Date of Award

2017

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

Klaus H. Kaestner

Second Advisor

Jonathan P. Katz

Abstract

The intestinal epithelium is a continuously self-renewing tissue that must balance its function as the site of nutrient absorption with its role as a barrier between the body and the external environment. With its high cellular turnover and continual exposure to lumenal antigens, the intestinal epithelium is highly susceptible to developing diseases, such as inflammatory bowel disease (IBD) and cancer. Understanding the factors that control intestinal epithelial development are key to discovering disease etiology and to engineering new treatments. In mammals, studies have pointed to dysregulation of RNA Polymerase III as playing a role in oncogenic transformation. A deletion in the gene encoding Polr3b, a Pol III subunit, impairs development of the intestinal epithelial progenitor cells in zebrafish. Thus, I hypothesized that POLR3B also has an important role in the mammalian intestinal epithelium. I characterized mice expressing an intestinal epithelium-specific hypomorphic Polr3b mutation. Neonatal Polr3b mutants have severely reduced survival and growth, reduced intestinal epithelial proliferation, and lack normal crypt development. Thus, Pol III is essential for the establishment of the intestinal epithelium in mice. Given that normal Pol III is required for adequate protein production in order to sustain the high rate of proliferation in mammalian gut, this enzyme is a questionable target for cancer drugs. It was reported that Mucin2 (Muc2), the main component of intestinal mucus, is a direct transcriptional target of FOXA1 and FOXA2 in the mouse intestinal epithelium. I hypothesized that in addition to Muc2, the FOXA factors transcriptionally regulate other genes that are required for the maintenance of the intestinal mucosa. The Foxa1/a2-deficient colonic epithelium in mice of advanced age (>7 months) displays increased susceptibility to spontaneous colitis. I performed FOXA1 and FOXA2 chromatin immunoprecipitation assays followed by ultra-high throughput sequencing (ChIP-Seq) analysis on wild type mouse colonic crypts, as well as RNA-seq on Foxa1/a2-deficient and control aged colon. Of the FOXA1/A2-bound, differentially expressed genes, glycosyltransferases were overrepresented, including the IBD risk gene Fut2, which was downregulated in mutant colon.

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