4.6 Article

Strict De Novo Methylation of the 35S Enhancer Sequence in Gentian

Journal

PLOS ONE
Volume 5, Issue 3, Pages -

Publisher

PUBLIC LIBRARY SCIENCE
DOI: 10.1371/journal.pone.0009670

Keywords

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Funding

  1. Ministry of Education, Culture, Sports, Science, and Technology of Japan [20780005]
  2. Sumitomo Foundation [050356]
  3. Grants-in-Aid for Scientific Research [20780005] Funding Source: KAKEN

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A novel transgene silencing phenomenon was found in the ornamental plant, gentian (Gentiana triflora x 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.

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