ELUCIDATING THE ROLE OF POLYCOMB REPRESSIVE COMPLEX 2 IN LUNG STEM CELL FATE AND LUNG DISEASE

The average human lungs take ~22,000 breaths and inhale ~2,000 gallons of air each day. This organ is the primary conduit for the transfer of oxygen to the internal organs, making it crucial for the sustainment of life. Consequently, mechanical malfunction of the lungs and/or pollution of inhaled air negatively impact internal organ function and ultimately put survival in jeopardy. Epigenetics, by nature, is a plastic phenomenon in which environmental stimuli influence short term, long term, and generational gene expression patterns. Chronic exposure of harmful stimuli to the lung epithelium has also been shown to alter epigenetic pathways, engender aberrant lung cell differentiation, and manifest into long term diseases. Chronic Obstructive Pulmonary Disease (COPD) is a progressive, incurable lung disorder that is pathologically rooted in chronic inflammation and aberrant lung cell differentiation. Polycomb Repressive Complex 2 (PRC2) is a key epigenetic regulator of lung stem cell fate during development, but little is understood of its role in adult lung. In the following chapters, I will expound on how I utilized human patient samples, ex vivo lung organoids, and murine in vivo models to better elucidate how PRC2 controls lung cell biology and offer insight into potential therapeutic avenues to combat lung disease. To understand the role of PRC2 in COPD, we first analyzed patient and control lung tissues. Using quantitative immunohistochemistry and immunofluorescence, we observed a very significant decrease in the Polycomb Repressive Complex 2 catalytic mark, histone H3 lysine 27 tri-methylation (H3K27me3) in bronchiolar epithelium of COPD patients. Furthermore, H3K27me3 staining was strongly inversely correlated with markers of basal and goblet cells in patient samples. Next, we developed a new mouse model of conditional deletion of the Polycomb Repressive Complex 2 enzyme, EZH2, and used this model to interrogate lung stem cell function in organoid cultures and in vivo. Single cell RNA-sequencing of organoid cultures revealed the appearance of Krt17-negative basal cells and loss of the newly identified Krt8-positive progenitor cells in Ezh2-null cultures. Gene signatures associated with immune response were increased is Ezh2-heterozygous cultures, and genes enriched in EZH2 deficient organoids were enriched in human emphysemic lung. Furthermore, when we used this Ezh2 conditional knock-out mouse to interrogate the role of EZH2 in lung homeostasis and allergen response in vivo, we found that Ezh2-heterozygous mice had increased response to ovalbumin allergen and showed hallmarks of COPD including bronchiolar epithelial thickening and club to goblet cell transdifferentiation. Lastly, we sought to learn the mechanism of decreased PRC2 activity in COPD patients. We used human bronchiolar epithelial cells to interrogate the role of the redox-sensitive enzyme, cystathionine beta synthase (CBS), in controlling EZH2 levels and gene expression modulation. In the patient samples, we found that CBS was significantly higher in COPD lung. Excitingly, we identified shared transcriptional profiles between human bronchiolar epithelial cells that over-express CBS and mouse organoids with Ezh2 loss, offering insight into how redox stress may drive aberrant epigenetic reprogramming. Taken together, these findings suggests that PRC2 is integral to facilitating proper lung stem cell differentiation in adult humans and mice.