Date of Award

2015

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

Klaus H. Kaestner

Abstract

Patterns of DNA methylation are established and maintained by DNA methyltransferases (Dnmts), which have traditionally been subdivided into the ‘de novo’ methyltransferases, Dnmt3a and Dnmt3b, and the ‘maintenance’ methyltransferase, Dnmt1. Dnmt1 maintains DNA methylation patterns and genomic stability in several in vitro cell systems, but its function in tissue-specific development, homeostasis, and disease in vivo is only beginning to be investigated.

Recently, the Kaestner lab demonstrated that loss of Dnmt1 in the adult intestinal epithelium causes a two-fold expansion of the proliferative crypt zone, indicating that Dnmt1 and DNA methylation regulate proliferative processes in the intestine. I hypothesized that loss of Dnmt1 may impart similar effects during intestinal development and tumorigenesis, and employed distinct Cre-loxP mouse models to ablate Dnmt1 in progenitor cells during intestinal development and in the mature intestinal epithelium of cancer-prone ApcMin/+ mice.

In the first part of my thesis, I show that loss of Dnmt1 in intervillus progenitor cells in the developing intestine causes global hypomethylation, DNA damage, premature differentiation, and apoptosis. I confirm this novel role for Dnmt1 during crypt development using the in vitro organoid culture system, and illustrate a differential requirement for Dnmt1 in immature versus mature organoids. These results demonstrate an essential role for Dnmt1 in maintaining genomic stability during intestinal development and the establishment of intestinal crypts.

DNA methylation is thought to drive CRC progression by the repression of tumor suppressor genes via promoter methylation. In the second part of my thesis, I utilize inducible intestinal epithelial-specific gene ablation to determine the requirement of Dnmt1 in intestinal tumorigenesis. Surprisingly, I find that loss of Dnmt1 in cancer-prone ApcMin/+ mice results in accelerated, not decreased, intestinal tumor development. Dnmt1 deletion precipitates an acute response in mature intestinal epithelium characterized by hypomethylation of repetitive elements, genomic instability, and apoptosis, which is followed by remethylation with time. This recovery is entirely dependent on the activity of the de novo methyltransferase Dnmt3b. In light of these data, the current dogma regarding the role of DNA methylation in colon cancer needs to be revisited.

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