Date of Award

Summer 8-15-2019

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Molecular Genetics & Genomics)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

DNA damage is a major threat to genome stability, as it is estimated that a single cell may incur as many as one million DNA lesions per day. DNA double-strand breaks (DSBs) are the most precarious type of DNA lesion, since they result in a complete dissociation of two chromosomal ends. Broken DNA ends that are not repaired efficiently may induce cellular apoptosis or be aberrantly joined with other regions of the genome, forming translocations. Genomic rearrangements involving genic regions are especially dangerous as they potentially place translocated loci under abnormal regulatory constraints or create fusion genes, which can ultimately drive oncogenesis. Translocations involving gene fusions are found in haematological malignancies as well as solid tumors. Thus, the accurate and efficient repair of DSBs occurring in transcribed regions is critical to prevent carcinogenic transformations. Furthermore, as DNA serves as a substrate for transcription, DNA lesions at transcriptionally active loci threaten cell viability and normal cellular functions via the physical interruption of this metabolic process. It is well established that certain types of DNA lesions impede transcription. RNA polymerase II (RNAP II) serves as a sensor of bulky DNA adducts and UV photoproducts, and acts to recruit repair factors to the damage during transcription-coupled nucleotide excision repair (TC-NER). Recent literature points to a role for DNA damage signaling in regulating transcriptional activity in very close proximity to a DSB site. However, studies have been limited by available systems that employ cycling cells and/or DSBs that occur at restriction sites, most of which disrupt transcriptional promoters or gene bodies. Here, I describe an innovative approach for generating targeted DSBs at chosen genomic locations in non-cycling cells, a physiologic state most relevant to the vast majority of cells in mammals. Using this novel experimental system, I directly demonstrate that DSBs within and outside of the body of an endogenous gene silence its expression without disrupting regulatory or promoter elements. Importantly, our data establishes, for the first time, that a single DSB can repress either ongoing or inducible expression of a proximal gene, without affecting chromatin accessibility at nearby regulatory elements. Remarkably, and in contrast to prevailing models, this work demonstrates that a persistent DSB results in long-range repression of gene transcription, which spreads throughout the γ-H2AX domain and is independent of the precise break site within the region. Thus, our findings uncover an important functional consequence of the DDR signaling cascades that generate γ-H2AX domains. The novel experimental approach presented here will enable further investigations into DSB- driven alterations of local chromatin- based processes in a range of native, biological contexts.

Language

English (en)

Chair and Committee

Eugene M. Oltz Barry Sleckman

Committee Members

Sheila A. Stewart, Zhongsheng You, Peter M. Burgers, Nima Mosammaparast,

Comments

Permanent URL: https://doi.org/10.7936/v0kr-rh87

Available for download on Tuesday, August 15, 2119

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