Electronic Thesis and Dissertation Repository

Thesis Format

Monograph

Degree

Master of Science

Program

Biochemistry

Supervisor

Torchia, Joseph

Abstract

Thymine DNA Glycosylase (TDG) plays a key role in active demethylation by excising intermediates of 5-methylcytosine. The function of TDG is required for embryonic development, as Tdg-null embryos die at E11.5. To bypass this embryonic lethality, our lab generated conditional Tdg knockout (TDGCKO) mice. These mice develop late-onset hepatocellular carcinoma (HCC), partly due to impaired Farnesoid X Receptor (FXR) signaling. Interestingly, Fxr-knockout mice display a similar phenotype and transcriptional profile to TDGCKO mice, prompting us to investigate a role for TDG in FXR signaling. To this end, we generated Tdg/Fxr double-knockout (DKO) mice. We also generated a novel Fxr-null mouse model using CRISPR/Cas9, which facilitated the knockout of FXR through a 47-bp deletion event. We demonstrated that 3-week-old Fxr-null mice display impaired bile acid and glucose metabolism. Moreover, we demonstrated a novel interaction between TDG and FXR in vivo. Collectively, these findings implicate TDG as a coactivator of FXR signaling.

Summary for Lay Audience

DNA can be modified by a process known as methylation. This modification can be reversed by a counteracting process known as active demethylation. A key protein involved in active demethylation is Thymine DNA Glycosylase (TDG). Mouse studies have demonstrated that TDG is required for embryonic development. When TDG was deleted from birth in mouse embryos, these embryos died twelve days post-conception.

Since the deletion of TDG from birth is lethal, our lab deleted TDG eight weeks after birth to bypass this obstacle. This is known as a ‘conditional’ deletion; hence these mice are called conditional TDG-knockout (TDGCKO) mice. Our lab found that TDGCKO mice develop late-onset liver cancer, partly due to an increase in bile acids (BAs). Excessive amounts of BAs can cause damage to the liver. Consequently, BAs are tightly regulated. The main protein involved in regulating BAs is Farnesoid X Receptor (FXR). Interestingly, FXR-knockout (FXRKO) mice develop late-onset liver cancer, which is similar to TDGCKO mice. To this end, I aimed to generate TDG/FXR double-knockout (DKO) mice by breeding FXRKO and TDGCKO mice together, predicting that DKO mice will develop a more accelerated form of liver cancer. However, to generate DKO mice in this manner, the genes for TDG and FXR would need to be on separate chromosomes. Incidentally, TDG and FXR are on the same chromosome. Consequently, we used a gene-editing technique that allowed us to bypass this hindrance. With this technique, we generated a new FXRKO model which enabled us to subsequently generate DKO mice. This technique functions by introducing various mutations into a gene of interest. I showed that the specific mutation that occurred in our FXRKO mice was a deletion of 47 base pairs. I found that our FXRKO mice have significantly more BAs in the liver compared to normal mice. Collectively, the preliminary data generated from our FXRKO model is consistent with the published data from previous FXRKO models.

Finally, I investigated whether TDG plays a role in FXR function. I demonstrated that TDG and FXR interact in mouse liver. Altogether, my results suggest that TDG plays a coactivating role in FXR function.

Creative Commons License

Creative Commons Attribution 4.0 License
This work is licensed under a Creative Commons Attribution 4.0 License.

Available for download on Saturday, December 24, 2022

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