Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Molecular Genetics and Genomics

Language

English (en)

Date of Award

8-29-2012

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Sarah CR Elgin

Abstract

Heterochromatin packages a large portion of the genome in eukaryotes, and plays important roles in mitosis, telomere homeostasis, and transposon: TE) control, all necessary to ensure genome stability. A precisely controlled mechanism to designate which regions of the genome should be so packaged is critical to the fitness of an organism. Mis-localized heterochromatin can lead to mis-regulation of nearby and even distant genes, with pleotropic consequences. How cells: and hence organisms) precisely control the location and extent of heterochromatin formation is an intriguing question. Models involving small RNAs, epigenetic inheritance of histone modifications, DNA binding proteins targeting specific motifs, and other mechanisms have been proposed.

Small RNA targeting of heterochromatin formation is an established mechanism in fission yeast: Schizosaccharomyces pombe) and the flowering plant, Arabidopsis thaliana. However, a parallel mechanism in the animal kingdom has not been clearly established. My thesis work focuses on this issue, using the fruit fly, Drosophila melanogaster. Heterochromatin silencing in flies can be monitored using a reporter exhibiting variegating pigmentation in the eyes, i.e. Position Effect Variegation: PEV). The PEV phenotype is known to be quite variable, and I have found that much of the variation has a genetic basis: Chap. 2). Using PEV reporter lines I have investigated a subtype of heterochromatin, the telomeric region of the Y chromosome short arm: Ys). I found that telomeric Ys has a unique response profile to PEV modifiers and appears to employ a distinct type of targeting mechanism: Chap. 3). To further address the question of targeting, I have engineered knockdown mutations of Piwi, a nuclear-localized small RNA binding protein, and demonstrated a loss of silencing and a corresponding loss of heterochromatin formation at targeted TE's. Genetic manipulations of Piwi and other components of the system confirm that small RNA targeting appears to be one of the mechanisms used in determining the regions subject to heterochromatin formation: Chap. 4). Moreover, Piwi appears to silence transposons through additional mechanisms as well, including playing a cytoplasmic role: Chap. 4, 5).

From my thesis work, I conclude that Piwi has a role in piRNA- based transcriptional silencing of transposons. However, how this mechanism translates to overall heterochromatin formation in the fly genome awaits further investigation. The packaging of the eukaryotic genome appears to be more complex than the picture delineated by the euchromatin/heterochromatin dichotomy. A more complex targeting mechanism, as is suggested by this work, is therefore needed to adequately describe the packaging process.

Comments

Permanent URL: http://dx.doi.org/10.7936/K71J97SP

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