach and Grafi 2007). At least three of these MBD proteins,
AtMBD5, AtMBD6 and AtMBD7, can bind to CG sequences in
a methylation-dependent manner in vitro, while AtMBD5
also binds to methylated sites in a CHH context (Ito et al.
2003, Zemach and Grafi 2003, Schebba et al. 2003). AtMBD5,
AtMBD6 and AtMBD7 are localized in a chromocenter in a
highly methylated region (Scebba et al. 2003, Zemach et al.
2005). Two of the proteins, AtMBD5 and AtMBD6, are preferentially localized in a region adjacent to rDNA gene clusters,
suggesting that they act on rDNA silencing (Zemach et al.
2005). In a similar manner to mammalian MBD proteins,
AtMBD6 interacts with protein complexes with histone deacetylase activity (Zemach and Grafi 2003). However, downregulation of AtMBD6 and AtMBD7 by T-DNA insertion does
not produce an overt phenotype (Zemach and Grafi 2007).
This is also the case in mammals; thus, for example, mice
with a knockout of an MBD gene do not show a severe phenotype (Chen et al. 2001, Guy et al. 2001, Hendrich et al. 2001,
Millar et al 2002, Zhao et al. 2003). As a result of the decrease
in DNA methylation by DNA methyltransferases, plant met1
and mammal Dnmt1 mutants show severe developmental defects (Li et al. 1992, Mathieu et al. 2007). Studies of these mutants suggest that MBD proteins have functional redundancy or
that other proteins have more pivotal roles for reading DNA
methylation.
AtMBD9, one of the MBD proteins in Arabidopsis, has a
unique feature in that it works as a transcriptional activator.
Mutants of AtMBD9 showed an early flowering phenotype
which is caused by transcriptional repression of FLC, a major
flowering repressor (Peng et al. 2006). A decrease in histone
acetylation and an increase in DNA methylation were observed
in the FLC locus in the mutants, which is consistent with in vitro histone acetylation activity of AtMBD9 recombinant
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H. Saze et al.
protein (Yaish et al. 2009). AtMBD9 is the largest member of
the Arabidopsis MBD protein family possessing multiple
chromatin-associated domains such as a PHD finger (Peng
et al. 2006). Study of AtMBD9 indicates functional diversity
of MBD proteins, although methyl DNA-binding activity has
not been known yet. In mammals, MBD1 and MeCP2 are
known to have specific functions in neurogenesis, indicating
functional diversity in this protein family (Chen et al. 2001,
Guy et al. 2001, Zhao et al. 2003).
The second class of MBPs includes proteins with an SRA
domain, which is responsible for recognizing DNA methylation.
As mentioned above, the plant histone methyltransferase
family, SUVH, has an SRA domain, suggesting that proteins in
this family translate DNA methylation into histone methylation
(Johnson et al. 2007). Plant VIM/ORTH and mammal UHRF1
proteins have an SRA domain together with PHD and RING
fingers. Mutations of VIM/ORTH and UHRF1 proteins produce
similar phenotypes to those displayed by met1 and Dnmt1
mutants, respectively (Sharif et al. 2007, Woo et al. 2007,
Woo et al. 2008). However, VIM/ORTH and UHRF1 proteins
appear to work in maintaining DNA methylation levels rather
than for reading and interpretation of signals, because the DNA
methylation level is decreased in the mutants. Consistent with
this, UHRF1 preferentially binds to hemimethylated DNA
and associates with Dnmt1 during replication (Bostick et al.
2007). UHRF1 also associates with histone methyltransferase
and deacetylase (Unoki et al. 2004, Karagianni et al. 2008),
while the PHD finger can bind to H3K9 methylase (Sharif
et al. 2007), suggesting a role in the maintenance of histone
modifications.
A third class of MBPs includes proteins with zinc finger domains that bind to methylated DNA. Mammal Kaiso, ZBTB4
and ZBTB38 are known to belong to this protein group; however, no zinc finger proteins that bind to methylated DNA have
yet been reported in plants (Prokhortchouk et al. 2001, Filion
et al. 2006). Kaiso interacts with a protein complex that has
histone deacetylase activity and works as a transcriptional repressor (Prokhortchouk et al. 2001). The mouse knockout of
Kaiso does not show any developmental defects, and even the
additional knockout of two MBD proteins, MeCP2 and MBD2,
does not cause any greater effect, suggesting that Kaiso is dispensable in development (Prokhortchouk et al. 2006, Caballero
et al. 2009).
MBPs are expected to be the main regulators for translating
DNA methylation signals on the status of histone modifications
into effects on gene expression. However, other as yet unknown
downstream mechanisms for translating DNA methylation signals cannot be excluded. There may be other classes of MBPs
and other downstream mediators of DNA methylation signaling in mammals and plants. Genetic studies using plant mutants have identified a candidate factor that might be involved
in the downstream mechanisms for interpretation of DNA
methylation signals. Mutations in Arabidopsis MOM1, a plant
relative of mammalian CHD3 chromatin remodeling factors,
cause the release of silencing of highly methylated repeats
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including pericentromeric and inactive 5S rDNA, transposons
and transgenic loci (Amedeo et al. 2000, Steimer et al. 2000,
Habu et al. 2006, Vaillant et al. 2006, Čaikovski et al. 2008,
Numa et al. 2010, Yokthongwattana et al. 2010). However,
the chromatin status, including DNA methylation, is not changed in mom1, while mutants of other silencing factors influence
gene expression by changing the DNA methylation status
(Amedeo et al. 2000, Probst et al. 2003, Habu et al. 2006,
Vaillant et al. 2006). These fascinating features of mom1
imply that MOM1 acts on independent and/or downstream
pathways of DNA methylation. Genetic analyses with RdDM
factors showed that MOM1 acts in either a redundant or a
cooperative manner on gene silencing and in a locusdependent manner with the RdDM pathway (Numa et al.
2010, Yokthongwattana et al. 2010). In addition, MOM1 is suggested to interpret DNA methylation signals into H3K9 methylation at the SDC locus, which is a target of RdDM (Numa et al.
2010). The molecular action of MOM1 is still unknown, but
elucidating how MOM1 works may be the key to solving how
DNA methylation affects gene silencing.
Genome-wide analyses of DNA methylation have shown
that in eukaryotes the coding regions of transcribed genes are
highly methylated, so-called gene body methylation (Suzuki
and Bird 2008). Their correlation with active gene expression
suggests that they may be involved in mechanisms for reading
DNA methylation different from those that act on gene repression in promoter regions. Common factors may interpret DNA
methylation signals in both the promoter and gene body, but
output from these factors could be different. The roles of gene
body methylation are still elusive, but it tends to be observed in
long and functionally important genes (Takuno and Gaut
2011), implying significance for gene body methylation.
Although studies on the roles of gene body methylation have
just started, it is likely that they will unveil how DNA methylation influences the status of both chromatin and gene
expression.
DNA Methylation and Transposon Regulation
Transposons as major components of genomes
Transposons were discovered in maize by Barbara McClintock
(McClintock 1965). Analysis of eukaryotic genome sequences
revealed that a large fraction of the genome consists of transposons. In Arabidopsis, rice and maize, transposons occupy 12,
40 and 85% of the genomes, respectively (Arabidopsis Genome
Initiative 2000, International Rice Genome Sequencing Project
2005, Schnable et al. 2009). Transposons are divided into
two major classes based on their structure and transposition
mechanism (Fig. 3). Retrotransposons (class I) involve a reverse
transcription process with an RNA intermediate, and the transposition results in an increase in the copy number. DNA transposons (class II) translocate from the integrated site to a new
site in the genome, and the copy number remains stable except
when the element moves in the S or G2 phase of the cell cycle
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DNA methylation in plants
Fig. 3 Structures of representative transposons described in this review. The arrowheads represent terminal-inverted (closed) or terminal-direct
(open) repeats. The horizontal bars and gray circles indicate integrated genome and target site duplications that are generated by the transposon
insertion events. Colored boxes in transposon boxes and arrows indicate exons and transcripts for transposition-related genes. Dotted lines
indicate the relationship between the original elements and their derivatives. Ds6 is shown as a representative element of several structurally
altered Ds elements (Fedoroff 1989). In LTR retrotransposons, the gag gene encodes structural proteins that form a virus-like particle as well as IN,
integrase; PR, protease; and RT, reverse transcriptase (Havecker et al. 2004). The large or small dotted boxes in Karma are the 50 - or
30 -untranslated regions superimposed on Line-1 (Cordaux and Batzer 2009). The black boxes in Alu indicate promoter regions for RNA polymerase III (Mills et al. 2007).
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H. Saze et al.
(Engels et al. 1990). While an autonomous element encodes the
transposase protein required for transposition, a nonautonomous element does not encode a transposase. Thus,
non-autonomous elements use the supplied transposase for
transposition. Transposons can cause mutation by disruption
of functional genes and can also induce chromosomal instability. Indeed, McClintock first identified transposable elements
in a study of chromosome breaks at specific sites in maize,
which she showed were caused by the transposition of a
non-autonomous element, Dissociation (Ds), under the control
of an autonomous element, Activator (Ac) (McClintock 1951).
Since the original investigation, it has become clear that organisms have evolved epigenetic regulatory systems to inactivate
transposition activity, because of its potential to damage
the host genome (Slotkin and Martienssen 2007, Lisch 2009).
In several plants, genome-wide analysis of DNA methylation
showed that transposon sequences are heavily methylated
(Liu et al. 2009, Zemach et al. 2010). Here, we describe the
characteristic features of representative transposons and the
regulation of transposition by DNA methylation.
Epigenetic regulation of DNA transposons
The correlation between cytosine DNA methylation and
transposon activity was examined in the transposon superfamilies, CACTA, hAT and Mutator, using methylation-sensitive
restriction enzymes (Chandler and Walbot 1986, Chomet et al.
1987, Banks et al. 1988). The Enhance/Suppressor-mutator
(En/Spm) transposon belonging to the CACTA superfamily
generates several mature transcripts (TnpA–TnpD) from a primary transcript by alternative splicing; two of these transcripts,
TnpA and TnpD, are required for transposition. In addition to
its role in transposition, the TnpA protein mediates demethylation of its promoter region (Fedoroff et al. 1995). It is suggested
that TnpA induces DNA demethylation by binding to the newly
replicated hemimethylated promoter regions (Cui and Fedoroff
2002). Ac/Ds is a member of the hAT superfamily and shows
high transposition activity shortly after DNA replication in the
unmethylated state (Ros and Kunze 2001). When Ac/Ds or its
modified systems are used for transgenic gene tagging lines in
various plants, inactivation of elements that were active in earlier generations has been reported (Izawa et al. 1997, Szeverenyi
et al. 2006). Conversely, previously inactive Dart1 elements belonging to the hAT family in rice can translocate from integrated sites in transgenic Arabidopsis by demethylation
associated with the cloning process (Shimatani et al. 2009).
The transposition of non-autonomous nDart1-0 is induced
by treatment with 5-azacytidine, a DNA methylation inhibitor,
in a rice line carrying the pale yellow leaves mutation without
the active autonomous element (Tsugane et al. 2006) (Fig. 4).
Mutator (MuDR/Mu) elements exhibit a mutation frequency
of 103–105 per locus per generation in maize and are
characterized by high transposon activity (Walbot and
Rudenko 2002). Alterations of methylation status in Mu elements are observed at various development stages in maize.
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Fig. 4 Variegated rice mutant line, pyl-stb, induced by 5-azacytidine.
The pyl-stb line caused by insertion of the non-autonomous DNA
transposon nDart1 does not carry an active autonomous element
under natural growth conditions. The candidate(s) for the epigenetically silenced autonomous element is activated by demethylation
(Tsugane et al. 2006). (A) 5-Azacytidine-treated or untreated pyl-stb
mutants are shown in the lower and upper part of the image, respectively. (B) Magnified leaves of pyl-stb treated with 5-azacytidine (left)
and untreated (right).
Immature ears show an increased methylation of the Mu elements compared with young leaves, and, as a consequence of
the higher level, germinal transposition is restricted and results in transmission of a stable genome to the next generation
(Li et al. 2010). Mu killer (Muk), which is a natural derivative of
an inverted duplication of autonomous MuDR, is a strong repressor of the Mu element. Muk produces hairpin transcripts
that are processed to small RNAs by DCL3 processing. The small
RNAs homologous to MuDR induce DNA methylation and
post-transcriptional gene silencing of MuDR (Slotkin et al.
2005, Lisch 2009).
Epigenetic regulation of retrotransposons
Retrotransposons are subdivided into long terminal repeat
(LTR) elements and non-LTR elements. LTR retrotransposons
are the most abundant transposable elements in plant genomes, and the transposition mechanism exhibits significant
similarity with the replication process of retroviruses. Tos17,
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DNA methylation in plants
a well-characterized Ty1/copia LTR retrotransposon in rice, is
heavily methylated and immobilized under normal plant
growth conditions (Cheng et al. 2006). However, Tos17 is activated in calli and gradually increases the copy number during
prolonged callus culture (Hirochika et al. 1996). Several recent
studies have shown that the transposition of Tos17 in calli or in
a DNA glycosylase/lyase-modified line is associated with DNA
hypomethylation (Cheng et al. 2006, Ding et al. 2007, La et al.
2011). An EVD element containing a copia structure was activated in Arabidopsis in a recombinant inbred line whose
genome was maintained over several generations at a low
methylation status by the met1 mutation. It was found that
the transposon activity of the EVD element was suppressed not
only transcriptionally but also post-transcriptionally in the wild
type (Mirouze et al. 2009). The Arabidopsis Athila elements,
that belong to the LTR family Ty3/gypsy, are only active in
vegetative nurse cells, which do not transmit genetic information to the next generation. The 21 nt siRNA from the Athila
elements accumulates in pollen. The findings suggested
that siRNA suppresses the activation of Athila elements
in the two sperm cells (Slotkin et al. 2009, Slotkin 2010).
Short-interspersed elements (SINEs), tRNA-derived retrotransposons, were originally discovered and studied in mammalian
systems (Shedlock and Okada 2000). In the human genome,
the Alu sequences (a SINE retrotransposon) are involved in
chromosome rearrangements that confer new functions
(Mills et al. 2006). In the ddm1 mutant background, an epigenetically controlled SINE-related sequence was identified in the
promoter region of the FWA gene (Kinoshita et al. 2004) that
was originally identified as the gene responsible for a
late-flowering phenotype (Koornneef et al. 1991). The FWA
gene, an imprinted gene, is suppressed in the apical meristem
and expressed in the endosperm (Kinoshita et al. 2004).
The direct tandem repeat sequences of the SINE-related
sequence in the promoter region of the FWA gene appear to
be the critical cis-element for the transcription start site
(Kinoshita et al. 2007). The methylation level of the transcription start site controls the expression of FWA in Arabidopsis
species (Fujimoto et al. 2011). Transposition of long interspersed nuclear elements (LINEs) is also regulated by cytosine
methylation. LINEs and SINEs are less abundant LTR retrotransposons in plant species, in sharp contrast to the situation in
mammals. Transposition of Karma (a LINE element) in rice is
affected by two factors. First, the induction of transcription
from Karma requires hypomethylation in the callus. Secondly,
for integration into the genome, a prolonged culture process is
needed for the release of post-transcriptional regulation
(Komatsu et al. 2003). Studies on active transposons show
that they are regulated at each developmental stage by the
host genome.
Transpositions induced by ddm1 mutation
The Arabidopsis genome has a low transposon content
compared with other plant genomes. Although the reasons
for the compact genome of Arabidopsis have not been resolved,
Zhang and Wessler (2004) proposed that inhibition of an
increase in transposon copy number rather than a large
genomic deletion was responsible. In the global low DNA
methylation state caused by the ddm1 mutation, transposition
of various DNA transposons (Mutator superfamily, VANDAL21;
and CACTA superfamily, CACTA1) and retrotransposons (gypsy
family, ATGP3; and copia family, ATCOPIA13, ATCOPIA21 and
ATCOPIA93) are observed (Miura et al. 2001, Tsukahara et al.
2009). Interestingly, following its activation by the ddm1 mutation, CACTA1 remained mobile in the absence of the mutation.
Therefore, inactivation of the transposon was not the responsibility of DDM1 (Kato et al. 2004). While most transposons are
located at the centromeric regions, new transposon insertions
in the Arabidopsis ddm1 mutant were not restricted to the
heterochromatin.
Transposon burst
Transposons contribute to genome evolution and diversity.
However, host genomes appear to have developed epigenetic
mechanisms to suppress bursts of transposition activity, possibly to the disadvantage of the host organisms. Despite the
existence of a variety of inhibitory mechanisms, active movement of the DNA transposons mPing and nDart1-0 results in
an increased copy number under natural growth conditions
(Naito et al. 2009, Hayashi-Tsugane et al. 2011). DNA methylation is a key event associated with transposon activity. Several
studies on transposons have shown changes in the methylation
status during plant development (Banks and Fedoroff 1989,
Slotkin et al. 2009, Li et al. 2010). This phenomenon may also
be an effective strategy by the host to suppress a burst of transposon activity. A fuller understanding of epigenetic regulation
and the DNA methylation status of transposons could provide
important clues to plant evolution and adaptation.
Perspective
Recent advances in the methodologies for analysis of plant
genomes have provided a better understanding of the mechanisms that regulate DNA methylation. More particularly, they
have enabled the identification of new components of regulation such as the RdDM pathway; however, the molecular functions of the various factors and their interrelationships are still
largely unknown. To remedy this lack of knowledge, it will be
necessary to make much greater use of biochemical approaches
in addition to genetic methods. Genome-wide analyses of mutants has provided insights into new aspects of the regulation
of DNA methylation. The identification of locus-dependent
methylation and demethylation using genome-wide analyses
has stimulated a search for previously unknown mechanisms
and cellular machineries that recruit regulatory proteins to
their target loci. In addition, the methylation of transcribed
genes, discovered by genome-wide analyses, implies a targeting
mechanism and function that had not been previously
Plant Cell Physiol. 53(5): 766–784 (2012) doi:10.1093/pcp/pcs008 ! The Author 2012.
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H. Saze et al.
anticipated. There are still many questions in this field, and
most of these have not been addressed even by investigations
in mammals. Studies using plants have some advantages with
regard to genetic investigations and, in combination with the
rapid development of genomic technologies, will undoubtedly
help to elucidate the answers to many of these key questions. In
some cases, the achievements of plant research will precede
those from mammals.
Funding
This work was supported by the Japan Science and Technology
Agency [PRESTO program (to H.S., T.K. and T.N.)]; the Ministry
of Education, Culture, Sports, Science, and Technology of Japan
(grant No. 22780007 to K.T.).
Acknowledgments
We are grateful to the editor and anonymous reviewers whose
comments have greatly improved this paper.
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