[Intellectual contribution][Advances in Technology]

DNA elements preventing transcriptional gene silencing isolated by a novel screening strategy

Naoki Kishimoto1, Yuko Ohashi2, Ichiro Mitsuhara3
1Plant Genome Engineering Research Unit, 2Plant Microbes Interaction Research Unit, 3Division of Plant Sciences
[Abstract]
We developed a successful novel screening strategy using transgenic tobacco for isolation of DNA elements that could prevent transcriptional gene silencing (TGS) to protect a flanking transgene from TGS. We isolated three different DNA sequences with anti-silencing function from Lotus japonicus.
[Keywords]
transgene inactivation, transcriptional gene silencing, transgenic plant, plant transformation, cauliflower mosaic virus 35S promoter (P35S)

[Background]

Transcriptional gene silencing (TGS)—a phenomenon observed in endogenous genes/transgenes in eukaryotes—is a huge hindrance to transgenic technology and occurs mainly when the genes involved share sequence homology in their promoter regions. TGS depends on chromosomal position, suggesting that its suppression is associated with the existence of specific genomic elements. However, no systematic approach to identify such DNA elements has yet been reported. Here, we developed a successful novel screening strategy to identify such elements (anti-silencing regions, ASRs) based on their ability to protect a flanking transgene from TGS.
[Results and Discussion]
  1. We found a silenced transgenic tobacco plant in which a subsequently introduced transgene undergoes obligatory promoter-homology dependent TGS in trans allowed the DNA elements to prevent TGS and used it as an efficient screening strategy. We then isolated ASRs from DNA libraries constructed with selectable-marker (hygromycin-resistance gene–HPT)-harboring vectors. If P35S::HPT construct containing genomic fragments with no ASR activity is supertransformed into explants of the TGS plant, the P35S would be silenced and no supertransformant would be obtained on hygromycin-containing medium (Fig. 1, left). In contrast, supertransformants from explants of the TGS plant can be regenerated on the selection medium if the supertransformed P35S::HPT is protected from TGS by an adjacent ASR (Fig. 1, right).
  2. We identified three different ASRs with lengths of 3 Kb, 0.3 Kb and 0.2 Kb, from a genomic DNA library of Lotus japonicus. The anti-silencing activity of these ASRs was confirmed using a transgenic plant in which transformed P35S-driven gene are prone to TGS (Fig. 2).
  3. Two of the ASRs also showed anti-silencing activity in other plant species such as Ipomoea batatas and Arabidopsis thaliana.
  4. This is the first report of a screening strategy to identify anti-TGS elements.
[Future prospects]
  1. ASRs would facilitate the development of transformation-vectors that could prevent TGS and the production of less TGS-prone transgenic crops.
  2. Characterization of ASRs including ASR-binding proteins will provide new insights in understanding epigenetic genome regulation.

Fig.1. Identification of anti-silencing regions (ASRs) preventing TGS. Strategy to creen ASRs from a genomic library.


Fig.2. Anti-silencing activity of ASRs in preventing promoter-homology dependent TGS. The effects of ASRs on expression of a GUS construct driven by P35S were examined when a transgenic plant carrying a P35S-driven transgene was transformed. CT: control transformants using wild type; CST: control supertransformants; ASR ST: supertransformants with one of the ASRs

 

[Reference]

  1. Kishimoto N, Nagai J, Kinoshita T, Ueno K, Ohashi Y, Mitsuhara I (2013) DNA elements reducing transcriptional gene silencing revealed by a novel screening strategy PLoS ONE 8(1): e54670
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