Trans‐generation inheritance of methylation patterns in a tobacco transgene following a post‐transcriptional silencing event

Abstract

SummaryWe have studied the inheritance of the epigenetic state of tobacco transgenes whose expression was posttranscriptionally silenced by an invertedly repeated silencer locus. We show that, in hybrids, the coding region of the target neomycin phosphotransferase (nptII) gene was almost exclusively methylated at CG configurations, and dense non-CG methylation occurred in the 3¢ untranslated region. Homologous sequences in the silencer locus were heavily methylated at both CG and non-CG motifs. After segregation of the silencer locus, the CG methylation but not the non-CG methylation of the target genes was transmitted to the progeny. In the segregants, we observed an overall increase of CG methylation in the target genes, associated with a re-distribution from the 3¢ end of the coding region towards the middle. This pattern was inherited with some fluctuation for at least two additional generations in the absence of a detectable T-DNA-derived small RNA fraction. Thus CG methylation is not cleared during meiosis and may be inherited over generations without RNA signals being present. These epi-allelic variants re-expressed the reporter gene immediately after segregation of the trigger, showing that relatively dense CG methylation (approximately 60-80%) imprinted on most of the coding region (>500 bp) did not reduce expression compared with the parental non-methylated locus. We propose that the genic CG methylation seen in euchromatic regions of the genome may originate from ancient post-transcriptional gene silencing events as a result of adventitiously produced methylationdirecting RNA molecules.

Viroid-induced DNA methylation in plants

“…Notably, this maintenance of methylation was not associated with histone H3 dimethylation and/or H3 acetylation (41). These data were in agreement with observations made in other systems (42), and they clearly showed that transgenic coding/ transcribed regions can also retain CG methylation in the absence of RdDM guide molecules.…”

Hairpin transcription does not necessarily lead to efficient triggering of the RNAi pathway

“…Variable maintenance of methylation and contradictory results of previous reports (Jones et al 2001;Lunerova-Bedrichova et al 2008), argue for a model of a sequence-specific mechanism for maintenance of RdDM. Homogeneous/heterogenous distribution of cytosines and/or high/low GC-context all may account for this variation.…”

“…However, in contrast to promoter regions, coding region methylation was thought to be meiotically lost in the absence of an RdDM trigger (Jones et al 2001). Nevertheless, in a recent study, CG methylation was maintained in a transgene coding region (Lunerova-Bedrichova et al 2008). Here, besides CG, we observed maintenance also of CHG methylation.…”

Inhibition of SAH-hydrolase activity during seed germination leads to deregulation of flowering genes and altered flower morphology in tobacco

“…On the other hand, the CG hypomethylation elicited with increased concentration of the drug remained stably inherited throughout plant development and could potentially influence developmental processes. As a consequence, the CG epialleles could be more stable than those of non-CG (Lunerova-Bedrichova et al 2008). However, this may not be absolute since a stable non-CG epiallele (SUP) rescued from the hypomethylated background was reported in Arabidopsis (Jacobsen and Meyerowitz 1997).…”