PhD: Molecular and genetic requirements of a defence mechanism that silences foreign DNA inserts
- Centre for Plant Sciences, University of Leeds, Leeds, UK
Description: Transgenic plants are widely used in basic research to study gene function and in modern agriculture to improve crop performance. An essential requirement for the reliability and success of these applications is the stable activity of the transgene when it has been inserted into the host genome. Unfortunately, however, many transgenes are subject to ‘gene silencing’, which results in complete or partial inactivation of their expression. The molecular causes of gene silencing are various epigenetic effects that either alter the chromatin structure of transgenes, rendering them inaccessible to the transcription machinery, or that generate small RNA molecules that degrade transgene-specific transcripts or inhibit their translation.
Several strategies have been developed to overcome or avoid gene silencing effect, which includes the selection of single copy transgenes, as repeat structures favour small RNA production, the use of host lines with compromised small RNA functions, or simply the screening of large numbers of transformants under various conditions to select candidates with stable transgene activity. While these strategies have significantly improved transgene stability, they don’t answer the interesting question what makes transgenes such prominent targets for gene silencing mechanisms.
To address this issue, we have selected a DNA element (the RPS element) that functions as a ‘hot spot’ for DNA methylation. The methylation of cytosine residues is a common feature of genomic regions that have acquired a condensed (heterochromatic) structure, which inhibits efficient transcription. DNA methylation, which is often accompanied by modifications of the histones associated with the DNA, is a hallmark of silent genes and of genes prone to become silent at a later stage. A large number of transposons, retrotransposons and pseudogenes illustrates that eukaryotic genomes have a history of gene integration events. Most of these ‘foreign’ inserts have been methylated and silenced, most likely due to molecular defence mechanisms that aim to protect the cell from detrimental consequences of their transcription. We are using the RPS element as a model to examine how ‘foreign’ DNA is singled out by these defence systems.
Our current work is based on three observations: (i) When an RPS element is integrated into the plant genome, DNA methylation is initiated within a specific region, which we labelled the methylation trap (MT). We therefore want to design deletion and substitution constructs to examine if this region contains a specific sequence or secondary structure that marks it as a target for DNA methylation. (ii) Following initial DNA methylation within the MT region, methylation spreads in both directions. We are using a series of mutants that are impaired in different epigenetic functions to test their involvement in the spreading effect. This should help us to understand how silent states are propagated and if and how their spreading is controlled. (iii) Because the RPS element is such an efficient target for DNA methylation, it is also a powerful tool to search for genomic regions where its integration does not trigger to DNA methylation, which may make these regions ideal target areas for transgene insertion. We have so far found one methylation-free RPS transformant and intend to search for more and to map the corresponding integration regions.
The project will include recombinant DNA techniques, plant transformation, transcript analysis, genomic sequencing, chromatin immunoprecipitation and in silico mapping.
Application Details: This project will be available for UK undergraduates and for students from EU member states. Overseas students from non EU member states need to secure their own funding to cover fees and living expenses.
See the links below for full application information.
Contact: Professor Peter Meyer
More Information: Description and Application details
More Information: Meyer Lab
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