Cell Culture-Induced Gradual and Frequent Epigenetic Reprogramming of Invertedly Repeated Tobacco Transgene Epialleles  

Abstract

Using a two-component transgene system involving two epiallelic variants of the invertedly repeated transgenes in locus 1 (Lo1) and a homologous single-copy transgene locus 2 (Lo2), we have studied the stability of the methylation patterns and trans-silencing interactions in cell culture and regenerated tobacco (Nicotiana tabacum) plants. The posttranscriptionally silenced (PTGS) epiallele of the Lo1 trans-silences and trans-methylates the target Lo2 in a hybrid (Lo1/Lo2 line), while its transcriptionally silenced variant (Lo1E) does not. This pattern was stable over several generations in plants. However, in early Lo1E/Lo2 callus, decreased transgene expression and partial loss of Lo1E promoter methylation compared with leaf tissue in the parental plant were observed. Analysis of small RNA species and coding region methylation suggested that the transgenes were silenced by a PTGS mechanism. The Lo1/Lo2 line remained silenced, but the nonmethylated Lo1 promoter acquired partial methylation in later callus stages. These data indicate that a cell culture process has brought both epialleles to a similar epigenetic ground. Bisulfite sequencing of the 35S promoter within the Lo1 silencer revealed molecules with no, intermediate, and high levels of methylation, demonstrating, to our knowledge for the first time, cell-to-cell methylation diversity of callus. Regenerated plants showed high interindividual but low intraindividual epigenetic variability, indicating that the callusinduced epiallelic variants were transmitted to plants and became fixed. We propose that epigenetic changes associated with dedifferentiation might influence regulatory pathways mediated by trans-PTGS processes.

Tissue Culture-Induced Novel Epialleles of aMybTranscription Factor Encoded bypericarp color1in Maize

“…In a previous study, attenuated expression of cell culture-derived epialleles of an invertedly repeated transgene in tobacco was attributed to methylation mosaicism at the cellular level (Krizova et al 2009). Our study, however, provides evidence that such intermediate expression might also be due to mosaic methylation patterns of individual gene copies.…”

Epigenetics in plant tissue culture

“…Euchromatin, in which active genes reside, was hypermethylated in culture, but only a small percentage of genes obtained heterochromatic marks, which might lead to long-term silencing. Krizova et al (2009) studied the fate of an epigenetically silenced LO1 transgenic allele in tobacco taken through cell culture and regeneration from callus. They observed partial loss of Lo1E 35S promoter methylation compared with leaf tissue in the parent plant.…”

“…Mistargeted de novo methylation would allow the establishment and perpetuation of DNA hypermethylation of promoters, which would create epialleles, and hypomethylation of repetitive DNA, which may lead to activation of mobile elements. Krizova et al (2009) examined the development of a tobacco cell culture into callus followed by plant regeneration. They observed the loss of parental imprinting in 25% of the regenerated tobacco plants, regardless of whether the epiallele was originally methylated or unmethylated.…”

“…The methylationdependent restriction enzyme McrBC can be used to separate methylated from non-methylated DNA, followed by detection using whole genome microarray hybridisation (Vaughn et al 2007). Bisulfite-treated DNA can be sequenced and compared to untreated DNA, thus producing detailed information on the methylation of cytosines on both strands of the DNA (Krizova et al 2009;Zhang et al 2009). Histone modifications are studied using chromatin immunoprecipitation (ChIP) of associated DNA followed by polymerase chain reaction (PCR) of cDNA or whole genome microarray hybridisation (Matarazzo et al 2004;Roh et al 2004;Shi et al 2003).…”

Effect of orientation of transcription of a gene in an inverted transferred DNA repeat on transcriptional gene silencing in rice transgenics—a case study

“…However, nuclear runon transcription analysis revealed that the gus as well as OSM and hph genes were subjected to TGS. An IR locus, locus1 in tobacco, epigenetically switched from PTGS to TGS upon prolonged vegetative propagation in tissue culture (Fojtova et al 2003;Krizova et al 2009). The P35S promoter of the TGS silenced nptII gene was hypermethylated.…”