PROJECT SUMMARY Future Research Questions As a graduate student in the Toxicology and Cancer Biology Department of University of Kentucky, I seek to understand the complex molecular processes that drive development of lung diseases. The goal of my dissertation research is to determine how the role of Polycomb Repressive Complex 2 (PRC2) directs proper lung stem cell fate, and how this process is dysregulated in lung disease. My central hypothesis is that PRC2 normally represses basal and goblet cell fates in bronchiolar epithelium, and that loss of PRC2-mediated repression of these lineages leads to phenotypes observed in bronchiolar epithelium of Chronic Obstructive Pulmonary disease (COPD), including basal cell and goblet cell metaplasia. Background and Methodology Polycomb Repressive Complex 2 (PRC2) modulates gene expression through histone modification. Recent unpublished data from the Brainson lab suggests that PRC2 activity is dysregulated in chronic obstructive pulmonary disease (COPD). These findings fit with others in the field that demonstrated loss of PRC2 in developing lung causes aberrant expression of basal cell markers including KRT5 and p63 in distal lung airways in mice. Together these data suggest that improper PRC2 activity is important for cell development, fate, and function. They also warrant further investigation into PRC2's role in disease development and propagation, particularly those involving lung functions. Methodology The Aims of my proposal are to: 1) Elucidate the cellular changes in bronchiolar epithelial cells observed in in vitro and in vivo after genetic modulation of the PRC2 complex and 2) Confirm that loss of proper PRC2 gene silencing leads to expansion of basal cell and goblet cell fates in human bronchiolar epithelial cells. For Aim 1, I am knocking out one or two alleles of Ezh2, the gene encoding the enzyme for PRC2, in adult mouse lung for four months, and challenging the mice with ovalbumin to induce goblet cell metaplasia. My hypothesis that loss of PRC2 will prevent cells from resolving allergic response will be tested by lung histology. In parallel, I will use lung organoid cultures and single cell RNA-sequencing to examine lung cell fates when PRC2 is dysregulated in mouse lung cells. For Aim 2, I will use an EZH2 inhibitor, short hairpin RNAs against PRC2 components, and cigarette smoke extract on human bronchial epithelial cells in air-liquid interphase cultures to examine how perturbation of PRC2 changes lung cell fate decisions. I will also establish new cultures from COPD patients, and finally explore the transcriptional changes in these cultures and how PRC2 influences these changes by chromatin immunoprecipitation experiments. Career Potential My ultimate goal is to influence national and global health policy by effectively investigating the physiological changes that are currently taking and have taken place as a result of environmental changes. My personal interest in stem cell biology a...