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. 2012 Sep;7(9):1071-8.
doi: 10.4161/epi.21644. Epub 2012 Aug 6.

Transgenerational Maintenance of Transgene Body CG but Not CHG and CHH Methylation

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Transgenerational Maintenance of Transgene Body CG but Not CHG and CHH Methylation

Athanasios Dalakouras et al. Epigenetics. .
Free PMC article


In plants, RNA-directed DNA methylation (RdDM) can target both transgene promoters and coding regions/gene bodies. RdDM leads to methylation of cytosines in all sequence contexts: CG, CHG and CHH. Upon segregation of the RdDM trigger, at least CG methylation can be maintained at promoter regions in the progeny. So far, it is not clear whether coding region methylation can be also maintained. We showed that the body of Potato spindle tuber viroid (PSTVd) transgene constructs became densely de novo methylated at CG, CHG and CHH sites upon PSTVd infection. In this study, we demonstrate that in viroid-free progeny plants, asymmetric CHH and CHG methylation was completely lost. However, symmetric CG methylation was stably maintained for at least two generations. Importantly, the presence of transgene body methylation did not lead to an increase of dimethylation of histone H3 lysine 9 or a decrease of acetylation of H3. Our data supports the view that CG methylation can be maintained not only in promoters but also in the body of transgenes. They further suggest that maintenance of methylation may occur independently of tested chromatin modifications.


Figure 1. Southern blot analysis of DNA samples of Nt-SB2, Nt-SB2/Nbdi, Nt-SB2* and Nt-SB2** plants. (A) Physical map of the SB2 and Nbdi locus. LB, T-DNA left border; P35S, Cauliflower mosaic virus 35S promoter; PSTVd, Potato spindle tuber viroid cDNA either carrying a 26-bp deletion (indicated by an X in the SB2 locus) or full-length (Nbdi locus); TNOS, terminator of the nopaline synthase gene; RB, T-DNA right border. The arrows in the transgene body denote PSTVd cDNA orientations. DraI, HpaII, HaeIII and AluI sites and relative fragment sizes are presented. (B) Southern blot analysis using the methylation-insensitive DraI and methylation-sensitive HpaII (left panel), HaeIII (middle panel) and AluI (right panel). Cleaved DNA was separated on 1% agarose gels and hybridized with full-length PSTVd cDNA (HpaII and HaeIII) and TNOS (AluI) probe.
Figure 2. Northern blot analysis of total RNA from Nt-SB2 and Nt-Nbdi (A), Nt-SB2/Nbdi (B) and Nt-SB2* and Nt-SB2** (C). Total RNA was separated on 1.2% agarose/formaldehyde gels (high molecular weight RNA) or 15% acrylamide gels (small RNAs) and hybridized with full-length PSTVd cDNA as a probe. Samples from Nicotiana tabacum wild type (Nt-WT) plants were used as negative controls. Upper panels show SB2 transcript accumulation, middle panels vd-siRNA accumulation, and lower panels the ethidium bromide (EtBr) stained gels, which served as RNA loading controls. In Nt-SB2, the 666-nt transcript is not processed. In Nt-Nbdi, the 718-nt primary transcript is processed into 359-nt mature viroid molecules.
Figure 3. Methylation analysis by bisulfite sequencing. (A) Sequencing results from 14 clones for the SB2 locus in Nt-SB2, Nt-SB2/Nbdi, Nt-SB2* and Nt-SB2** are shown. Filled and empty boxes indicate methylated and unmethylated Cs, respectively. The analyzed fragment is depicted as a black bar in Figure 1A. The SB2 sequence is underlined, the non-underlined sequence represents the terminator and CG dinucleotides are printed in bold. The SB2 26-bp deletion (compared with the full-length mature PSTVd molecule) is indicated by lower-case letters (ag) in Italic. (B) Histograms representing the percentage of methylation (y-axis) as determined from bisulfite sequencing of SB2 locus in Nt-SB2, Nt-SB2/Nbdi, Nt-SB2* and Nt-SB2** (x-axis).
Figure 4. Chromatin immunoprecipitation analysis in Nt-SB2, Nt-SB2/Nbdi and Nt-SB2* plants. (A) PCR reaction for SB2 locus. (B) PCR reaction for actin (GQ339768.1), reference for euchromatin. (C) PCR reaction for centromere (AB376965.1), reference for heterochromatin. PCR products were analyzed on 1.5% agarose gel and visualized with ethidium bromide staining under UV.

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