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. 2012 Sep 21;24(9):3603–3612. doi: 10.1105/tpc.112.103119

Mutations in the Arabidopsis H3K4me2/3 Demethylase JMJ14 Suppress Posttranscriptional Gene Silencing by Decreasing Transgene Transcription[C],[W]

Ivan Le Masson 1, Vincent Jauvion 1, Nathalie Bouteiller 1, Maud Rivard 1, Taline Elmayan 1, Hervé Vaucheret 1,1
PMCID: PMC3480290  PMID: 23001035

This article reveals that the H3K4me2/3 demethylase JMJ14 likely is unable to demethylate transgene loci, thus promoting high levels of transcription, which favor the triggering of posttranscriptional transgene silencing. Removing JMJ14 may allow other H3K4 demethylases to reduce transgene transcription and prevent the triggering of posttranscriptional transgene silencing.

Abstract

Posttranscriptional gene silencing (PTGS) mediated by sense transgenes (S-PTGS) results in RNA degradation and DNA methylation of the transcribed region. Through a forward genetic screen, a mutant defective in the Histone3 Lysine4 di/trimethyl (H3K4me2/3) demethylase Jumonji-C (JmjC) domain-containing protein14 (JMJ14) was identified. This mutant reactivates various transgenes silenced by S-PTGS and shows reduced Histone3 Lysine9 Lysine14 acetylation (H3K9K14Ac) levels, reduced polymerase II occupancy, reduced transgene transcription, and increased DNA methylation in the promoter region, consistent with the hypothesis that high levels of transcription are required to trigger S-PTGS. The jmj14 mutation also reduces the expression of transgenes that do not trigger S-PTGS. Moreover, expression of transgenes that undergo S-PTGS in a wild-type background is reduced in jmj14 sgs3 double mutants compared with PTGS-deficient sgs3 mutants, indicating that JMJ14 is required for high levels of transcription in a PTGS-independent manner. Whereas endogenous loci regulated by JMJ14 exhibit increased H3K4me2 and H3K4me3 levels in the jmj14 mutant, transgene loci exhibit unchanged H3K4me2 and decreased H3K4me3 levels. Because jmj14 mutations impair PTGS of transgenes expressed under various plant or viral promoters, we hypothesize that JMJ14 demethylation activity is prevented by antagonistic epigenetic marks specifically imposed at transgene loci. Removing JMJ14 likely allows other H3K4 demethylases encoded by the Arabidopsis thaliana genome to act on transgenes and reduce transcription levels, thus preventing the triggering of S-PTGS.

INTRODUCTION

RNA silencing regulates gene expression through the action of small RNAs. RNA silencing also serves as a eukaryotic defense response that thwarts RNA derived from invading transposons, viruses, and transgenes (Mallory and Vaucheret, 2006; Ding and Voinnet, 2007; Kloc and Martienssen, 2008; Voinnet, 2009). In plants, 24-nucleotide small interfering RNAs (siRNAs) mediate transcriptional gene silencing (TGS) through DNA methylation and chromatin modifications, whereas 21-nucleotide siRNAs and microRNAs mediate posttranscriptional gene silencing (PTGS) through RNA cleavage and translational inhibition (Mallory and Vaucheret, 2006; Ding and Voinnet, 2007; Kloc and Martienssen, 2008; Voinnet, 2009). Transgene-based forward genetic screens identified mutants defective for TGS or PTGS and have served as important models for deciphering endogenous RNA silencing pathways.

PTGS specifically directed against an endogenous gene could be achieved using inverted repeat transgenes producing self-complementary transcripts that naturally form double-stranded RNA (dsRNA). In this form of PTGS (called IR-PTGS), dsRNAs are processed into siRNA duplexes by DCL4 without the need for an RNA-dependent RNA polymerase (Smith et al., 2000). siRNAs are methylated at their 3′ end by the methyltransferase HUA ENCHANCER1 (HEN1) (Boutet et al., 2003; Li et al., 2005; Yu et al., 2005) before loading onto ARGONAUTE1 (AGO1), which cleaves complementary target RNAs (Dunoyer et al., 2005; 2007). Forward genetic screens based on an inverted repeat trigger expressed specifically in the phloem implicated CLASSY1 (CLSY1), NUCLEAR RNA POLYMERASE IV largest subunit (NRPD1a), and RNA-DEPENDENT-RNA-POLYMERASE2 (RDR2), which are typical components of the endogenous 24-nucleotide siRNA TGS pathway (Dunoyer et al., 2005; 2007; Smith et al., 2007; Dunoyer et al., 2010). Their exact roles in IR-PTGS remain elusive.

PTGS could also be achieved using sense transgenes that are expected to only produce single-stranded RNA. During this form of PTGS (called S-PTGS), primary siRNAs are produced by an unknown mechanism and methylated at their 3′ end by the methyltransferase HEN1 (Boutet et al., 2003; Li et al., 2005; Yu et al., 2005) before loading into AGO1, which then cleaves complementary target RNAs (Morel et al., 2002; Baumberger and Baulcombe, 2005). AGO1-mediated cleavage generates RNA fragments that escape degradation by 5′→3′ and 3′→5′ exoribonucleases (Gazzani et al., 2004; Souret et al., 2004; Gy et al., 2007) because of the protecting activity of SUPPRESSOR OF GENE SILENCING3 (SGS3) and are transformed into dsRNA by RDR6 (Dalmay et al., 2000; Mourrain et al., 2000; Yoshikawa et al., 2005; Elmayan et al., 2009). These dsRNAs are processed into siRNA duplexes by DCL4 to produce secondary siRNA (Dunoyer et al., 2005; Blevins et al., 2006; Bouché et al., 2006; Deleris et al., 2006; Fusaro et al., 2006). These secondary siRNAs are loaded onto AGO1, which cleaves complementary transgene mRNAs, resulting in an amplification loop that reinforces silencing and contributes to the systemic propagation of S-PTGS from cell-to-cell (short-distance signaling) and through the vasculature (long-distance signaling) (Palauqui et al., 1997; Voinnet et al., 1998; Brosnan et al., 2007).

Forward genetic screens based on the line L1, which carries a posttranscriptionally silent p35S:GUS sense transgene, and the line 2a3, which carries a p35S:NIA2 sense transgene that triggers cosuppression of the endogenous genes NIA1 and NIA2, identified a series of S-PTGS–deficient mutants that defined 15 independent suppressor of gene silencing (sgs) loci. Previous analyses revealed the functions mutated in sgs2/sde1/rdr6, sgs3/sde2, sgs4/ago1, sgs5/hen1, sgs6/met1, sgs7/sde5, sgs9/hpr1, and sgs13/sde3 (Elmayan et al., 1998; Fagard et al., 2000; Morel et al., 2000; Mourrain et al., 2000; Dalmay et al., 2001; Hernandez-Pinzon et al., 2007; Jauvion et al., 2010); however, the functions mutated in sgs1, sgs8, sgs10, sgs11, sgs12, sgs14, and sgs15 remained unknown. Reverse genetics also identified a role for the chromatin-remodelling protein DDM1 in S-PTGS (Morel et al., 2000), suggesting that transgene chromatin state plays a role in this form of silencing. Here we show that sgs8 impairs both L1 and 2a3 S-PTGS and carries a mutation in Jumonji-C (JmjC) domain-containing protein14 (JMJ14), which encodes a Histone3 Lysine4 di/trimethyl (H3K4me2/3) demethylase (Jeong et al., 2009). Plants defective in JMJ14 exhibit reduced Histone3 Lysine9 Lysine14 acetylation (H3K9K14Ac) levels, reduced polymerase II (Pol II) occupancy, and reduced transgene transcription, consistent with the hypothesis that high levels of transgene transcription are required to trigger S-PTGS.

RESULTS

sgs8 Is Defective in S-PTGS but Not in the Trans-Acting siRNA Pathway

The sgs8 mutant was recovered from the L1 screen. Genetic analyses revealed that sgs8 behaves as a single recessive mutation (i.e., L1/L1 sgs8/sgs8 plants exhibited β-glucuronidase [GUS] activity), whereas L1/L1 sgs8/SGS8 plants were as silenced as L1/L1 SGS8/SGS8 plants. Bulk analysis of 14-d-old sgs8 seedlings revealed that GUS siRNAs are below detectable levels, whereas they accumulate in silenced L1 controls. Consistently, GUS mRNA and GUS activity were higher in L1/sgs8 than in L1 but lower than in L1/sgs3 plants that are totally defective in S-PTGS (Figure 1A).

Figure 1.

Figure 1.

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Analysis of S-PTGS and tasiRNA Pathways in jmj14 Mutants.

(A) LMW and HMW RNA gel blots of aerial parts of 14-d-old seedlings of the indicated mutant plants were probed with an RNA GUS probe. 25S rRNA and U6 small nuclear RNA hybridizations served as loading controls. GUS activity quantification is in fluorescence units per minute and per microgram of total protein. EtBr, ethidium bromide.

(B) LMW and HMW RNA gel blot analyses of mature rosette leaves of the indicated mutant plants. LMW RNA gel blots were probed with DNA oligonucleotides complementary to TAS2 major product (siRNA F). HMW RNA gel blots were probed with DNA complementary to the TAS2 precursor. The expected migration positions of primary TAS RNA precursor and the 5′ and 3′ cleavage products generated after miR173-guided cleavage are indicated. 25S rRNA and U6 small nuclear RNA hybridizations served as loading controls. Col-0, ecotype Columbia.

(C) Percentage of 35S:NIA2 and endogenous NIA1 and NIA2 cosuppressed plants at the adult stage in the indicated genotypes. Cosuppression frequency was scored as the number of plants that died from NIA cosuppression. 100 plants were analyzed.

Most mutants identified in the L1 screen are also impaired in the endogenous trans-acting small interfering RNA (tasiRNA) pathway (Yoshikawa et al., 2005); therefore, TAS2 precursor and cleavage products as well as mature TAS2 tasiRNA levels were analyzed in the sgs8 mutant. Unlike in ago1, hen1, hpr1, rdr6, sde5, and sgs3 mutants, no change in mature tasiRNA level was observed in sgs8 (Figure 1B). Moreover, no change in TAS2 precursor and cleavage products was observed, indicating that SGS8 does not participate in the tasiRNA pathway.

sgs8 affects L1 S-PTGS but not the tasiRNA pathway; therefore, we tested whether it affects other transgenes that undergo S-PTGS. L1/sgs8 plants were crossed to 2a3 plants, which carry a 35S:NIA2 transgene that triggers cosuppression of the endogenous genes NIA1 and NIA2 (Elmayan et al., 1998). F2 plants homozygous for sgs8 and 2a3 were identified. Analysis of 100 F3 plants revealed that the sgs8 mutation totally suppressed 2a3 S-PTGS (Figure 1C), similar to ago1, hen1, hpr1, rdr6, sde5, and sgs3 mutants that were also identified in the L1 screen. These results suggest that SGS8 encodes a general actor in the S-PTGS pathway that is not shared with the tasiRNA pathway.

SGS8 Encodes the H3K4me2/3 Demethylase JMJ14

The sgs8 mutant did not exhibit obvious developmental defects, with the exception of an early flowering phenotype (Figure 2A). The sgs8 mutation mapped to a C→T nucleotide change in the ninth exon of the JMJ14 gene (also known as PKDM7b [Yang et al., 2010]), which causes a Pro→Ser amino acid change at position 789 of the encoded H3K4me2/3 demethylase (Figure 2C). The sgs8 mutation is located at the junction between the two FY-rich domains that are conserved among histone methyltransferases (García-Alai et al., 2010) (see Supplemental Figure 1 online). To determine whether the sgs8 mutation actually abolishes JMJ14/PKDM7b activity, the pkdm7b-2 mutant, which exhibits early flowering like sgs8, was crossed to either the L1 control or the L1/sgs8 mutant. All L1/sgs8 × pkdm7b-2 hybrids flowered earlier than L1 × pkdm7b-2 (Figure 2B), indicating that sgs8 is allelic to pkdm7-2.

Figure 2.

Figure 2.

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Developmental Phenotype and Analysis of IR-PTGS in jmj14 Mutants.

(A) Pictures of the indicated mutants. L1/sgs8 plants flower earlier than L1.

(B) Allelism test between sgs8 and pkdm7b-2 as scored by flowering time.

(C) Schematic representation of the SGS8/JMJ14/PKDM7b gene. Black boxes represent exons, gray boxes represent untranslated regions, and thin lines represent introns and intergenic regions. Nucleotide and amino acid changes in sgs8/jmj14-4 are indicated.

(D) Allelism test between sgs8 and jmj14-3 as scored by JAP3 IR-PTGS phenotype.

[See online article for color version of this figure.]

Proof that the sgs8 mutant is impaired in PTGS through JMJ14 impairment was obtained by allelism tests using jmj14-3. This mutant was recovered from a forward genetic screen using the JAP3 locus, which triggers IR-PTGS of the endogenous PDS gene (Searle et al., 2010). First, the L1 locus was introduced into jmj14-3. L1/jmj14-3 plants flowered earlier than L1 and exhibited increased GUS activity, similar to L1/sgs8 (see Supplemental Figure 2 online). Then, the JAP3 locus was introduced into the sgs8 mutant. JAP3/sgs8 plants exhibited impaired PDS silencing similar to JAP3/jmj14-3 (Figure 2D). Moreover, crosses between JAP3/jmj14-3 and JAP3/sgs8 generated F1 plants that exhibited impaired PDS silencing similar to their parents (Figure 2D), indicating that sgs8 corresponds to a novel jmj14 allele, hereafter referred to as jmj14-4, and that JMJ14 acts in PTGS.

SGS8/JMJ14 Is Required for Efficient Pol II Occupancy during Transgene Transcription

GUS mRNA and GUS activity are higher in L1/jmj14-4 than in L1, although lower than in the S-PTGS–deficient mutant sgs3 (Figure 1A). This raised the question of whether this intermediate GUS mRNA level is caused by residual S-PTGS activity or reduced transgene transcription in jmj14-4. The absence of detectable GUS siRNAs in jmj14-4 (Figure 1A) suggests that S-PTGS is not occurring. Therefore, the intermediate transgene mRNA level observed in L1/jmj14-4 plants could result from reduced transgene transcription. To test this hypothesis, we examined Pol II occupancy, which is a hallmark of transcriptional activity. Pol II occupancy was examined by chromatin immunoprecipitation (ChIP) on 14-d-old seedlings using Pol II antibodies, followed by quantitative PCR (qPCR) using primers located on either the 35S promoter or the 5′ or the 3′ end of the GUS transcribed region (see Supplemental Figure 3 online). Pol II occupancy was reduced in L1/jmj14-3 compared with L1 in both the 35S promoter and the 5′ or the 3′ end of the GUS transcribed region (Figure 3A). The same effect was observed in L1/jmj14-4 (Figure 3B), indicating that transcription is reduced all along the p35S:GUS transgene in jmj14 mutants.

Figure 3.

Figure 3.

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Pol II Occupancy in jmj14 Mutants.

ChIP experiments using anti-Pol II antibodies of the indicated locus in the indicated genotypes. The mean of qPCR is reported relative to the STM gene control in the same genotype (see Methods). Graphical representation shows the fold change as the mean of the different biological repeats. Error bars represent the sd.

To determine whether the change in Pol II occupancy results in qualitative or quantitative changes in GUS mRNA production, we mapped the 5′ end of the GUS mRNA in L1 and L1/jmj14-4 plants by 5′ rapid amplification of cDNA ends (RACE). No change was observed (see Supplemental Figure 4 online), indicating that the absence of JMJ14 quantitatively affects the level of transcription of GUS mRNA but not the location of the transcription start.

To determine whether jmj14 mutations affect the transcription of other transgenes, we determined 35S:NIA2 nuclear pre-mRNA levels in 2a3 and 2a3/jmj14-4 plants by RT-PCR. Indeed, the amount of nuclear pre-mRNA also gives a good indication of transcription rate, but this method could not be applied to transgenes that lack introns, such as the p35S:GUS transgene carried at the L1 locus. Consistent with the hypothesis that the jmj14 mutation generally reduces transgene transcription, 35S:NIA2 nuclear RNA levels are lower in 2a3/jmj14-4 plants than in 2a3 controls (Figure 4A). It is assumed that transcription above a threshold level triggers S-PTGS; therefore, a reduction in transgene transcription would likely be sufficient to prevent the triggering of S-PTGS in L1/jmj14 and 2a3/jmj14 plants.

Figure 4.

Figure 4.

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Expression Analysis in jmj14 Mutants.

(A) RT-PCR analysis of the 35S:NIA2 pre-mRNA transcript level from 14-d-old-seedlings in sgs8/jmj14-4 mutant. EF1a served as a standard for RT-PCR. Col-0, ecotype Columbia.

(B) HMW RNA gel blot analysis of aerial parts of 14-d-old seedlings of the indicated backgrounds was probed with a GUS probe. 25S rRNA hybridizations served as loading control.

(C) GUS activity in the indicated backgrounds. GUS activity quantification is in fluorescence units per minute and per microgram of total protein.

sgs8/jmj14 Mutations Affect Transgene Transcription Independent of S-PTGS

To gain further insight on the effect of the jmj14-4 mutation on transgene transcription, we generated the L1/jmj14/sgs3 double mutant to quantify L1 expression in a genetic background impaired in S-PTGS. RNA gel blot and GUS fluorimetric analyses revealed that GUS mRNA and GUS activity are lower in L1/jmj14/sgs3 compared with L1/sgs3 (Figures 4B and 4C). Moreover, Pol II occupancy is reduced in L1/sgs3-1/jmj14-4 compared with L1/sgs3-1 in both the 35S promoter and the 5′ or the 3′ end of the GUS transcribed region (Figure 3D), indicating that JMJ14 is required for efficient L1 transgene transcription, independent of a PTGS-efficient or PTGS-deficient genetic background.

To determine whether JMJ14 specifically promotes high levels of transcription at transgenes that have the capacity to undergo S-PTGS in a wild-type background, the 6b4 locus, which stably expresses a p35S:GUS sense transgene in wild-type plants, was introduced into the jmj14-4 mutant. RNA gel blot and GUS fluorimetric analyses revealed that GUS mRNA and GUS activity are lower in 6b4/jmj14-4 compared with 6b4 (Figures 4B and 4C). Moreover, Pol II occupancy is reduced in 6b4/jmj14-4 compared with 6b4 in both the 35S promoter and the 5′ or the 3′ end of the GUS transcribed region (Figure 3C), These results therefore indicate that JMJ14 is required for efficient transgene transcription independent of S-PTGS.

Reduced Transcription in sgs8/jmj14 Correlates with Reduced H3K9K14Ac and Increased DNA Methylation in the 35S promoter

Histone acetylation is a hallmark of transcription. Reduced Pol II occupancy is observed in jmj14 mutants; therefore, we examined H3K9K14Ac levels by ChIP on 14-d-old seedlings using anti-H3K9K14Ac antibodies followed by qPCR using primers located on either the 35S promoter or the 5′ end of the GUS transcribed region. H3K9K14Ac levels were reduced in L1/jmj14-3 compared with L1 (Figure 5A). Similar results were observed in L1/jmj14-4 compared with L1 (Figure 5B), in L1/jmj14-4/sgs3-1 compared with L1/sgs3-1 (Figure 5D), and in 6b4/jmj14-4 compared with 6b4 (Figure 5C), consistent with reduced transcription of the p35S:GUS transgene in jmj14 mutants.

Figure 5.

Figure 5.

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Histone Acetylation Analysis in jmj14 Mutants.

ChIP experiments using anti-H3K9K14-Ac antibodies at the indicated locus in the indicated genotypes. The mean of qPCR is reported relative to the STM control in the same genotype (see Methods). Graphical representation shows the fold change as the mean of the different biological repeats. Error bars represent the sd.

Increased DNA methylation in the promoter region is a hallmark of reduced transcription (Deleris et al., 2010). Reduced H3K9K14Ac levels, reduced Pol II occupancy, and reduced transgene mRNA accumulation in jmj14 mutants suggest reduced transgene transcription; therefore, we expected to find increased DNA methylation in the 35S promoter. Consistent with this hypothesis, DNA gel blot analysis using methylation-sensitive restriction enzymes HpaII and MspI revealed increased DNA methylation in the 35S promoter in L1/jmj14-3 and L1/jmj14-4 mutants compared with L1. Increased methylation was similar using both HpaII and MspI, indicating that methylation mostly affects CNG sites (Figure 6). Similar observation was made in L1/sgs3-1/jmj14-4 mutants compared with L1/sgs3-1, and in 6b4/jmj14-3 and 6b4/jmj14-4 mutants compared with 6b4 (Figure 6), indicating that the absence of JMJ14 promotes increased DNA methylation independent of S-PTGS.

Figure 6.

Figure 6.

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DNA Methylation Analysis in jmj14 Mutants.

(A) Partial map of the 35S:GUS locus and expected digestion fragments. Methylation-insensitive EcoRI, HindIII, and methylation-sensitive HpaII/MspI sites are indicated, as well as the expected digestion fragments detected by hybridizing with the 35S probe.

(B) DNA of 15-d-old seedlings was digested with EcoRI, HindIII, and MspI (indicated as M) or EcoRI, HindIII, and HpaII (indicated as H) and probed with a DNA 35S probe.

Mutations in SGS8/JMJ14 Correlate with Decreased Transgene H3K4me3 Levels

JMJ14 encodes an H3K4me3 demethylase (Jeong et al., 2009; Lu et al., 2010); therefore, global levels of H3K4me3 are supposed to increase in a jmj14 mutant. At first, we quantified global H3K4me1, H3K4me2, and H3K4me3 levels in L1 and L1/jmj14-4 plants. As previously reported for other jmj14 mutants (Jeong et al., 2009; Lu et al., 2010), levels of H3K4me1 were mostly unchanged in jmj14-4, whereas levels of H3K4me2 and H3K4me3 were increased (Figure 7A), indicating that jmj14-4 is an actual loss of JMJ14 function and that JMJ14 demethylates both H3K4me3 and H3K4me2.

Figure 7.

Figure 7.

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Global and Targeted H3K4 Methylation Levels in jmj14 Mutants.

(A) Immunoblot analysis of H3K4me1, H3K4me2, and H3K4me3 whole-genome levels using the corresponding antibodies in the indicated genotypes. β-tubulin (b-tub) served as a loading control.

(B) ChIP experiments using anti-H3K4me3 antibodies of the indicated locus in the indicated genotypes. The FWA endogenous locus was previously shown to be a JMJ14 target, whereas STM served as an insensitive control.

Previous reports revealed several endogenous targets of JMJ14 (Deleris et al., 2010; Lu et al., 2010; Searle et al., 2010). To ensure that the jmj14-4 mutant isolated from the S-PTGS screen behaves as other jmj14 mutants, H3K4me3 levels were measured at the FWA locus. ChIP was performed on 14-d-old seedlings using H3K4me3 antibodies, followed by qPCR using primers located on FWA. STM was used as a negative control. H3K4me3 levels were increased at FWA but not STM (Figure 7B), confirming that jmj14-4 is an actual loss of JMJ14 function.

Then, we quantified H3K4me3 and H3K4me2 levels at the L1 and 6b4 loci in wild-type plants and in the jmj14 mutant. Unexpectedly, H3K4me3 levels were reduced in L1/jmj14-4 compared with L1 (Figure 8A). Moreover, a similar reduction was observed in 6b4/jmj14-4 compared with 6b4 (Figure 8B). The reduction in H3K4me3 levels was visible in the 35S promoter and in the GUS transcribed region, indicating that H3K4me3 levels are globally reduced all along the p35S:GUS transgene, independent of whether S-PTGS takes place. By contrast, H3K4me2 levels were unchanged or only slightly reduced (Figures 8C and 8D).

Figure 8.

Figure 8.

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ChIP Analysis of H3K4me2 and H3K4me3 Levels in jmj14 Mutants.

ChIP experiments using anti-H3K4me3 ([A] and [B]) or anti-H3K4me2 ([C] and [D]) antibodies at the indicated locus in the indicated genotypes. The mean of qPCR is reported relative to the STM gene control in the same genotype (see Methods). Graphical representation shows the fold change as the mean of the different biological repeats. Error bars represent the sd.

jmj14 Mutations Impair PTGS of Transgenes Driven by Either Plant or Viral Promoters

The results presented above indicate that jmj14 mutations affect transgene loci carrying a 35S-driven GUS transgene undergoing S-PTGS (L1) or insensitive to PTGS (L1/sgs3 and 6b4). They also impair IR-PTGS triggered by the JAP3 locus carrying a pSUC2-driven inverted repeat PDS transgene, suggesting that the effect of jmj14 mutations is not specific to exogenous promoters, such as the 35S promoter. However, p35S:GUS and pSUC:IR-PDS transgenes are not directly comparable, because they differ in both promoter and transcribed sequences. Therefore, we examined the effect of jmj14 on p35S:AGO1 and pAGO1:AGO1 constructs, which differ only by the promoter and which both efficiently trigger endogenous AGO1 cosuppression through PTGS (Mallory and Vaucheret, 2009). Transformation of jmj14-4 by either p35S:AGO1 or pAGO1:AGO1 constructs resulted in strongly reduced AGO1 cosuppression efficiency compared with ecotype Columbia (see Supplemental Figure 5 online). These results confirm that the effect of jmj14 mutations is not specific to exogenous promoters and point to a specific effect on certain transgene loci.

DISCUSSION

In Arabidopsis thaliana, JMJ14 has been described as an H3K4me2/3 demethylase. Consistent with this, loss of JMJ14 activity leads to a global increase in H3K4me2 and H3K4me3 levels in the Arabidopsis genome (Jeong et al., 2009; Lu et al., 2010), confirmed by ChIP experiments at known JMJ14 endogenous targets (Deleris et al., 2010; Yang et al., 2010). Increased H3K4me2 and H3K4me3 levels correlate with increased transcription at these endogenous loci. Surprisingly, we identified JMJ14 in a screen for mutants defective in S-PTGS. This was unexpected, because S-PTGS likely depends on the transcription of aberrant RNAs at high levels. Thus, only mutations causing reduced transgene transcription were expected to prevent the triggering of S-PTGS. Our results indicate that jmj14 mutants could be recovered in a PTGS screen because JMJ14 unexpectedly promotes high levels of transgene transcription. We found that JMJ14 action on transgene transcription is PTGS-independent, because jmj14 mutations not only reduce the expression of transgenes that undergo S-PTGS but also of transgenes that do not trigger S-PTGS. Moreover, expression of transgenes that undergo S-PTGS in a wild-type background was reduced in jmj14 sgs3 double mutants compared with PTGS-deficient sgs3 mutants, confirming that JMJ14 is required for high levels of transcription in a PTGS-independent manner. Finally, we observed that the effect of jmj14 mutations was not specific to exogenous promoters, such as the 35S promoter. Indeed, they also affected transgenes driven by the Arabidopsis SUC2 or AGO1 promoter, suggesting that JMJ14 is required for efficient transgene expression.

Reduced transgene mRNA accumulation could definitely be attributed to reduced transcription, because we observed reduced Pol II occupancy at these transgene loci. Also consistent with reduced transcription, reduced levels of H3K4me3 were observed all along the transgene loci, whereas H3K4me2 levels were mostly unchanged. Although the reduced transcription explains the finding of jmj14 mutants in a PTGS screen, it raises the question of how a loss of JMJ14 activity could cause an effect on transgenes opposite to that observed for endogenous genes regulated by JMJ14. The fact that transgene H3K4me3 and H3K4me2 levels do not increase in jmj14 mutants suggests that JMJ14 is unable to demethylate H3K4me3 and H3K4me2 at the examined transgene loci. Moreover, it is likely that JMJ14 prevents other H3K4me3 demethylases encoded by the genome (Lu et al., 2008) to demethylate transgene loci, because H3K4me3 levels at transgene loci decrease in jmj14 mutants. To explain these results, we propose that epigenetic marks are specifically imposed on certain transgene loci, which do not prevent the binding of JMJ14 but do prevent the demethylating activity of JMJ14 on these loci. The nature of such marks remains to be identified, and the ability of JMJ14 to bind but not demethylate such transgene loci remains to be demonstrated.

In yeast, it has been proposed that H3K4me2 recruits the Set3 Histone Deacetylase Complex (HDAC) at the 5′ end of transcribed regions (Kim and Buratowski, 2009). Deacetylation of the 5′ region likely limits histone acetylation to spread from promoters into transcribed regions and may be used for repressing transcription of cryptic promoters along the coding sequence of the genes (Pinskaya and Morillon, 2009). Examples of cryptic transcripts that trigger TGS have been described already (e.g., at the Ty1 retrotransposon [Berretta et al., 2008]). The Arabidopsis genome contains several possible orthologs of Set3 HDAC members (Berr et al., 2011). However, the existence of this complex in plants and its recruitment by H3K4me2 has not been described yet. In jmj14 mutants, transgene H3K4me2 levels remain unchanged or are only slightly reduced at the 5′ end of the transcribed region. Consistent with this, the transgene transcription start remains unchanged, confirming that transgene transcription is changed quantitatively but not qualitatively. Therefore, the absence of PTGS likely results from a general reduction in transcription and not to the specific erasure of aberrant RNAs.

In addition to H3K4me2, which recruits HDAC, H3K4me3 recruits Chromodomain- or PHD domain-containing proteins (Taverna et al., 2007), which are present in complexes that activate transcription (Berr et al., 2011). A coordinated deposition of H3K4me2 and H3K4me3 is required to remove histone deacetylases, facilitate histone acetylation, and recruit transcriptional activators, allowing transcription to occur properly (Bannister and Kouzarides, 2011). The transgene transcription defects observed in jmj14 mutants could be the consequence of an accumulation of HDAC at the promoter, thus blocking transcription. Indeed, a direct correlation between H3 acetylation and transcriptional activation has been described (Kuo et al., 1996; Liu et al., 2012). We found that H3K9K14Ac levels in the transgene promoter are drastically reduced in the jmj14-4 mutant. This low level of acetylation is consistent with an HDAC accumulation and likely explains the low level of transgene transcription observed in jmj14 mutants.

METHODS

Plant Material

The L1, 2a3, and JAP3 lines (Elmayan et al., 1998; Smith et al., 2007) and the rdr6 (sgs2-1), sgs3-1, jmj14-3, and pkdm7b-2 (Elmayan et al., 1998; Mourrain et al., 2000; Morel et al., 2002; Searle et al., 2010; Yang et al., 2010) mutants have been described previously. Analyses were performed on in vitro seedlings grown on Bouturage media (Duchefa) for 14 d in controlled growth chambers. All plants were grown in standard long-day conditions (16 h of light, 8 h of dark) at 20°C.

Molecular Analyses

DNA sequencing was performed as described before (Gy et al., 2007; Mallory and Vaucheret, 2009). RNA extraction, gel blot analyses, and quantification of GUS activity were performed as described before (Gy et al., 2007; Mallory and Vaucheret, 2009). All RNA gel blot analyses were performed using 5 to 10 μg of total RNA. GUS, TAS, U6, miR173, and 25S probes have been described before (Gy et al., 2007; Smith et al., 2007; Elmayan et al., 2009; Mallory and Vaucheret, 2009). TAS2 probe allowed detection of precursors as well as both 5′ and 3′ cleavage products (Elmayan et al., 2009). Hybridization signals were quantified using a Fuji phosphor imager and normalized to a U6 oligonucleotide probe and a 25S DNA probe for low molecular weight (LMW) and high molecular weight (HMW) gel blot analyses, respectively. For cDNA synthesis, RNAs were extracted with the RNeasy Plant Mini Kit (Qiagen), treated with DNaseI (Invitrogen), and 2 μg of DNA-free RNA was reverse transcribed with oligo-dT (Invitrogen). PCR was performed using Taq DNA polymerase (Invitrogen) according to the manufacturer’s protocol. Each reaction was performed on 5 μL of 1:60 dilution of the cDNA and was synthesized in a 25-μL total reaction. Specific oligonucleotide pairs are listed in Supplemental Table 1 online. Forty cycles of amplification were used at a 58°C annealing temperature for the 35S:NIA2 transgene, and 24 cycles of amplification at a 62°C annealing temperature were used for EF1∝. The results were standardized to the expression level of EF1∝. For 5′ RACE, total RNA was extracted from 15-d-old plantlets using RNeasy Plant Mini Kit (Qiagen). Experimentation was done on 5 μg of RNA as described in the 5′ RACE System Kit (Invitrogen). cDNA was amplified using GUS-3′-rev and abridged anchor primer kit–specific oligo. The major band from nested amplification with abridged anchor primer and GUS-5′-rev was cloned using the TOPO TA Cloning Kit (Invitrogen). Seven clones from each condition were sequenced.

ChIP

ChIP was performed on chromatin from 2 g of crosslinked in vitro plantlets 15 d after germination as previously described (Moehs et al., 1988; Gendrel et al., 2002). After 2 × 5 cycles of sonication (30 s “ON”, 30 s “OFF”) with a Bioruptor UCD200 (Diagenode), the chromatin solution was diluted 10-fold to a final volume of 4 mL with ChIP dilution buffer. A total of 50 μL of Dynabeads Protein A (Invitrogen) was washed twice and resuspended in 50 μL of ChIP dilution buffer. Antibodies were added (2 μL of H3 [Abcam], 5 μL of H3K4me3 [Millipore], 3 μL of H3K4me2 [Millipore], 5 μL of Pol II [Abcam], and 5 μL of H3KAc [Millipore]) and incubated for at least 3 h at 4°C with gentle rotation on a wheel. After three washes, 1 mL of the chromatin solution was added to the antibodies plus beads and was incubated overnight at 4°C with gentle rotation for histone capture.

The washing of beads was performed as described (Gendrel et al., 2005). After the last TE wash, the reverse crosslinking (5 h at 65°C) and elution were performed using IPure kit (Diagenode). The final elution was performed in 60 μL, and the chromatin was stored at −20°C until analysis.

qPCR

The ChIP was analyzed by qPCR on 2 μL of the chromatin. Each oligo pair (primers are listed in Supplemental Table 1 and Supplemental Figure 3 online) was used four times on each chromatin sample. SHOOT MERISTEMLESS (STM) was used as an internal control (Angel et al., 2011). The mean of four qPCRs (with sd < 0.5 cycle threshold) was used for each point. For each oligo pair and each histone modification, results are represented as the ratio of (H3Kmodif1 oligocouple1 / H3 oligocouple1) to (H3Kmodif1 STM / H3 STM). Either two or three biological replicates were analyzed each time. Results show the mean and sd of the independent biological replicates.

Immunoblot Analysis

A total of 50 μg of protein extracted from a purified nucleus from the first step of chromatin purification after sonication were loaded after denaturation on a 15% SDS-PAGE on a 1× Laemmli buffer. After transfer onto membrane, blots were reveled using the same antibodies as the ChIP experiment (diluted 1/2000). Loading control was anti-α-tubulin (clone B5-1-2; Sigma-Aldrich) (diluted 1/10000).

Accession Numbers

Sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL databases under the following accession numbers: EF1a, NM_125432.3; FWA, NM_118685.2; GUS (uidA), NC_000913.2; JMJ14, NM_118159.2; NIA2, NM_103364.2; STM, NM_104916.3.

Supplemental Data

The following materials are available in the online version of this article.

Acknowledgments

We thank David Baulcombe for the JAP3 line and jmj14-3 mutant, Institute National de la Recherche Agronomique for the FLAG_188F08 (pkdm7b-2) mutant, and Hervé Ferry, Bruno Letarnec, and Philippe Maréchal for plant care. This study was supported by the Fondation Louis D. de l’Institut de France and the Program Saclay Plant Sciences (ANR-10-LABX-40) grant to H.V. and by a PhD fellowship from the Région Ile-de-France to V.J.

AUTHOR CONTRIBUTIONS

I.L.M., V.J., and H.V. designed the research. All authors performed the research. I.L.M., V.J., T.E., and H.V. analyzed the data. H.V. wrote the article with the help of I.L.M.

Glossary

S-PTGS

posttranscriptional gene silencing mediated by sense transgenes

Pol II

polymerase II

PTGS

posttranscriptional gene silencing

siRNA

small interfering RNA

TGS

transcriptional gene silencing

dsRNA

double-stranded RNA

IR-PTGS

posttranscriptional gene silencing mediated by inverted repeat transgenes

GUS

β-glucuronidase

tasiRNA

trans-acting small interfering RNA

qPCR

quantitative PCR

RACE

rapid amplification of cDNA ends

LMW

low molecular weight

HMW

high molecular weight

ChIP

chromatin immunoprecipitation

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