EZH2, the functional enzymatic component of the Polycomb Repressive Complex 2 (PRC2), is a histone methyl transferase that tri-methylates histone H3 at lysine 27 (H3K27me3). This protein plays a significant role in maintaining tissue specificity by silencing gene programs that control lineage fate. We hypothesized that PRC2 controls cell fate of the lung stem cells. In order to test this hypothesis, we generated mice in which we can conditionally delete Ezh2 upon ingestion of doxycycline. To examine growth, differentiation and self-renewal potentials of different lung epithelial cells, we FACS-isolated distal lung bronchioalveolar stem cells (BASCs) and cultured them in air-liquid-interface Matrigel cultures. We observed a 2fold decrease in organoid number of EZH2 null vs wild type bronchiolar cells and a significant difference in organoid diameter between Ezh2 wild type, heterozygous and null bronchiolar cells. To test in vivo potential of EZH2 depleted epithelium to repair, we performed naphthalene injury, which specifically injures the bronchiolar club cells. We observed that 7 days post naphthalene injury, Ezh2 knock-out lungs had significantly less repair, as measured by repopulation of bronchiolar lung cells. Given that we observed that lung stem cells were impaired when PRC2-mediated gene repression was perturbed, we sought to learn if PRC2 is also dysregulated in Chronic Obstructive Pulmonary Disease (COPD). We first performed Gene Set Enrichment Analysis and found that genes up-regulated in COPD patients relative to healthy smokers were also up-regulated in Ezh2 knock-out adenocarcinoma cells. We next obtained lung tissue from healthy patients and patients with COPD, and performed immunohistochemistry for the PRC2 mark H3K27me3 and immunofluorescence for basal and club cells. There was a significant decrease in H3K27me3, coupled with an increase in basal cells and goblet cells that contained high expressions of club cell markers in COPD lung compared to healthy controls. Finally, to determine if loss of PRC2 function drives basal cell fate and goblet cell differentiation, EZH2 transcripts were knocked down with short hairpins in human bronchiolar epithelial cells, and basal cell differentiation capacity was analyzed. We observed an increased expression of a goblet cell marker and decreased expression of a club cell marker in EZH2 knockdown cultures. Together our results indicate that PRC2 plays an important role in lung stem cell function as well as club cell differentiation fate. These mechanisms may also be perturbed in COPD patients. ChIP-seq and RNA-seq studies to understand the transcriptional and epigenetic changes leading to these phenotypes are underway Support or Funding Information NIH grant K22 CA 20103, Molecular Mechanisms of Toxicity Training Grant T32ES07266 There was a 2-fold decrease in organoid number of EZH2 null vs wild type bronchiolar cells. There was a significant difference in organoid diameter between Ezh2 wild type, heterozygous and null bronchiolar cells. Immunohistochemistry staining of EZH2 shows distinct differences in EZH2 expression between wild type, heterozygous and null organoids. Naphthalene exposure to mouse lung has been shown to kill club cells in the trachea and bronchioles. This injury typically results in club cell regeneration and subsequent lung cell repopulation. Club cells without EZH2 were unable to regenerate and repopulate the airway after naphthalene injury. Club cells that did regenerate in the Ezh2-null lungs lacked the GFP tag, indicating that these cells were likely not responding to doxycycline to delete Ezh2. Gene Set Enrichment Analysis was used to compare gene expression between murine Ezh2-deleted adenocarcinoma cells and COPD patients. It revealed that a significant number of the same genes are highly expressed in both states when compared to healthy lungs or the Ezh2 WT setting. Immunohistochemistry shows significantly lower abundance of H3K27me3 in the nuclei of COPD bronchiolar cells compared to normal. Immunofluorescence staining of club (CCSP and SOX2) and basal cells (KRT5 and SOX2) in normal vs COPD lung airway shows a mixture of metaplasia and hyperplasia along with collapsed airways in COPD airway compared to normal. There is an increase in basal cells and goblet cells that also contain high expressions of CCSP (a club cell marker). EZH2 transcripts were knocked down in immortalized, Human Bronchiolar Epithelial Cells (HBEC) which were, then, prompted to differentiate in air-liquid-interface cultures. Acetylated-Mucin5 was highly expressed while club cell secretory protein (CCSP) was lowly expressed in EZH2 knockdown cells compared to wild type. This suggest that EZH2 plays a role in club and goblet cell differentiation. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.