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|Title:||Biochemical and Genomic Analyses of Ciliate Chromatin Structure and Function|
|Authors:||Beh, Yee Ming Leslie|
|Advisors:||Landweber, Laura F|
|Contributors:||Ecology and Evolutionary Biology Department|
|Publisher:||Princeton, NJ : Princeton University|
|Abstract:||Ciliates are single-celled eukaryotes, defined by the presence of two structurally and functionally distinct nuclei. The somatic macronucleus constitutes the transcriptionally active focus of the cell, being responsible for all RNA polymerase II-mediated transcription. Separately, the germline micronucleus contains genetic information that is used to build the new somatic macronucleus upon sexual reproduction. My PhD work seeks to understand how ciliate macronuclear genomes are organized as chromatin, and how this in turn may affect gene regulation. To do so, I applied a combination of genomic and biochemical approaches to map chromatin organization in two distantly related ciliates, Oxytricha trifallax and Tetrahymena thermophila. These organisms, having diverged > 1 billion years ago, exhibit vastly different macronuclear genome architectures: Oxytricha exhibits “nano”chromosomes that ~average 3kb in length, while Tetrahymena thermophila contains megabase-sized chromosomes. Together, this comparative analysis illuminated novel principles and molecules underlying chromatin organization. Chapter 1 consists of an introduction to the ciliate life cycle, nuclear duality, and chromatin organization. Chapter 2 describes the discovery of DNA-guided nucleosome organization in the AT-rich macronculear genome of Tetrahymena thermophila. A combination of in vitro and in vivo MNase-seq was used to dissect the contributions of cis- and trans-factors to nucleosome organization in Tetrahymena. Chapter 3 reports the genomic organization of nucleosomes, transcription start sites, and DNA adenine methylation (6mA) in Oxytricha nanochromosomes, and discovery of the corresponding 6mA methyltransferase. We found that 6mA is localized in nucleosome linker regions, near transcription start sites. Biochemical fractionation identified a novel 6mA methyltransferase – MTA1 – necessary for methylation. Mutation of MTA1 resulted in a genome-wide loss of 6mA methylation and enabled the functional assessment of the consequences of this perturbation on nucleosome organization and gene expression.|
|Alternate format:||The Mudd Manuscript Library retains one bound copy of each dissertation. Search for these copies in the library's main catalog: catalog.princeton.edu|
|Type of Material:||Academic dissertations (Ph.D.)|
|Appears in Collections:||Ecology and Evolutionary Biology|
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