Date of Award

5-2014

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Mariano Labrador

Committee Members

Bruce McKee, Albrecht von Arnim, Ranjan Ganguly, David Brian

Abstract

Eukaryotic chromatin insulators play an essential role in regulating gene expression and modifying nuclear architecture by organizing the higher-order chromatin structure in response to cellular and developmental cues. The details on how insulators function in this capacity are not completely understood.

Five different types of insulators have been identified in Drosophila. Each functional insulator consists of an insulator DNA response element bound by an insulator protein, which recognizes specific DNA sequences. Each type of insulator functions individually as well as collaboratively. Except for the Su(Hw) insulator protein, the other insulator proteins are necessary for viability considering loss of Su(Hw) only interrupts insulator function and causes female sterility. It has been suggested that Su(Hw) may play a separate role in these two functions. To gain a better understanding of Su(Hw), its functions were studied using female germline development as a model system, and the mechanisms of its regulation were studied using an in vitro cell culture system.

This study examined the critical function of Su(Hw) in both gene regulation and genome organization during oogenesis. Chapter one describes a remarkable ring canal developmental defect phenotype first identified and characterized in su(Hw) mutants, and this phenotype may contribute to female infertility. Chapter two details a newly discovered role of Su(Hw) in maintaining the genome integrity of germline cells. Loss of Su(Hw) causes accumulation of double strand breaks (DSBs), further triggering DNA damage signaling. Genome instability causes developmental defects resulting in incomplete oogenesis and consequent female sterility.

To understand insulator function regulatory mechanisms, Drosophila Schneider 2 cells (S2 cells), were used as the system for investigation. Chapter three demonstrates that Su(Hw) was identified at a novel sub-cellular location within the midbody during mitotic telophase, and SUMOylation and phosphorylation may play a role in the functional regulation and sub-cellular localization of Su(Hw).

Our work proposes that in spite of regulating gene expression, the Su(Hw) insulator also plays a critical role in maintaining genome stability by directing higher-order organization of the chromatin structure. Moreover, the protein-protein interactions and sub-cellular localization of Su(Hw) may regulate its function, and this functionality control may be fine-tuned by post-translational modifications.

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