Download Epigenetic Inactivation of Chalcone Synthase-A

Plant Cell Physiol. 48(4): 638–647 (2007)
doi:10.1093/pcp/pcm028, available online at www.pcp.oxfordjournals.org
ß The Author 2007. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
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Short Communication
Epigenetic Inactivation of Chalcone Synthase-A Transgene Transcription
in Petunia Leads to a Reversion of the Post-Transcriptional Gene Silencing
Phenotype
Akira Kanazawa
1,
*, Michael O’Dell
2
and Roger P. Hellens
3
1
Graduate School of Agriculture, Hokkaido University, Sapporo, 060-8589 Japan
Department of Genetics, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK
3
Gene Discovery and Function Department, HortResearch, Private Bag 92 169, Auckland, New Zealand
2
of DNA methylation in the affected promoters, as well as
changes in histone modification. The siRNAs may also
direct DNA methylation in the nucleus (for a review,
see Matzke et al. 2004). Like siRNAs, small RNAs called
micro RNAs also negatively regulate the expression of
endogenous genes through either RNA cleavage or arrest of
translation, which is another pathway of RNA silencing
(reviewed by Baulcombe 2004).
Overexpression of the chalcone synthase-A (CHS-A)
gene under the control of the cauliflower mosaic virus
(CaMV) 35S promoter and the nopaline synthase (NOS)
terminator causes the production of white sectors or
completely white flowers in transformed petunia (Petunia
hybrida) plants, and was one of the first examples of PTGS
(also termed co-suppression; Napoli et al. 1990, Van der
Krol et al. 1990). The production of wild-type pigment is
inhibited because CHS performs an essential step in the
biosynthesis of anthocyanins. Various silencing patterns
have been observed in the petunia CHS-A silencing system
(Jorgensen 1995, Jorgensen et al. 1996), and patterns can be
transmitted both somatically and germinally (Jorgensen
1995). Using a transgene-induced silencing system that
exploits genes involved in anthocyanin biosynthsis, Sijen
et al. (2001) demonstrated that a transgene that expresses
dsRNA can induce PTGS when the coding sequence is
transcribed, and can induce TGS when the promoter
sequence is transcribed. A flower color pattern in nontransgenic petunia plants has also been attributed to
sequence-specific degradation of the CHS-A RNA
(Koseki et al. 2005), suggesting that similar mechanisms
are involved in the induction of CHS-A silencing in
transgenic and non-transgenic petunia plants.
We have maintained silenced lines of petunia including
the line C001, which produces completely silenced white
flowers (for details of plant materials, see Materials
and Methods). A C001 plant is male-sterile and has
been maintained through backcrossing as a dominant
heterozygous trait for410 generations. The purple-flowered
Petunia plants that exhibit a white-flowering phenotype
as a consequence of chalcone synthase transgene-induced
silencing occasionally give rise to revertant branches that
produce flowers with wild-type pigmentation. Transcription
run-on assays confirmed that the production of white flowers
is caused by post-transcriptional gene silencing (PTGS), and
indicated that transgene transcription is repressed in the
revertant plants, providing evidence that induction of PTGS
depends on the transcription rate. Transcriptional repression
of the transgene was associated with cytosine methylation
at CpG, CpNpG and CpNpN sites, and the expression was
restored by treatment with either 5-azacytidine or
trichostatin A. These results demonstrate that epigenetic
changes occurred in the PTGS line, and these changes
interfere with the initiation of transgene transcription, leading
to a reversion of the PTGS phenotype.
Keywords: Bisulfite sequencing analysis — Cauliflower
mosaic virus 35S promoter — Chalcone synthase —
Cytosine methylation — Petunia hybrida — Posttranscriptional gene silencing.
Abbreviations: CaMV, cauliflower mosaic virus; CHS,
chalcone synthase; dsRNA, double-stranded RNA; Npt, neomycin
phosphotransferase; NOS, nopaline synthase; OCS, octopine
synthase; PTGS, post-transcriptional gene silencing; RdDM,
RNA-directed DNA methylation; RT–PCR, reverse transcription–
PCR; siRNA, short interfering RNA; TGS, transcriptional gene
silencing.
The silencing of genes induced by the presence of
homologous double-stranded RNA (dsRNA) sequences is
termed RNA silencing. RNA silencing in plants includes
post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS; for a review, see Baulcombe
2004). PTGS involves sequence-specific degradation of
RNA in the cytoplasm and is mediated by short interfering
RNAs (siRNAs). TGS is the repression or inactivation
of transcription and is associated with an increased level
*Corresponding author: E-mail, [email protected]; Fax, þ81-11-706-4933.
638
Epigenetic changes suppressing PTGS
C002 line arose from a C001 plant as a spontaneous
revertant; the mechanism of the reversion is unknown. The
CHS-A mRNA levels differ greatly in the two lines, as
demonstrated by RNA gel-blot analysis and reverse
transcription–PCR (RT–PCR) (Metzlaff et al. 1997).
The levels of CHS-A mRNA from both the CHS-A
transgene and the endogenous CHS-A gene (CHS-A
endogene) are very low in C001 flowers and leaves, as
expected from its white flower color. In C002, transcripts
from both the CHS-A transgene and the CHS-A endogene
are high in flowers, as predicted from the purple flower
color, whereas leaves contain low levels of the CHS-A
endogene transcript and no observed CHS-A transgene
transcript (Metzlaff et al. 1997, Metzlaff et al. 2000). In this
study, we investigated the mechanisms underlying the
reversion of the PTGS phenotype (white flowers) to the
normal phenotype (purple flowers) by comparing the C001
and C002 lines.
Transcription run-on assays were performed to
determine the activity of both the CHS-A transgene and
the CHS-A endogene in leaf and flower tissues of the C001
(silenced; white flowers) and C002 (revertant; purple
flowers) lines (Fig. 1). We were able to distinguish the
transcriptional activity of the two genes by hybridization of
probes specific to an intron sequence (endogene specific)
or the NOS terminator sequence (transgene specific) relative
to a positive control (Kanazawa et al. 2000; for probes, see
Fig. 1B). The hybridization to CHS-A exon 1 (common
to both the endogene and the transgene) reflects the
transcription from both genes. The pBluescript plasmid
and the Antirrhinum majus ubiquitin gene (EMBL:
AMUBIMRP X67957) were used as negative and positive
controls, respectively. Significant levels of CHS-A transgene
transcription were detected in both flowers and leaves of
C001 (Fig. 1A). Northern blot analysis showed that little to
no CHS-A mRNA accumulated in the flowers or leaves of
this line, and the steady-state mRNA levels of both the
CHS-A endogene and the CHS-A transgene have also been
shown to be very low by RT–PCR (Metzlaff et al. 1997).
The run-on data demonstrate that the mechanism of the
silencing in C001 is post-transcriptional, as was previously
shown in other transgenic petunia lines (Van Blokland et al.
1994, Stam et al. 1998).
There was no significant difference in the transcription
rate of the CHS-A endogene in C001 and C002 flower
tissues. However, differences were observed in the transcription rate of the CHS-A transgene in the two lines: only
a weak signal hybridizing to the NOS terminator sequence
was observed in C002 flower tissues, and no signal was
detected in C002 leaf tissues (Fig. 1A). This result shows
that transcription of the transgene was repressed during the
conversion from C001 to C002. This observation was
confirmed by the intensity of hybridization to the CHS-A
A
V26
639
C001
C002
J-type
(silenced) (revertant) (silenced)
pBS
CHS exon 1
CHS intron
Flower
NOS ter
Ubiquitin
pBS
CHS exon 1
Leaf
CHS intron
NOS ter
Ubiquitin
B
CHS-A endogene
Exon 1
Exon 2
Intron
CHS exon 1 probe
CHS intron probe
0.1 kb
CHS-A transgene
CaMV 35S pro
NOS T
CHS exon 1 probe
NOS terminator probe
Fig. 1 The production of white flowers in the transgenic petunia
plant is caused by PTGS, and transgene transcription is repressed in
the revertant. (A) Run-on transcription assay using isolated nuclei
from flower and leaf tissues of V26, C001, C002 and junction-type
(J-type) plants. Nuclei were isolated from flowers and leaves of
these plants, and used to synthesize radiolabeled run-on transcripts. The labeled RNA was hybridized to DNAs blotted on the
filter that contain pBluescript plasmid (negative control), exon 1 of
the CHS-A gene (hybridizes to transcripts from both the CHS-A
transgene and the CHS-A endogene), the intron of the CHS-A gene
(hybridizes to transcripts from the CHS-A endogene), the NOS
terminator (hybridizes to transcripts from the CHS-A transgene) and
the ubiquitin gene (positive control). For each material, the run-on
assay was repeated three times, and the results of one of the
experiments are shown. (B) Structure of the CHS-A endogene and
the CHS-A transgene and positions of probes for the run-on assay.
Exons of the CHS-A gene are indicated by hatched boxes. The
positions of probes are indicated by lines. A dotted line indicates a
portion of the CHS-A exon 1 probe that is hybridized with the
intron of the CHS-A endogene transcript but not with the CHS-A
transgene transcript.
exon 1 sequence: in C002, very similar hybridization
intensities to those observed for wild-type V26 flowers
and leaves were observed, indicating that the transgene did
not contribute to the labeled RNA run-on transcription
pool. Quantification of the radioactivity of hybridization
signals (CHS-A exon 1 vs. the ubiquitin gene) indicated that
the total transcription activity of the CHS-A genes in flower
tissues of C002 was reduced to 52.6 5.3% of the activity in
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
B
−
+
CHS-A transgene/tubulin
A
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0
5-azacytidin
C
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
D
−
+
Trichostatin A
−
+
5-azacytidin
CHS-A transgene/tubulin
flower tissues of C001. These data explain the results
of Northern blot and RT–PCR analyses (Metzlaff et al.
1997, Metzlaff et al. 2000) including the lack of CHS-A
transgene transcript in C002 leaf tissues where no transcriptional activity of the gene was detected (see Fig.1A).
A transcription run-on assay was also performed using
nuclei isolated from a transgenic plant line that produces
purple flowers with white centers of various sizes, which
is called a ‘junction’-patterned flower phenotype (see
Jorgensen 1995). This line contains a single-copy transgene,
which may possibly account for the less stable phenotype of
PTGS than in C001 that produces completely silenced
flowers (discussed below). The transcription rate of the
CHS-A transgene in the junction-type (J-type) plant was
slightly lower than the transcription rate in C001,
due possibly to the lower copy number of the gene, and
was apparently higher than that in C002 (Fig. 1A). This
result also indicates that the transgene transcription is
repressed in C002.
We suspected that the reversion to a purple-flowering
phenotype was epigenetic, since regenerated calli from
C002 leaves produced some white-flowering individuals
(R. P. Hellens et al. unpublished data). This result suggests
that rather than resulting from a mutation at the DNA
level, the change from C001 to C002 is reversible, most
probably involving a heritable epigenetic modification.
We therefore examined whether the level of mRNA from
the CHS-A transgene was affected by treatments of plants
with the demethylating agent 5-azacytidine, or with
trichostatin A, a specific inhibitor of histone deacetylase.
Trichostatin A is also known to cause demethylation in
Neurospora (Selker 1998) and plants (Lawrence et al. 2004),
as well as down-regulation of DNA methyltransferase in
human cells (Januchowski et al. 2006). Surface-sterilized
seeds of C002 were sown on a solid medium containing
20 mM 5-azacytidine or 2 mM trichostatin A. Twenty plants
were grown on the medium in a plate, and RNA was
extracted from whole seedlings of plants grown on a plate.
This experiment was repeated three times.
Changes in the levels of mRNAs from the CHS-A
endogene and the CHS-A transgene were analyzed by
real-time RT–PCR. The levels of these mRNAs were
quantified relative to the level of a-tubulin mRNA.
The level of the mRNA from the CHS-A transgene was
found to increase prominently in C002 plants after
treatment with 5-azacytidine, whereas the mRNA level of
the CHS-A endogene was unchanged (Fig. 2A, B). Similar
results were obtained when plants were treated with
trichostatin A (Fig. 2C, D). No PTGS was induced in
seedlings by these treatments, although the transcription of
the transgene appeared to be restored. This may be due to
the intrinsically low level of transcription of the CHS-A
endogene in plants of this developmental stage, and/or
CHS-A endogene/tubulin
Epigenetic changes suppressing PTGS
CHS-A endogene/tubulin
640
5.0
4.0
3.0
2.0
1.0
0.0
−
+
Trichostatin A
Fig. 2 Effects of 5-azacytidine and trichostatin A on the mRNA
levels of the CHS-A endogene and the CHS-A transgene in young
plants of C002. Real-time RT–PCR was conducted to analyze the
mRNA levels of the CHS-A endogene (A and C) and the CHS-A
transgene (B and D) in plants treated with 5-azacytidine (A and B)
and trichostatin A (C and D). The mRNA levels were quantified
relative to the mRNA level of the a-tubulin, and the relative values
of plants treated with 5-azacytidine or trichostatin A and untreated
control plants were compared. The value of untreated plants was
set at 1. The data represent the mean and standard errors obtained
from three replicates of the analysis.
partial restoration of transgene transcription, which may
not be sufficient to induce the RNA degradation reactions
of PTGS. In any case, the results indicate that the
mechanisms of transcriptional repression of the CHS-A
transgene in C002 involve epigenetic changes including
methylation of genomic DNA.
We next examined the methylation status of
the transgene promoter in C001 and C002 by bisulfite sequencing analysis. For each material, 20–22 clones obtained
from four independent PCR amplifications from bisulfitetreated DNA templates were sequenced. The data were
compiled in Fig. 3 and Table 1. The results indicated that
cytosine residues in the 298 to 47 region of the CaMV
35S promoter were more methylated in C002 (Fig. 3B) than
in C001 (Fig. 3A) at CpG, CpNpG and CpNpN sites.
Cytosine methylation was detected in 10/10, 7/7 and 38/51
instances of CpG, CpNpG (symmetrical positions) and
CpNpN (non-symmetrical positions) sequences, respectively, in the PCR products from C002. The frequency of
cytosine methylation was 73.6, 60.4 and 23.5% at CpG,
CpNpG and CpNpN sites, respectively, in C002. On the
other hand, the frequency was 8.0, 0 and 10.5% at CpG,
CpNpG and CpNpN sites, respectively, in C001 (Table 1).
The presence of more methylcytosine residues overall in the
Epigenetic changes suppressing PTGS
A
C001
641
CpG
1
CpNpG
CpNpN
0.75
0.5
0.25
0
−298
−248
−198
−148
C002
B
−98
CpG
1
As-1
CpNpG
−48
CpNpN
0.75
0.5
0.25
0
−298
C
−248
−198
−148
C002 (+ 5-azacytidine)
−98
CpG
1
As-1
CpNpG
−48
CpNpN
0.75
0.5
0.25
0
−298
D
−248
−198
−148
C002 (+ trichostatin A)
−98
CpG
1
As-1
CpNpG
−48
CpNpN
0.75
0.5
0.25
0
−298
−248
−198
−148
−98
As-1
−48
Fig. 3 DNA methylation status of the CaMV 35S promoter. Sequencing data of PCR products amplified from bisulfite-treated DNA have
been compiled. (A) C001 plants, (B) C002 plants, (C) C002 plants treated with 5-azacytidine, (D) C002 plants treated with trichostatin A.
The height of the vertical lines shows the frequency of methylcytosines at respective positions per total PCR clones sequenced. Red, green
and blue lines indicate frequencies of methycytosine at CpG, CpNpG and CpNpN sites, respectively. For C001, C002, 5-azacytidinetreated C002 and trichostatin A-treated C002 plants, 20, 22, 21 and 22 clones, were sequenced, respectively. Clones of each material
contain products of four independent PCR amplifications from bisulfite-treated DNA templates. Numbers below the line indicate
nucleotide positions relative to the transcription start site of the CaMV 35S promoter. The analyzed sequences cover the 298 to 47
region of the promoter. The position of two CpG sites in the as-1 element is indicated by arrows.
642
Table 1
Epigenetic changes suppressing PTGS
A summary of bisulfite sequencing analysis of the CaMV 35S promoter in C001 and C002 plants
Plant lines
and treatments
Frequency of cytosine methylation
No. of sites at which cytosine
methylation was detected
Frequency of
methylation
at two
CpG sites
in the as-1
element (%)
CpG
CpNpG
CpNpN
Total
CpG
CpNpG
CpNpN
Total
C001
8.0%
(16/200)
0%
(0/140)
10.5%
(107/1020)
9.0%
(123/1360)
3/10
0/7
21/51
24/68
25.0
C002
73.6%
(162/220)
60.4%
(93/154)
23.5%
(264/1122)
34.7%
(519/1496)
10/10
7/7
38/51
55/68
81.8
C002
(þ 5-azacytidine)
62.9%
(132/210)
55.8%
(82/147)
15.5%
(166/1071)
26.6%
(380/1428)
10/10
6/7
23/51
39/68
64.3
C002
(þ trichostatin A)
44.1%
(97/220)
46.1%
(71/154)
13.0%
(146/1122)
21.0%
(314/1496)
10/10
7/7
29/51
46/68
50.0
Data obtained from 20, 22, 21 and 22 clones of C001, C002, 5-azacytidine-treated C002 and trichostatin A-treated C002 plants,
respectively, were compiled. Clones of each material contain products of four independent PCR amplifications from bisulfite-treated
DNA templates.
promoter region in C002 is consistent with the notion that
cytosine methylation in the promoter interferes with
transcription directly or indirectly.
The frequency of cytosine methylation decreased when
C002 plants were treated with 5-azacytidine (Fig. 3C) or
trichostatin A (Fig. 3D). The frequencies of cytosine
methylation at CpG/CpNpG/CpNpN sites were reduced
to 62.9%/55.8%/15.5% and 44.1%/46.1%/13.0% by treatments with 5-azacytidine and trichostatin A, respectively.
The CaMV 35S promoter contains a 21 bp element (83
to 63) designated as the activation sequence-1 (as-1) that
binds basic leucine zipper-type transcription factors
(Benfey and Chua 1990). The binding of nuclear factors
to the as-1 element is influenced by cytosine methylation
in vitro (Kanazawa et al. 2007). The frequency of CpG
methylation at 79 and 67 in the as-1 element was higher
in C002 (81.8%) than in C001 (25.0%), and was also
reduced to 64.3 and 50.0% by treatments with 5-azacytidine
and trichostatin A, respectively. These results are consistent
with the restoration of the mRNA levels of the CHS-A
transgene by these agents (see Fig. 2), and support the
notion that methylation of cytosines in the promoter is
involved in the transcriptional repression of the CHS-A
transgene in C002. It appeared that 5-azacytidine was
slightly more effective than trichostatin A in the restoration
of the mRNA level, but was less effective than trichostatin
A in demethylation. This implies that the transcriptional
repression may also be mediated by chromatin modification
rather than DNA methylation alone, and the mechanisms
of the transcriptional restoration by these agents may not
be identical.
To examine whether the structure of the transgene locus
has a specific feature that may account for PTGS of the
CHS-A genes and/or transcriptional repression of
the CHS-A transgene, we analyzed the genomic DNA
of the transgene locus (Fig. 4). Using a Southern blot
strategy previously reported (Cluster et al. 1996), two
sequences, the neomycin phosphotransferase II (NptII)
gene and the CaMV 35S promoter, were used as probes for
the analysis. This analysis distinguishes structural organizations of the Agrobacterium-transferred DNA in the genome
including three possible orientations of the DNA copies as
repeated sequences (Fig. 4C–E) as well as those dispersed in
the genome (Fig. 4B). Hybridization signals of 6.6 and 7.2 kb
were detected with the NptII probe when C001 DNA was
digested with HindIII and XbaI, respectively. In addition,
two signals were detected with the CaMV 35S promoter
probe for both HindIII- and XbaI-digested DNA (Fig. 4F).
These results indicate that the Agrobacterium-transferred
DNA (Fig. 4A) is integrated in the genome as an inverted
repeat centered on the left border (Fig. 4D).
We also looked for the presence or absence of siRNA,
a hallmark of the occurrence of RNA silencing. A Northern
blot analysis of the small RNA fraction revealed that
siRNAs corresponding to the CHS-A coding sequence
accumulated in the C001 line, but not the C002 line
(Fig. 4G). In contrast, no siRNA corresponding to the
CaMV 35S promoter sequence was detected in either C001
or C002 lines (data not shown).
The results of the transcription run-on assays
confirmed previous observations that showed that the
white phenotype of C001 flowers is caused by PTGS
Epigenetic changes suppressing PTGS
A
F
NOS pro
1 kb
OCS ter NOS ter
NptII
CHS-A
LB
LB
aI
Xb
II
I
nd
Hi
aI
Xb
35S pro
HindIII XbaI
B
III
nd
Hi
643
kb
19.3
RB
7.7
6.2
RB
4.2
XbaI
3.5
HindIII
C
LB
RB
XbaI
2.7
LB
5.2 kb XbaI
HindIII 5.8 kb HindIII
D
RB
LB
Probe: NptII
G
E
XbaI
7.2 kb
XbaI
HindIII
6.6 kb
HindIII
LB
RB LB
XbaI
HindIII
6.2 kb
Probe: 35S pro
RB
6
V2
C0
01 002
C
siRNA
RB
XbaI
20 mer
tRNA +
5S rRNA
6.2 kb HindIII
Fig. 4 Structural organization of Agrobacterium-transferred DNA and accumulation of CHS-A siRNA in the transgenic petunia plants.
(A) Structure of transgenes used for transformation. Restriction sites of HindIII and XbaI are indicated below the map. Directions of
transcription are indicated by arrows. NOS pro, nopaline synthase promoter; OCS ter, octopine synthase terminator; LB, left border;
RB, right border. (B–E) Possible organization of Agrobacterium-transferred DNA in the petunia genome and expected sizes of HindIII- or
XbaI-digested DNA fragments. (B) A dispersed copy; (C) a right border-centered inverted repeat; (D) a left border-centered inverted repeat;
(E) a direct repeat. White and black arrows indicate the CHS-A transgene and the NptII gene, respectively. Positions of sequences
hybridizing with the probes for the CaMV 35S promoter and the NptII gene are indicated by open and filled boxes, respectively, below the
arrows. The expected sizes of HindIII- or XbaI-digested DNA fragments are shown. The sizes of other fragments depend on the distance to
the nearest flanking genomic restriction site. (F) Southern blot analysis of C001 DNA. HindIII- or XbaI-digested DNA was hybridized with
probes specific for the NptII gene or the CaMV 35S promoter sequences. Note that the observed hybridization profiles were consistent with
the left border-centered inverted repeat structure (D): the NptII probe hybridized with a 6.6 kb HindIII-digested fragment and a 7.2-kb XbaIdigested fragment, and the CaMV 35S promoter probe hybridized with two HindIII-digested fragments and two XbaI-digested fragments.
(G) Northern blot analysis of low molecular weight RNA of flower tissues from V26, C001 or C002, probed for the CHS-A gene. A DNA
oligonucleotide (20-mer) was also loaded as a size control; its position is indicated on the left. Ethidium bromide-stained tRNA and 5S
rRNA bands are shown as a loading control.
(Metzlaff et al. 1997, Metzlaff et al. 2000). Despite the
extensive understanding of proteins involved in PTGS,
primary factor(s) that are responsible for initiation of
transgene-mediated silencing have not been fully understood. In particular, PTGS caused by transcripts from
inverted repeat DNA remains one of the least understood
RNA silencing processes in plants (reviewed by Brodersen
and Voinnet 2006). The different transcription rates in C001
and C002 flowers (see Fig. 1) appear to be responsible for
determining whether PTGS is triggered. This resembles an
observation that the transgene transcription rate was
reduced in revertants of co-suppressed nitrate reductase
genes in tobacco (Vaucheret et al. 1997). Although it is
unknown why a partial reduction in transgene transcription
leads to a complete loss of PTGS, this phenomenon is
consistent with the RNA threshold model (Smith et al.
1994), in which mRNA degradation is triggered when the
mRNA accumulates to a certain level. This notion is similar
to that in a report in which the frequency of PTGS
occurrence in petunia is correlated with the strength of the
promoter of a transgene (Que et al. 1997). On the contrary,
lack of correlation between the transcription rate of a
transgene and induction of PTGS has also been reported
based on comparisons between independently transformed
644
Epigenetic changes suppressing PTGS
plant lines (Van Blokland et al. 1994, English et al. 1996).
In the present study, we compared C001 and C002 plants
whose genetic backgrounds (e.g. locus and copy number of
transgene) are the same. This eliminated possible genetic
differences between materials, and the results consequently
provided strong support for the threshold model. Neither
swapping the CaMV 35S promoter with a weaker promoter
nor swapping the NOS terminator with a less stable
terminator caused CHS-A PTGS in petunia (R. P. Hellens
et al. unpublished data). In light of these results, the present
study suggests that the triggering of PTGS depends on
the amount of CHS-A transgene transcript that has
accumulated and/or the transcription rate of the CHS-A
transgene at a certain stage of development.
An inverted repeat structure of a gene has often been
implicated in efficient silencing of the gene (reviewed by
Muskens et al. 2000). It is possible that the repeated
structure of the CHS-A transgene may account for the
stable induction of PTGS that causes production of
completely white flowers in C001 if read-through transcription occurs over the repeated transgene copies and,
consequently, produces dsRNA. In this scenario, de novo
methylation of the corresponding coding sequence via an
RNA intermediate (reviewed by Wassenegger 2000, Matzke
et al. 2004) is conceivable. A secondary and more efficient
long-term response may be to spread this methylation
(see Wassenegger 2000, Fojtova et al. 2003, Mishiba et al.
2005) to promoter regions, which results in the activation
of TGS. RNA-directed DNA methylation (RdDM) of
the transgene promoter may also occur directly if unexpected transcription from upstream of the promoter occurs,
together with the read-through transcription. These transcription events may cause the production of dsRNA
corresponding to the CaMV 35S promoter sequence.
DsRNA corresponding to even a portion of the promoter
might induce TGS: we have demonstrated that a 120 bp
portion of the CaMV 35S promoter cloned in an RNA virus
vector can induce both RdDM and TGS of a reporter gene
driven by the promoter (Otagaki et al. 2006). Although
we have not detected siRNA corresponding to the CaMV
35S promoter sequence, it is possible that extremely
small amounts of siRNA corresponding to the CaMV 35S
promoter sequence are present. RdDM mediated by
siRNAs whose level is below the limit of detection of
a standard gel-blot method has also been postulated in the
silencing system of PAI genes in Arabidopsis thaliana (for
a review, see Mathieu and Bender 2004). In addition
to these RNA-mediated mechanisms, de novo methylation
in the transgene promoter sequences may also be caused
by DNA–DNA interaction between inverted repeats of the
transgene (see Muskens et al. 2000).
Bisulfite sequencing analysis revealed an elevated
frequency of cytosine methylation in the transgene
promoter in C002 (Fig. 3). Unlike the methylation status
of most of the cytosine residues analyzed in the present
study, a slightly higher level of methylation was detected
in C001 at a restriction site located far upstream (1.2 kb)
of the promoter (O’Dell et al. 1999). One plausible
explanation for the hypermethylation at the site may be
that RNA-directed de novo methylation preferentially
occurred at limited cytosines as a consequence of transgene
transcription in C001. It is also possible that the methylated
state at the site was established and maintained in C001,
whereas the state was destabilized in C002. Although
whether the methylation status at the site reflects methylation frequency in its surrounding region remains to be
examined, these results suggest that cytosine methylation in
the distal region is not directly involved in transcriptional
repression. In contrast, a higher frequency of cytosine
methylation overall within 0.3 kb upstream of the transcription start site of the promoter has been detected in various
transformed plants that exhibit TGS (see, for example,
Meyer et al. 1994) as observed in the present study.
We presume that the cytosine methylation that primarily
affects promoter activity may be limited to the proximal
region of the promoter in C001 and C002.
The observed changes in DNA methylation status may
be closely associated with changes in chromatin structure.
A model of TGS suggests that a methylcytosine-binding
protein (such as MeCP2) recruits Sin3 and histone
deacetylases to the chromatin containing methylated DNA
to which it is bound, subsequently leading to an alteration
in the chromatin structure (reviewed by Kass et al. 1997,
Ashraf and Ip 1998). A recent report also suggested that
heterochromatin formation marked by site-specific histone
modifications may in turn lead to DNA methylation by
DNA methyltransferase recruited by heterochromatin
protein 1 (HP1; Jackson et al. 2002). It is known that
heterochromatin-mediated silencing can spread (Noma
et al. 2004), although a direct relationship between
spreading of DNA methylation and heterochromatin
formation has not yet been understood.
Our findings have shown that epigenetic change(s)
occurred during the process that resulted in the generation
of the C002 line from the C001 line, as evidenced by the
difference in the methylation statuses of the transgene
promoter as well as the restoration of transgene expression
with either 5-azacytidine or trichostatin A in C002 plants.
The results demonstrate that at least three events were
involved in the change: (i) epigenetic changes including cytosine methylation of the transgene promoter;
(ii) repression of transgene transcription; and (iii) suppression of the specific degradation of the CHS-A mRNA. If the
epigenetic changes occur, such as in C002, there is a very
low rate of transcription of the transgene, and PTGS can
no longer initiate (Fig. 5). Somatic conversion from PTGS
Epigenetic changes suppressing PTGS
CHS-A transgene
Epigenetic
changes
C ··· C
m5
m5
C ··· C
High rate of
transcription
PTGS
Low rate of
transcription
(co-suppression)
no PTGS
White flower
Purple flower
Fig. 5 Schematic diagram of the generation of the C002 revertant
line from the C001 PTGS line. In C001 plants, the CHS-A transgene
is transcribed at a high rate that causes PTGS (co-suppression).
Epigenetic changes that involve cytosine methylation in the CaMV
35S promoter occurred during the generation of C002, in which the
CHS-A transgene is transcribed at a very low rate that does not
cause PTGS.
to TGS may explain at least some of the various phenotypes
related to co-suppression and the changeable gene silencing
phenotypes sometimes observed during development
(see Jorgensen 1995). A similar change in a transgene
conferring drug resistance was observed in a long-term
tobacco callus culture (Fojtova et al. 2003). Our present
results indicate that conversion from PTGS to TGS can
occur during the growth of a plant as a consequence of the
presence of a transgene homologous to an endogenous gene
in the plant genome.
Materials and Methods
Transgenic petunia lines C001 and C002 (Metzlaff et al. 1997,
O’Dell et al. 1999, Kanazawa et al. 2007) were used. These plants
were obtained by the transformation of the wild-type plant V26
with the CHS-A transgene controlled by the CaMV 35S promoter
and the NOS terminator (Napoli et al. 1990). C001 and C002 refer
to pedigree numbers of a transformant CHS38 (Jorgensen et al.
1996), which was originally designated 218.38 (Napoli et al. 1990),
and have also been designated CHS38W and CHS38P, respectively
(R. A. Jorgensen, personal communication). C001 shows a silenced
phenotype with white flowers, while C002 is a spontaneous
revertant plant from C001 with purple flowers. A junction-type
plant is a descendant of the CHS223 line (Cluster et al. 1996,
Jorgensen et al. 1996) and contains a single-copy CHS-A
transgene. Flowers (35–45 mm long) and young leaves of these
plants were used for run-on transcription assays. The isolation of
nuclei and transcription run-on assay were performed using the
645
mini-scale method described by Kanazawa et al. (2000). For each
material, the experiment was repeated three times. For quantitative
assays, membranes were exposed to imaging plates (Fuji Film,
Tokyo, Japan) and radioactivity was quantified with a Bio-imaging
analyzer (Fujix BAS 2000; Fuji Film).
For treatment of plants with 5-azacytidine or trichostatin A,
surface-sterilized seeds of C002 were sown on plates containing
half concentration standard MS medium (Murashige and Skoog
1962) that contained 10 g l1 sucrose. The pH of the medium was
adjusted to 5.7 before autoclaving. Media were solidified with
0.8% (w/v) agar. 5-Azacytidine or trichostatin A was added to the
medium just before plates were poured. 5-Azacytidine (dissolved
in water) was added to a final concentration of 20 mM. Trichostatin
A (dissolved in methanol) was added to a final concentration of
2 mM. After sowing seeds, plates were incubated at a photoperiod
of 16 h light/8 h dark, 248C for 1 month. We found that these
treatments slightly affected the development of plants (e.g. delay
in germination). Total RNA was isolated from plants as described
by Napoli et al. (1990), except that we removed genomic DNA
from the RNA fraction using DNase I (TAKARA BIO INC.,
Otsu, Japan). A 1 mg aliquot of the total RNA was used as the
template for cDNA synthesis. The cDNA synthesis reaction
mixture was prepared by mixing 4 ml of 5 reaction buffer
[250 mM Tris–HCl (pH 8.3), 375 mM KCl, 15 mM MgCl2], 2 ml
of 0.1 M dithiothreitol (DTT), 1 ml of 100 mM oligo(dT)20 primer,
4 ml of 2.5 mM dNTPs, the total RNA solution, and water to a final
volume of 19 ml. The mixture was heated at 658C for 5 min and
rapidly cooled on ice. After the addition of 1 ml of reverse
transcriptase (M-MLV, Invitrogen, Carlsbad, CA, USA), the
cDNA synthesis was performed at 428C for 1 h. The reverse
transcriptase was inactivated by heating the sample at 998C for
1 min. Real-time RT–PCR was carried out using a 1 ml aliquot of
the reaction mixture and SYBR Premix Ex Taq kit (TAKARA
BIO INC.) with a DNA Engine Opticon 2 System (MJ Research,
Waltham, MA, USA). The PCR cycle was 958C for 30 s, 588C for
30 s, 728C for 30 s and 788C for 2 s. This cycle was repeated
40 times. Fluorescence quantification was carried out before and
after the incubation at 788C to monitor for the formation of
primer dimers. A reaction mixture without reverse transcriptase
was used as a control to confirm that no amplification
occurred from genomic DNA contaminates in the RNA sample.
Primers for RT–PCR were as follows: CHS-A endogene,
50 -GATACTTACACTTGTCACGTA-30 (‘995’) and 50 -GTGCTT
TGATCAACACAGTTTG-30 (‘2350’); CHS-A transgene, 50 -ACA
CGCTCGAGCTCATTTC-30 (‘996’) and primer ‘2350’; a-tubulin,
50 -GCCACCATCAAGACCAAGC-30 (‘tubulin F’) and 50 -ACCT
CAGCAACACTGGTTGA-30 (‘tubulin R’). Primers ‘995’ and
‘996’ anneal the 50 -untranslated sequences of the CHS-A endogene
and the CHS-A transgene, respectively, and thereby allow
amplification of transcripts specific to the CHS-A endogene and
the CHS-A transgene, respectively.
For analysis of DNA methylation by bisulfite sequencing,
DNA was isolated from young seedlings of C001, C002,
5-azacytidine-treated C002 and trichostatin A-treated C002
plants using a Nucleon PhytoPure DNA extraction kit
(Amersham Biosciences, Piscataway, NJ, USA). The protocol
of bisulfite treatment in this study is based on the methods of
Frommer et al. (1992) and Paulin et al. (1998). DNA was cleaved
with the restriction enzyme HindIII, extracted with phenol/
chloroform, and precipitated by ethanol. The cleaved DNA was
alkali denatured in 0.3 M NaOH at 378C for 20 min. Denatured
DNA was incubated in a total volume of 600 ml with freshly
prepared 6.4 M urea/3.1 M sodium bisulfite/0.5 mM hydroquinone,
646
Epigenetic changes suppressing PTGS
pH 5.0, at 608C for 30 h under mineral oil. DNA was then
recovered by a Qiaquick PCR purification kit (Qiagen GmbH,
Hilden, Germany). NaOH was added to the DNA solution to
a concentration of 0.3 M and then incubated at 378C for 20 min.
Glycogen and ammonium acetate were added to the solution to
final concentrations of 0.16 mg ml1 and 2.64 M, respectively.
DNA was then precipitated with ethanol and dissolved in 20 ml
of TE (pH 8.0) buffer. Two rounds of PCR were carried out
for amplification. The first round of the PCR was carried out in
a total volume of 50 ml using 1 ml of bisulfite-treated DNAs as a
template. The second round of the PCR was carried out using
a 1 ml aliquot of the reaction mixture from the first round of PCR.
To amplify target sequences, primers ‘35S –346F bisulfite T’
(50 -TATTGAGATTTTTTAATAAAGGGTAA-30 ) and ‘35S þ 1R
bisulfite A’ (50 -TCCTCTCCAAATAAAATAAACTTC-30 ) were
used for the first round PCR, and primers ‘35S –323F bisulfite T’
(50 -TAATATTTGGAAATTTTTTTGGATT-30 ) and ‘35S –21R
bisulfite A’ (50 -TTCCTTATATAAAAAAAAAATCTTAC-30 )
were used for the second round of PCR. The PCR cycling
conditions were: 948C for 30 s, 528C for 30 s and 728C for 1 min.
This cycle was repeated 40 times, and the reaction mixture was then
further incubated at 728C for 10 min. The PCR products were
cloned into the pGEM-T Easy vector (Promega, Madison, WI,
USA) and were subjected to sequence analysis. As a control to
ensure that bisulfite treatment was complete, DNA isolated from
Arabidopsis thaliana leaves was simultaneously treated. A region of
the A. thaliana ASA1 gene that is not methylated was amplified as
previously reported (see Kusaba et al. 2003). All five cloned
sequences of PCR products showed complete conversion of
cytosines to thymidines.
The structural organization of transgenes in the petunia
genome was analyzed by DNA gel-blot analysis. Total DNA was
isolated from leaves as described by Kanazawa and Tsutsumi
(1992). DNA was digested with restriction enzymes and fractionated by electrophoresis on a 1% (w/v) agarose gel. After
electrophoresis, the DNA was transferred to a nylon membrane
(Hybond Nþ, Amersham Biosciences) and allowed to hybridize
with labeled probes. Labeling of probes, hybridization, washing of
membranes and detection of signals were carried out using the
Alkphos Direct nucleic acid labeling and detection system
(Amersham Biosciences). The CaMV 35S promoter sequence
amplified by PCR using primers ‘35S –345F’ and ‘35S þ 1R’
(Kanazawa et al. 2007) and the NptII gene sequence amplified by
PCR using primers 50 -GAATGAACTCCAAGACGAGG-30 and
50 -AAGAACTCGTCAAGAAGGCG-30 were labeled for use as
hybridization probes. The CHS-A siRNAs were detected by
Northern blot analysis of the small RNA fraction using a
digoxigenin-labeled probe as described by Goto et al. (2003).
Acknowledgments
We would like to express our appreciation to Cathie Martin
for giving us helpful advice and constructive comments throughout
this research and on the manuscript. We are grateful to Richard B.
Flavell for valuable suggestions and financial support, and Michael
Metzlaff for helpful discussions. We are also grateful to Peter
Shaw, Alison Beven and Nadia S. Al-Kaff for technical advice on
transcription run-on assay, Tomoki Matsuyama, Mineo Senda and
Makoto Kusaba for technical advice on bisulfite sequencing
analysis, Chikara Masuta and Kazunori Goto for valuable
information on virus-induced gene silencing, Andrea Davies,
Akiko Takahashi, Maiko Koseki and Shungo Otagaki for
technical assistance, Sayuri Tsukahara for her help in constructing
a bisulfite sequencing system, and Neal Gutterson and Richard
Jorgensen for plant materials. This work was supported in part by
Grants-in-Aid for Scientific Research from the Ministry of
Education, Culture, Sports, Science and Technology of Japan.
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(Received October 30, 2006; Accepted February 17, 2007)