Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Biochemistry

Supervisor

Torchia, Joe

Abstract

Precise control over transcriptional regulation is required for normal cell function. Errors in transcriptional regulation underpin many diseases including cancer. Thymine DNA Glycosylase (TDG) is a base excision repair protein and a coregulator that has been implicated in a diverse set of fundamental biological processes including embryonic development, nuclear receptor signaling and Wnt signaling. Importantly, TDG has been shown to play an important role in transcriptional regulation in a wide variety of systems. Details surrounding the mechanism through which TDG acts remain unclear. In this thesis we explore the role of TDG in Estrogen Receptor (ER)-dependent signaling and in cellular senescence.

To characterize the role of TDG in ER mediated signaling we first mapped β-Estradiol (E2)-dependent DNA binding of TDG in the MCF7 breast cancer cell line using ChIP-Seq. Using bioinformatics in conjunction with more traditional biochemistry techniques I established that a significant component of TDG binding occurs at enhancers, where it was able to mediate the production of enhancer RNA (eRNA) and 3-dimensional reorganization of transcriptional units. Knockdown of TDG disrupts E2-mediated upregulation of ER-targets and inhibits growth. Remarkably, in addition to behavior mimicking that of an oncogene, I find that TDG knockdown and depletion result in a much more aggressive phenotype, revealing its role as a potential potent tumor suppressor.

To explore the role of TDG in cellular senescence we induced senescence in IMR90 human fibroblasts using hydrogen peroxide (H2O2) and monitored markers of senescence, including proliferation and β-galactosidase staining. I found that while senescence was readily inducible in this cell line using H2O2, knockdown of TDG was able to significantly impede the process. Using ChIP, I found that TDG was recruited to a CpG island overlapping the CDKN2A promoter, a tumor suppressor important for senescence. Further studies including ChIP, bisulfite sequencing and conventional assays revealed that TDG is required for H2O2-mediated transcription of CDKN2A in a CBP-dependent and active-demethylation independent manner.

Collectively, these studies extend the role of TDG in transcriptional regulation, implicating it as a mediator of cellular senescence and as a mediator of eRNA transcription and 3-dimensional re-organization in hormone signaling.

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