The regulation of transcription within eukaryotic genomes is complex and depends upon many pathways working in concert. Plants face a unique set of circumstances, from both their inability to physically escape from stressors, to large transposable element loads, and a complex reproductive strategy. These biological difficulties manifest themselves in the ways that plant genomes regulate transcription. At the epigenetic level, modifications to genomic DNA and the ways in which it’s packaged within a cell affect transcription without changing the underlying sequence present in the DNA. One of the main mechanisms behind transcriptional gene silencing (TGS) in plant genomes is RNA directed DNA methylation (RdDM). RdDM functions through the action of multiple plant-specific RNA polymerases and results in the production of 24-nt siRNAs, the majority size class of small RNAs in plants, and culminates in DNA methylation. DNA methylation recruits further chromatin modifications, and results in TGS.
RdDM has most frequently been studied in the context of silencing transposable elements. These elements, if left unchecked, may multiply throughout genomes and disrupt the function of essential genes. Additionally, RdDM has largely been studied in Arabidopsis thaliana. In this model system, despite the presence of 24-nt siRNAs that are specific to, and highly expressed in, reproductive structures, there appears to be no gross developmental or reproductive consequence for loss of RdDM. In my work we have moved outside of A. thaliana to Brassica rapa, where there is a specific reproductive phenotype associated with loss of RdDM. In B. rapa, RdDM mutant seeds abort at approximately 15 days post-fertilization.
To understand the reproductive role for RdDM we characterized RdDM’s function in B. rapa seeds and their constituent tissues. This led to our discovery of maternal-specific small RNAs produced from genomic regions called siren loci. We found that most small RNAs in seeds accumulate at a relatively small number of siren loci, and this is the case both before fertilization in the integument tissues and after fertilization in the seed coat. Additionally, siren loci accumulate small RNAs strongly in endosperm, and they are maternal in origin, suggesting movement and/or imprinting of siren RNAs. Siren loci also exhibit tissue specific RdDM.
In order to analyze data to support these biological insights we took advantage of next generation sequencing technologies. This enabled us to assay small RNAs and DNA methylation genome-wide. The analysis of sequencing data requires an understanding of computational methods and computer systems, and therefore a significant part of my work was in developing solutions for analysis of this data. Several computational tools developed in the course of this work have been made available to the public and have greatly increased the efficiency of our work. These tools automate long computational workflows and ensure reproducibility. Automation and reproducibility are critical in bioinformatics, and the usage of workflow managers, containers, and high-performance computing systems together helps to facilitate research at scales much larger than could otherwise be possible.
|Advisor:||Mosher, Rebecca A.|
|Commitee:||Schwartz, Jacob C., Lyons, Eric H., Palanivelu, Ravishankar, Beilstein, Mark A.|
|School:||The University of Arizona|
|Department:||Molecular & Cellular Biology|
|School Location:||United States -- Arizona|
|Source:||DAI-B 82/4(E), Dissertation Abstracts International|
|Subjects:||Bioinformatics, Plant sciences, Molecular biology|
|Keywords:||DNA Methylation, Epigenetics, Genomics, RNA, Seed development, Small RNA|
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