De novo DNA methylation of the 35S enhancer revealed by high-resolution methylation analysis of an entire T-DNA segment in transgenic gentian

Abstract

We have found that cauliflower mosaic virus (CaMV) 35S promoter-specific transgene silencing is mediated by DNA methylation in gentian (Gentiana triflora ϫ G. scabra). De novo methylation of asymmetric cytosines (CpHpH; where H is A, C, or T) sequence has been detected at the enhancer region (Ϫ148 to Ϫ85) of the 35S promoter in transgenic gentians, and is thought to be responsible for the silencing mechanism. To clarify the concept of de novo methylation, the present study examined the detailed DNA methylation profile of the entire T-DNA sequence (ca. 4 kb) integrated into transgenic gentians. Although highly methylated cytosines at CpG and CpWpG (W is A or T) sequences were broadly distributed, except in the sGFP coding region, highly methylated cytosines at CpHpH and CpCpG sequences were mainly limited to the 35S enhancer region. In addition to the previously identified de novo methylation peak (Ϫ148 to Ϫ85), another peak was discovered at Ϫ298 to Ϫ241. Electrophoretic mobility shift assays showed that gentian nuclear extracts could bind to the corresponding probes (Ϫ149 to Ϫ124 and Ϫ275 to Ϫ250), and that the probes could compete with one another for binding. Thus, a nuclear factor might be involved in the de novo methylation of the two regions. In addition, the present data indicated that the methylation patterns at CpCpG sites could be categorized as CpHpH methylation rather than CpWpG methylation.

Progressive 35S promoter methylation increases rapidly during vegetative development in transgenic Nicotiana attenuata plants

“…Unwanted or unintended transgene silencing was commonly associated with an increase in methylation within the promoter region of the transgene [24,26,29,63]. Since we found evidence for epigenetic gene silencing (intermediate stages of sensitivity and high variability among replicates), we analyzed promoter methylation levels in the transgenic cassette by bisulfite sequencing.…”

“…Erratic occurrence and variegated phenotypes are commonly reported phenomena of transgene silencing and have been shown in many different plant species [22,25,27,29,71]. This was recently illustrated for N. benthamiana plants, transformed with a 35S:GFP construct [58,59].…”

“…However, the pattern of establishment and prerequisites for the methylation process remain elusive [21]. Characteristic symptoms of unwanted transgene silencing are spatially variegated or transient gene expression levels, patterns which have been observed in several different plant taxa including Nicotiana tabacum [22-24], Petunia hybrida [25,26], Arabidopsis thaliana [27,28], Gentiana triflora X G. scabra [29] and even in some transgenic woody plants such as grapevine ( Vitis spp.) [30] and birch trees ( Betula platyphylla ) [31].…”

Levels of DNA methylation and transcript accumulation in leaves of transgenic maize varieties

“…Although bisulfite sequencing of single clones has been regarded as the gold standard of DNA methylation analysis for the past few years [50], there is no consensus of how many clones are adequate to provide reliable results. Distinct studies have used five [7, 51], six [52, 53] or ten clones [54, 55]; other studies do not even mention it within their “Methods” section [56, 57]. …”

Beyond the blue rose: Modification of floral architecture with plant-specific chimeric repressors

“…Plant-specific transgene inactivation by promoter methylation in the Japanese gentian is effectively avoided using Arabidopsis actin2 promoter, thus leading to the production of the first bi-color flower . Intensive analyses of this 35S promoter-specific methylation are also reported by Yamasaki et al (2011) in this issue. Such in-depth examination of phenotypes and technical improvements focusing on flowers as commercial crops will accelerate the practical use of GM flowers.…”