The trans-silencing capacity of invertedly repeated transgenes depends on their epigenetic state in tobacco

Abstract

We studied the in trans-silencing capacities of a transgene locus that carried the neomycin phosphotransferase II reporter gene linked to the 35S promoter in an inverted repeat (IR). This transgene locus was originally posttranscriptionally silenced but switched to a transcriptionally silenced epiallele after in vitro tissue culture. Here, we show that both epialleles were strongly methylated in the coding region and IR center. However, by genomic sequencing, we found that the 1.0 kb region around the transcription start site was heavily methylated in symmetrical and non-symmetrical contexts in transcriptionally but not in posttranscriptionally silenced epilallele. Also, the posttranscriptionally silenced epiallele could trans-silence and trans-methylate homologous transgene loci irrespective of their genomic organization. We demonstrate that this in trans-silencing was accompanied by the production of small RNA molecules. On the other hand, the transcriptionally silenced variant could neither trans-silence nor trans-methylate homologous sequences, even after being in the same genetic background for generations and meiotic cycles. Interestingly, 5-aza-2-deoxy-cytidine-induced hypomethylation could partially restore signaling from the transcriptionally silenced epiallele. These results are consistent with the hypothesis that non-transcribed highly methylated IRs are poor silencers of homologous loci at non-allelic positions even across two generations and that transcription of the inverted sequences is essential for their trans-silencing potential.

Figure 1

Schematic representation of the genomic organization of transgenic loci and physical maps of the restriction sites. The transgenic locus 1 has been previously reported as locus X (29). The methylation analysis of the promoter involved the three TaiI sites; the diagnostic sites for the analysis of the nptII-transcribed region were SmaI and BamHI, and Eco47III for the non-transcribed sequences at the right border. Evidence for the IR character of the T-DNA insertions in locus 1 and locus 1E has been given elsewhere (18). EcoRV, BglII, and HindIII enzymes were used to dissect particular subregions of T-DNA. P_35S, promoter of the cauliflower mosaic virus; nptII, neomycin phosphotransferase II gene; RB, T-DNA right border; 3′chs, transcription termination sequence from the 3′-untranslated region of the chalcone synthase gene from snapdragon (Antirrhinum majus).

Figure 2

Distribution and density of cytosine methylation in the posttranscriptionally (HeLo1) and transcriptionally (HeLo1E) silenced epialleles of tobacco. (A) Genomic sequencing showing the methylation distribution along the 35S promoter and the 5′-nptII-coding region. The sequences obtained were processed through the Methtools software (24). The colored dots and horizontal grey bar indicate the positions of the methylated cytosines in trinucleotide contexts and the position of the core 35S promoter, respectively. TSS, transcription start site. (B) Percentages of methylated cytosines in symmetrical (CG, CNG) and non-symmetrical (CNN) contexts for the promoter (−400/+1) and the 5′-nptII-transcribed region (+1/+400). Data were assembled from 21 HeLo1 and 9 HeLo1E clones, each representing a pattern in individual cell.

Figure 3

Expression analysis of the nptII reporter gene in the parental and hybrid lines. (A) Representative RNA gel-blot hybridizations of the nptII transcripts in silenced and non-silenced lines. Five micrograms of total RNA was loaded per lane and hybridized to the nptII DNA probe. RNA samples from paternal HeLo2 and HeB plants have been loaded to better compare hybridization bands intensities. The 25S rRNA ethidium bromide-stained bands are shown as loading controls. (B) NPTII protein accumulation level determined by ELISA. The silenced lines generally had NPTII protein levels below the detection limit of the method (5 ng/mg protein). The data represent averages from two to three ELISAs with two different protein extracts isolated from the same plant.

Figure 4

Methylation analysis of transgene loci in parental lines, F1 hybrids and subsequent generations. DNA gel-blot hybridization was carried out using the nptII gene probe. Genomic DNAs were predigested with methylation-insensitive enzymes (−) to obtain locus-specific bands (indicated by arrows). Absence of digestion of locus-specific bands with methylation-sensitive enzymes (+) is indicative of transgene methylation. Expected lengths of restriction fragments are given in Figure 1. (A–C) Progeny of plants that inherited epiallellic variants of locus 1 and locus 2 (Lo1/Lo2 and Lo1E/Lo2). (D) Progeny of plants that inherited epiallelic variants of locus 1 and locus B (Lo1/LoB and Lo1E/LoB). (A) CG methylation in the nptII-coding sequence. DNAs were digested with methylation-insensitive EcoRV followed by methylation-sensitive SmaI. (B) CG methylation in the non-transcribed downstream region. DNAs were digested with methylation-insensitive EcoRV followed by methylation-sensitive Eco47III. (C) Non-symmetrical methylation in the nptII-coding sequence. DNAs were digested with methylation-insensitive EcoRV followed by methylation-sensitive BamHI. The sequence context C within the BamHI site is CCC and CCT on the top and bottom strand, respectively. (D) CG methylation in the nptII-coding sequence. To separate the loci in Lo1E/LoB and Lo1/LoB hybrids, genomic DNA was digested with the HindIII/BglII enzymes and afterwards with the methylation-sensitive SmaI.

Figure 5

Absence of trans-methylation of promoter sequences in F1 hybrids carrying PTGS and TGS epialleles. Cytosine methylation was studied in the 35S promoter by DNA gel-blot hybridization of restricted DNAs. The sizes of the restriction fragments were calculated from the T-DNA sequence (Figure 1). The 0.8 kb fragment corresponds to the non-methylated BglII/TaiI genomic fragment. The positions of the locus-specific bands after separation of the loci with methylation-insensitive BglII enzyme are marked by arrows. Symbols above the blots indicate digestion with the methylation-insensitive enzyme only (−) or in combination with the methylation-sensitive TaiI enzyme (+).

Figure 6

Detection of smRNAs. Low-molecular weight RNA fractions were blotted on a Hybond-XL membrane and hybridized with the hydrolyzed sense-specific nptII riboprobe. A 20 nt oligomer was used as a size marker. The loading control was a non-specific high molecular band. Approximately 50 µg of RNA material was loaded into each lane. SR1, non-transgenic control; HeLo2, non-silenced line; HoLo1, PTGS single locus line; HeLo1E, TGS single locus line; Lo1/Lo2 and Lo1/LoB, PTGS F1 hybrids; Lo1E/Lo2 and Lo1E/LoB, non-silenced F1 hybrids.

Figure 7

Expression and methylation analysis in hypomethylated Lo1E/Lo2 plants. (A) RNA gel-blot hybridization of the nptII RNA isolated from 5-aza-2-deoxy-cytidine (azaC)-treated (1–5) and non-treated (C) plants. The numbers below the lanes indicate relative signal intensities of the nptII-specific band. The signals were normalized by ethidium bromide fluorescence of the 25S rRNA gene (bottom panel). The signal in the control lane (C) was arbitrarily chosen as 100%. (B) DNA methylation in the endogenous HRS60 satellite analyzed by HpaII restriction enzyme. (C) DNA methylation in the 35S promoters analyzed by TaiI restriction enzyme (+) as described in Figure 5. DNAs were predigested with BglII to separate both loci. The ratio between methylated/non-methylated fragments are indicated below the lanes.