plosPLoS GenetplosgenPLoS Genetics1553-73901553-7404Public Library of ScienceSan Francisco, USAPGENETICS-D-10-0026510.1371/journal.pgen.1002055Research ArticleBiologyGeneticsEpigeneticsDNA modificationHistone modificationChromatinRNA interferenceGene expressionDNA modificationHistone modificationChromatinRNA interferencePlant geneticsGenomicsGenome expression analysisMolecular cell biologyTransposonsPlant sciencePlant geneticsPlant genomicsGenetics and GenomicsMolecular BiologyPlant BiologyArabidopsis HDA6 Regulates Locus-Directed
Heterochromatin Silencing in Cooperation with MET1HDA6 Regulates Gene Silencing with
MET1ToTaiko Kim12KimJong-Myong1MatsuiAkihiro1KuriharaYukio1MorosawaTaeko1IshidaJunko1TanakaMaho1EndoTakaho3KakutaniTetsuji4ToyodaTetsuro3KimuraHiroshi5YokoyamaShigeyuki2ShinozakiKazuo6SekiMotoaki17*Plant Genomic Network Research Team, RIKEN
Plant Science Center, Yokohama, Kanagawa, JapanGraduate School of Science, The University of
Tokyo, Tokyo, JapanBioinformatics and Systems Engineering
Division, RIKEN Yokohama Institute, Yokohama, Kanagawa, JapanDepartment of Integrated Genetics, National
Institute of Genetics, Mishima, Shizuoka, JapanGraduate School of Frontier Biosciences, Osaka
University, Suita, Osaka, JapanGene Discovery Research Group, RIKEN Plant
Science Center, Yokohama, Kanagawa, JapanKihara Institute for Biological Research,
Yokohama City University, Yokohama, Kanagawa, JapanFerguson-SmithAnne C.EditorUniversity of Cambridge, United Kingdom* E-mail: mseki@psc.riken.jp
Conceived and designed the experiments: TKT JMK MS. Performed the
experiments: TKT JMK JI MT. Analyzed the data: TKT JMK AM YK TM TE TT SY KS
MS. Contributed reagents/materials/analysis tools: TK HK. Wrote the paper:
TKT JMK MS.
The authors have declared that no competing interests exist.
42011284201174e1002055311201012320112011To et alThis is an open-access article distributed under the
terms of the Creative Commons Attribution License, which permits unrestricted use,
distribution, and reproduction in any medium, provided the original author and
source are credited.
Heterochromatin silencing is pivotal for genome stability in eukaryotes. In
Arabidopsis, a plant-specific mechanism called
RNA–directed DNA methylation (RdDM) is involved in heterochromatin
silencing. Histone deacetylase HDA6 has been identified as a component of such
machineries; however, its endogenous targets and the silencing mechanisms have
not been analyzed globally. In this study, we investigated the silencing
mechanism mediated by HDA6. Genome-wide transcript profiling revealed that the
loci silenced by HDA6 carried sequences corresponding to the RDR2-dependent
24-nt siRNAs, however their transcript levels were mostly unaffected in the
rdr2 mutant. Strikingly, we observed significant overlap of
genes silenced by HDA6 to those by the CG DNA methyltransferase MET1.
Furthermore, regardless of dependence on RdDM pathway, HDA6 deficiency resulted
in loss of heterochromatic epigenetic marks and aberrant enrichment for
euchromatic marks at HDA6 direct targets, along with ectopic expression of these
loci. Acetylation levels increased significantly in the hda6
mutant at all of the lysine residues in the H3 and H4 N-tails, except H4K16.
Interestingly, we observed two different CG methylation statuses in the
hda6 mutant. CG methylation was sustained in the
hda6 mutant at some HDA6 target loci that were surrounded
by flanking DNA–methylated regions. In contrast, complete loss of CG
methylation occurred in the hda6 mutant at the HDA6 target loci
that were isolated from flanking DNA methylation. Regardless of CG methylation
status, CHG and CHH methylation were lost and transcriptional derepression
occurred in the hda6 mutant. Furthermore, we show that HDA6
binds only to its target loci, not the flanking methylated DNA, indicating the
profound target specificity of HDA6. We propose that HDA6 regulates
locus-directed heterochromatin silencing in cooperation with MET1, possibly
recruiting MET1 to specific loci, thus forming the foundation of silent
chromatin structure for subsequent non-CG methylation.
Author Summary
Eukaryotes are defended from potentially harmful DNA elements, such as
transposons, by forming inactive genomic structure. Chromatin, which consists of
DNA and histone proteins, is densely packed in the silent structure, and
chromatin chemical modifications such as DNA methylation and histone
modifications are known to be essential for this packing. In plants, small RNA
molecules have been thought to trigger DNA methylation and resulting silent
chromatin formation. We revealed that elimination of specific histone
modifications concomitant with DNA methylation is pivotal for the silent
chromatin. Furthermore, the histone deacetylase was shown to have more profound
target specificity than the DNA methyltransferase and is required for
locus-directed DNA methylation, implying the involvement of the histone
deacetylase for targeting the DNA methyltransferase to specific places on the
genome. These proteins and their functions for gene silencing are evolutionarily
conserved in higher eukaryotes, and several proteins involved in small RNA
production are plant-specific. Thus, we present a hypothesis that the plant
genome may build the protecting foundation by the conserved genome surveillance
in eukaryotes, and the reinforcing machinery involving small RNAs could be
evolutionarily added to the plant heterochromatin silencing system.
This research was supported by a grant from the RIKEN Plant Science Center (to
MS) and from Grants-in-Aid for Scientific Research on Priority Areas (no.
21027033) from the Ministry of Education, Culture, Sports, Science, and
Technology of Japan (to MS). The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the
manuscript.Introduction
Chromatin modification is epigenetic information that has evolved in diverse
eukaryotes adding another layer of information to the DNA code. In higher
eukaryotes, histone modification and DNA methylation are involved in numerous
biological processes such as development, regeneration, and oncogenesis [1], [2]. In addition, the
eukaryotic genome has evolved epigenetic mechanisms to silence potentially harmful
transposable elements (TEs) and the repetitive elements that constitute a large
proportion of the genome [3]. Heterochromatin formation, a striking function of the
eukaryotic genome, is intricately controlled through repressive histone modification
and DNA methylation [4]. Thus, mutations that affect the status of chromatin
structure often result in strong phenotypic alterations or inviability, because of
aberrant regulation of gene expression or distorted genome stability [5]–[8].
The flowering plant, Arabidopsis thaliana, is a model organism
particularly suited for epigenetic research due to the availability of viable and
heritable null mutants of histone modifying enzymes and DNA methyltransferases.
Recent genome-wide studies on epigenetic marks of gene silencing in plants have
focused on DNA methylation or repressive histone methylations [9]–[13]. Few studies, however, have
focused on histone deacetylation, which is crucial for epigenetic regulation in
eukaryotes [14],
[15].
Investigating histone deacetylation and DNA methylation in
Arabidopsis could contribute not only to our understanding of
plant biology, but also to a broad range of essential biological processes in
mammals and therapeutic applications in humans [16], [17].
Gene silencing has been investigated extensively in Arabidopsis.
Plants have evolved gene silencing machinery called RNA-directed DNA methylation
(RdDM). Plant-specific RNA POLYMERASE IV (Pol IV), RNA-DEPENDENT RNA POLYMERASE 2
(RDR2) and DICER-LIKE 3 (DCL3) are involved in the production of 24-nt small
interfering RNAs (siRNAs) that guide DNA methyltransferases, DOMAINS REARRANGED
METHYLTRANSFERASES 1/2 (DRM1/2), to the corresponding genomic DNA for de
novo DNA methylation in all cytosine contexts (CG, CHG, CHH; H: A, T,
or C; [18]).
METHYLTRANSFERASE 1 (MET1), a homolog of mammalian DNMT1, is primarily responsible
for the maintenance of genome-wide CG methylation [19]–[22]. KRYPTONITE (KYP), a member of
the Su(var)3–9 class of histone methyltransferases, contributes an epigenetic
mark of constitutive heterochromatin, histone H3 Lys 9 dimethylation (H3K9me2) [23], [24].
CHROMOMETHYLASE 3 (CMT3), a plant-specific DNA methyltransferase, maintains CHG
methylation via H3K9me2 dependence mediated by KYP [23], [25], [26]. Histone Deacetylase 6
(HDA6), a homolog of yeast RPD3 and mammalian HDAC1, is involved in gene silencing
and RNA-directed DNA methylation [27]–[30].
Of the 16 Arabidopsis histone deacetylases [31], the importance of HDA6 in gene
silencing was discovered by identification of HDA6 in three
independent genetic screens of gene silencing [27], [32], [33]. In each case,
hda6 mutant plants lacking histone deacetylase activity
(sil1, axe1, and rts1) were
shown to exhibit reactivation of transcription on target transgenes. Analyses of the
endogenous function of HDA6 have been limited, thus far, to the regulation of
chromatin at repetitive sequences such as rDNA loci [28], [30], [34], [35], transposable elements and
centromeric satellite repeats [29], [36]. However, the positions of the loci silenced by HDA6 have
yet to be determined genome-wide.
Various effects of the hda6 mutations on cytosine methylation have
been observed previously. Several transposable elements were hypomethylated in
sil1[36]. Reduction
of DNA methylation has been reported for the siRNA-directed NOS promoter in
rts1, predominantly at CG and CHG sites [27]. Similarly, a reduction in CG
and CHG methylation was observed in axe1-5, sil1,
and rts1 mutants at rDNA repeats, although the demethylation was
much less than that observed in the DNA hypomethylation mutant ddm1[28], [30]. In contrast to
these observations, a drastic reduction in CHG methylation, but not CG methylation,
was observed in a Sadhu-type transposable element in axe1-5[37], and 5S
rDNA in sil1[35].
Furthermore, few changes in DNA methylation were observed in the centromeric repeats
or transgene region in sil1, although their silencing was lost
[29], [38]. These various
effects of the hda6 mutations on DNA methylation might be due to
locus dependence rather than differences in the mutations themselves, because
similar effects were observed between the mutants [28], [30]. Because previous studies have
focused on only a few specific loci, precisely how the hda6
mutation influences DNA methylation in general remains obscure. Therefore, a
genome-wide analysis of HDA6 target loci is vital to improve our understanding of
the mechanistic basis for HDA6-mediated gene silencing via DNA methylation and
histone modification.
In this study, aimed at understanding the silencing mechanism mediated by HDA6, we
identified HDA6 transcriptionally repressed loci across the genome and determined
the direct targets of HDA6. We also studied the regulation mechanisms involved in
histone modification and DNA methylation on HDA6 direct targets. Our data show that
the hda6 mutation causes loss of heterochromatic marks and aberrant
enrichment for euchromatic epigenetic marks at HDA6 direct targets. Furthermore, we
present evidence that the upregulated loci in hda6 overlapped with
those in met1, and that the hda6 mutation causes
the complete loss of DNA methylation on some HDA6 target loci. These results suggest
that a strong functional connection between HDA6 and MET1 exists. Remarkably,
hypomethylation only occurred in hda6 on the HDA6 target loci where
surrounding MET1 targets were absent. We propose, therefore, that HDA6 is required
for gene silencing and that it acts in cooperation with MET1 to build the
infrastructure of heterochromatin.
ResultsGenome-Wide Identification of Loci Derepressed in
axe1-5
To identify target loci for HDA6 binding in the Arabidopsis
genome, we first performed a genome-wide comparison of RNA accumulation between
the wild-type plant (DR5) and the hda6 mutant,
axe1-5[33], using a
whole-genome tiling array. This approach identified 157 statistically
significant loci that were transcriptionally upregulated in
axe1-5 compared with wild-type plants (>3 fold,
p-initial<10−6, FDR α = 0.05)
(Figure 1A and 1B).
RT–PCR of a random selection of these loci was used to confirm their
up-regulation in the axe1-5 mutant (Figure S1).
Among these loci, nearly half (81 genes; see Table S1)
were annotated by the Arabidopsis Genome Initiative (http://www.arabidopsis.org/; hereafter referred to as AGI
genes). The other half (76 genes; Table S2) were intergenic non-AGI annotated
transcriptional units (non-AGI TUs) identified using the ARTADE program [39] (Figure 1B). It is noteworthy
that only a small fraction of transcripts (5 loci: 3% of all
differentially expressed loci) were classified as having reduced levels of
expression in axe1-5 (Figure 1A, Table S3).
We consistently found that the loci upregulated in axe1-5 were
strongly silenced in wild-type plants (Figure 1C) and consisted predominantly of TE
fragments and genes for unknown proteins (79%) (Figure 1D). A survey of the TE fragments
[40],
mapping on or around the loci upregulated in axe1-5 (from 1 kb
upstream to 1 kb downstream), showed that a significant number of the fragments
(342 TE fragments) were located on or around such loci (Table S1
and S2).
These results show that HDA6 regulates gene silencing on a genome-wide
scale.
10.1371/journal.pgen.1002055.g001
HDA6 is required for genome-wide gene silencing of TE fragments and
other silenced loci.
Transcript profiling of the hda6 mutant,
axe1-5, using a tiling array revealed that the
hda6 mutation resulted in derepression of 157 loci
including TE fragments and other silenced regions. (A) The fractions of
the loci upregulated (orange) or downregulated (grey) in
axe1-5. The 157 upregulated loci accounted for
97% of differentially expressed loci. (B) The fraction of AGI
genes (red) and non-AGI TUs (blue) out of the loci upregulated in
axe1-5. (C) The fraction of silenced loci (signal
intensity <500) in wild-type plants (grey) out of the loci
upregulated in axe1-5. (D) Functional classification of
upregulated AGI genes in axe1-5. TE fragments (red;
37%); genes for unknown proteins (orange; 42%); ncRNA
(yellow; 6%); genes for known proteins (green; 15%).
HDA6-Mediated Gene Silencing Is Mostly Independent of the RdDM
Components
Forward genetic screens for plants deficient in RNA-mediated transcriptional
silencing identified HDA6 as an essential component of the RdDM
pathway [27],
[41]. To
address whether the endogenous HDA6 target loci were also directed by the RdDM
pathway, siRNAs from the ASRP database [42] were mapped to the loci
derepressed in axe1-5. Consistent with knowledge that
24-nt-long siRNAs are required for the establishment of RdDM, the most abundant
siRNAs mapping to upregulated loci in axe1-5 are 24-nt long
(Figure
S2A). These 24-nt siRNAs were hardly found in the
rdr2 and dcl3 mutants (Figure
S2B), suggesting that loci derepressed in axe1-5 contain
siRNA sequences produced by RDR2 and DCL3-dependent pathways, as previously
predicted [27], [41]. Thus, our previous study for the targets of RDR2
[43],
that were identified using same growth conditions and array technology as in
this study, were compared with the genes derepressed in axe1-5.
This revealed, surprisingly, despite the loss of 24-nt siRNAs from those genes
were observed in the rdr2 mutant (Figure
S2B), the majority of the loci derepressed in axe1-5 are
kept in a silenced state in the rdr2 mutant (Figure 2A). In fact, elevated
transcript levels were not detectable at many loci in the mutants deficient in
siRNA production (rdr2, and nrpd1; 10 and 11
respectively, out of 13 genes tested; Figure 2B). There also was evidence that
small subsets of the HDA6-mediated gene silencing showed dependence on RdDM
pathway and the overlapped genes between axe1-5 and
rdr2 was confirmed for their accumulated transcripts
(AT3TE60310, At1g67105, and At3g28899) in the
RdDM mutants (rdr2 and nrpd1; Figure 2B). Interestingly,
larger overlap to the triple mutant drm1 drm2 cmt3
(ddc) involved in siRNA-directed non-CG methylation was
observed (5 out of 13 genes; Figure
2B). Taken together, these results indicate the partial involvement
of RdDM pathway in HDA6-mediated endogenous gene silencing.
10.1371/journal.pgen.1002055.g002
HDA6-mediated gene silencing requires MET1 but not RdDM
pathway.
(A) A Venn diagram showing the small fraction of overlap between the AGI
genes upregulated in axe1-5, compared with those in
rdr2. The AGI genes identified as upregulated in
rdr2 in a previous genome-wide transcriptional
profiling experiment [43], were compared with the AGI genes upregulated
in axe1-5 identified in this study. (B) Various mutants
deficient in gene silencing, such as the chromatin modifying enzymes
(hda6, met1, ddc,
and kyp) and siRNA production (rdr2
and nrpd1) were examined for the activation of
representative HDA6 target loci (AT3TE60310, At1g67105,
At3g28899, AT3TE63935, AT3TE76225, AT4TE03410,
AT5TE39590, At2g15555, At3g44070,
At3g54730, At5g41660,
G447 and G683) by RT-PCR analysis.
ACT2 served as a control. The MET1 requirement for
repression of HDA6 target loci is highlighted. (C) A
Venn diagram showing the significant overlap between the AGI genes
upregulated in axe1-5 and in met1-3
that were identified in genome-wide transcriptional profiling. (D) The
DNA methylation status in wild-type Col-0 for the genes upregulated in
axe1-5. The fraction of AGI genes associated with
DNA methylation at more than 50% of methylated cytosines around
TSS (−500 to +500 from TSS) was determined using publicly
available datasets of methylcytosine immunoprecipitation [13].
(E) Cytosine methylation was investigated in each sequence context (CG,
CHG, and CHH) in wild-type Col-0, met1-3, and
ddc using publicly available datasets of cytosine
methylation (methylC-seq, [13]). The percentages
of methylated cytosines of the AGI genes upregulated in
axe1-5 are shown.
HDA6 and the CG DNA Methyltransferase MET1 Share Common Target Loci for
Epigenetic Silencing
We also examined the effects of mutations in other chromatin modifying enzymes on
the silencing of putative HDA6 target loci. Strikingly, 10 out of 13 of the
putative HDA6 target loci were also upregulated in the met1-3
mutant (Figure 2B). To
address whether HDA6 and MET1 share common target loci genome-wide, we also
identified differentially regulated loci in met1-3 using a
tiling array (Tables S4, S5, S6 and S7), and
compared the upregulated loci in met1-3 with those in
axe1-5. A significant overlap of upregulated loci in
axe1-5 to those in met1-3 was observed
(Hypergeometric distribution,
P = 1.08E−54; Figure 2C). Furthermore, the DNA methylation
status of the loci derepressed in axe1-5 was also investigated
using publicly available DNA methylation datasets [13]. Most of the genes
upregulated in axe1-5 (i.e. 70% of the upregulated AGI
genes) were substantially methylated in the wild-type plants with more than
50% of all cytosines at regions surrounding transcriptional start sites
methylated (Figure 2D).
Cytosine methylation in the wild-type plants was predominantly found at CG, to a
lesser extent at CHG, and least of all at the CHH sites of derepressed AGI genes
in axe1-5 (Figure
2E). A large proportion of the cytosine methylation on derepressed
AGI genes in axe1-5 appears to be highly dependent on MET1
because the drastic reduction in cytosine methylation was observed not only at
CG, but also CHG and CHH sites (Figure 2E). In contrast, CG methylation in the ddc
mutant remained at similar levels as the wild-type plants (Figure 2E). Thus, these data demonstrate that
the CG DNA methyltransferase MET1 is required for HDA6-mediated epigenetic gene
silencing.
Identification of the Direct Targets of HDA6
Identification of the direct targets of HDA6 is a crucial step in providing
mechanistic insight into HDA6 function in transcriptional control, chromatin
regulation, and DNA methylation. To determine the HDA6 target loci using
chromatin immunoprecipitation (ChIP), we raised a specific antibody against
HDA6. The epitope for the antibody was designed against the C-terminal region of
the HDA6 protein that is absent in axe1-5. We verified that the
peptide sequence was not similar to any other sequence of annotated
Arabidopsis proteins. Thus, comparisons made between
wild-type and axe1-5 for the level of enrichment of
immunoprecipitated DNA using the antibody allowed us to exclude any non-specific
signals to identify HDA6 binding sites (Figure 3A). Western blot analysis confirmed
the specificity of the antibody, which detected a unique band of 53 kDa in the
wild-type, but not in axe1-5 (Figure 3B).
10.1371/journal.pgen.1002055.g003
Identification of direct targets of HDA6 using an HDA6-specific
antibody.
(A) Schematic illustration of HDA6-specific antibody raised in this
study. The point mutation in axe1-5[33],
is indicated by a red cross and the resulting C-terminal truncation of
the HDA6 protein in axe1-5 is indicated by a dashed
box. The 17 amino acid (aa) peptide selected for the epitope is
indicated by a red box, which is absent in axe1-5. (B)
Western blot analysis indicates high specificity of the HDA6 antibody
for its target. Total protein extracts from wild-type (WT) and
axe1-5 plants were analyzed to check the
specificity of the HDA6 antibody. (C) Screening of HDA6 direct targets
by ChIP-qPCR assay. The transcribed regions of 22 selected loci from
those derepressed in axe1-5 (Figure
S1), including 9 AGI annotated TE fragments, 9 non-TE AGI
genes and 4 non-AGI TUs were used in this study. AGI annotated TE
fragments are as follows: AT3TE60310 (At3g42658),
AT3TE63935 (At3g44042), AT3TE76225
(At3g50625), AT4TE06710
(At4g02960), AT4TE09845 (At4g04293),
AT5TE36235 (At5g27927), AT5TE39590
(At5g28880), AT5TE41870
(At5g30480), and AT5TE43385
(At5g32566). The loci down-regulated in
axe1-5 (AT4TE42860: At4g16870),
and a gene with no apparent transcriptional difference between
axe1-5 and WT (At5g55670) were
included in this screening. Equal amounts of the input DNA and the
immunoprecipitates were analyzed and normalized against the input DNA.
The values obtained were normalized with ACT2 and the
relative enrichment of HDA6-binding in the wild-type plants compared
with axe1-5 is shown as the mean and standard
deviations obtained from three independent immunoprecipitates (orange,
WT; green, axe1-5).
Using the HDA6 antibody, we performed ChIP assays and quantitative PCR (qPCR).
Three genes, AT3TE60310 (At3g42658), AT3TE76225
(At3g50625) and At5g41660 were selected
from genes upregulated in axe1-5, representing RdDM dependent,
MET1 independent, and MET1 dependent genes, respectively (Figure S1).
Three primer sets were designed for each gene within the promoter, 5′ and
3′ regions of the genes (Figure S3). HDA6 binding levels in the
wild-type plants were significantly higher than in axe1-5 for
all of the genes tested, regardless of the dependence on RdDM pathway or MET1.
This indicates that HDA6 binds directly to all such genes. In addition,
preferential binding of HDA6 was observed within the 5′ regions of the
genes. Therefore, a further screening of HDA6 target loci was performed for the
5′ regions of selected loci from those upregulated in
axe1-5 (Figure S1; Figure 3C). As a negative control, we tested
one gene that exhibited no apparent transcriptional change
(At5g55670, Figure S1), and found only a minimal
difference. Our experiments show that HDA6 binding levels were enriched by 2 to
12-fold in the wild-type plants relative to axe1-5, with
statistical significance observed for 17 of the loci (Figure 3C). 5 loci did not show significant
differences between the wild-type and axe1-5 mutant.
HDA6 Is Required for Heterochromatic Silencing, and the Mutation Results in
Loss of Heterochromatic Histone Modification Along with Aberrant Enrichment for
Euchromatic Modification at HDA6 Targets
Heterochromatic or repressive regions are associated with H3K9me2 and/or
H3K27me3, whereas euchromatic or transcriptionally active regions are associated
with H3K4me3 and H4 tetra-acetylation (H4 tetra-acetylated on K5, K8, K12, and
K16) [44].
Furthermore, many endogenous RdDM targets are known to be associated with
euchromatic modification H3K4me3 [45]. To elucidate HDA6
function and chromatin status, the effects of the hda6 mutation
on histone modification was analyzed using ChIP-qPCR on its direct targets. The
results show that, regardless of the dependence on siRNAs or positions on the
chromosome, weak H4 tetra-acetylation and H3K4me3 and significantly high levels
of heterochromatic modification, H3K9me2, were observed in the wild-type plants
at all of the loci tested (Figure
4A, 4B and 4C; Figure S4). In the axe1-5
mutant, the active marks strongly increased (H4 tetra-acetylation at range 5 to
30 fold and H3K4me3 at 8 to 78 fold, respectively), and the levels of H3K9me2
were drastically reduced (range 2 to 53 fold) compared with the wild-type plants
(Figure 4A, 4B and 4C;
Figure
S4). H3K27me3, another repressive modification, are highly enriched
in wild-type plants predominantly on genes within the chromosome arm regions,
such as At1g67105 and At5g41660, and
drastically reduced in axe1-5 (Figure 4D). Consistent with this observation,
the enrichment of H3K27me3 in the euchromatic arm regions was also seen in the
previous genome-wide studies of H3K27me3 [11], [12]. These results indicate that
the hda6 mutation caused an alteration of the chromatin status
from a heterochromatic to euchromatic state that was concomitant with
transcriptional release of HDA6 target loci.
10.1371/journal.pgen.1002055.g004
HDA6 mutation causes abolishment of heterochromatic
mark and elevation of euchromatic modifications.
ChIP-qPCR assays were performed for quantitative analysis of histone
modifications, using antibodies against; (A) H4 tetra-acetylation, (B)
H3K4me3, an active euchromatic mark, (C) H3K9me2, and (D) H3K27me3, a
constitutive heterochromatic or repressive mark. Equal amounts of input
DNA and the immunoprecipitates were analyzed and normalized against
input DNA. The values obtained were normalized again using
ACT2 and are shown as the means and standard
deviations from three independent immunoprecipitated DNA analyses (blue,
WT; red, axe1-5). At5g55670 with no
apparent transcriptional difference between axe1-5 and
WT was used as a negative control.
HDA6 deacetylase activity has been reported for H3K9, H3K14, H4K5 and H4K12, as
well as for H4 tetra-acetylation [34]. The activity at other
residues, however, is currently unknown. To assess the HDA6 deacetylase activity
at other lysine residues, we investigated all of the potential acetylation sites
of the H3 and H4 N-tails, including pre-determined sites using ChIP-qPCR. Three
HDA6 target loci (AT3TE76225, At5g41660, and
G683) were examined. The results showed that in
axe1-5, the acetylation levels significantly increased at
H3K9, H3K14, H3K18, H3K23, H3K27, H4K5, H4K8, and H4K12 residues relative to
those in wild-type plants at all three loci tested (Figure 5A, 5B and 5C, Figure S5).
Interestingly, among these residues, H3K23ac levels showed the highest
enrichments in axe1-5, for the three loci, AT3TE76225,
At5g41660 and G683 (at 10, 14, 5 fold
respectively). The acetylation levels were not significantly altered in
axe1-5 for the control genes At5g55670
(Figure 5D) and
ACT2 (Figure S5). It is noteworthy that the
deacetylase activity observed for HDA6 at H3K27ac as well as H3K9ac, are both
likely to be important for the subsequent histone methylation of H3K27me3 and
H3K9me2, respectively [46]. Interestingly, residues that showed increased levels
of acetylation in axe1-5, were identical to the target residues
in yeast RPD3 deacetylation [47]. These results indicate that the deacetylase activity
of HDA6 occurred on all of the lysine residues in H3 and H4 N-tails, except
H4K16.
10.1371/journal.pgen.1002055.g005
Elevated acetylation at all H3 and H4 lysines except H4K16 in
axe1-5.
ChIP-qPCR assays for all of the potential deacetylation substrates of
histone H3 and H4 N-tails (acetylation sites, K9, K14, K18, K23, K27 of
H3 and K5, K8, K12, K16 of H4) were examined on (A) AT3TE76225, (B)
At5g41660, (C) G683 and a negative
control gene (D) At5g55670. Specific antibodies against
each acetylated lysine were used (See Materials and Methods for detail). The normalized
acetylation enrichments in axe1-5, relative to those in
the wild-type plant are shown as the mean plus standard deviation
obtained from three independent immunoprecipitated DNA experiments
(blue, WT; red, axe1-5).
The Effect of the hda6 Mutation on DNA Methylation
The different effects that hda6 mutations impose on DNA
methylation have been reported as described above. To assess the function of
HDA6 on DNA methylation and the relationship between HDA6 and MET1, the DNA
methylation status of HDA6 direct targets was investigated. We used the
endonuclease McrBC (Figure
6A), which preferentially cleaves methylated DNA, and a Chop-PCR
assay using methylation sensitive restriction enzymes (Figure 6B), whose cleavage is blocked by DNA
methylation.
10.1371/journal.pgen.1002055.g006
Differential impacts of the hda6 mutation on DNA
methylation at HDA6 target loci.
The effect of the hda6 mutation on cytosine methylation
at HDA6 direct targets was determined by; (A) McrBC assays and, (B)
Chop-PCR assays. (A) McrBC-digested genomic DNA was amplified by PCR.
(B) Chop-PCR assays were conducted using the methylation sensitive
restriction enzymes HpaII (which reports CG
methylation), MspI (which reports CHG methylation), and
HaeIII (which reports CHH methylation). Genomic DNA
digested with each enzyme was amplified by PCR. NA indicates that the
amplified sequences do not include the HaeIII
recognition sequence GGCC. In each assay, undigested genomic DNA was
used as a PCR control. ACT2 served as a control for
enzymatic digestion. (C) The DNA methylation status for flanking regions
of HDA6 target loci was determined using McrBC assays. The regions from
around 2 kb to 0.5 kb (i.e. 1.5 kb) of upstream (left panel, red
arrows), and downstream (right panel, blue arrows), of HDA6 target loci
were assessed. Absence of flanking methylated regions around the HDA6
target loci correlated with the loss of CG methylation in
axe1-5. Primers are listed in Table
S8.
In the McrBC assay shown in Figure
6A, no strong bands were detected in the wild-type plants after McrBC
digestion, indicating that the direct targets of HDA6 are highly DNA methylated
in the wild-type plants. This is consistent with dense DNA methylation on the
loci upregulated in axe1-5 (Figure 2D and 2E). However, in
axe1-5, we observed two types of McrBC sensitivity among
the HDA6 direct target loci. The Group A genes substantially lost cytosine
methylation, as demonstrated by the presence of strong bands of similar
intensity in both Group A genes and the non-digested control (Figure 6A, left panel). In
contrast, the genes in Group B retained DNA methylation in
axe1-5 in common with the wild-type plants as no strong
bands were detected (Figure
6A, right panel).
We also performed Chop-PCR assays to investigate in which cytosine contexts are
dependent on HDA6 (Figure
6B). The methylation sensitive restriction enzymes used were
HpaII, MspI and HaeIII,
which reports CG, CHG, and CHH methylation, respectively. From each group
categorized in Figure 6A,
four representative HDA6 target loci were tested and using ACT2
as a negative control. PCR amplification of ACT2 was
undetectable regardless of the genotypes or the enzymes tested (Figure 6B), confirming
substantial cleavage by the enzymes had occurred. In the wild-type plants,
strong amplification, at a similar level as the undigested control was detected
after digestion with HpaII (CG methylation). Strong
amplification was observed with MspI (CHG methylation), but it
was mostly less than the undigested control; least amplification of all was
observed with HaeIII (CHH methylation) (Figure 6B). The results of this experiment
are consistent with the results shown in Figure 2E and Figure 6A, where HDA6 target loci were shown
to be significantly methylated in wild-type plants, predominantly at CG sites,
and to a similar or lesser extent at CHG sites, but least of all at CHH
sites.
In common with the results obtained for the McrBC assay (Figure 6A); the results from the Chop-PCR
assays split the HDA6 target loci into two separate groups. In Group A
(AT3TE60310, AT3TE76225, At3g54730 and
At5g41660), complete demethylation in
axe1-5 occurred in all sequence contexts and amplification
after digestion of each enzyme was undetectable (Figure 6B, Group A). In the Group B genes
(AT3TE63935, AT5TE43385, At1g67105, and
At3g44070), for axe1-5, however, CG
methylation was mostly sustained at the similar level as the wild-type (Figure 6B, lower panel,
HpaII digest), although a drastic reduction in CHG and CHH
methylation was detected [Figure 6B, lower panel CHG (MspI) and CHH
(HaeIII)]. Bisulfite sequencing analyses of wild-type,
axe1-5 and met1-3 further confirmed that
Group A genes (At5g41660 and AT3TE76225) lost DNA methylation
in all sequence contexts in axe1-5, and a Group B gene
(AT3TE63935) lost CHG and CHH methylation but sustained CG methylation in
axe1-5 (Figure S6). Collectively, the methylation
analyses of the HDA6 targets demonstrated that, 1) CG and CHG sites were
predominantly highly methylated in the wild-type plants; 2) CHG and CHH
methylation was substantially reduced at all target loci in
axe1-5; and, 3) CG methylation was lost at
some loci but sustained at others in axe1-5.
An Absence of DNA Methylated Regions around HDA6 Target Loci Is Correlated
with the Loss of CG Methylation in axe1-5
Why were two different CG methylation states observed in axe1-5?
Interestingly, according to the public database for DNA methylation [9], a clear
correlation was observed between a loss of CG methylation on the HDA6 targets in
axe1-5 and the absence of methylated DNA regions around the
HDA6 target loci (Figure S7). HDA6 target loci in Group A (with
loss of DNA methylation) were shown to be isolated from other methylated DNA
regions, whereas HDA6 target loci in Group B (with persistent CG methylation)
were surrounded by other methylated DNA regions. We confirmed the presence or
absence of DNA methylated regions around HDA6 target loci (Figure 6C). The 1.5 kb regions upstream and
downstream of the HDA6 target loci were analyzed to determine their DNA
methylation status using McrBC assays. We found evidence of robust DNA
methylation around the HDA6 target loci in Group B (with persistent CG
methylation in axe1-5) (Figure 6C, Group B). These methylated regions
often contained other TE fragments adjacent to HDA6 target loci. These TEs were
densely DNA methylated dependently on MET1 but independently of HDA6, since the
PCR amplification after McrBC digestion was detected only in
met1-3 (Figure S8). We also confirmed that these
adjacent TE fragments were not targeted by HDA6 using ChIP-qPCR assays; these
results showed no enrichment of HDA6 binding to adjacent TE fragments in the
wild-type plants compared with axe1-5 (Figure S9).
As a result, HDA6 target loci with sustained CG methylation in
axe1-5 (Group B) must harbor the flanking TE fragments that
are highly DNA methylated by MET1 independently of HDA6. On the other hand, we
found that the target loci in Group A (with loss of DNA methylation in
axe1-5) were isolated from other DNA methylated regions as
no substantial methylation was detected around the target loci (Figure 6C, Group A). We
confirmed the absence of DNA methylation around the HDA6 target loci in Group A
by bisulfite sequencing analysis of the upstream region of a Group A gene,
At5g41660 (data not shown). Thus, we deduced a requirement
for HDA6 involvement in CG methylation by MET1 for HDA6 target loci, in the
absence of other flanking DNA methylated regions.
Discussion
We have identified 157 loci that require HDA6 for epigenetic silencing in
Arabidopsis. This is the first report to identify derepressed
loci in axe1-5 on a genome-wide scale. Our study revealed several
interesting features of HDA6 target loci, mapped large numbers of TE fragments and
DNA methylation sites in wild-type Arabidopsis plants. The
derepressed loci in axe1-5 overlapped significantly with the
derepressed loci in met1-3, a CG DNA methyltransferase mutant,
rather than the RdDM deficient mutants rdr2 and
ddc, suggesting that HDA6 plays an important role in gene
silencing, in cooperation with MET1. We also identified 17 direct targets of HDA6
using ChIP-qPCR assays with a HDA6 specific antibody. We found that HDA6 was
required for heterochromatic histone modifications and DNA methylation in the target
loci. Interestingly, HDA6 deficiency resulted in aberrant enrichment for euchromatic
epigenetic marks and DNA hypomethylation at HDA6 targets, along with ectopic
expression of these loci. DNA hypomethylation at CG sites in axe1-5
occurred at some HDA6 target loci, but only where isolated from other MET1 target
loci, possibly indicating the requirement of HDA6 for the recruitment of MET1 to
specific loci.
We showed that all of the HDA6 direct targets tested in this study colocalized with a
constitutive heterochromatin mark, histone H3K9me2 (7 out of 7; Figure 4C), rather than another mark of
repressive chromatin, H3K27me3 (Figure
4D, 3 out of 7). We saw no evidence of euchromatic modification H3K4me3
(0 out of 7; Figure 4B).
Moreover, our results suggested the deacetylase activity of HDA6 against H3K9ac and
H3K27ac, will be important for subsequent histone methylations of H3K9me2 and
H3K27me3, as well as H3K14ac, H3K18ac, H3K23ac, H4K5ac, H4K8ac and H4K12ac. These
results suggest that H3K9 and H3K27 deacetylation by HDA6 is essential for the
establishment of the heterochromatic and repressive marks mediated by H3K9me2 and
H3K27me3, and HDA6 deficiency resulted in loss of heterochromatic histone marks and
aberrant enrichment for euchromatic marks at HDA6 target loci. It is noteworthy that
the enrichments observed on H3K23ac were the highest among the possible acetylation
sites, which may indicate the importance of H3K23 deacetylation on heterochromatic
gene silencing. The abundance of silenced TE fragments and genes for unknown
proteins among the loci derepressed in axe1-5 (Figure 1C and 1D) supports the role of HDA6 in
silencing at heterochromatic regions. In addition, an important role for HDA6 in CHG
methylation is indicated by the observation that all of the direct targets of HDA6
tested in this study lost CHG methylation in axe1-5 (Figure 6B; Figure S6).
Several papers report that CHG methylation maintained by CMT3 is dependent on
H3K9me2 and that CMT3 is recruited to methylated histones [24]–[26]. Taken
together, this indicates that HDA6 deacetylase activity against its target loci is
required for establishment of the heterochromatic and repressive marks H3K9me2 and
H3K27me3, and CHG methylation by CMT3.
The separation of endogenous HDA6 target loci from endogenous RdDM target loci were
investigated in this study. Our results show that endogenous HDA6 target loci were
associated with a constitutive heterochromatic mark, H3K9me2 (Figure 4C), but not a euchromatic mark, H3K4me3
(Figure 4B). However, many
of the endogenous target loci of RdDM components (such as Pol V and DRD1) were found
to be associated with euchromatic histone modification H3K4me3, but not H3K9me2
[45].
Surprisingly, genome-wide identification of the loci derepressed in
axe1-5 revealed that these loci overlap with only a small
fraction of the genes upregulated in rdr2 (Figure 2A). However, considering the recent
studies proposing the role of siRNAs in re-establishment of DNA methylation and gene
silencing when DNA methylation was lost in the DNA methylation deficient mutants
like met1 and ddm1[48]–[50], the siRNAs
found on the HDA6 target loci might also have a role in this mechanism, and
therefore the double mutants of hda6 and siRNA deficient mutants
might result in larger release of gene silencing. In addition, cell-type specific
regulation of siRNAs and TEs silencing especially in the gametes has been proposed
recently [51]. In
this case, DDM1 expression is downregulated in the pollen
vegetative nucleus, which accompanies the sperm cells. Considering that HDA6 has
common features with DDM1 [18], [29], [36]–[38], it would also be interesting to see if HDA6 also have a
role in regulating transposon silencing in gametes.
An important functional connection between HDA6 and MET1 was revealed by observing
the significant overlap of the loci upregulated in axe1-5, with the
loci upregulated in met1-3 (Figure 2B and 2C). Indeed, we found several HDA6
target loci (AT3TE60310, AT3TE76225, At3g54730 and
At5g41660) that require HDA6 for MET1 CG methylation (Figure 6B; Group A). Thus, we
propose that HDA6 acts in cooperation with MET1, possibly as a recruiter or as a
component of the silencing machinery with MET1 (Figure 7). Actually we observed the loss of HDA6
binding on several HDA6 targets in the met1-3 mutant (Figure S10). It
indicates the requirement for MET1 and/or CG methylation to facilitate HDA6 binding,
suggesting the cooperative interplay between HDA6 and MET1. Further support for this
hypothesis, is the observation that HDA6 targets isolated from other flanking MET1
targets experienced a loss of CG methylation in the absence of HDA6 (Figure 6B and 6C). It is also
supported by several papers evidencing the physical interactions that occur between
histone deacetylases and DNA methyltransferases in mammals [52]–[54].
10.1371/journal.pgen.1002055.g007
Mechanistic model for epigenetic heterochromatin silencing regulated by
HDA6.
In our hypothetical model, HDA6 and MET1 cooperate in the initial step of
epigenetic silencing. HDA6 directs deacetylation of all lysine residues in
H3 and H4 N-tails, except H4K16. The CG DNA methyltransferase MET1 probably
requires HDA6 for its recruitment to the HDA6 target loci isolated from
other MET1 target loci. Once MET1 is recruited it directs CG DNA
methylation. After deacetylation by HDA6 and CG methylation by MET1, H3K9me2
is established by Histone Methyl Transferase(s) (HMT), followed by
H3K9me2-dependent CHG methylation by CMT3. In hda6, all
repressive modifications such as H3K9me2, histone deacetylation, and all DNA
methylation in all sequence contexts are lost. This results in
transcriptional derepression, indicating that HDA6 may trigger a series of
epigenetic modifications at heterochromatin, possibly as a recruiter, or as
a component of the silencing machinery in conjunction with MET1.
The sustained CG methylation status of the other HDA6 targets in the
axe1-5 mutant was found to correlate with the existence of
other flanking MET1 target loci in neighboring regions of the HDA6 targets (Figure 6B and 6C, Group B; Figure S7,
S8, S9, S11). Thus it
appears likely, therefore, that MET1 could be recruited to the neighboring regions
of the HDA6 targets and pass through the HDA6 targets (Figure S11).
Another possibility is that MET1-dependent CG methylation is the primary repressive
modification to silence those HDA6 targets. There also were many loci that were
derepressed only in the met1-3 mutant (Figure 2C, Figure
S12).
It is noteworthy that the intensities of the RT-PCR bands were greater in
met1-3 than in axe1-5 for the genes with
sustained CG methylation (i.e. At1g67105, AT3TE63935,
At2g15555, At3g44070, and
G683; Figure
2B), indicating that sustained CG methylation on the HDA6 target loci can
be repressive to some extent. However, there is clear evidence that these genes are
transcriptionally derepressed strongly in the absence of HDA6 or MET1, regardless of
the CG methylation status. We deduce, therefore, that both HDA6 histone
deacetylation and MET1 CG methylation are essential for the silencing of HDA6 target
loci. In addition, for most of the HDA6 target loci, CHG and CHH methylation was
lost in both hda6 and met1 mutants (Figure 6B). A drastic reduction in
CHG and CHH methylation was also observed for the loci silenced by HDA6 in the
met1 mutant (Figure 2E). These observations indicate that CHG and CHH methylation on
HDA6 targets require the presence of epigenetically silent chromatin associated with
both histone deacetylation and CG methylation. Because HDA6 was
identified as RTS1 (RNA-mediated transcriptional silencing 1) and
MET1 as RTS2 in RdDM screens [22], [27], we strongly
suggest that these two genes are deeply connected to each other, as proposed
previously [22],
[36]. These
insights have an important evolutionary implication; that the histone deacetylase
superfamily, one of the most ancient enzymes in eukaryotes [16], may build the foundations for
gene silencing and concomitant CG DNA methylation. CG DNA methylation is a conserved
modification in higher eukaryotes, quite distinct from the siRNA derived CHG or CHH
methylation found only in plants [55], [56].
It is noteworthy that HDA6 has high specificity for target loci within the genome.
Moreover, we found that HDA6 binds only to its target loci, not the flanking TE
fragments (Figure
S9). Relatively low numbers of loci were derepressed in
axe1-5 (157 loci), contrasting with the met1-3
mutant, where a total of 1215 loci were derepressed. We consistently detected an
insignificant difference in the amount of total methylated DNA in
axe1-5, whereas a severe reduction was detected in the
met1-3 mutant compared with wild-type plants (Figure S13). It
was also reported that the total amount of histone H4 tetra-acetylation or H3K4me3
did not change between axe1-5 and the wild-type plants [28]. From this, we
conclude that the target specificity of HDA6 is likely to be precisely controlled,
with the effect of the hda6 mutation manifesting itself only in
local areas. Furthermore, HDA6, but not MET1, has the ability to trigger de
novo chromatin silencing, because only backcrossed
hda6/wild-type plants were able to restore DNA methylation, low
H3K4me3 levels and a silent transcriptional state, comparable with wild-type plants,
unlike met1/wt [36], [37]. These findings, taken together, indicate that HDA6 is a
regulator of locus-directed heterochromatin silencing in cooperation with MET1,
where it acts possibly as a recruiter or as a component of the chromatin silencing
machinery with MET1, thus establishing the foundations for silent chromatin status
for the subsequent heterochromatin mark H3K9me2 and non-CG methylation (Figure 7).
Because MET1 is the primary CG DNA methyltransferase encoded in
Arabidopsis, regulation of the proper and specific distribution
of MET1 CG methylation by employment of HDA6 and/or other possible factors would be
an efficient way for the Arabidopsis genome to adapt to several
developmental and environmental effects. It will be interesting to see if HDA6 and
MET1 form a complex as is the case in mammals [52]–[54], or what information is recognized
by these factors to trigger the sequential silencing mechanism. In this study, we
identified dozens of loci transcriptionally silenced by HDA6 and several loci
directly targeted by HDA6. These results will undoubtedly contribute to an
understanding of the complex interplay between histone deacetylation and DNA
methylation, revealing mechanistic insights into heterochromatin silencing in higher
eukaryotes.
Materials and MethodsPlants and Growth Conditions
Seeds were surface-sterilized and stratified for 4 days at 4°C in the dark.
The seeds were then grown in tissue culture plates on MS agar (0.8%)
medium supplemented with 1% sucrose under 16 h light/8 h dark for 15 days
at 22°C. All experiments used axe1-5[33],
met1-3[21],
ddc (drm1-2 drm2-2 cmt3-11, [57]),
kyp (SALK_069326 [58]), rdr2-1
(SAIL_1277_H08 [42]), nrpd1a-3 (SALK_128428 [59]) mutants
or DR5 [33]
and Col-0 wild-type plants. All plants were the Columbia ecotype.
Whole-Genome Tiling Array Analysis
The GeneChip Arabidopsis tiling array set (1.0F Array and 1.0R Array, Affymetrix)
was used. Total RNA was extracted using Isogen reagent (Nippon Gene). Probe
synthesis, hybridization, detection, data evaluation with U-test (FDR
α = 0.05) and P initial value
(P<10−6) was conducted essentially as described
previously [60]. Three independent biological replicates were
performed for each strand array. Detection of intergenic transcribed units was
performed as described previously [39], [60] based on the TAIR8
annotation. A threefold increase or decrease in RNA accumulation was taken as
additional criteria for defining the loci upregulated or downregulated in the
axe1-5 and met1-3 mutants. Tiling array
data are available at the GEO website under the accession number GSE23950.
RT-PCR Analysis
Total RNA was extracted using Plant RNA Purification Reagent (Invitrogen) and
subjected to cDNA synthesis using the QuantiTect Reverse Transcription Kit
(Qiagen), according to the manufacturer's instructions. The PCR conditions
were as follows; pre-incubation for 5 min at 94°C, 30 cycles at 94°C for
30 sec, 58°C for 20 sec, 72°C for 40 sec and a final extension at
72°C for 4 min. Primers are listed in Table S8.
The amplified DNA was visualized on a 2% agarose gel stained with
ethidium bromide.
Generation of the HDA6 Antibody and Western Blot Analysis
Antibodies against HDA6 were generated as follows; a peptide (DEMDDDNPEPDVNPPSS)
corresponding to the C-terminus of HDA6 was synthesized, HPLC purified,
conjugated to Bovine Serum Albumin (BSA) and used to immunize two rabbits
(Scrum). The antiserum obtained was affinity-purified and used for ELISA and
western blot analysis. Total protein extraction was performed on 15-day-old
seedlings. Seedlings were ground in liquid nitrogen, suspended in PBS
supplemented with 1 mM PMSF, centrifuged, and the supernatant used as a total
protein extract. The protein concentration was analyzed using the BioRad
Bradford reagent and 50 ug of protein was used for western blot analysis.
Western blots prepared by the iBlot Dry Blotting system (Invitrogen) were
blocked and incubated with the HDA6 antibody diluted at 1∶500, washed, and
incubated with anti-rabbit IgG HRP-conjugated antibodies (GE Healthcare) diluted
1∶5000. The results were visualized using ECL Plus Western Blotting
Detection Reagents (GE Healthcare).
Chromatin Immunoprecipitation
ChIP assays were performed essentially as described previously [61]. The
antibodies used in this study were: anti-H3K4me3 and H3K9me2 [62];
anti-H3K9ac (ab4441) and H3K14ac (ab1191) from Abcam; anti-H4 tetra-acetylation
(06-866), H3K27me3 (07-449), H3K18ac (07-328), H3K23ac (07-355), H3K27ac
(07-360), H4K5ac (07-327), H4K8ac (07-328), and H4K12ac (07-595) from Millipore,
and H4K16ac (CB-SC-8662-R) from Santa Cruz. The precipitates were analyzed with
quantitative PCR (Power SYBR real time reagent and ABI Prism 7000, Applied
Biosystems) and the relative amount of each modification was estimated as
described previously [63]. Statistical significance of the wild-type plants
compared with axe1-5 was determined by Kruskal–Wallis
test (P<0.05). The primers used are listed in Table
S8.
DNA Methylation Analysis
Genomic DNA was extracted using a Phytopure DNA extraction kit (GE Healthcare)
and 5 µg of genomic DNA was linearlized with 20 U BamHI
for 3 hours at 37°C. McrBC assays were performed by incubating 30 U of McrBC
per 1 µg of BamHI digested genomic DNA at 37°C for 16
hours before PCR amplification as described for RT-PCR with a 1 min extension
time. Chop-PCR assays [30] were performed using the methylation sensitive
restriction enzymes HpaII, MspI, and
HaeIII (NEB). Linearlized genomic DNA was incubated with
the enzymes (30 U/µg) at 37°C for 3 hours and subjected to PCR
analysis. The amplified DNA was visualized on a 1.0% agarose gel stained
with ethidium bromide.
Supporting Information
Validation of up- or down-regulation of selected AGI genes and non-AGI TUs in
axe1-5 by RT-PCR. Several loci that were differentially
expressed in axe1-5 in the tiling array analysis were
randomly selected and their up- or down-regulation confirmed by RT-PCR. 24
AGI genes and 4 non-AGI TUs were used. ACT2 and
At5g55670, which showed no transcriptional change in
the tiling array analysis, were used as controls. Primers are listed in
Table
S8.
(TIF)
The numbers of siRNA sequences in wild-type plants that correspond to the
loci upregulated in axe1-5. siRNA sequences of
inflorescences of the wild-type, rdr2, and
dcl3 plants were retrieved from the ASRP database. (A)
The numbers of siRNA sequences in wild-type plants that correspond to the
loci upregulated in axe1-5. (B) The numbers of 24-nt siRNAs
and 21-nt siRNAs sequences in wild-type, rdr2 and
dcl3 plants corresponding to the loci upregulated in
axe1-5. (Black, Col-0; blue, rdr2;
yellow, dcl3).
(TIF)
Search for the binding position of HDA6 in target genes. Direct binding of
HDA6 within the promoter, 5′ and 3′ transcribed regions of three
representative derepressed genes, AT3TE60310, AT3TE76225, and
At5g41660 were examined using ChIP-qPCR assays with the
HDA6 antibody. Equal amount of input DNA and the immunoprecipitates were
analyzed and normalized against input DNA and ACT2. The
relative enrichment of HDA6-binding in the wild-type against that in
axe1-5 is shown as the mean of the results of repeated
experiments with three independent immunoprecipitated DNA preparations
(orange, WT; green, axe1-5). Error bars indicate the
standard deviation.
(TIF)
Histone modification status as determined by ChIP-PCR with specific
antibodies for H4 tetra-acetylation, H3K4me3, H3K9me2 and H3K27me3. Three
representative HDA6 direct targets (AT3TE60310, AT3TE76225, and
At5g41660) were examined. ACT2 served
as a control. Equal amounts of the input and the immunoprecipitated DNA were
subjected to 30 cycles of PCR amplification using the same primers as in
Figure 3C. PCR
amplicons were analyzed by 6% polyacrylamide gel electrophoresis.
(TIF)
Enrichments of histone acetylation in axe1-5 were examined
by ChIP-PCR with specific antibodies for all the possible substrates of HDA6
deacetylation at H3 and H4 N-tails. The acetylation levels of AT3TE60310,
AT3TE76225 and At5g41660 were analyzed using
ACT2 as a control. Equal amount of the input and the
immunoprecipitated DNA were subjected to 30 cycles of PCR using the same
primers used in Figure
3C. The PCR products obtained were analyzed by 6%
polyacrylamide gel electrophoresis.
(TIF)
DNA methylation status of HDA6 target loci in wild-type,
axe1-5 and met1-3 determined by
bisulfite sequencing. Representative HDA6 target loci from each group
(At5g41660 and AT3TE76225 from Group A; AT3TE63935 from
Group B) were analyzed for their DNA methylation status by bisulfite
sequencing analysis. (A) The percentage total cytosine methylation is shown
as the mean and standard deviation of 10 independent sequencing reads (red,
CG methylation; blue, CHG methylation; green, CHH methylation). (B) The
percentage of methylated cytosine at each site was analyzed using publicly
available software: Kismeth (http://katahdin.mssm.edu/kismeth). The primers used are
listed in Table S8. Bisulfite treatment was performed using BisulFast DNA
modification Kit for Methylated DNA Detection (TOYOBO). The modified DNA was
amplified as follows by PCR: pre-incubation step of 1 min at 94°C, 40
cycles at 94°C for 20 sec, 50 to 54°C for 20 sec, 72C for 1 min and
a final extension for 4 min. at 72°C, using Ex-Taq polymerase (Takara
Bio). The amplified DNA was cloned into pCR4 using a TOPO TA cloning kit
(Life Technologies), transformed into E. coli DH5α
cells and plasmid DNA purified from single colonies for sequencing.
(TIF)
DNA methylation status of HDA6 target loci and their surrounding regions. The
DNA methylation status of the HDA6 direct targets and their surrounding
regions were investigated by reference to GBrowse (http://gbrowse.arabidopsis.org), which shows the DNA
methylation datasets of HMBD [9]. Upstream and downstream regions of representative
HDA6 target loci from each group are shown. The yellow arrow indicates the
HDA6 target loci.
(TIF)
DNA methylation of the TE fragments located adjacent to the HDA6 target loci
in Group B loci is dependent on MET1, but not HDA6. The DNA methylation
status of the TE fragments located adjacent to the HDA6 target loci in Group
B was determined using McrBC assays. The TE fragments located adjacent to
the HDA6 target loci in Group B are shown in the schematic diagram (orange
box). The inside of the TE fragments were investigated (green arrow).
McrBC-digested genomic DNA was PCR amplified using the primer sets listed in
Table
S8.
(TIF)
ChIP-qPCR assay showing that some TE fragments located adjacent to the HDA6
targets are not directly targeted by HDA6. HDA6 binding to the TE fragments
located adjacent to the HDA6 target loci in Group B (AT3TE63935, AT5TE43385,
At1g67105, and At3g44070) was examined
using ChIP-qPCR. Relative enrichments of HDA6-binding in wild-type plants
versus axe1-5 are shown as the mean plus standard deviation
of three independent immunoprecipitates.
(TIF)
ChIP-qPCR assay for the HDA6 binding at some HDA6 direct targets in the
met1-3 mutant. HDA6 binding to the HDA6 direct targets
(AT3TE60310, At3g54730, G683 and
G1136) was examined using ChIP-qPCR. Relative
enrichments of HDA6-binding in wild-type plants versus
met1-3 are shown.
(TIF)
Model for the epigenetic mechanism of heterochromatin silencing regulated by
HDA6 on HDA6 target loci surrounded by other MET1 target loci. HDA6 is
required for the maintenance of epigenetic chromatin modifications such as
H3K9me2, CHG and CHH DNA methylation, but not required for the recruitment
of MET1, when MET1 target loci are located adjacent to HDA6 target loci.
HDA6 directs deacetylation of all lysine residues in H3 and H4 N-tails,
except H4K16. H3K9me2, CHG and CHH DNA methylation were all dependent on
HDA6. However, once MET1 is recruited near HDA6 target loci, MET1 directs CG
DNA methylation on or around the HDA6 target loci even in the absence of
HDA6. Thus, in hda6, repressive modifications such as
H3K9me2, histone deacetylation, and non-CG methylation are lost; only CG
methylation was retained on the HDA6 target loci. Even though CG methylation
may or may not be sustained, transcriptional derepression occurred in
hda6 and met1, indicating the
requirement for both histone deacetylation by HDA6 and CG methylation by
MET1 for establishment of the silent heterochromatin status.
(TIF)
Validation of genes upregulated only in
met1-3 by RT-PCR. Several loci that
were upregulated in met1-3 but not in
axe1-5 in the tiling array analysis were selected and
their upregulation was confirmed by RT-PCR. 4 AGI genes were used. Primers
are listed in Table S8.
(TIF)
Dot blot assay to determine total DNA methylation in wild-type plants and
axe1-5, met1-3 and
ddc mutants. Equal amounts of genomic DNA (400 ng) from
each genotype was blotted onto Nylon membrane (Hybond N+; GE
Healthcare) and incubated with an antibody against 5- Methylcytidine
(BI-MECY-0100; EUROGENTEC), followed by a secondary antibody conjugated to
HRP. The luminescence of HRP was detected with an ECL detection kit and
Hyperfilm ECL (GE Healthcare).
(TIF)
AGI annotated genes upregulated in axe1-5. The AGI genes
that were transcriptionally upregulated in axe1-5 compared
with wild-type plants (>3 fold, p-initial<10−6, FDR
α = 0.05) are listed.
(XLS)
Non-AGI TUs upregulated in axe1-5. Among the Non-AGI TUs
identified using the ARTADE program, the non-AGI TUs that were
transcriptionally upregulated in axe1-5 compared with
wild-type plants (>3 fold, p-initial<10−6, FDR
α = 0.05) are listed.
(XLS)
AGI annotated genes downregulated in axe1-5. The AGI genes
that were transcriptionally downregulated in axe1-5
compared with wild-type plants (<1/3 fold,
p-initial<10−6, FDR
α = 0.05) are listed.
(XLS)
AGI annotated genes upregulated in met1-3. The AGI genes
that were transcriptionally upregulated in met1-3 compared
with wild-type plants (>3 fold, p-initial<10−6, FDR
α = 0.05) are listed.
(XLS)
Non-AGI TUs upregulated in met1-3. Among the Non-AGI TUs
identified using the ARTADE program, the non-AGI TUs that were
transcriptionally upregulated in met1-3 compared with
wild-type plants (>3 fold, p-initial<10−6, FDR
α = 0.05) are listed.
(XLS)
AGI annotated genes downregulated in met1-3. The AGI genes
that were transcriptionally downregulated in met1-3
compared with wild-type plants (<1/3 fold,
p-initial<10−6, FDR
α = 0.05) are listed.
(XLS)
Non-AGI TUs downregulated in met1-3. Among the Non-AGI TUs
identified using the ARTADE program, the non-AGI TUs that were
transcriptionally downregulated in met1-3 compared with
wild-type plants (<1/3 fold, p-initial<10−6, FDR
α = 0.05) are listed.
(XLS)
List of primers. The primers used in this study are listed.
(XLS)
We thank Drs Craig S. Pikaard for axe1-5 and DR5, Hidetoshi Saze for
met1-3, Steve Jacobsen for ddc, James C.
Carrington for rdr2-1, and the Arabidopsis Biological Resource
Center for kyp (SALK_069326) and nrpd1a-3
(SALK_128428). We also thank Drs. Yoshiki Habu, Alessandra Devoto, and Fiona
Robertson for invaluable comments on the paper.
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