Interplay among RNA polymerases II, IV and V in RNA-directed DNA methylation at a low copy transgene locus in Arabidopsis thaliana

Abstract

RNA-directed DNA methylation (RdDM) is an epigenetic process whereby small interfering RNAs (siRNAs) guide cytosine methylation of homologous DNA sequences. RdDM requires two specialized RNA polymerases: Pol IV transcribes the siRNA precursor whereas Pol V generates scaffold RNAs that interact with siRNAs and attract the methylation machinery. Recent evidence also suggests the involvement of RNA polymerase II (Pol II) in recruiting Pol IV and Pol V to low copy, intergenic loci. We demonstrated previously that Pol V-mediated methylation at a transgene locus in Arabidopsis spreads downstream of the originally targeted region by means of Pol IV/RNA-DEPENDENT RNA POLYMERASE2 (RDR2)-dependent 24-nt secondary siRNAs. Here we show that these secondary siRNAs can not only induce methylation in cis but also in trans at an unlinked target site, provided this sequence is transcribed by Pol II to produce a non-coding RNA. The Pol II transcript appears to be important for amplification of siRNAs at the unlinked target site because its presence correlates not only with methylation but also with elevated levels of 24-nt siRNAs. Potential target sites that lack an overlapping Pol II transcript and remain unmethylated in the presence of trans-acting 24-nt siRNAs can nevertheless acquire methylation in the presence of 21-24-nt hairpin-derived siRNAs, suggesting that RdDM of non-transcribed target sequences requires multiple size classes of siRNA. Our findings demonstrate that Pol II transcripts are not always needed for RdDM at low copy loci but they may intensify RdDM by facilitating amplification of Pol IV-dependent siRNAs at the DNA target site.

Fig. 1

T + S transgene silencing system and model for production of secondary siRNAs. a The two-component transgene silencing system comprises a Target locus (T) and a Silencer locus (S). The T locus contains a GFP reporter gene under the control of a minimal promoter (hatched) and an enhancer (ENH) that drives GFP expression in shoot and root meristem regions. The S locus contains an inverted DNA repeat (IR) of target enhancer sequences (opposing blue arrows) that is transcribed from the cauliflower mosaic virus 35S promoter by Pol II (Kanno et al. 2008). b The resulting RNA hairpin from the S locus is processed by DICER-LIKE3 (DCL3) into 24-nt primary siRNAs (blue dashes) that induce Pol V-mediated methylation of the target enhancer (blue ‘m’) leading to TGS of the GFP reporter gene (step 1). An additional feature of this system (step 2) is that methylation spreads (red ‘m’) from the originally targeted enhancer into the downstream region (red shaded bar) through the presence of secondary siRNAs (red dashes), which rely on Pol IV and RDR2 for their biogenesis (Daxinger et al. 2009). Key to secondary siRNA production is a ‘nascent’ RNA that extends through the target enhancer region (black arrow). In a hypothetical model, the nascent RNA is transcribed by Pol IV and following cleavage by ARGONAUTE 4 (AGO4), is copied by RDR2 into double stranded RNA that is processed by DCL3 into 24-nt secondary siRNAs (Daxinger et al. 2009)

Fig. 2

SD construct and methylation analysis of 88 bp target region. a The SD construct contains the 88 bp-Ubi-pro-DsRed sequence (Supplementary Fig. 1) in the orientation shown relative to the T-DNA left and right borders (LB and RB, respectively) together with a PAT (phosphinothricin acetyl transferase) gene encoding resistance to phosphinothricin under the control of the mannopine synthase promoter (MASpro) and octopine synthase terminator (OCSter) for selection of transformed plant cells (construct not drawn to scale). The black arrow indicates the predicted DsRed transcript initiating from the Ubi-pro. b Bisulfite sequencing analysis of methylation in the 88 bp target sequence (red bar) and immediate flanking sequences (left black bar represents vector sequence; right grey bar represents Ubi-pro sequence). Note that these fragments are shown in 5′–3′ orientation, which is reverse to that shown in Part A. The 88 bp target sequence contains 19 cytosines: four in a CHG context (blue lines), 15 in a CHH context (red lines) and none in a CG context (black lines) (Supplementary Fig. 1). Percent cytosine methylation is shown for T + S + SD lines #1 and #4. Methylation was first observed in the T1 generation (left), in which the SD locus is hemizygous, and it persisted and even increased by the T3 generation (right) when the SD locus is homozygous. In T + S + SD lines #2 and #3, no methylation was detected. In T + S + SD line #5, sparse methylation was observed in T1 plants, but CHH methylation did not persist into the T3 generation (data not shown). The results from at least 15 cloned sequences are shown

Fig. 3

RT-PCR analysis of Pol II transcripts and Pol II occupancy. a Positions of primers used for RT-PCR analysis to detect transcripts from the 88 bp-Ubi-pro-DsRed sequence (short red arrows) and to study Pol II occupancy (short blue arrows) (see also Supplementary Fig. 1). Longer black arrows indicate transcripts potentially detected in RT-PCR reactions. Only the ‘short’ and dsREDfor + dsREDrev transcripts were detected (Part B). The short black vertical arrow indicates the approximate position of the predicted transcription start site of the DsRed transcript, which is around 150 bp downstream of the Ubipro oligo (Supplementary Fig. 1). b Reverse transcriptase (RT) reactions to synthesize first strand cDNA were carried out using either an Ubi-pro primer or oligo(dT) (right of each gel image). The primers used for the PCR reaction are shown under each gel image. The T + S + SD lines (#1 through #5) are labeled at the top. The ‘short’ transcript (A) overlapping the 88 bp region and extending into the Ubi-pro was observed only in lines #1 and #4 using either the Ubi-pro or oligo(dT) primer for RT and RTfor + RTrev primers for PCR (see minus RT controls in part C). The absence of the ‘long’ transcript [oligo(dT) in RT reaction and RTfor + Ubi-pro oligo for PCR] indicates that the non-coding transcript ends somewhere after the Ubi-pro oligo. In the RT reaction, the oligo(dT) is probably priming at A stretches upstream of the Ubi-pro oligo (Supplementary Fig. 1). A DsRed transcript (primers dsREDfor + dsREDrev) was observed in all five lines. However, expression of DsRed protein was only very weak or not detectable in these plants (data not shown). gDNA, genomic DNA. ACTIN was used as a constitutive control. c The ‘short1’ and ‘short2’ transcripts in lines T + S + SD #1 and #4 (part B) are not detected in minus RT (‘−’ sign) controls. ‘+’ sign indicates reactions with RT. D. The ‘short’ non-coding RNA overlapping the 88 bp target sequence was detectable in wild-type (WT) plants of T + S + SD line #4 but not in nrpd1 or nrpe1 mutant backgrounds. Two plants of each genotype were tested. The Ubi-pro primer was used for the RT reaction and the primer pair RTfor + RTrev for PCR. Plus and minus signs at the top indicate reactions with and without RT, respectively. e Pol II occupancy in the vicinity of the 88 bp target sequence. ChIP was performed using anti-Pol II antibody, and Pol II co-purified DNA was quantified by real-time PCR. Positions of primers used for the short (Ubi ChIP short for + rev) and long (Ubi ChIP long for + rev) fragments (top graph) are shown in Part A. Positive and negative controls for Pol II occupancy are Actin and IGN5 (bottom graph). Mock precipitations without antibody were used to judge background levels of ChIP samples. Two biological replicates were performed and SD were calculated from three technical repeats

Fig. 4

Methylation of the 88 bp target region in nrpd1 and nrpe1 mutants. Percent cytosine methylation in the 88 bp target sequence (red bar) and immediate flanking sequences (left black bar, vector sequence; right grey bar, Ubi-pro sequence) in lines T + S + SD #1 (left) and #4 (right) in nrpd1 (top) and nrpe1 (bottom) mutant backgrounds as determined by bisulfite sequencing. Methylation in CHH (red lines) and CHG (blue lines) nucleotide groups is substantially reduced relative to wild-type levels (see Fig. 2b). Major methylation is maintained only in a CG dinucleotide (black line) in the Ubi-pro region. The results from at least 15 cloned sequences are shown

Fig. 5

Northern blot analysis of siRNAs. a A probe specific for the 88 bp target sequence was used on Northern blots to detect siRNAs in T + S + SD lines #1 though #4 (left blot) as well as the original T + S line (T + S) and SD(#2) carrying the 88 bp-HP construct (SD(#2) + HP) (right blot). The arrow to the left indicates the position of the 24-nt size class. B. The 88 bp-specific probe was used to detect 24-nt siRNAs (arrow, left) in the original T + S line as well as T + S + SD line #4 in an nrpd1 mutant background (#4-nrpd1) and line SD(#4). In a and b, the control lane (C) contains RNA isolated from non-transgenic plants. Ethidium bromide staining of the major RNA on the gel is shown at the bottom of each blot as a loading control. In b, the middle panel shows the 24-nt size class of siRNAs (arrow) on the stained gel. This size class disappears in the nrpd1 mutant (lane #4 nrpd1) confirming the genotype of this plant

Fig. 6

Analysis of Pol II transcript and methylation of 88 bp target sequence in line SD(#4). a RT-PCR analysis of the transcript overlapping the 88 bp target region in the line SD(#4). Four individual plants were tested. Positions of the primers used are shown in the Fig. 3a. An oligo(dT) primer was used for the RT reaction and primers for PCR were either RTfor + RTrev, which detects the ‘short’ transcript (Fig. 3a, b) or RTfor + Ubipro Oligo, which does not detect a transcript (Fig. 3b). ‘Minus RT’ indicates reactions without reverse transcriptase. ACTIN was used as a positive control for expression. gDNA, genomic DNA. b Percent cytosine methylation in the 88 bp target sequence (red bar) and immediate flanking sequences (left black bar, vector sequence; right grey bar, Ubi-pro sequence) in line SD (#4) as determined by bisulfite sequencing. Results from at least 15 cloned sequences from two individual plants are shown. CG, CHG and CHH are indicated by the black, blue and red lines, respectively. c The graphs show the comparison of overall levels of methylation in CG (black), CHG (blue) and CHH (red) nucleotide groups between line T + S + SD line #4 (left) and line D(#4) (right). Original bisulfite data for line T + S + SD line #4 are shown in Fig. 2b

Fig. 7

Methylation of 88 bp target sequence induced by 88 bp-HP construct and absence of Pol II transcript in line SD(#2). a Percent cytosine methylation in the 88 bp target sequence (red bar) and immediate flanking sequences (left black bar, vector sequence; right grey bar, Ubi-pro sequence) in line SD(#2) in T1 and T3 generations when the 88 bp-HP locus is hemizygous and homozygous, respectively. CG, CHG and CHH are indicated by the black, blue and red lines, respectively. The results from at least 15 cloned sequences are shown. b RT-PCR analysis of the transcript overlapping the 88 bp target region in line SD(#2). Either a Ubi-pro or oligo(dT) primer was used for the RT reaction (top) and primers for PCR were RTfor + RTrev, which detects the ‘short’ transcript (Fig. 3a, b). ‘Minus’ lanes 2 and 4 indicate reactions without reverse transcriptase. ACTIN was used as a positive control for expression. gDNA, genomic DNA

Fig. 8

Model for roles of RNA polymerases II, IV and V in RdDM at the 88 bp target sequence. Step 1 Initiating at an unidentified promoter in flanking plant DNA, Pol II transcribes a non-coding RNA that overlaps the 88 bp target region and terminates in the Ubi-pro. Step 2 Pol II transcription (or transcripts) recruits Pol IV to transcribe through the 88 bp target region. Step 3 Trans-acting secondary siRNAs (short red bar) matching the 88 bp target sequence may guide AGO4 cleavage of the ‘aberrant’ Pol IV transcript, thus initiating siRNA amplification at the 88 bp target region by providing substrates for RDR2, which produces double stranded RNA that is processed by DCL3 to 24-nt siRNAs. Step 4 The amplified siRNAs (thick red bars) reach a sufficiently high local concentration to induce Pol V-mediated methylation (red ‘+m’) of the 88 bp target sequence. The results leading to this model provide experimental validation of the RNA silencing cascade model proposed previously (Baulcombe 2006)