DNA methylation maintenance consolidates RNA-directed DNA methylation and transcriptional gene silencing over generations in Arabidopsis thaliana

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This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/tpj.12630 This article is protected by copyright. All rights reserved.

Received Date : 28-May-2014 Revised Date : 21-Jul-2014 Accepted Date : 24-Jul-2014 Article type : Original Article DNA methylation maintenance consolidates RNA-directed DNA methylation and

transcriptional gene silencing over generations in Arabidopsis thaliana

Markus Kuhlmann§1,3, Andreas Finke§1,4, Martin Mascher2 and Michael Florian Mette*1,5

§ These authors contributed equally to the work.

1 Research Group Epigenetics, Leibniz Institute of Plant Genetics and Crop Plant Research

(IPK), D-06466 Gatersleben, Germany;

2 Research Group Bioinformatics and Information Technology, Leibniz Institute of Plant

Genetics and Crop Plant Research (IPK), D-06466 Gatersleben, Germany;

3 Current affiliation: Research Group Abiotic Stress Genomics, Interdisciplinary Center for

Crop Plant Research (IZN), D-06120 Halle (Saale), Germany;

4 Current affiliation: Research Group Genome and Epigenome Maintenance, Max Planck

Institute for Plant Breeding Research, D-50829 Köln, Germany;

5 Current affiliation: Research Group Quantitative Genetics, Leibniz Institute of Plant

Genetics and Crop Plant Research (IPK), D-06466 Gatersleben, Germany;

*Author for correspondence: email [email protected], phone ++49 39482 5181, fax

++49 39482 5137;

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This article is protected by copyright. All rights reserved.

Email contacts of other authors: [email protected], [email protected],

[email protected];

Running title: Consolidation of RdDM and TGS

Keywords (up to 10): RNA-directed DNA methylation, transcriptional gene silencing, DNA

methylation maintenance, transgenerational effect, Arabidopsis thaliana, DRM2, At5g14620;

Accession numbers: PRJEB6549 (whole genome shotgun reads of Col-0 and nrd3-2)

Summary

In plants, 24-nucleotide short interfering RNAs serve as a signal to direct cytosine

methylation at homologous DNA regions in the nucleus. If the targeted DNA has promoter

function, this RNA-directed DNA methylation can result in transcriptional gene silencing. In

a genetic screen for factors involved in RNA-directed transcriptional silencing of a ProNOS-

NPTII reporter transgene in Arabidopsis thaliana, we captured alleles of DOMAINS

REARRANGED METHYLTRANSFERASE 2, the gene encoding the DNA methyltransferase

mainly responsible for de novo DNA methylation in the context of RNA-directed DNA

methylation. Interestingly, reporter gene ProNOS methylation was not erased completely in

these mutants, but persisted in symmetric CG context, indicating that RNA-directed DNA

methylation had been consolidated by DNA methylation maintenance. Taking advantage of

the segregation of the transgenes giving rise to ProNOS short interfering RNAs and carrying

the ProNOS-NPTII reporter in our experimental system, we found that ProNOS DNA

methylation maintenance became first evident after two generations of ongoing RNA-

directed DNA methylation, and then increased in its extent with further generations. As

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ProNOS DNA methylation already had reached its final level in the first generation of RNA-

directed DNA methylation, our findings suggest that setting up DNA methylation at a

particular region can be divided into distinct stages. An initial phase of efficient, but still fully

reversible de novo DNA methylation and transcriptional gene silencing is followed by the

transition to efficient maintenance of cytosine methylation in symmetric sequence context

accompanied by persistence of gene silencing.

Introduction

Proper temporal and spatial regulation of gene expression is essential for all complex

organisms. In eukaryotes, confinement of gene transcription is supported by chromatin

marks. One of the most prominent marks in plants is methylation at position 5 of cytosines in

genomic DNA, which is mainly associated with transcriptionally silent repetitive sequences

and transposable elements and is typical for heterochromatin. DNA methylation in plants can

not only be found in CG but also in CHG and CHH (with H standing for C, A, T) sequence

context (Cokus et al,. 2008; Lister et al., 2008). In A. thaliana DNA preparations,

approximately 7% of all cytosines are methylated (Rozhon et al., 2008), with methylation

levels amounting to 24% in CG, 6.7% in CHG and 1.7% in CHH context (Cokus et al.,

2008).

Cytosine methylation is introduced after DNA replication by DNA methyltransferases

(DMTases). The A. thaliana genome encodes four classes of genes displaying sequence

homology to conserved DMTase domains, the METHYLTRANSFERASE 1 (MET1) group

belonging to the Dnmt1 family, the DOMAIN REARRANGED METYHLTRANSFERASE

(DRM) group sharing similarity to Dnmt3, the CHROMOMETHYLASE (CMT) group specific

to plants and one putative member of the Dnmt2 family (Finnegan and Kovac, 2000).

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Different DMTase families are thought to be responsible for cytosine methylation in different

sequence contexts.

Members of the DRM group are required for de novo DNA methylation in all sequence

contexts and for the propagation of CHH context methylation. Only DRM1 and DRM2 genes

encode proteins that contain all invariant residues necessary for catalytic activity (Cao et al.,

2000; Cao and Jacobsen, 2002; Naumann et al., 2011; Jullien et al., 2012), while DRM3 is

not expected to display catalytic activity and most likely has auxiliary functions (Henderson

et al., 2010). Sequence specificity is conferred to DRM1 and DRM2-mediated methylation by

short interfering (si) RNAs (Zhong et al., 2014) in a mechanism termed RNA-directed DNA

methylation (RdDM) that was first observed in tobacco plants infected with the RNA

pathogen potato spindle tuber viroid (Wassenegger et al., 1994).

A prerequisite for the generation of a siRNA signal guiding RdDM in plants is the formation

of double-stranded (ds) RNA, as was demonstrated by means of Pol II-mediated transcription

of inverted repeat structures (Mette et al., 2000). Resulting transcripts with partial self-

complementarity, able to fold intra-molecularly to form dsRNA stems, could efficiently

trigger DNA hypermethylation of homologous sequences in trans, which, if targeted to

promoter sequences, could lead to transcriptional silencing of affected gene(s) (Mette et al.,

2000; Aufsatz et al., 2002b; Mette et al,. 2005). However, the extent of promoter RdDM and

related RNA-directed transcriptional gene silencing (RdTGS) at different target loci was

reported to vary depending on transgene structure (Khaitova et al., 2011) and insertion

context (Fischer et al., 2008). Once established, DNA methylation can persist to variable

degrees after removal of the inducing trigger (Jones et al., 2001; Lunerova-Bedrichova et al.,

2008; Khaitova et al., 2011). A number of RdDM-reporter systems making use of naturally

occurring as well as engineered transcribed inverted repeats were designed and employed in

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forward and reverse genetic screens to dissect the molecular pathways underlying RdDM and

RdTGS (Aufsatz et al., 2002a; Bologna and Voinnet, 2014).

Semi-conservative DNA replication in the course of the cell cycle diminishes DNA

methylation in all contexts as the newly synthesised DNA strand is free of methylation.

However, there are mechanisms that allow the re-establishment of pre-replication

methylation patterns at cytosines in symmetric context. In A. thaliana, like in mammals, CG

context sites show a stark correlation of the methylation on one strand with the methylation

on the opposite (Bird, 2002; Cokus et al., 2008). The re-establishment of full CG context

methylation on hemi-methylated DNA is thought to involve the activity of DMTase MET1,

chromatin remodelling factor DECREASE IN DNA METHYLATION1 (DDM1) and

VARIANT IN METHYLATION (VIM) proteins 1 to 3 (Vongs et al., 1993; Woo et al,.

2007; Woo et al,. 2008). Furthermore, the interaction of MET1 with HISTONE

DEACETYLASE 6 (HDA6) is necessary to maintain CG context methylation at various loci

(Aufsatz et al., 2002b; To et al., 2011; Liu et al., 2012; Blevins et al., 2014). However, the

details of the molecular mechanisms underlying CG context methylation maintenance in

plants are still only partly understood.

The maintenance of DNA methylation in symmetric CHG context is unique to plants and

differs significantly from CG methylation maintenance (Cokus et al., 2008). In A. thaliana,

the majority of CHG context methylation depends on the plant-specific DMTase

CHROMOMETHYLASE3 (CMT3) and on the H3-K9-specific SUPPRESSOR OF

VARIAGATION3-9 HOMOLOG (SUVH) histone methyltransferases SUVH4, SUVH5,

SUVH6 (Jackson et al., 2002; Malagnac et al., 2002; Ebbs and Bender, 2006; Pontvianne et

al., 2012). Experiments that showed an in vitro affinity of the chromodomain of CMT3 to

K9-methylated H3-derived peptides and a decrease of CHG methylation in H3K9me2-

deficient mutants suggest that H3-K9-methylation serves as a label for CMT3 target sites

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(Lindroth et al., 2004), which is also consistent with results from the genome-wide

superimposition of CMT3 binding sites and H3K9me2-marked nucleosomes (Du et al.,

2012). Moreover, the involvement of histone deacetylase HDA6 in CHG methylation

maintenance at specific loci is also well documented (To et al., 2011; Liu et al., 2012;

Blevins et al., 2014).

Here we employ a transgene system undergoing efficient RdDM and RdTGS in order to

analyse the interdependence of de novo DNA methylation and DNA methylation

maintenance in A. thaliana. By means of newly identified drm2 alleles defective in RNA-

directed DNA methylation and by taking advantage of the segregation of transgene loci to

remove the promoter-siRNA source from the target promoter, we demonstrate that DNA

methylation induced by RdDM can be consolidated by CG context DNA methylation

maintenance if RdDM is acting for more than one generation. The initiation of the silenced

state per se by RdDM and the maintenance of CG context DNA methylation are not

associated with histone-H3-lysine-9-dimethylation (H3K9me2) that is otherwise typical for

repressed chromatin in plants.

Results

RNA-directed transcriptional gene silencing is released in nrd3 mutants

We have performed a screen for ethyl methanesulfonate (EMS)-induced mutants (no rna-

directed transcriptional silencing, nrd) that reactivate expression of a silenced reporter

transgene in A. thaliana (Finke et al., 2012a) . The line K/K;H/H submitted to mutagenesis

was doubly homozygous for a SILENCER (H) transgene on chromosome 4 containing an

inverted repeat (IR) of the NOPALIN SYNTHASE promoter (ProNOS) sequence under control

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of the cauliflower mosaic virus 35S promoter (Pro35) (Aufsatz et al., 2002a) and a TARGET

(K; Kchr1-10 in Fischer et al., 2008) transgene on chromosome 1 containing a NEOMYCIN

PHOSPHOTRANSFERASE II (NPTII) reporter gene controlled by a ProNOS conferring

resistance to kanamycin (Figure 1a) that was previously found to show efficient DNA

methylation and silencing in the presence of the SILENCER (Fischer et al., 2008). Thus,

K/K;H/H plants homozygous for TARGET and SILENCER are sensitive to kanamycin (Figure

1b). F4 K/K;H/H seeds were incubated with EMS, sown on soil and grown to M1 plants,

which were allowed to self-pollinate (Figure S1). The resulting M2 seeds, the first generation

in which an EMS-induced mutation can be homozygous and thus, if recessive, show its

impact on the phenotype, were germinated on medium containing 200 mg/l kanamycin to

screen for individuals that became kanamycin resistant (KanR). Presence and integrity of

TARGET and SILENCER transgenes in resulting KanR M2 K/K;H/H nrd candidate plants

were verified via PCR using primer combinations specific for different parts of the

transgenes. For sake of readability, in the following “nrd” will be used in the sense of “KanR

K/K;H/H nrd” unless specified otherwise. M2 nrd plants were allowed to self-pollinate and

kanamycin resistance was verified for the resulting M3 generation. M3 seedlings of

independent mutant lines nrd3-1 and nrd3-2 showed consistent resistance when grown on

medium containing kanamycin (Figure 1b).

As kanamycin resistance can also arise in A. thaliana mutants by loss of chloroplast-localised

transporter proteins required for kanamycin uptake (Aufsatz et al., 2009; Conte et al., 2009)

rather than by reactivated NPTII expression, NPTII protein levels in mutants were tested by

ELISA (Figure 1c). M3 nrd3-1 and nrd3-2 plants showed clearly more NPTII than K/K;H/H

plants, indicating that their kanamycin resistance was due to a reactivation of NPTII

expression. However, NPTII levels measured in M3 nrd3-1 and nrd3-2 did not reach that of

K/K control plants. To address whether nrd3-1 and nrd3-2 mutations release RdDM, we

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analysed ProNOS DNA methylation at the ProNOS-NPTII reporter gene in the TARGET

transgene by methylation-sensitive restriction enzyme cleavage-quantitative PCR (msRE-

qPCR) in M3 mutant plants (Figure 1d). The result showed an almost complete loss of

cytosine methylation in non-CG context (NheI, Alw26I), but only partial reduction in CG

context (Psp1406I), which is a hallmark of mutations affecting RdDM (Kanno et al., 2004;

Kanno et al., 2005; Kanno et al., 2008; Daxinger et al., 2009; Finke et al., 2012a; Finke et

al., 2012b). Cleavage with NcoI, whose recognition site lies outside the region targeted by

RdDM and thus is not methylated, served as control for the accessibility of genomic DNA

(Fischer et al., 2008). To check for possible effects on ProNOS-IR-derived siRNAs, RNA

preparations enriched for small RNAs from M3 nrd3-1 and nrd3-2 were analysed in Northern

blots in comparison to RNA from wild-type K/K;H/H plants and a nrpd2a-55 mutant

defective in the second-largest subunit of Pol IV as a representative for a mutation affecting

RdDM (Finke et al., 2012a). No gross differences in SILENCER transgene-derived 24nt, 22nt

and 21nt siRNAs were detected (Figure 1e). This indicates a defect downstream of siRNA

formation in the analysed nrd3-1 and nrd3-2 mutants.

Map-based cloning and next generation sequencing identifies nrd3-1 and nrd3-2 as new drm2

alleles

M3 nrd3-1 plants were crossed with A. thaliana accession Ler and F2 generation seedlings

obtained from these crosses were screened for individuals resistant to hygromycin (HygR,

SILENCER present) and kanamycin (KanR, TARGET present, homozygous for mutation

releasing RdTGS) (Methods S1). Genotyping of these HygR KanR F2 plants using an Illumina

GoldenGate® genotyping assay (Figure S2) identified a section on chromosome 5 (Figure 2a)

including genes DRM2 (At5g14620) and DRM1 (At5g15380) known to have roles in de novo

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DNA methylation in RdDM (Cao and Jacobsen, 2002; Cao et al., 2003; Jullien et al.; 2012;

Naumann et al., 2011). PCR amplification and Sanger sequencing of DRM2 in five M3

individuals of nrd3-1 identified consistently a C to T transition at position 2908 in exon 9

according to the TAIR 10 gene model, causing a premature stop codon at position 547 of the

protein (Figure 2b). The resulting protein lacks motives III, IV and V of the DMTase domain,

which are highly conserved among DMTases (Cheng, 1995; Cao et al., 2000) and therefore is

most likely not functional. That nrd3-1 is allelic to drm2 was verified via complementation,

for which the intact coding region of DRM2 under the control of its native promoter

(ProDRM2:DRM2) was amplified from wild-type plants via PCR and introduced into M3

plants of nrd3-1 via Agrobacterium tumefaciens mediated transformation (Figure 2c). The

mutation in nrd3-2 was determined by whole genome sequencing of a M3 nrd3-2 plant in

comparison to a non-mutagenized control K/K;H/H plant using Illumina HiSeq2000®

technology. Sequence comparison identified 1071 unique SNPs between the M3 nrd3-2 plant

and the non-mutagenized control. 80 of these SNPs were predicted to cause non-synonymous,

homozygous mutations in open reading frames of protein coding genes (Table S1). One SNP

was found at position 2467 (relative to A of the START codon) in exon 9 of DRM2 according

to the TAIR 10 gene model (Figure 2b). This mutation causes a premature stop codon at

position 435 of the protein, thus resulting in a protein lacking all conserved DNMTase motifs.

The presence and homozygous nature of the mutation was confirmed by PCR amplification

and Sanger sequencing of DRM2 of five M3 nrd3-2 plants. Thus, in nrd3-2 an additional

drm2 allele was identified.

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CG context methylation is maintained to different degrees at the TARGET ProNOS and

endogenous RdDM tragets in nrd3 mutants

As methylation sensitive restriction cleavage-qPCR (msRE-qPCR) (Figure 1d) can test

methylation only at the suitable restriction sites available, the overall methylation of the

ProNOS in the TARGET ProNOS-NPTII reporter gene in wild-type K/K;H/H as well as in M3

nrd3-1 and nrd3-2 plants carrying drm2 alleles was determined by bisulfite sequencing

(Figure 3a). The results showed that cumulative cytosine methylation in the ProNOS region

undergoing RdDM that is 68% in wild-type K/K;H/H plants is reduced to 27% and 19% in

nrd3-1 and nrd3-2, respectively. This reduction is primarily due to extensive loss of CHH

context and less pronounced loss of CHG context methylation. ProNOS methylation in CG

context, which was 83% in wild-type K/K;H/H plants, is with 74% almost unchanged in M3

nrd3-1 and with 52% moderately reduced in M3 nrd3-2. The persistence of CG context

methylation is in particular illustrated by the Psp1406I site in the TARGET ProNOS, where in

M3 nrd3-1 and nrd3-2 methylation is present in more than a half of DNA molecules

according to msRE-qPCR (Figure 1d) and bisulfite sequencing (Figure S3). In K/K;H/H

plants, methylation is distributed evenly over the whole length of the TARGET ProNOS

despite of a prevalence of cytosines in CG context in its 5’ and in CHH context in its 3’ half

(Figure S3). As persistence of CG context methylation was rather equal over all CG context

sites within the ProNOS, more remaining methylation was found in the 5’ than the 3’

ProNOS half in nrd3-1 and nrd3-2 mutants.

To test whether this effect was specific for the analysed transgenic ProNOS of TARGET Kchr1-

10 (Fischer et al., 2008) or a general feature, DNA methylation at the endogenous RdDM

targets AtSN1 (Zilberman et al., 2003; Kuhlmann and Mette, 2012) (Figure 3b), MEA-ISR

(Cao and Jacobsen, 2002) (Figure 3c) and AtMU1 (Bäurle et al., 2007) (Figure 3d) was

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determined in M3 nrd3-1 and nrd3-2 by bisulfite sequencing. In addition, DNA methylation

at AtCOPIA4 was analysed as a RdDM-independent control (Johnson et al., 2007) (Figure

3e). In M3 nrd3-1 and nrd3-2 plants, DNA methylation is reduced at AtSN1 in CG, CHG and

CHH context, which differs from the pattern observed for the TARGET ProNOS. In contrast,

CG context methylation is well maintained in nrd3-1 and nrd3-2 at MEA-ISR and in nrd3-1 at

AtMU1, while CHG and CHH context methylation are reduced. Methylation at AtCOPIA4 is

unaltered in all contexts in M3 nrd3-1 and nrd3-2 plants. Thus, methylation patterns at

transgenic and endogenous RdDM targets are affected by the loss of de novo methylation in

nrd3 mutants in a selective way, with AtSN1 acting distinct from the TARGET ProNOS,

MEA-ISR and AtMU1.

Persistence of CG context methylation at the TARGET ProNOS increases with extended

exposure to RNA-directed DNA methylation

The high persistence of CG context methylation at the TARGET ProNOS in M3 nrd3-1 and

nrd3-2 mutants was striking, as this was already the second generation in which the analysed

plants had been homozygous for the nrd3 mutant alleles and thus defective in RNA-directed

DNA methylation (Figure S1). This prompted us to approach the persistence of TARGET

ProNOS CG context methylation in a systematic way by taking advantage of the independent

segregation of the genetically unlinked transgene loci TARGET Kchr1-10 on chromosome 1 and

SILENCER loci H on chromosome 4, respectively, in our system (Fischer et al., 2008). If

K/K;H/- plants homozygous for the TARGET and hemizygous for the SILENCER are allowed

to self-pollinate, 25% of the progeny will be of genotype K/K;H/H, 50% of K/K;H/- and 25%

of K/K, having lost the SILENCER by segregation. In these K/K* individuals (*indicating

that the TARGET transgene was exposed to the SILENCER in the previous generation),

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maintenance of methylation in absence of the inducing signal can be analysed in wild-type

plants (Figure 4a). The K/K;H/- plants in such a segregating population can serve as a source

for the next generation of K/K* individuals, in which the TARGET now has been exposed for

one generation more to the SILENCER. Parental (P) plants homozygous for either TARGET K

or SILENCER H where crossed and the obtained F1 plants hemizygous for both transgenes

(K/-;H/-) were allowed to self-pollinate. Among the resulting F2 and their follow up progeny,

individuals with the desired K/K;H/- and K/K* genotypes were identified by PCR and

reporter gene activity tests.

TARGET ProNOS methylation was analysed in DNA preparations from rosette leaves of

individual mature plants by msRE-qPCR for members of the F1, F3, F4, and F5 generation

(Figure 4b) and bisulfite sequencing for members of the F1, F2, F3, and F4 generation (Figure

4c). ProNOS DNA methylation in the TARGET transgene reached its final level already when

encountering the SILENCER in the F1 generation and then stayed essentially the same in the

following F2 to the F5 in presence of the SILENCER (Figure 4b top, Figure 4c top, Figure S4).

This applied in particular to CG context methylation (F1 78%, F2 82%, F3 77%, F4 80%)

determined by bisufite sequencing. If at all, there might have been a limited decrease in non-

CG context methylation over generations, as seen at the sites recognised by NheI and Alw26I

in the assay using methylation-sensitive restriction enzyme cleavage and for CHH context

methylation (F1 67%, F2 59%, F3 47%, F4 51%) in bisulfite sequencing data. Further, no

change in the distribution pattern of DNA methylation over the cytosine sites of the TARGET

ProNOS was seen in bisulfite sequencing data over generations (Figure S4). However, we

found indication for slight spreading of DNA methylation into the area 5´ of the region

covered by ProNOS siRNAs. In particular, the first CG site adjacent to the 5énd of the

region covered by ProNOS siRNAs showed methylation after 3 generations in the presence

of the SILENCER (Figure S4, F3 K/K;H/-, F4 K/K;H/-) that was not observed earlier (Figure

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S4, F1 K/-;H/-). No DNA methylation spread into the area 3´ of the region covered by

ProNOS siRNAs (Figure S4). This is in line with the results from msRE-qPCR for enzyme

NcoI, for which no consistent shift to cleavage inhibition was seen (Figure 4b).

In K/K* individuals that lost the SILENCER by segregation, persisting ProNOS methylation

was first detected at a low level in F2 plants and then at stepwise increasing levels in F3, F4

and F5 plants with increasing numbers of generations of the TARGET having been exposed to

the SILENCER (Figure 4b bottom, Figure 4c bottom). Thus, DNA methylation persistence on

the TARGET ProNOS accumulates over generations. Mainly methylation in symmetric CG

context was inherited, which was consistently detected by msRE-qPCR for Psp1406I (F3, F4,

F5) and by bisulfite sequencing (F2 12%, F3 39%, F4 43%) (Figure S5). Again, analysis of the

Psp1406I site was particularly informative, with methylation detected in more than a half of

DNA molecules in F4 and F5 K/K* plants according to msRE-qPCR (Figure 4b) and F3 and F4

K/K* according to bisulfite sequencing (Figure S5). Persistence of CG context cytosine

methylation was rather equal over all sites present in the TARGET ProNOS, thus showing a

slight preference in the 5´ part of the region that had been covered by SILENCER-derived

ProNOS siRNAs due to the higher prevalence of CG context cytosines (Figure S4; Figure

S5). Further, in F3 and F4 K/K* plants, persistence of some CHG methylation could be

observed at the 5énd of the ProNOS siRNA-target region. Similar as in the analysed F4

K/K;H/- plants, in F4 K/K* plants some DNA methylation was found at the first CG site

adjacent to the 5énd of the ProNOS siRNA-target region. In contrast, the methylation at

asymmetric context cytosines in flanking regions did not persist. In comparison, in control

K/K and K/- plants containing the naive TARGET, only minor apparent ProNOS methylation

was indicated by both methods, most likely resulting from incomplete restriction cleavage or

incomplete chemical conversion of actually unmethylated DNA.

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Inherited TARGET ProNOS CG context methylation compromises NPTII expression.

The 43% of CG context methylation together with the lower levels of CHG and CHH

methylation at the TARGET ProNOS detected by bisulfite sequencing in F4 K/K* plants

accounted to in total 14% of methylated cytosines. To see whether this is sufficient to affect

ProNOS-NPTII reporter gene expression, NPTII protein levels in extracts from mature rosette

leaves were measured by ELISA (Figure 5). NPTII levels in F4 K/K* plants were reduced in

comparison to K/K control plants that contained the naive TARGET, showing that even

inheritance of moderate levels of promoter methylation can affect gene expression.

The silenced TARGET ProNOS does not accumulate histone modification H3K9me2

As proposed above, an explanation for the increasing heritability of CG context methylation

at the TARGET ProNOS would be that the ProNOS undergoing RdDM attracts some mark

that accumulates over generations. This is most likely not cytosine methylation itself, as

DNA methylation already reaches its final level in F1 K/-;H/- plants, while little methylation

inheritance is seen in F2 K/K* derived from them. Histone-H3-lysine-9-dimethylation

(H3K9me2) can be associated with transcriptionally silenced transgenes in plants (Foerster et

al., 2011) and thus represents an obvious candidate for such a mark. Chromatin

immunoprecipitation combined with quantitative PCR (ChIP qPCR) was performed to

quantify H3K9me2 relative to total H3 associated with different regions of the TARGET

ProNOS in one-week-old F4 K/K and F4 K/K;H/H seedlings (Figure 6). The tightly silenced

transposable element Ta3 known to be associated with H3K9me2 was used as a reference,

while the active housekeeping gene PHOSPHOFRUCTOKINASE (PFK) served as a negative

control (Johnson et al., 2002; Mathieu et al., 2003; Mathieu et al., 2005). However, no

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increase of TARGET ProNOS-associated H3K9me2 was visible for the transcriptionally

silenced state, indicating that RdTGS and RdDM of the transgene promoter are independent

of histone modification H3K9me2. Consistently, introgression of mutant suvh4R302 allele

known to affect DNA methylation via formation of H3K9me2 at endogenous silencing

targets (Jackson et al., 2002; Ebbs and Bender, 2006) did not affect TARGET ProNOS

silencing and DNA methylation persistence (Figure S6).

Discussion

Utilising a two-component transgene system with a ProNOS-NPTII target gene highly

susceptible for RdTGS (Fischer et al., 2008; Finke et al., 2012a), two no rna-directed

transcriptional silencing 3 (nrd3) mutants with suppressed RdTGS and RdDM were isolated.

These showed restored resistance to kanamycin and presence of NPTII protein combined

with a strong reduction of asymmetric DNA methylation in the targeted ProNOS while

ProNOS-derived siRNAs were still present in unchanged levels. All these features are

characteristic for mutants defective in RdDM (Law and Jacobsen, 2010; Eun et al., 2012;

Matzke and Mosher, 2014). This was confirmed by the reduced DNA methylation (mainly in

non-CG context) found at the endogenous RdDM targets AtSN1 (Zilberman et al., 2003;

Kuhlmann and Mette, 2012), MEA-ISR (Cao and Jacobsen, 2002) and AtMU1 (Bäurle et al.,

2007). Map-based cloning and next generation sequencing identified in nrd3-1 and nrd3-2

the two new drm2 alleles nrd3-1/drm2-8 and nrd3-2/drm2-9 carrying premature translational

stop codons upstream of the essential DMTase domain of DOMAIN REARRANGED

METYHLTRANSFERASE 2. The identification of DRM2 in the screen for mutants defective

in RdDM is consistent with the predicted predominant role of DRM2 in comparison to DRM1

in RdDM (Cao and Jacobsen, 2002; Cao et al., 2003) and its repeated identification in

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independent mutagenesis studies (Naumann et al., 2011). The maintenance of up to 70% of

symmetric context ProNOS DNA methylation in our nrd3 lines to the second generation (M3)

is remarkable and was associated with a reduction of NPTII accumulation, but it is unclear

whether this was due to persistent transcriptional silencing or to pleiotropic effects of

mutagenesis. Similar maintenance of RdDM-induced CG context methylation at transgenic

reporter genes in RdDM-releasing mutants has been observed previously (Aufsatz et al.,

2002b; Daxinger et al., 2009; Finke et al., 2012a; Sasaki et al., 2014), but was not followed

up in detail.

At the endogenous RdDM targets AtSN1, AtMU1, and MEA-ISR, CG context methylation

was maintained to different degrees in nrd3 mutants, while CHG and CHH context

methylation was clearly reduced. This is consistent with previous observations for mutants

affected in RdDM, for example nrd1/idn2 or nrd2/nrpd2a (Finke et al., 2012a) or a suvh2

suvh9 double mutant (Kuhlmann et al., 2012). For ATSN1, increased accumulation of

transcripts in the presence of remaining DNA methylation has been seen in several mutants

affecting RdDM (He et al., 2009; Gao et al., 2010), including the suvh2 suvh9 double mutant

showing remaining AtSN1 CG and CNG context methylation levels similar to the ones in

nrd3-1 and nrd3-2 (Kuhlmann et al., 2012). However, as, in contrast to transgenic reporter

genes, completely unmethylated endogenous RdDM targets that would allow the

determination of reference transcript levels are usually not available, the effect of persistent

DNA methylation on transcript accumulation cannot be determined. The maintenance of

symmetric TARGET ProNOS methylation in our material must have been established within

the four generations between the first encounter of TARGET and SILENCER in the F1 and the

M2 generation equivalent to F5 in which the RdDM pathway became first defective.

However, as the level of siRNAs with ProNOS-homology was unaltered in drm2 plants, it

could not be distinguished whether ProNOS CG context methylation was maintained by a

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mechanism solely based on DNA methylation per se (e.g. via MET1) independent from the

siRNA signal or by a so far unknown mechanism dependent on siRNAs but independent of

DRM2.

In order to address this point and to get insights into a possible cumulative effect of multiple

generations of exposure to the siRNA signal also reported by others (Lunerova-Bedrichova et

al., 2008; Khaitova et al., 2011), persistence of DNA methylation at the TARGET ProNOS

was analysed over successive generations with the SILENCER transgene presence or after

segregating out the SILENCER transgene as the source of ProNOS siRNAs in sexual

reproduction. Consistent with previous observations (Fischer et al., 2008), the particular

TARGET transgene used in our study showed already in the first generation (F1) of encounter

with the SILENCER the final level of approx. 80% cytosine methylation in all contexts. In the

following F3 to F5 generations with the SILENCER present, TARGET ProNOS methylation

stayed essentially the same, with a slight tendency to reduced asymmetric DNA methylation

in later generations. Such a possible partial mutual exclusion of DNA methylation

maintenance (via MET1 at CG context sites only) and RdDM-mediated de novo methylation

(in all contexts) has been discussed previously based on observations made in met1 mutants

(Mathieu et al., 2007). After losing the SILENCER in the F2 by segregation after one

generation of encounter, only little DNA methylation in mainly CG context persisted at the

TARGET ProNOS. However, persisting DNA methylation increased with each following

generation of joint presence of TARGET and SILENCER transgenes. At latest in the F4

generation, more than half of the DNA molecules in the TARGET ProNOS maintained CG

context methylation after segregation of the SILENCER. Thus, from this stage on, it can be

excluded that only one of the two copies present in plants homozygous for the TARGET

remained methylated, for example due to differential inheritance of CG context methylation

via male versus the female gametophytes (Saze et al., 2003). In the F4 generation, the

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persisting approx. 40% DNA methylation were sufficient to reduce the TARGET ProNOS-

NPTII gene expression by approx. 30%, proving that maintained symmetric context promoter

methylation can substantially reduce gene expression levels. Despite the presence of some

CHG methylation in F3 and F4 K/K* plants, we did not find evidence that the persisting DNA

methylation and its effect on gene expression was related to histone modification H3K9me2,

as the silenced ProNOS was not associated with increased H3K9me2 and a suvh4 mutant

background did not affect reporter gene silencing. This is consistent with the rather limited

involvement of H3K9me2 in RdDM reported by others, for example in the case of trans-

chromosomal methylation induction (Greaves et al., 2014). Loss of histone acetylation via

histone deacetylase HDA6 might be an alternative candidate for involved histone

modifications (Blevins et al., 2014).

In summary, our observations made using a transgene-based experimental system suggest

that setting up DNA methylation at a particular region in the A. thaliana genome can be

divided into an initial phase of immediate, but still fully reversible de novo methylation and

transcriptional gene silencing, that is followed by the setting of so far unknown additional

chromatin mark(s) maintenance over several generations, resulting in efficient maintenance

of cytosine methylation in symmetric sequence context connected with consolidation of gene

silencing (Figure 7). This is important for the role of RNA-directed DNA methylation in

genome-wide epigenetic regulation (Bond and Baulcombe, 2014). In terms of using RNA-

directed DNA methylation of endogenous promoter regions in plant biotechnology (Okano et

al., 2008; Kasai and Kanazawa, 2013), persistence of DNA methylation and transcriptional

silencing after a number of generations would allow “non-transgenic” lines with modulated

gene expression after removal of the SILENCER transgene. Differences in the time scale

needed for the initial de novo methylation to be consolidated by methylation maintenance

might affect the way in which RNA-directed DNA methylation contributes to heterosis

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(Birchler et al., 2010) via trans-chromosomal changes of DNA methylation (Greaves et al.,

2012; Greaves et al., 2014). Further, it is tempting to speculate that separating short-term

responses from establishing long-term epigenetic memory would allow the plant to integrate

external stress exposure over extended time periods (Luna and Ton, 2012; Thellier and

Lüttge, 2013).

Experimental procedures

Additional detailed information is provided online in supporting experimental procedures

(Methods S1).

Plant material and EMS mutant screen

Transgenic A. thaliana double homozygous for TARGET and SILENCER transgenes (Kchr1-

10/Kchr1-10;H/H) in accession Col-0 have been described in Fischer et al. (2008). Plant

cultivation, testing for antibiotics resistances and PCR-based detection of transgenes was

performed as indicated there. Seed EMS (ethyl-methanesulfonate) mutagenesis was

performed by Lehle Seeds. No rna-directed transcriptional silencing (nrd) mutations were

screened for as described in Finke et al. (2012a) and (2012b).

NPTII protein quantification

Amounts of NPTII protein were determined by ELISA using an Agdia PathoScreen Kit for

NPTII (Agdia), with total protein in the same extracts determined using a Pierce BCA Protein

Assay kit (Pierce).

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DNA methylation analysis and small RNA analysis

ProNOS DNA methylation analysis by methylation sensitive restriction cleavage-qPCR was

done according to Finke et al. (2012a) and (2012b) with data analysis using the ΔΔCt method

according to Pfaffl (2001). Primers are listed in Table S2. Analysis by bisulfite genomic

sequencing was done as described in Kuhlmann and Mette (2012) and Finke et al. (2012a)

using primers listed in Table S3. DNA methylation patterns were analysed using CyMATE

software (Hetzl et al., 2007). For analysis of ProNOS-derived siRNAs and Mir167 as control

(for probe, see Table S2), a RNA preparation enriched in small RNAs was submitted to

Northern blot analysis as described previously (Mette et al., 2005; Finke et al., 2012a).

Mapping of mutations and complementation by Agrobacterium-mediated transformation

Mapping of nrd3-1 was performed using Illumina VeraCode GoldenGate® genotyping (Table

S4) and InDel markers (Salathia et al., 2007; Table S5) as described (Finke et al., 2012a).

The wild-type DRM2 ORF including the 3’UTR and around 450 bp upstream of the

transcriptional start site was amplified from A. thaliana accession Col-0 and cloned in binary

vector pCMBL2 (Finke et al., 2012a) to yield pCMBL2+DRM2. pCMBL2+DRM2 was

introduced into A. tumefaciens strain pGV2260 and transferred to M3 nrd3-1 via the floral dip

method (Clough and Bent, 1998).

Next generation sequencing and bioinformatics analysis

Next generation sequencing (NGS) of plant genomic DNA was performed by IPK’s in-house

sequencing facility using an Illumina HiSeq2000 device according to manufacturer’s

protocols. Sequencing was performed in paired-end read mode (2 times 100 bp). In total, 47

194 081 (M3 nrd3-2) and 62 130 438 (control K/K;H/H) paired-end reads (2 times 100 bp)

were obtained. Sequencing reads were mapped to the A. thaliana reference sequence (version

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TAIR10; http://www.arabidopsis.org/) using BWA 0.6.1 (Li and Durbin, 2009). The

parameter "-q 15" was used for "bwa aln" to trim off low quality bases. 30 158 845 (63.9%;

M3 nrd3-2) and 56 782 470 (91.4%; control K/K;H/H) reads were mapped as proper pairs to

the A. thaliana nuclear genome to reach on average 22-fold and 41-fold coverage,

respectively. SNP calling was performed with the SAMtools mpileup/bcftools pipeline

version 0.1.18 (Li, 2011) using default parameters. The program snpEff 2.1b (Cingolani et

al., 2012) was used for variant annotation and effect prediction based on the TAIR10 genome

annotation. For the identification of mutations in M3 nrd3-2 in comparison to the non-

mutagenized K/K;H/H control, the respective SNP lists were imported into MS Excel and

compared manually. SNPs occurring in both, mutant and control plants were considered as

pre-existing in the transgenic line submitted to mutagenesis and excluded from further

analysis. Further, SNPs affecting promoters, introns, 5’ and 3’ UTRs as well as “silent”

nucleotide changes in exons not expected to compromise gene function were excluded from

the initial search. The resulting list of gene loci potentially containing the mutation causative

for the release of transcriptional silencing in nrd3-2 is provided in Table S1. Original data

were deposited in the NCBI Short Read Archive under accession number PRJEB6549.

Chromatin immunoprecipitation-qPCR analysis

Chromatin immunoprecipitation (ChIP) was performed as outlined in Kuhlmann and Mette

(2012). Primers used for amplification of Ta3, ProNOS 5´part, ProNOS 3´part and PFK ChIP

are described in Table S6.

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Acknowledgements

We thank Christa Fricke, Inge Glaser, Beate Kamm, Doreen Stengel and Ines Walde for

excellent technical assistance, Lothar Altschmied, Axel Himmelbach and Uwe Scholz for

helpful discussions and Ingo Schubert and Renate Schmidt for critical comments on the

manuscript. This work received support from the German Research Foundation (DFG)

collaborative research centre (SFB) 648 “Molecular Mechanisms of Information Processing

in Plants” (M.K.) and IPK Gatersleben (A.F.).

Short legends for Supporting Information

Figure S1: Outline of the mutagenesis approach

Figure S2: Rough mapping of mutation nrd3-1

Figure S3: TARGET ProNOS methylation patterns in K/K;H/H wild-type control, M3 nrd3-1

and nrd3-2 plants

Figure S4: TARGET ProNOS methylation patterns in F1, F2, F3 and F4 plants in presence of

the SILENCER

Figure S5: TARGET ProNOS methylation patterns in F2, F3 and F4 plants after segregation of

the SILENCER

Figure S6: Mutation suvh4 does not affect TARGET ProNOS RNA-directed transcriptional

gene silencing and DNA methylation maintenance.

Table S1: Candidate genes for nrd3-2

Table S2: Primers for methylation-sensitive restriction cleavage-quantitative PCR and

miRNA hybridisation

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Table S3: Primers for bisulfite sequencing

Table S4: Illumina GoldenGate® markers for Col-0 / Ler polymorphisms

Table S5: Primers for PCR-based detection of Col-0 / Ler polymorphisms

Table S6: Primers for chromatin immunoprecipitation-quantitative PCR

Methods S1: Supporting experimental procedures

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Figure legends

Figure 1: Release of RNA-directed transcriptional gene silencing and RNA-directed DNA

methylation in nrd3 mutants.

(a) Transgene system: The SILENCER (H) transgene contains an inverted repeat (IR) of the

NOPALINE SYNTHASE promoter (ProNOS, big red arrows) sequence under control of the

strong constitutive cauliflower mosaic virus 35S promoter (Pro35S). Transcripts (thin red

arrow) of the ProNOS inverted repeat (IR) structure fold to form double-stranded RNA,

which is processed to short interfering RNAs providing a signal for in trans DNA

methylation and transcriptional silencing of the ProNOS copy that controls transcription of a

NEOMYCIN PHOSPHOTRANSFERASE II (NPTII, conferring kanamycin resistance) in the

unlinked TARGET (K) transgene. In addition, the SILENCER contains a HYGROMYCIN

PHOSPHOTRANSFERASE (HPT) gene conferring hygromycin resistance and the TARGET a

ß-GLUCURONIDASE (GUS) reporter gene. (b) Test for kanamycin resistance on medium

containing 200 mg/l kanamycin. (c) Quantification of NPTII protein by ELISA: NPTII

protein amounts in relation to total soluble protein were measured in extracts from leaves of

five KK, five KK;HH, ten M3 nrd3-1 and six M3 nrd3-2 8-week-old plants. Results are

displayed relative to the mean value for un-silenced expression in (K/K) plants (set to 1).

Column heights represent mean values, error bars standard deviations. (d) TARGET ProNOS

cytosine methylation was determined by quantitative PCR after cleavage of genomic DNA

from 8-week-old plants with methylation-sensitive restriction enzymes (C in recognition

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sequence underlined: methylation of cytosine blocks cleavage according to REBASE

(http://rebase.neb.com/rebase/rebase.html) Psp1406I (grey – symmetric CG context:

AACGTT), NheI (blue – CHG and CHH context: GCTAGC), Alw26I (orange – asymmetric

CHH context: GTCTC, GAGAC) and NcoI (yellow – asymmetric CHH context, control

outside of the methylated region: CCATGG). Per genotype, five individual plants were

tested. Results are displayed relative to the mean value for input DNA. Column heights

represent mean values, error bars standard deviations. (e) Northern blot for siRNAs derived

from transcription of ProNOS IR in the SILENCER. Blots were hybridised with a RNA probe

specific for sense ProNOS siRNAs (top). Comparable loading was confirmed by re-

hybridisation with miR167-specific probe after stripping (bottom).

Figure 2: Mutants nrd3-1 and nrd3-2 carry drm2 alleles.

(a) Physical map indicating markers and recombination events (numbers in parentheses, of

152 chromosomes in total) used to delineate the position of nrd3-1 on the upper arm of

chromosome 5. The position of the DRM2 gene is indicated by a red arrowhead. (b)

Nucleotide (top) and related amino acid changes (bottom) in nrd3-1 and nrd3-2 mutants in

the DRM2 gene model according to TAIR 10, with * indicating a stop codon. (c)

Complementation of nrd3-1 with transgenic DRM2. T2 seeds obtained by self-pollination of

T1 transformants from Agrobacterium-mediated transfer of binary vector containing intact

DRM2 or empty vector (EV) to M3 nrd3-1 were germinated on medium containing 200 mg/l

kanamycin. (d) NPTII protein amounts in relation to total soluble protein were measured in

extracts from leaves of 5-week-old plants (left). Results are displayed relative to the mean

value for unsilenced expression in (K/K) plants (set to 1). Per genotype, five individual plants

were tested. Column heights represent mean values, error bars standard deviations.

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Figure 3: Methylation at the TARGET ProNOS and endogenous DNA methylation tragets in

nrd3 mutants.

DNA methylation at (a) the ProNOS of the ProNOS-NPTII reporter gene and endogenous

RdDM targets (b) AtSN1, (c) MEA-ISR, (d) AtMU1, and (e) AtCOPIA4 were analysed in

detail by bisulfite sequencing. Cumulative methylation levels at all cytosines in the analysed

region (grey columns), cytosines in CG context (black columns), CHG context (blue

columns; H stands for A, C or T) and CHH context (red columns). N indicates the number of

clones sequenced per target and genotype.

Figure 4: TARGET ProNOS methylation persisting after segregation of the SILENCER.

(a) Pedigree scheme of plants types included in analysis. K/K* marked by an asterisk

indicates that the TARGET transgene was exposed to the SILENCER in the previous

generation(s) (b) TARGET ProNOS cytosine methylation was determined by quantitative

PCR after cleavage of genomic DNA from mature plants with methylation-sensitive

restriction enzymes Psp1406I (grey – symmetric CG context), NheI (blue – CHG and CHH

context), Alw26I (orange – asymmetric CHH context) and NcoI (yellow – asymmetric CHH

context, control outside of the methylated region, see legend of Figure 1). Per stage, three

individual plants were tested. Results are displayed relative to the mean value for input DNA.

Column heights represent mean values, error bars standard deviations. (c) DNA methylation

at the ProNOS of the ProNOS-NPTII reporter gene was analysed in detail by bisulfite

sequencing. Cumulative methylation levels at all cytosines in the analysed region (grey

columns), cytosines in CG context (black columns), CHG context (blue columns; H stands

for A, C or T) and CHH context (red columns). N indicates the number of clones sequenced

per stage.

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Figure 5: NPTII expression in presence of persisting TARGET ProNOS CG context

methylation

Quantification of NPTII protein by ELISA: NPTII protein amounts in relation to total soluble

protein were measured in extracts from leaves of 8-week-old plants. Results are displayed

relative to the mean value for un-silenced expression in K/K plants (set to 1). Per genotype,

four (F1 K/-;H/-, F4 K/K;H/-, F4 K/K*) or two (F4 K/K, F4 K/K;H/H) individual plants were

tested. Column heights represent mean values, error bars standard deviations.

Figure 6: H3K9me2 is not increased at the TARGET ProNOS undergoing RNA-directed

DNA methylation.

Chromatin immunoprecipitation combined with quantitative PCR was performed on 1 week

old seedling with antibodies specific to H3 and H3K9me2 and primers specific for transposon

Ta3, TARGET ProNOS 5´side and 3´side regions and housekeeping gene

PHOSPHOFRUCTOKINASE (PFK). Results were calculated separately as the ratio of the

H3K9me2-specific signal to the H3-specific signal in relation to the ratio obtained for Ta3

(set to 1.0) for each chromatin preparation. Column heights indicate the median, error bars

the absolute deviation from five (Ta3, ProNOS 5´side and 3´side) or four (PFK) biological

replicates, respectively.

Figure 7: Transition from immediate response to epigenetic memory over generations

In the first generation G1 of de novo DNA methylation of a promoter via RNA-directed DNA

methylation, transcriptional gene silencing sets in as an immediate response. However, this

silencing is still reversible. Only with the following generations G2, G3 and G4 of ongoing

RNA-directed DNA methylation, DNA methylation maintenance and thus an epigenetic

memory is established.

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K / K K / K ; H / H

nrd3-1 nrd3-2

(b)

K / KH / H

0

0.2

0.4

0.6

0.8

1.0

1.2

NP

TII

per

sol.

prot

ein

nrd3-1 nrd3-2K / K

(c)

(e)ProNOS

miR167

K / K K / KH / H

nrd3-1nrpd2a-55

nrd3-2

24 nt

21 nt

21 nt22 nt

SPro35SProNOS SONorPPro19SHPT35St

LB RB

SILENCER (H)

ProNOS NPTII Pro35SOCSt OCStGUS

LB RB

TARGET (K)

(a)

(d)

K / K K / KH / H

nrd3-1 nrd3-2

Psp1406I NheI Alw26I NcoI

0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

uncu

t re

l. to

inpu

t

Figure 1: Release of RNA-directed transcriptional gene silencing and RNA-directed DNA methylation in nrd3 mutants.

(a) Transgene system: The SILENCER (H) transgene contains an inverted repeat (IR) of the NOPALINE SYNTHASE promoter

(ProNOS, big red arrows) sequence under control of the strong constitutive cauliflower mosaic virus 35S promoter (Pro35S).

Transcripts (thin red arrow) of the ProNOS inverted repeat (IR) structure fold to form double-stranded RNA, which is processed to

short interfering RNAs providing a signal for in trans DNA methylation and transcriptional silencing of the ProNOS copy that controls

transcription of a NEOMYCIN PHOSPHOTRANSFERASE II (NPTII, conferring kanamycin resistance) in the unlinked TARGET (K)

transgene. In addition, the SILENCER contains a HYGROMYCIN PHOSPHOTRANSFERASE (HPT) gene conferring hygromycin

resistance and the TARGET a ß-GLUCURONIDASE (GUS) reporter gene. (b) Test for kanamycin resistance on medium containing

200 mg/l kanamycin. (c) Quantification of NPTII protein by ELISA: NPTII protein amounts in relation to total soluble protein were

measured in extracts from leaves of five KK, five KK;HH, ten M3 nrd3-1 and six M3 nrd3-2 8-week-old plants. Results are displayed

relative to the mean value for un-silenced expression in (K/K) plants (set to 1). Column heights represent mean values, error bars

standard deviations. (d) TARGET ProNOS cytosine methylation was determined by quantitative PCR after cleavage of genomic DNA

from 8-week-old plants with methylation-sensitive restriction enzymes (C in recognition sequence underlined: methylation of cytosine

blocks cleavage according to REBASE (http://rebase.neb.com/rebase/rebase.html) Psp1406I (grey – symmetric CG context:

AACGTT), NheI (blue – CHG and CHH context: GCTAGC), Alw26I (orange – asymmetric CHH context: GTCTC, GAGAC) and NcoI

(yellow – asymmetric CHH context, control outside of the methylated region: CCATGG). Per genotype, five individual plants were

tested. Results are displayed relative to the mean value for input DNA. Column heights represent mean values, error bars standard

deviations. (e) Northern blot for siRNAs derived from transcription of ProNOS IR in the SILENCER. Blots were hybridised with a RNA

probe specific for sense ProNOS siRNAs (top). Comparable loading was confirmed by re-hybridisation with miR167-specific probe

after stripping (bottom).

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DRM2

nrd3 n = 76

At5g05780 At5g25370

(a) (b)

0.25 kb

At5g17620 (DRM2)

ATG TGA

31501

MTase

IX X I II III IV V

U U U1 626

100 aa

435

*

547

*

(c)

T2 nrd3-1 + DRM2 T2 nrd3-1 + EV

T2

nrd3-1+ EV

K / K K / KH / H

T2

nrd3-1 + DRM2

nrd3-10

0.2

0.4

0.6

0.8

1.0

1.2

NP

TII

per

sol.

prot

ein

(d)

DRM1

Figure 2: Mutants nrd3-1 and nrd3-2 carry drm2 alleles.

(a) Physical map indicating markers and recombination events (numbers in parentheses, of 152 chromosomes in total) used to

delineate the position of nrd3-1 on the upper arm of chromosome 5. The position of the DRM2 gene is indicated by a red arrowhead.

(b) Nucleotide (top) and related amino acid changes (bottom) in nrd3-1 and nrd3-2 mutants in the DRM2 gene model according to

TAIR 10, with * indicating a stop codon. (c) Complementation of nrd3-1 with transgenic DRM2. T2 seeds obtained by self-pollination of

T1 transformants from Agrobacterium-mediated transfer of binary vector containing intact DRM2 or empty vector (EV) to M3 nrd3-1

were germinated on medium containing 200 mg/l kanamycin. (d) NPTII protein amounts in relation to total soluble protein were

measured in extracts from leaves of 5-week-old plants (left). Results are displayed relative to the mean value for unsilenced

expression in (K/K) plants (set to 1). Per genotype, five individual plants were tested. Column heights represent mean values, error

bars standard deviations.

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020406080

100

020406080

100

TARGET ProNOS

AtSN1

MEA-ISR

AtCOPIA4

N = 19 N = 17 N = 19

N = 22 N = 13 N = 25

N = 21 N = 15 N = 15

K / KH / H

nrd3-1 nrd3-2

(b)

(c)

(d) AtMU1

N = 20 N = 12

02040

6080

100100

80

60

40

20

0

% m

eth

ylat

ed

(a)

020406080

100%

met

hyla

ted

100

80

60

40

20

0

100

80

60

40

20

0

% m

eth

ylat

ed

(e)

% m

eth

ylat

ed

100

80

60

40

20

0

% m

ethy

late

d

100

80

60

40

20

0N = 19 N = 15 N = 9

C

CG

CHG

CHH

Figure 3: Methylation at the TARGET ProNOS and endogenous DNA methylation tragets in nrd3 mutants.

DNA methylation at (a) the ProNOS of the ProNOS-NPTII reporter gene and endogenous RdDM targets (b) AtSN1, (c) MEA-ISR, (d)

AtMU1, and (e) AtCOPIA4 were analysed in detail by bisulfite sequencing. Cumulative methylation levels at all cytosines in the

analysed region (grey columns), cytosines in CG context (black columns), CHG context (blue columns; H stands for A, C or T) and

CHH context (red columns). N indicates the number of clones sequenced per target and genotype.

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Figure 4: TARGET ProNOS methylation persisting after segregation of the SILENCER.

(a) Pedigree scheme of plants types included in analysis. K/K* marked by an asterisk indicates that the TARGET transgene was

exposed to the SILENCER in the previous generation(s) (b) TARGET ProNOS cytosine methylation was determined by

quantitative PCR after cleavage of genomic DNA from mature plants with methylation-sensitive restriction enzymes Psp1406I

(grey – symmetric CG context), NheI (blue – CHG and CHH context), Alw26I (orange – asymmetric CHH context) and NcoI

(yellow – asymmetric CHH context, control outside of the methylated region, see legend of Figure 1). Per stage, three individual

plants were tested. Results are displayed relative to the mean value for input DNA. Column heights represent mean values, error

bars standard deviations. (c) DNA methylation at the ProNOS of the ProNOS-NPTII reporter gene was analysed in detail by

bisulfite sequencing. Cumulative methylation levels at all cytosines in the analysed region (grey columns), cytosines in CG

context (black columns), CHG context (blue columns; H stands for A, C or T) and CHH context (red columns). N indicates the

number of clones sequenced per stage.

(b)(a)

uncu

t re

lativ

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inpu

t

(c)

K / K

K / K

K / K

K / K

F5

K / K X - / -

K / -

K / K X H / H

K / K ; H / -

K / - ; H / -

K / K ; H / -

K / K ; H / -

K / K ; H / -

F4

P

F1

F2

F3 *

*

*

*

Psp1406I NheI Alw26I NcoI

% m

ethy

late

d

100

80

60

40

20

0

C CG CHG CHH

n.d.

0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

PH / H

n.d.

F1

K / -H / -

F2

K / KH / -

F3

K / KH / -

F4

K / KH / -

F5

K / KH / -

0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

PK / K

F1

K / -F2

K / KF3

K / KF4

K / KF5

K / K

n.d.

* * * *

100

80

60

40

20

0

F1 K / -

N=7

F3 K / K

N=21

F4 K / K

N=22

F2 K / K

N=10

* * *

F1 K / - ; H / -

N=7

F3 K / K ; H / -

N=12

F4 K / K ; H / -

N=10

F2 K / K ; H / -

N=10

SILENCERpresent

SILENCERabsent

SILENCERpresent

SILENCERabsent

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Figure 5: NPTII expression in presence of persisting TARGET ProNOS CG context methylation

Quantification of NPTII protein by ELISA: NPTII protein amounts in relation to total soluble protein were measured in extracts from

leaves of 8-week-old plants. Results are displayed relative to the mean value for un-silenced expression in K/K plants (set to 1).

Per genotype, four (F1 K/-;H/-, F4 K/K;H/-, F4 K/K*) or two (F4 K/K, F4 K/K;H/H) individual plants were tested. Column heights

represent mean values, error bars standard deviations.

0

0.2

0.4

0.6

0.8

1.0

1.2

F1

K / -H / -

F4

K / KH / -

F4

K / K

F4

K / K

F4

K / KH / H

NP

TII

per

solu

ble

prot

ein

*

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F4 K / K F4 K / K ; H / H

Ta3 ProNOS

5´side

ProNOS

3´side

PFK

1.2

1.0

0.8

0.6

0.4

0.2

0H3K

9me2

/ H

3 re

lativ

e to

Ta3

Figure 6: H3K9me2 is not increased at the TARGET ProNOS undergoing RNA-directed DNA methylation.

Chromatin immunoprecipitation combined with quantitative PCR was performed on 1 week old seedling with antibodies specific to

H3 and H3K9me2 and primers specific for transposon Ta3, TARGET ProNOS 5´side and 3´side regions and housekeeping gene

PHOSPHOFRUCTOKINASE (PFK). Results were calculated separately as the ratio of the H3K9me2-specific signal to the H3-

specific signal in relation to the ratio obtained for Ta3 (set to 1.0) for each chromatin preparation. Column heights indicate the

median, error bars the absolute deviation from five (Ta3, ProNOS 5´side and 3´side) or four (PFK) biological replicates,

respectively.

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Figure 7: Transition from immediate response to epigenetic memory over generations

In the first generation G1 of de novo DNA methylation of a promoter via RNA-directed DNA methylation, transcriptional gene

silencing sets in as an immediate response. However, this silencing is still reversible. Only with the following generations G2, G3

and G4 of ongoing RNA-directed DNA methylation, DNA methylation maintenance and thus an epigenetic memory is established.

de novomethylation

methylationmaintenance

generation G1 G2 G3 G4

memoryresponse

Documents

DNA methylation maintenance consolidates RNA-directed DNA methylation and transcriptional gene silencing over generations in Arabidopsis thaliana