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Maintenance of DNA Methylation during the Arabidopsis
Life Cycle Is Essential for Parental Imprinting
W
Pauline E. Jullien,a Tetsu Kinoshita,b Nir Ohad,c and Frédéric Bergera,1
a Chromatin and Reproduction Group, Temasek Lifesciences Laboratory, National University of Singapore, Singapore
117604, Republic of Singapore
b Integrated Genetics, National Institute of Genetics, Mishima 411-8540, Japan
c Department of Plant Sciences, Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel
Imprinted genes are expressed predominantly from either their paternal or their maternal allele. To date, all imprinted genes
identified in plants are expressed in the endosperm. In Arabidopsis thaliana, maternal imprinting has been clearly
demonstrated for the Polycomb group gene MEDEA (MEA) and for FWA. Direct repeats upstream of FWA are subject to DNA
methylation. However, it is still not clear to what extent similar cis-acting elements may be part of a conserved molecular
mechanism controlling maternally imprinted genes. In this work, we show that the Polycomb group gene FERTILIZATIONINDEPENDENT SEED2 (FIS2) is imprinted. Maintenance of FIS2 imprinting depends on DNA methylation, whereas loss of
DNA methylation does not affect MEA imprinting. DNA methylation targets a small region upstream of FIS2 distinct from the
target of DNA methylation associated with FWA. We show that FWA and FIS2 imprinting requires the maintenance of DNA
methylation throughout the plant life cycle, including male gametogenesis and endosperm development. Our data thus
demonstrate that parental genomic imprinting in plants depends on diverse cis-elements and mechanisms dependent or
independent of DNA methylation. We propose that imprinting has evolved under constraints linked to the evolution of plant
reproduction and not by the selection of a specific molecular mechanism.
INTRODUCTION
Reproduction of flowering plants is characterized by a double
fertilization event, which involves two identical sperm cells and
two female gametes, the egg cell and the central cell. Fertilization
of the egg cell leads to the development of the embryo, whereas
the second sperm cell independently fertilizes the central cell,
giving rise to the endosperm (Guignard, 1899; Maheshwari,
1950; Berger, 2003). The endosperm controls the supply of
maternal nutrients to the developing embryo and does not contribute genetic material to the next generation. In Arabidopsis
thaliana and in maize (Zea mays), parental imprinted expression
has been detected only in the endosperm for a few genes
expressed only from their paternal or their maternal allele
(Berger, 2004; Gehring et al., 2004; Köhler and Grossniklaus,
2005). In Arabidopsis, imprinting was demonstrated using allelespecific RT-PCR for the paternally expressed gene PHERES1
(Köhler et al., 2005) and the maternally expressed genes FWA
(Kinoshita et al., 2004) and MEDEA (MEA) (Kinoshita et al., 1999;
Vielle-Calzada et al., 1999). FWA and MEA are expressed during
neither vegetative development nor male gametogenesis. During
1 To
whom correspondence should be addressed. E-mail fred@tll.
org.sg; fax 65-68727007.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described
in the Instructions for Authors (www.plantcell.org) is: Frédéric Berger
([email protected]).
W
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female gametogenesis, FWA and MEA become transcriptionally
active, and both genes are expressed specifically in the central
cell (Choi et al., 2002; Kinoshita et al., 2004). After fertilization,
only the maternal alleles of FWA and MEA are expressed during
endosperm development, whereas their paternal alleles remain
silenced.
Imprinting of a gene thus results from a combination of silencing
of the paternal allele and activation of expression of the maternal
allele. Silencing of the FWA paternal allele is likely correlated with
methylation of cytosine residues located in the direct repeats of
the 59 region mediated by the DNA methyltransferase MET1
(Kinoshita et al., 2004). The transcriptionally active FWA maternal
allele in endosperm is characterized by demethylation of the
direct repeats. This demethylation may occur in the central cell
and might result from the activity of the DNA glycosylase DEMETER (DME). DME creates single-strand 59 nicks, and such DNA
breaks would be recognized by the DNA repair machinery, which
in turn replaces the methylated cytosine residue with unmethylated ones (Choi et al., 2002; Gehring et al., 2006). Based on the
available knowledge regarding FWA imprinting, it was possible to
model a cycle for plant imprints (Berger, 2004; Kinoshita et al.,
2004). According to the model, FWA is silenced during the
vegetative phase by MET1. DME releases FWA silencing in the
central cell. After fertilization, the activated maternal FWA allele is
inherited by endosperm and is transcribed, whereas the paternal
allele remains silenced because it was inherited in an inactive
state from the male gamete. MET1 is active during male gametogenesis (Saze et al., 2003), but its involvement in silencing of the
FWA paternal allele during male gametogenesis and endosperm
development has not been demonstrated. Moreover, it is not clear
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whether the mode of regulation of FWA imprinting could apply to
other imprinted genes.
Regulation of MEA imprinting has been analyzed, but as
detailed below, the mechanism controlling MEA imprinting is
still unclear. DME is also essential for MEA transcriptional activation (Choi et al., 2002), and methylated CpG residues have
been identified in the MEA promoter (Xiao et al., 2003; Gehring
et al., 2006), suggesting that MEA imprinting is controlled by DNA
methylation similar to FWA. DNA methylation could target putative CpG islands and transposons at the MEA locus (Xiao et al.,
2003; Spillane et al., 2004). However, such elements are not essential for MEA imprinting, as suggested by conflicting genetic
experiments, which casts doubt on the role of MET1 in this
process (Vielle-Calzada et al., 1999; Luo et al., 2000; Vinkenoog
et al., 2000; Xiao et al., 2003). Although MET1 might play a
regulatory role in the control of imprinting of several genes
involved in endosperm development (Adams et al., 2000), its
direct involvement in the control of MEA imprinting remains to be
tested.
Besides DNA methylation, histone methylation was shown
recently to be involved in the control of MEA imprinting (Gehring
et al., 2006; Jullien et al., 2006) and likely also controls the
imprinting of PHERES1, encoding a MADS box transcription
factor (Köhler et al., 2005). MEA imprinting depends on transcriptional repression of its paternal allele by methylation of the
Lys-27 of Histone3 by several Polycomb group complexes active
during the plant life cycle (Gehring et al., 2006; Jullien et al., 2006).
In endosperm, the PRC2 complex contains MEA (Grossniklaus
et al., 1998; Kiyosue et al., 1999; Luo et al., 1999) and a core of three
additional proteins, the VEFS domain protein FERTILIZATIONINDEPENDENT SEED2 (FIS2) (Luo et al., 1999), the WD40 protein FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) (Luo
et al., 1999; Ohad et al., 1999), and the WD40 protein MULTICOPYSUPPRESSOR OF IRA1 (MSI1) (Köhler et al., 2003; Guitton
et al., 2004). The mutants mea, fie, msi1, and fis2 exhibit similar
maternal defects in endosperm, and as a consequence, FIE,
MSI1, and FIS2 are anticipated to be subject to one or more of
the genetic mechanisms that regulate imprinting, including the
one presented for MEA (Chaudhury et al., 1997; Grossniklaus
et al., 1998; Kiyosue et al., 1999). Involvement of imprinting is
also supported for FIE and FIS2 by the monoallelic maternal
expression of transcriptional reporters for FIE–green fluorescent
protein (GFP) (Yadegari et al., 2000) and FIS2–b-glucuronidase
(GUS) (Luo et al., 2000), respectively. However, this transcriptional control may affect only the corresponding transcriptional
reporter. Silencing in endosperm has been shown for the paternal copy of reporter constructs inserted at several loci, leading to
the hypothesis of global silencing of the paternal genome in the
first days after fertilization (Vielle-Calzada et al., 2000). Alternatively, the maternal effect associated with the loss-of-function
mutations could originate from abnormal development of the
female gametophyte or from dosage effects. Hence, the imprinted
status of FIS2, FIE, and MSI1 remains unclear.
In this work, we show that FIS2 is subject to parental genomic
imprinting. We study in detail the role of DNA methylation in the
transcriptional control of FIS2 and MEA and conclude that they
are distinct. We identify a cis-element upstream of FIS2 that is
targeted by MET1. This element is likely associated with the
control of FIS2 imprinting. Using a genetic approach, we investigated the role played by MET1 during the entire life cycle for the
control of FIS2 and FWA imprinting. To this end, we investigated
the effect of MET1 loss of function restricted to the vegetative
developmental phase (with the MET1as line) and the effect of
MET1 loss of function restricted to the gametogenesis phase
(with the met1-3/þ line). Based on our results, we propose a
revised model for the cycle of MET1-dependent imprinting in
plants.
RESULTS
FIS2 Expression Is Controlled by DNA
Methylation–Dependent Imprinting
To investigate the imprinted status of FIS2, we used polymorphisms in the FIS2 coding sequence between different wild-type
accessions to distinguish transcripts of each parental allele
(allele-specific RT-PCR) (Figure 1A). We performed allelespecific RT-PCR on seeds originating from crosses between
wild-type accessions C24 and Columbia (Col), used alternately
as male or female. One would expect that if FIS2 is not imprinted,
two transcripts should be detected. Contrary to this hypothesis,
we observed only expression of maternal FIS2 transcripts (Figure
1A). We observed monoallelic maternal expression of FIS2 up to
5 d after pollination (DAP) (see Supplemental Figure 1 online).
Maternal FIS2 transcripts stored in the ovules and inherited after
fertilization could be detected in developing seeds and mimic an
imprinted status. However, it is unlikely that such stored maternal
transcripts could be still present at 5 DAP. It is likely that we
detected transcripts from active postzygotic transcription of the
FIS2 maternal allele in the endosperm. A putative general paternal silencing has been observed for reporters of endospermexpressed genes for up to 4 DAP (Vielle-Calzada et al., 2000). It
could be hypothesized that such a general mechanism also
affects expression at genomic loci for genes expressed in
endosperm. During all stages of development examined, we
observed biparental expression of MINI3 (Figure 1A), a gene
expressed only in endosperm during the first 3 DAP (Luo et al.,
2005). These results and others (Berger, 2003; Kinoshita et al.,
2004) indicate that there is no general paternal silencing for
genes expressed in endosperm and that FIS2 monoallelic expression results from a specific silencing mechanism. In agreement with these findings, Luo et al. (2000) previously showed that
the FIS2-GUS translational reporter is expressed in endosperm
only from the maternal allele. We concluded that the endogenous
FIS2 gene is imprinted.
We investigated the potential role played by MET1 in FIS2
imprinting. To this end, we used the loss-of-function allele met1-3
in the Col background (Figure 1B). To avoid nonspecific cumulative effects of inbred met1 loss-of-function lines (Saze et al.,
2003; Xiao et al., 2006), we isolated met1-3/met1-3 plants from a
segregating population of selfed met1-3/þ plants. Loss of MET1
activity caused expression of the paternal allele of FIS2 in
crosses between wild-type female ovules and met1-3 pollen
(Figure 1B). To evaluate the impact of MET1 activity during
vegetative development, we used MET1as plants, which produce antisense MET1 mRNAs in vegetative tissues, leading to a
DNA Methylation and Imprinting
Figure 1. FIS2 Imprinting Is Controlled by DNA Methylation.
(A) Allele-specific RT-PCR on RNAs extracted from siliques obtained at
0.5, 1.5, and 3 DAP after crosses were made between wild-type Col and
wild-type C24. A 180-bp deletion in the FIS2 coding sequence of the
wild-type accession Col allows the distinction of the Col FIS2 transcript
from the FIS2 transcript in accession C24. Only the FIS2 maternal allele is
expressed in developing seeds. By contrast, MINI3 is expressed from
both parental alleles.
(B) Allele-specific RT-PCR on RNAs extracted from siliques obtained at
4 DAP after crosses between wild-type ovules and pollen from met1-3/
met1-3 plants (Col) show reduced activity of the maintenance DNA MET1
during the entire life cycle. In contrast with wild-type crosses, the FIS2
paternal allele is expressed in developing seeds, which inherit a paternal
allele from met1-3/met1-3 plants.
(C) Allele-specific RT-PCR on RNAs extracted from siliques obtained at
5 DAP after reciprocal crosses between wild-type plants and MET1as
plants. The FIS2 paternal allele is expressed in developing seeds when
pollen is provided by MET1as plants, which show reduced activity of the
maintenance DNA MET1 during the vegetative developmental phase
(Finnegan et al., 1996).
(D) Allele-specific RT-PCR on RNAs extracted from siliques obtained at
5 DAP after crosses made between wild-type plants and different mutants
affected for histone methylation or for DNA methylation. A 180-bp
deletion in the FIS2 coding sequence of the wild-type accession Col
allows the distinction of the Col FIS2 transcript from the FIS2 transcript in
accessions C24, Landsberg erecta (Ler), and Wassilewskija. C24 is
presented as a control. Only the maternal allele of FIS2 is expressed in
seeds resulting from reciprocal crosses between the wild type and
homozygous mutants for other pathways controlling DNA methylation in
Arabidopsis cmt3 (Lindroth et al., 2001) and drm1 and drm2 (Cao and
Jacobsen, 2002a). KYP methylates Histone3 Lys-9 residues (Jackson
et al., 2002; Tariq et al., 2003), whereas the Polycomb group complex
containing FIE methylates Histone3 Lys-27 residues (Bastow et al., 2004).
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loss of 70% of DNA methylation on symmetric CpG sites
(Finnegan et al., 1996). We observed that loss of MET1 activity
during vegetative development caused expression of the paternal allele of FIS2 in crosses between wild-type female ovules and
MET1as pollen (Figure 1C). We concluded that imprinting of FIS2
depends directly or indirectly on MET1. DNA methylation is
propagated through replication in a semiconservative manner
(Finnegan et al., 2000). Because in the MET1as line MET1
antisense RNAs are produced only during vegetative development, we propose that demethylation of FIS2 during vegetative
development is carried over to the endosperm, resulting in loss of
imprinting. DNA methylation likely interacts with mechanisms
that control other epigenetic marks (Tariq et al., 2003; Chan et al.,
2005; Takeda and Paszkowski, 2006; Vire et al., 2006), and the
effect of met1-3 and MET1as could be indirect. Thus, we tested
whether other epigenetic mechanisms might be involved, including DNA methylation and histone methylation. The DOMAINREARRANGED METHYLTRANSFERASEs DRM1 and DRM2
control de novo methylation (Cao and Jacobsen, 2002a), and
CHROMOMETHYLASE3 (CMT3), which is unique to plants,
maintains methylation at asymmetric sites (Lindroth et al.,
2001; Cao and Jacobsen, 2002b). Loss-of-function mutants in
these genes do not influence FIS2 imprinting (Figure 1D). Transcriptional gene silencing is also regulated by methylation of
histone H3. In Arabidopsis, Lys residues Lys-9 and Lys-27 of
histone H3 are methylated by KRYPTONITE (KYP) (Jackson et al.,
2002) and by Polycomb group complexes (Bastow et al., 2004;
Jullien et al., 2006), respectively. FIS2 imprinting was not altered
in crosses between wild-type ovules and pollen from mutant
plants carrying loss-of-function kyp or from FIE cosuppressed
plants (Katz et al., 2004) (Figure 1D). Because we could not find
any effect of loss of epigenetic marks likely linked with the
maintenance of DNA methylation, we conclude that MET1 directly silences FIS2.
DNA Methylation Targets a Specific CpG-Rich Domain
Upstream of the FIS2 Gene
Loss of MET1 activity caused ectopic expression of FWA, as
reported previously (Saze et al., 2003; Kinoshita et al., 2004), but
surprisingly, it did not result in ectopic expression of FIS2 in
vegetative tissues (Figure 2A). The absence of endospermspecific transcriptional activators could explain the absence of
ectopic expression of FIS2 in vegetative met1 tissues. Nevertheless, we hypothesized that loss of function of MET1 causes a
loss of DNA methylation marks in FIS2 genomic sequences. To
test this hypothesis, we analyzed DNA methylation in the 59 region of FIS2 in leaf tissues. Using the methylation-sensitive
restriction enzyme McrBC, we identified a region affected by
DNA methylation (Figure 2B). Moreover, bisulfite sequencing
analyses identified a region at ;2 kb upstream of the start codon
of FIS2 consisting of a 200-bp domain where most CpG sites
The paternal (p) and maternal (m) alleles are indicated. Glyceraldehyde3-phosphate dehydrogenase (GAPDH) was used as a loading control.
No-RT control assays were performed, but the data are not shown.
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Figure 2. MET1 Targets a CpG-Enriched Region at the FIS2 Locus.
(A) RT-PCR analysis shows that imprinted genes FWA and FIS2 are not expressed in vegetative tissues of the wild-type accession Col. Reduced
methylation of DNA in null met1-3 homozygous mutant plants causes ectopic expression of FWA but not of FIS2 in leaves. met1-3/þ plants, which show
no ectopic expression of FWA, were used in the experiments presented in Figure 5A. GAPDH was used as a loading control.
(B) Analysis of DNA methylation using the methylation-sensitive restriction enzyme McrBC in wild-type Col leaves identifies a methylated region
spanning 3104 to 1867 in the 59 region of FIS2. The sensitivity to McrBC is decreased in the met1/met1 background, which indicates that MET1
controls DNA methylation in the 3104 to 1867 region. FWA repeats, which are methylated by MET1, were used as controls (Kinoshita et al., 2004).
(C) Bisulfite sequencing of the FIS2 59 region, which contains a 200-bp domain enriched in methylated cytosine residues predominantly on CpGs (see
Supplemental Figure 2 online for the full sequence). Analysis was done with genomic DNA from rosette leaves. The structure of the FIS2 genomic
sequence is depicted, with the start codon indicated by 0. Exons are indicated by black boxes, and the 59 and 39 untranslated regions are indicated by
white boxes. The extent of the partial translational fusion FIS2-GUS is indicated below the schematic genomic structure.
were methylated (30 of 38) (Figure 2C; see Supplemental Figure 2
online). Methylation of CpG residues in this region was severely
reduced in met1-3 homozygous mutant leaves (see Supplemental Figure 2 online). We conclude that MET1 targets a specific
element in the 59 region that might control FIS2 silencing in leaves
as well as in pollen and endosperm.
Activation of the FIS2 Maternal Allele by DME in the
Central Cell
To investigate the role of DME on FIS2 expression, we tested the
effect of dme loss of function on FIS2 expression. To this end,
RT-PCR experiments were performed with mRNA extracted from
buds and flowers, where DME was shown to be expressed (Choi
et al., 2002). We used FWA as a control for the action of the dme-4
allele, which was isolated independently from other dme alleles
(Guitton et al., 2004). FWA is expressed in the wild type before
and after fertilization (Kinoshita et al., 2004), whereas in dme-4, a
strong reduction of FWA transcription was observed (Figure 3A),
as shown previously with dme-1 (Kinoshita et al., 2004). Similarly,
dme-4 caused a reduction of FIS2 expression (Figure 3A), but the
level of FIS2 expression was only partially reduced. The limited
reduction of FIS2 expression by dme-4 was confirmed by anal-
ysis of the expression of the FIS2-GUS reporter, which includes
most of the FIS2 locus and is only maternally expressed in wildtype endosperm (Figure 2C) (Luo et al., 2000). In self-pollinated
FIS2-GUS/FIS2-GUS, dme-4/DME plants, all ovules inherit FIS2GUS and 50% inherit the dme-4 mutation. Accordingly, we
observed a significant reduction of FIS2-GUS expression in 50%
of the ovules in which dme-4 was expected to be present (Figures
3B to 3D) compared with wild-type ovules (Figures 3B and 3E).
However, half of dme-4 ovules still expressed the reporter
at lower levels than wild-type ovules (Figures 3B and 3D). After
fertilization, we observed a similar reduction of FIS2-GUS expression in endosperm, which inherits a maternal dme-4 (Figure
3B). Homozygous dme-4/dme-4 mutants are viable (Guitton
et al., 2004), whereas dme-2/dme-2 causes seed developmental
arrest (Choi et al., 2002). Hence, partial reduction of FIS2
expression in the dme-4 background could result from incomplete penetrance of the dme-4 mutation. However, a similar
partial reduction of FIS2-GUS expression was obtained with
the strong allele dme-2 (Choi et al., 2002) (Figure 3B). We
conclude that DME is required for the expression of FIS2 in
the central cell as well as in the endosperm. Persisting FIS2
expression in a fraction of dme ovules could be accounted for by
either redundant functions for DME or by additional activation
DNA Methylation and Imprinting
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Figure 3. Maternal Control of FIS2 Expression by DME.
(A) RT-PCR analyses performed on mRNA from buds (stages 11 and 12) and flowers (stage 13) (Smyth et al., 1990) show a lack of FWA expression and
reduced FIS2 expression in the dme-4 homozygous mutant compared with the wild-type C24 accession. GAPDH was used as a control.
(B) to (E) Effects of dme on FIS2-GUS expression.
(B) Percentage of ovules expressing the FIS2-GUS reporter construct in dme-2/þ and dme-4/þ before pollination (BP) and at 2 DAP. Three different
classes are observed: no expression (white), little expression (light blue), and wild-type expression (dark blue). Error bars correspond to the SD
associated with each set of measurements.
(C) to (E) Micrographs showing the three categories of FIS2-GUS expression in the dme-4/þ mutant background: no expression (white; [C]), little
expression (light blue; [D]), and wild-type expression (dark blue; [E]). Bars ¼ 20 mm.
mechanisms sufficient to overcome the repressive effect of DNA
methylation.
Imprinting of FWA and FIS2 in Endosperm Requires
the Maintenance of Silencing by MET1 during
Male Gametogenesis
In contrast with female gametogenesis, male gametogenesis
does not promote the expression of FWA, FIS2, and MEA (Luo
et al., 2000; Kinoshita et al., 2004). We have investigated how
silencing of FWA and FIS2 is maintained during male gametogenesis. Expression of MET1 has been detected in pollen, by
transcriptome analysis (Honys and Twell, 2004), and there is
evidence for MET1 function in the silencing of exogenous transcriptional reporters during male gametogenesis (Saze et al.,
2003). Thus, we tested whether the loss of MET1 function
affected the expression of FWA and FIS2 paternal alleles in
pollen and later in endosperm. To this end, we used heterozygous met1-3/þ plants, which exhibit reduced MET1 activity only
during gametogenesis. In met1-3/þ plants, the mutant met1
allele is inherited by half of the microspores, which develop in
pollen grains after two cell divisions (McCormick, 2004). The
generative cell is expected to inherit a hemimethylated copy and
divides further to produce two male gametes, in which the
genomic DNA is demethylated (Figure 4A). Accordingly, we
observed ectopic expression of the transcriptional reporter
FWA-GFP in segregating pollen from met1-3/þ, FWA-GFP/
FWA-GFP plants (Figure 4B) (47.4% of pollen bearing FWA-GFP
fluorescence, SD ¼ 6.7, n ¼ 1372). Expression of FWA-GFP was
observed in the sperm cells, which indicates that met1 pollen
grains are able to provide a transcriptionally active FWA-GFP
copy (Figure 4B, inset).
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Figure 4. Maintenance of FIS2 and FWA Silencing during Male Gametogenesis.
(A) During pollen development in met1-3/þ plants, half of haploid microspores produced by meiosis inherit the met1-3 mutation, and the other half
inherit the wild-type MET1 allele. All microspores inherit a methylated copy of the genome (gray rectangles). DNA replication in met1-3 microspores
produces hemimethylated DNA from the methylated template (gray/white rectangles). Hemimethylated DNA is inherited after mitosis by the vegetative
nucleus (V) and the nucleus of the generative cell (G), which divides a second time and produces the two sperm cells (S). After this division, one sperm
cell inherits a fully demethylated DNA copy (white rectangles) and the other sperm cell inherits hemimethylated DNA. The vegetative nucleus does not
participate in fertilization. During sperm cell maturation, DNA replicates (Durbarry et al., 2005), and at fertilization, all sperm from met1-3 microspores is
expected to contain at least one hemimethylated copy.
(B) Segregating population of pollen grains from FWA-GFP/FWA-GFP, met1-3/þ plants (confocal section). The inset represents a confocal section of a
pollen grain expressing FWA-GFP in the vegetative cell (V) and in the two sperm cells (s). Bar ¼ 5 mm.
(C) Ectopic expression of FWA and FIS2 observed by RT-PCR in RNA purified from stamens. GAPDH was used as a control to assess equal amounts of
RNA loaded. DUO1 is expressed specifically in pollen (Rotman et al., 2005) and was used as a quality control for pollen mRNA extraction.
Our cytological observations were supported by RT-PCR
experiments showing ectopic expression of FWA in mRNA populations extracted from stamens of met1-3/þ plants (Figure 4C).
Thus, we used met1-3/þ plants to test whether loss of function of
MET1 during male gametogenesis was sufficient to allow the
expression of FWA paternal alleles in endosperm. In seeds resulting from crosses between wild-type ovules and met1-3/þ
pollen, FWA transcripts were detected from both the paternal
and maternal alleles (Figure 5A; note that polymorphism for Col is
represented by a double band). Accordingly, the paternal copy of
FWA-GFP is expressed in endosperm of seeds produced by
crosses between wild-type ovules and pollen from met1-3/þ,
FWA-GFP/FWA-GFP plants (Figure 5C) (46.7%, SD ¼ 5.2, n ¼
358). By contrast, no expression was observed when FWA-GFP
was provided from a paternal wild-type background (Figure 5B;
n ¼ 126) (Kinoshita et al., 2004). We conclude that during
gametogenesis, MET1 maintains FWA silencing and that FWA
imprinting in endosperm requires MET1 activity during male
gametogenesis. The MEA paternal allele is imprinted in endosperm (Kinoshita et al., 1999; Vielle-Calzada et al., 1999) and is
predominantly expressed from its maternal allele during the first
days after pollination (Jullien et al., 2006). We tested the effect of
MET1 on MEA paternal allele expression in endosperm. MEA
paternal allele expression was examined in seeds resulting from
crosses between wild-type ovules and met1-3/þ pollen. In such
crosses, we detected a predominance of maternal MEA allele
expression, which is similar to the result obtained when crosses
were made with wild-type male accessions. We concluded that
MEA imprinting was not affected by loss of DNA methylation
during male gametogenesis (Figure 5D). A similar result was
recently obtained independently (Gehring et al., 2006). Thus,
MET1 does not appear to play a major role in MEA silencing
comparable to its role in FWA and FIS2 imprinting.
Similarly, we tested the impact of met1 loss of function during
male gametogenesis on FIS2 silencing in pollen and imprinting in
endosperm. FIS2 was ectopically expressed in stamens from
met1-3/þ plants (Figure 4C). Pollination of wild-type ovules with
met1-3/þ plants caused expression of the FIS2 paternal allele in
endosperm (Figure 5E), which indicates that FIS2 silencing by
MET1 during male gametogenesis is required for FIS2 imprinting.
Previously, it was shown that maternal inheritance of fis2 loss-offunction alleles causes major defects in endosperm development, including overexpression of the KS117 marker (Sørensen
et al., 2001). In addition, endosperm produced by fis2 mothers
fails to cellularize during the syncytial stage (Chaudhury et al.,
1997). In crosses between fis2 ovules and met1 pollen, the wildtype FIS2 paternal allele is activated by met1 and is expected to
rescue the maternal loss-of-function fis2 allele. We next tested
whether in met1-3/þ plants the demethylated expressed paternal allele of FIS2 is sufficient to rescue the inactive mutant fis2
allele provided maternally to the developing endosperm. Heterozygous met1-3/þ plants produce 50% met1 pollen. The KS117
expression pattern was rescued in 49% of seeds produced by
crosses with pollen from met1-3/þ plants and fis2/fis2, KS117/
KS117 plants used as females (SD ¼ 5.3, n ¼ 459) (Figure 5G). In
these crosses, 19% of seeds showed cellular endosperm similar
to wild-type seeds (SD ¼ 5.1, n ¼ 365) (Figure 5J). Hence, met1
loss of function during pollen development is sufficient to remove
DNA Methylation and Imprinting
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the repressive epigenetic marks from the FIS2 paternal allele,
which remains active in endosperm.
We conclude that MET1 activity is essential to maintain the
silencing of FIS2 and FWA during male gametogenesis. If MET1
activity is absent during pollen development, the sperm cells
deliver a transcriptionally active paternal allele to endosperm and
imprinting of FIS2 and FWA in endosperm is compromised.
Regulation of MEA imprinting appears to depend on a distinct
regulatory mechanism independent of MET1. Instead, recent
evidence supports the notion that MEA silencing relies on histone
methylation by Polycomb group complexes (Gehring et al., 2006;
Jullien et al., 2006).
MET1 Is Required for Silencing the FWA
Paternal Allele in Endosperm
We have shown that FIS2 and FWA imprinted status in endosperm requires MET1 activity before fertilization. However, the
requirement of MET1 after fertilization and during endosperm
development has not yet been reported. Although the paternal
FWA-GFP copy provided by met1 pollen becomes expressed in
endosperm, its expression gradually decreases in signal intensity
Figure 5. Imprinting of FWA and FIS2 in Endosperm Relies on the
Maintenance of Their Silencing during Male Gametogenesis.
The imprinting status of FWA, MEA, and FIS2 was analyzed in developing
seeds resulting from crosses between wild-type ovules and pollen from
met1-3/þ plants.
(A) Allele-specific RT-PCR detecting expression of the paternal and
maternal alleles of FWA in seeds resulting from crosses between wildtype ovules (Ler accession) and pollen from met1-3/þ plants. (For the Col
accession, two bands were obtained because digestion of the restriction
polymorphism was partial; the lowest band was Col-specific [3 DAP].)
We used met1-3/þ plants, which did not show expression of FWA in
vegetative tissues (see Figure 2A). Although crosses between wild-type
Ler and Col showed only expression of the maternal FWA allele, the FWA
paternal allele was expressed in crosses with met1-3/þ pollen.
(B) and (C) Accordingly, at 2 DAP, the FWA-GFP reporter was expressed
paternally in endosperm when provided by pollen from met1-3/þ plants
(C) but not from wild-type plants (B).
(D) Allele-specific RT-PCR performed on crosses between wild-type
accessions RLD and Col detected expression of the maternal MEA allele,
typical of MEA imprinting (1 DAP). MEA imprinting was not altered if
met1-3/þ plants provided the MEA paternal allele.
(E) Crosses between wild-type accessions C24 and Col showed expression only from the maternal FIS2 allele typical of FIS2 imprinted
status in wild-type endosperm. By contrast, allele-specific RT-PCR
showed expression of the paternal and maternal alleles of FIS2 in seeds
resulting from crosses between ovules from wild-type C24 and pollen
from met1-3/þ plants (Col accession; 3 DAP).
(F) to (J) Rescue of endosperm development upon removal of the FIS2
paternal allele imprint in met1-3 pollen.
(F) and (H) GFP fluorescence of the enhancer trap marker KS117 was
increased uniformly in endosperm of the fis2 homozygous mutant (F)
compared with the wild type (H), in which it was restricted to the
endosperm posterior pole at 5 DAP.
(G) After pollination of fis2/fis2, KS117/KS117 plants by pollen from
met1-3/þ plants, half of the seeds showed a pattern of KS117 expression similar to the KS117 pattern in wild-type endosperm (asterisks).
Such seeds also showed a rescue of cytological alteration in fis2
endosperm ([I] and [J]).
(I) The phenotype conferred by fis2 is characterized by the absence of
cellularization of the syncytial endosperm and the enlargement of the
posterior pole opposite the embryo (Guitton et al., 2004).
(J) Rescue of such phenotypic traits was obtained in 19% of seeds
resulting from crosses between fis2/fis2 plants and met1-3/þ pollen.
Bars ¼ 50 mm ([B], [C], [I], and [J]) and 200 mm ([F] to [H]). GAPDH was
used as a control for the RT-PCR analyses ([A], [D], and [E]).
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The Plant Cell
between 2 and 4 DAP (Figures 6A to 6C) and becomes gradually
undetectable after 6 DAP (data not shown). We hypothesized
that this resulted from progressive silencing of the paternal FWAGFP copy by maternally provided MET1 activity in met1/þ
endosperm. According to this hypothesis, a loss of function of
MET1 in met1/met1 endosperm is expected to sustain the
expression of a paternally provided copy of FWA-GFP. Transmission of met1-3 through male and female gametes follows
Mendelian proportions (see Supplemental Table 1 online), and
we expected production of 25% met1/met1 seeds in crosses
between met1/þ plants. We performed crosses between ovules
of met1-3/þ plants and pollen from met1/þ, FWA-GFP/FWAGFP plants, leading to 25% met1/met1 seeds and 25% met1/þ
seeds with a paternal FWA-GFP copy from met1 pollen. We
observed two distinct classes of seeds expressing FWA-GFP
either in met1-3/þ or in met1/met1 endosperm. One-quarter of
seeds showed low expression levels comparable to those of
seeds with met1/þ endosperm (Figures 6D and 6E), and another
one-quarter of seeds showed high levels of FWA-GFP expression at 4 DAP (Figures 6D and 6F). Given the Mendelian proportion of each seed class, the latter class presumably corresponds
to met1/met1 endosperm with no MET1 activity, in which progressive silencing of a demethylated FWA-GFP paternal copy
does not occur. These results suggest that MET1 is continuously
required for silencing of the paternal FWA allele during endosperm
development to maintain FWA imprinted status.
DISCUSSION
Imprinting of FIS2 and FWA Relies on MET1 Activity during
Male Gametogenesis
We have shown that FIS2 undergoes monoallelic maternal
expression in endosperm. A gene is considered to be imprinted
Figure 6. Maintenance of FWA Imprinting in Endosperm Depends on MET1.
(A) At 2 DAP, after fertilization of wild-type ovules with pollen from met1-3/þ, FWA-GFP/FWA-GFP plants, expression of the paternally provided allele of
FWA-GFP is observed in endosperm in half of the seeds.
(B) and (C) The intensity of FWA-GFP expression decreases during endosperm development from 2 DAP (B) to 4 DAP (C).
(D) In contrast with the gradual silencing of FWA-GFP paternal expression in met1-3/þ endosperm, one-quarter of the seeds show expression of the
FWA-GFP paternal allele in crosses between ovules from met1-3/þ plants and pollen carrying met1-3/þ and FWA-GFP/FWA-GFP.
(E) and (F) One-quarter of the seeds express low levels of FWA-GFP (E) and are presumed to be heterozygous for met1-3. Another one-quarter of the
seeds show higher sustained expression of FWA-GFP (F) and are presumably homozygous for met1-3 in endosperm.
Bars ¼ 80 mm ([B], [C], [E], and [F]). The number of seeds observed for each cross is indicated above each bar. SD is indicated for each measurement.
HO, homozygous.
DNA Methylation and Imprinting
when it is expressed after fertilization and primarily from one of its
two parental alleles (Vielle-Calzada et al., 1999). We detected
FIS2 maternal transcripts up to 5 d after pollination and showed
that the FIS2 paternal allele can be expressed in endosperm
during met1 loss of function in gametogenesis. Thus, FIS2 is actively transcribed after fertilization and is expressed only from its
maternal allele. We conclude that FIS2 is a maternally expressed
imprinted gene. MET1 activity is necessary during endosperm
development for sustained silencing of the paternal copy of FWA.
We anticipate a similar dependence of the FIS2 paternal allele
silencing on MET1 activity in endosperm. In conclusion, when
MET1 activity is removed, as in met1-3 mutants, the expression
of MET1-dependent imprinted genes is maintained beyond the
wild-type schedule for both parental alleles.
Continuous DNA methylation by MET1 at specific sites during
vegetative development maintains the silencing of both parental
FWA and FIS2 alleles. The imprinted genes are expressed during
female gamete maturation, whereas they remain silent during
male gametogenesis. This imbalance of gene expression in
gametes prefigures imprinting in endosperm after fertilization.
In the central cell, silencing of imprinted genes expressed in
endosperm would be removed via DNA demethylation mediated
by the glycosylase DME. This was shown for FWA (Kinoshita
et al., 2004), and our data indicate that DME also acts on
FIS2 together with other unknown activators, because FIS2
expression is only partially suppressed in dme ovules. We have
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shown that MET1 is required during male gametogenesis
to maintain FWA and FIS2 silencing. This provides an example
for the role played by MET1 during male gametogenesis, which
was originally demonstrated with an exogenous reporter construct (Saze et al., 2003). Furthermore, we have shown that
sperm cells deficient for met1 provide activated FIS2 paternal
alleles, which are able to rescue endosperm defects caused
by the maternally provided fis2 null alleles. In conclusion, imprinting results from the maintenance of DNA methylation
in sperm cells, whereas DNA methylation is removed in the
female gamete (Figure 7). Our model thus extends and complements the model proposed previously (Berger, 2004; Kinoshita
et al., 2004).
Similarly, in mammals, loss of function of de novo DNA
methylation during gametogenesis caused a lack of imprints
(epigenetic marks responsible for silencing) establishment in
mice (Monk, 1990; Kaneda et al., 2004). Thus, during gametogenesis, crucial steps are made necessary for imprinting genes
in both mammals and plants, but the establishment of imprints
occurs through distinct DNA methylation mechanisms. In contrast with plants, extensive demethylation occurs in the mammalian germ line and erases parental imprints (Delaval and Feil,
2004). Imprints are reestablished in the male and female germ
lines by a specific de novo DNA methyltransferase (Kaneda et al.,
2004) and not by the maintenance DNA methyltransferase, as in
flowering plants.
Figure 7. Model for the Dual Control of Parental Genomic Imprinting by DNA Methylation in Plants.
The genes FIS2 and FWA are imprinted in endosperm and silenced by the continuous action of MET1, which targets methylation (pink circles) to an
element in the promoter specific for each gene. MET1-dependent silencing of FIS2 and FWA is required during the vegetative phase, male
gametogenesis, and endosperm development, when it maintains silencing of the paternal (p) allele. FIS2 and FWA expression is activated during female
gametogenesis by the DNA glycosylase DME, leading to the expression of their maternal (m) allele in endosperm after fertilization. In endosperm, the
paternal copy remains silenced through the continuous action of MET1, whereas the maternal copy is active, leading to an imprinted expression.
10 of 13
The Plant Cell
MET1 Controls Imprinting in Arabidopsis via
Diverse cis-Elements
In spite of the similarities between the expression patterns of
maternally imprinted genes identified in Arabidopsis, we show
that imprinting mechanisms of different genes are distinct. Our
results demonstrate that MET1 plays a major role in FIS2 and
FWA imprinting, but some aspects of MEA imprinting do not
appear to depend critically on MET1. Silencing of the MEA
paternal allele is not mediated by MET1 but rather depends on
the continuous action of histone methylation mediated by Polycomb group complexes (Gehring et al., 2006; Jullien et al., 2006).
Nevertheless, activation of the maternal allele of MEA depends
on the removal of MET1-dependent DNA methylation (Gehring
et al., 2006). It is not clear, however, whether DME also leads to
the removal of Polycomb group–dependent histone methylation
marks. The regulation of MEA imprinting also could involve
DDM1, a SWI2/SNF2 factor, which interferes with MEA function
(Vielle-Calzada et al., 1999) and interacts with DNA methyl
binding proteins (Zemach et al., 2005).
MET1 was shown to target tandem repeats in the promoter of
FWA and to regulate its imprinting (Kinoshita et al., 2004), although a direct connection between these two events remains to
be demonstrated. Additional silencing mechanisms are involved
in the silencing of FWA (Chan et al., 2004), but it is not clear
whether they control FWA imprinting. Upstream of the FIS2
coding sequence, we could detect neither repetitive sequence
nor transposons with bioinformatics analysis, which successfully
detected such elements in the MEA promoter (Spillane et al.,
2004). Rather, we show that methylation is directed to a 200-bp
region upstream of the FIS2 coding sequence. A putative MET1targeted control element has been detected upstream of one
of the maize homologues of FIE, which is also imprinted and
expressed in endosperm (Danilevskaya et al., 2003). This element consists of CpG-enriched sequences different from the
cis-elements identified in Arabidopsis for FIS2. Based on the
limited data available, we are able to conclude that imprinting in
flowering plants relies on several types of cis-elements and
several silencing mechanisms. It is also becoming clear that
imprinting in mammals does not rely on a single type of epigenetic transcriptional regulation. Both maintenance and de novo
DNA methyltransferase are involved, as well as Polycomb group
activities.
DNA methylation targets several types of elements in imprinting control regions, which can be related to altered transposable
elements and CpG-enriched regions (Constancia et al., 2004;
Delaval and Feil, 2004). The diversity of cis control elements and
trans-acting mechanisms involved in imprinting found in flowering plants and mammals contrasts with the relative similarity of
functions for some targets of imprinting. Most imprinted genes
identified in flowering plants play an essential role in endosperm
development and thus indirectly control embryo nutrition in the
developing seed. In animals, several imprinted genes were found
to have an essential function in reproduction and to affect
interactions between the mother and the developing embryo or
infant (Constancia et al., 2004). It is thus likely that the evolution of
imprinting did not depend on selection of a molecular epigenetic
mechanism but more likely stemmed from biological constraints
linked to reproductive innovations. Selective mechanisms might
have used available silencing machineries and targeted potential
cis-elements already present at the loci of some genes important
for reproduction. This evolutionary process eventually led to the
actual state of diverse cis and trans control elements associated
with imprinting.
METHODS
Plant Materials and Growth Conditions
The MET1 antisense line of Arabidopsis thaliana was provided by J.
Finnegan (Finnegan et al., 1996). This line is characterized by a loss of
70% CpG methylation and by a dominant effect of the MET1 antisense in
vegetative diploid tissues. As the line is maintained by selfing, it likely
accumulates epimutations of unknown nature, which may interfere with
our experiments. Hence, for most experiments, we used the met1-3 (Col)
line provided by J. Paszkowski (Saze et al., 2003) and took care to
propagate the met1-3 line as a heterozygote. Homozygous mutants
cmt3, drm1, drm2, and kyp (Ler) came from S.E. Jacobsen’s laboratory
(Lindroth et al., 2001; Cao and Jacobsen, 2002a; Jackson et al., 2002).
The mutant dme-2 (Col) was provided by R. Fischer (Choi et al., 2002). The
mutants dme-4 and fis2-6 (C24) were characterized previously in the
laboratory of F.B. (Guitton et al., 2004). FIEcs (Col) plants were characterized previously by N.O.’s group (Katz et al., 2004). We used met1-3/þ
plants in which neither FIS2 nor FWA transcripts could be detected in
vegetative tissues (Figure 2A), which indicated that the parent plants were
not affected by the mutation during the vegetative phase but only during
the production of male gametes.
The KS117 line was identified after a screen in Jim Haseloff’s enhancer
trap line collection (Haseloff, 1999). The transgenic reporter line FIS2GUS was kindly provided by A. Chaudhury (Chaudhury et al., 1997; Luo
et al., 2000). The FWA-GFP fusion was described previously (Kinoshita
et al., 2004).
After 3 d at 48C in the dark, seeds were germinated and grown on soil.
Plants were cultured in a growth chamber under short days (8 h of light at
208C/16 h of dark at 168C; 60 to 70% RH) until rosettes were formed.
Plants were transferred to long days at 208C (14 h of light/10 h of dark) to
induce flowering and grown until seeds were harvested. Crosses were
performed by emasculating flowers and pollinating them manually. Siliques were harvested at various times after pollination, as indicated in
figure legends and in the text.
RT-PCR Analyses
Sample tissues were collected from Arabidopsis plants and immediately
frozen in liquid nitrogen. Tissues were ground, and total RNA was
prepared using the RNeasy mini kit (Qiagen). DNase treatment was
done on 2 mg of total RNA using the DNase free kit (Ambion).
For reverse transcription, 500 ng of total RNA was incubated for 1 h at
428C with 200 units of RevertAid Moloney murine leukemia virus reverse
transcriptase (Fermentas) in a 20-mL reaction mixture containing 4 mM
oligo(dT) 12 primer, appropriate reaction buffer, 1 mM deoxynucleotide
triphosphate, and 40 units of recombinant RNasin ribonuclease inhibitor
(Promega). The reaction was stopped by incubation at 708C for 10 min.
Equal amounts of RT products were used to perform subsequent
PCRs. Primers used to amplify FIS2 were Fis2-R5018 (59-CCTGCATTGTTTGGGAGTGATAGAA-39) and Fis2-F3412 (59-GGATGATGTAGGAAATCCCCAATTGAGCCCTTTG-39). Allele-specific RT-PCR of FWA was
performed as described (Kinoshita et al., 2004). Allele-specific RT-PCR
of MEA was performed as described (Kinoshita et al., 1999). Primers used
to amplify FWA were FWA-RTf2 (59-GTTACATGGATTGAACAAGCGG-39)
DNA Methylation and Imprinting
and FWA-RTr2 (59-ACCTTGAATGAGTGCAGCAGTTG-39). Primers
used to amplify the control, GAPDH RNA, were GAPDH39 (59-GTAGCCCCACTCGTTGTCGTA-39) and GAPDH59 (59-AGGGTGGTGCCAAGAAGGTTG-39). A negative control without RT was used for each
RT-PCR experiment. We designed primers spanning introns, such that
genome-specific and cDNA-specific PCR products could easily be
distinguished by size differences.
Microscopy
FIS2-GUS expression was analyzed as described by Jullien et al. (2006).
KS117 GFP fluorescence was analyzed in 5-DAP seeds with a stereomicroscope (MZ16FA; Leica). GUS-stained developing seeds or pistils
cleared with a derivative of Hoyer’s medium were observed with differential interference contrast optics. In developing seeds mounted in 50%
glycerol solution, FWA-GFP fluorescence was analyzed with a GFPspecific filter set with a 320 planapo objective (DM6000 B; Leica). Images
were acquired with a DXM1200F digital camera (Nikon) and processed
using Metamorph (version 6.2; Universal Imaging). The settings for observation were saved and applied to all seeds observed to allow comparison of fluorescence intensities at 2 and 4 DAP. Pollen was prepared
as described (Rotman et al., 2005). In pollen, FWA-GFP fluorescence was
analyzed by confocal microscopy (LSM 510) with a 340 water-immersion
objective, and images were further processed with Adobe Photoshop and
Adobe Illustrator.
For each measurement of FWA-GFP and FIS2-GUS in developing
seeds, counting was established on individual siliques. Measurements of
FWA-GFP expression in pollen were performed by placing several anthers on a slide. Fifteen individual fields were observed, each containing
20 to 50 pollen grains, which were scored, thus establishing the data set
for determining the percentage of positive expressing pollen. Standard
deviation was calculated for each genetic background used based on the
average obtained per silique or per field of pollen grains observed.
11 of 13
(Jeddeloh et al., 1998). We used met1-3 homozygous mutants segregated from met1-3/þ heterozygotes because these plants do not accumulate epimutations, in contrast with MET1as selfed plants.
Accession Numbers
Arabidopsis Genome Initiative numbers for the genes used in this
study are At1g02580 (MEA), At2g35670 (FIS2), At4g25530 (FWA), and
At5g04560 (DME).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Analysis of FIS2 Imprinting at 3 and 5 DAP.
Supplemental Figure 2. Bisulfite Sequencing of the FIS2 59 Region
Contains a 200-bp Domain Enriched in Methylated Cytosine Residues
Predominantly on CpGs.
Supplemental Table 1. Resistance to BASTA Is Associated with
met1-3.
ACKNOWLEDGMENTS
We thank all contributors of materials cited in Methods and the
Nottingham Arabidopsis Resource Centre and ABRC. P.E.J. and F.B.
were supported by the Temasek Lifesciences Laboratory and by the
National University of Singapore. N.O. was supported by the Israel
Science Foundation (Grant 574-04) and by the U.S.–Israel Binational
Agricultural Research and Development Fund (Grant IS-3604-04c).
Received January 16, 2006; revised March 24, 2006; accepted April 11,
2006; published April 28, 2006.
McrBC Analysis
DNA methylation status in the FIS2 promoter was analyzed by PCR
amplification of DNA that had been pretreated with McrBC endonuclease,
which digests methylated DNA (New England Biolabs). One hundred
nanograms of genomic DNA from rosette leaves of met1-3 and Col-0
plants was digested overnight with 10 units of McrBC, according to the
manufacturer’s instructions. Ten nanograms of template DNA was then
amplified by PCR with ExTaq polymerase (Takara) using the following
primers: F3697Tf (59-AAAGAGTTATGGGYYGAAG-39) and F3697Tr
(59-GGGCAGAAACATGGTCCA-39), FIS2.-3104BisBf (59-ACAARTCACAACCAAAACCTTAA-39) and FIS2.-2312r (59-TGCCAAATAGCACAATGAGGA-39), FIS2.-3104BisBf and FIS2.-1867r (59-ATGTTGCGCCTTCACCACTT-39), and FIS2.-1218BisBf (59-TCCARTCCACTATTCTTTACTCTT-39)
and FIS2.-16r (59-GTAGTTGAATCTTATTTTCCCACCTGA-39).
Bisulfite Sequencing
Bisulfite sequencing was performed as described (Paulin et al., 1998).
DNA was purified from rosette leaves. After chemical bisulfite reaction,
the top strand of the FIS2 promoter from 2185 to 1768 (relative to the
translation start) was amplified with primers FIS2.-2185BisTf (59-AGGTYYAATYGYATATTTATTTAGGGTTTYGGGT-39) and FIS2.-1768BisTr
(59-TCCTACATTTTAATAAAATATTACTRAATCTAARCA-39), and the bottom
strand from 2110 to 1670 was amplified with primers FIS2.-2110BisBf
(59-TACCAAACCCRAARAARAAAAATTTACAA-39) and FIS2.-1670BisBr
(59-TGATGGYAGTAGAGATTATAAGAAAAGA-39). The amplified PCR
fragments were gel-purified and cloned into pT7Blue plasmid (Novagen),
and then six to nine independent clones were sequenced. The ASA1 gene
was used as a positive control for the bisulfite chemical reaction
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Maintenance of DNA Methylation during the Arabidopsis Life Cycle Is Essential for Parental
Imprinting
Pauline E. Jullien, Tetsu Kinoshita, Nir Ohad and Frédéric Berger
Plant Cell; originally published online April 28, 2006;
DOI 10.1105/tpc.106.041178
This information is current as of June 17, 2017
Supplemental Data
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