Strict De Novo Methylation of the 35S Enhancer Sequence in Gentian Kei-ichiro Mishiba1*., Satoshi Yamasaki1., Takashi Nakatsuka2, Yoshiko Abe2, Hiroyuki Daimon 1 , Masayuki Oda1, Masahiro Nishihara2 1 Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Osaka, Japan, 2 Iwate Biotechnology Research Center, Kitakami, Iwate, Japan Abstract A novel transgene silencing phenomenon was found in the ornamental plant, gentian (Gentiana triflora 6 G. scabra), in which the introduced Cauliflower mosaic virus (CaMV) 35S promoter region was strictly methylated, irrespective of the transgene copy number and integrated loci. Transgenic tobacco having the same vector did not show the silencing behavior. Not only unmodified, but also modified 35S promoters containing a 35S enhancer sequence were found to be highly methylated in the single copy transgenic gentian lines. The 35S core promoter (290)-introduced transgenic lines showed a small degree of methylation, implying that the 35S enhancer sequence was involved in the methylation machinery. The rigorous silencing phenomenon enabled us to analyze methylation in a number of the transgenic lines in parallel, which led to the discovery of a consensus target region for de novo methylation, which comprised an asymmetric cytosine (CpHpH; H is A, C or T) sequence. Consequently, distinct footprints of de novo methylation were detected in each (modified) 35S promoter sequence, and the enhancer region (2148 to 285) was identified as a crucial target for de novo methylation. Electrophoretic mobility shift assay (EMSA) showed that complexes formed in gentian nuclear extract with the 2149 to 2124 and 2107 to 283 region probes were distinct from those of tobacco nuclear extracts, suggesting that the complexes might contribute to de novo methylation. Our results provide insights into the phenomenon of sequence- and species- specific gene silencing in higher plants. Citation: Mishiba K-i, Yamasaki S, Nakatsuka T, Abe Y, Daimon H, et al. (2010) Strict De Novo Methylation of the 35S Enhancer Sequence in Gentian. PLoS ONE 5(3): e9670. doi:10.1371/journal.pone.0009670 Editor: Peter Meyer, University of Leeds, United Kingdom Received December 10, 2009; Accepted February 19, 2010; Published March 23, 2010 Copyright: 2010 Mishiba et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by Grant-in-Aid for Scientific Research 20780005 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (http://www.mext.go.jp/english/), and by Grant for Basic Science Research Projects 050356 from The Sumitomo Foundation (http://www.sumitomo.or.jp/e/ index.htm). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: mishiba@plant.osakafu-u.ac.jp . These authors contributed equally to this work. Introduction Epigenetic regulation of gene expression has been uncovered by recent progress in genome research. In higher plants, many regulatory factors for epigenetic regulation have been isolated, mainly from Arabidopsis [1,2]. Studies on transgene silencing in higher plants have contributed to the elucidation of epigenetic regulation [3]. In plants, transgenes can be silenced by both post- transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS), according to presence or absence of transcription [4]. Vaucheret and Fagard [4] classified TGS into trans-TGS, which is often induced by endogenous sequences in the genome similar to transgenes, and cis-TGS, which occurs in cases where the transgene is duplicated or is inserted into a transcriptionally inactive region subject to silencing. In both cases, hypermethyla- tion of the transgene generally occurs in the transgene promoter region. That is, de novo methylation must have been triggered in such TGS silencing at some point after integration of the transgene into the host genome. The mechanisms of sequence specific targeting of DNA methylation have been investigated by various researchers in recent years [1,5,6]. RNA-directed DNA methylation (RdDM), which is associated with the establishment of DNA methylation patterns and the initiation of gene silencing, has been discovered in plants [7]. Although there are potential alternative mechanisms to RdDM for introducing sequence specific DNA methylation, the causal factors for such phenomena are still unclear [8,9]. Even if the fundamental mechanisms of epigenetic regulation are shared in higher plant species, it is likely that particular characteristics of the epigenetic machinery have diversified in some plant species during evolution. One way to detect the divergence of epigenetic regulation is a comparative study of transgene silencing in different plant species; however, no study has focused on such an aspect. This could be due to the difficulty in providing a number of transformants sufficient for appropriate comparisons in most non-model plants. Nevertheless, different modes of transgene silencing might affect the screening efficiency of transformants in plant species, which is assumed to cause difficulty in obtaining transformants. This assumption is supported by a study of transgenic Mexican limes, which showed frequent transgene silencing after non-selective screening, whereas no silencing was found after selective culture [10]. Our previous study, using the ornamental floral plant, gentian (Gentiana triflora 6 G. scabra), showed that strict transgene silencing through cytosine methylation was observed in single copy T-DNA introduced transgenic plants [11]. No evidence of transgene PLoS ONE | www.plosone.org 1 March 2010 | Volume 5 | Issue 3 | e9670