Characterizing the de novo Methylation of an Exogenous Transposable Element
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Date
2016-05
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The Ohio State University
Abstract
Transposable elements (TEs) are DNA sequences that can duplicate and move from one location in the genome to another, inserting themselves into genes and causing mutations implicated in diseases like breast and colon cancer. DNA methylation is essential for silencing TE movement in plants and mammals. However, the establishment of de novo (new) TE DNA methylation on new or active TEs is poorly understood. To better understand this process, I analyzed DNA methylation levels achieved by an exogenous (foreign) TE promoter (LTR) construct, originally from tobacco, or an endogenous (native) gene promoter control construct inserted into several Arabidopsis thaliana lines. Using this experimental scheme, I was able to specifically assay the mechanism responsible for de novo DNA methylation targeted to a newly inserted TE. I performed bisulfite sequencing to obtain DNA methylation data, quantitative reverse-transcription PCR (qRT-PCR) to determine whether the TE promoter construct was transcribed, and inverse PCR coupled with BLAST to map the location of the constructs insertion. The bisulphite analysis showed that the promoter region of an exogenous TE, and not the control gene promoter, is sufficient to provoke significant de novo DNA methylation by the young seedling (about 1 week old) stage. Interestingly, several RNA degradation pathway mutants (rdr6, ago1, and dcl1) showed unexpectedly high levels of de novo methylation. Furthermore, the qRT-PCR experiments showed that the exogenous TE promoter becomes largely silenced in wild type plants, but is highly expressed in the rdr6 mutant. This, coupled with high de novo methylation values for the rdr6 mutant, suggests an undogmatic competitive interaction between RNA degradation, via RNAi, and de novo methylation, via RdDM. Preliminary data from the inverse PCR suggests that the insertion site of the exogenous construct into genic regions may have a significant impact on methylation achieved. As such, this thesis provides valuable information on the interactions between pathways thought to impact de novo DNA methylation and highlights the potential impact of TE location on de novo DNA methylation.
Description
OSU Undergraduate Research Scholarship
OSU Summer Undergraduate Research Fellowship
OSU Summer Undergraduate Research Fellowship
Keywords
Transposable element, Exogenous, de novo, Methylation, Silencing, Insertion site