Grants and Contracts Details
Bronchiolar dysplasia in COPD is governed by loss of Polycomb Repressive Complex 2 1. SPECIFIC AIMS Rationale: Chronic Obstructive Pulmonary Disease (COPD) is a progressive, lethal disease that hinders respiration at the lung’s conducting and respiratory zones. This disease is characterized by chronic bronchitis, emphysema, excessive airway mucus deposition, and profuse inflammatory cell infiltration into the lung airway and alveoli. Given these deleterious effects, it is important to elucidate the molecular mechanisms that drive COPD. However, while a number of exogenous and endogenous factors have been implicated in contributing to this disease, the epigenetic contributions are poorly understood. In the Brainson laboratory, we are investigating the role of Polycomb Repressive Complex 2 (PRC2) in the development and progression of lung disease. PRC2 is a multimeric, epigenetic enzyme that maintains cellular specificity by repressing unassociated genes. EZH2 is the subunit within this complex that tri-methylates histone H3 at lysine 27 (H3K27me3), marking DNA for transcriptional silencing and allowing expression of a single transcriptional profile. We recently fully knocked out Ezh2 in mice and observed a COPD-like phenotype in the airway. We also measured H3K27me3 in COPD patient lung cross-sections, and observed a significant decrease compared to normal human lung. Additionally, we conducted immunofluorescence staining on the COPD lung and observed a high number of KRT5+ basal cells and dually-expressing MUC5ac (goblet cell marker) and CCSP (club cell marker) cells in the lung airway. Our Central Hypothesis is that loss of EZH2-mediated gene repression reprograms lung bronchiolar epithelium from club cell fate to globlet and basal cell fates, and that this improper differentiation contributes to COPD. We will test this hypothesis using both mouse models and human cells. Aim 1: Characterize murine bronchiolar lung cell phenotypes in vitro and in vivo after Ezh2 ablation a) Examine the phenotype associated with Short-Term (in vitro) and Long-Term (in vivo) Ezh2 deletion in bronchiolar epithelium b) Perform RNA-seq of mouse bronchiolar epithelial cells with and without Ezh2 deletion For this Aim, a genetically engineered mouse model in which deletion of Ezh2 can be induced through doxycycline administration will be used. The effects of Ezh2 deletion on the differentiation potentials of lung epithelial cells will be tested in vitro using organotypic 3-dimensional cultures and in vivo through long-term doxycycline administration (4 months). Histology and immunostaining will be used to characterize the changes in lung epithelial differentiation, which will be quantified using the Nikon NIS software. RNA-sequencing on bronchiolar organoids that are Ezh2 wild-type, Ezh2 heterozygous and Ezh2 null will be used to understand the transcriptional states that are changed in response to EZH2 loss. Aim 2: Examine if EZH2 knock-down changes human bronchiolar epithelial cells to become more basal cell-like or more goblet cell-like a) Examine stem cell fate in Human Bronchial Epithelial Cell (HBEC) that are wild-type, knocked-down, or rescued for EZH2 expression b) Perform RNA-seq on HBEC cultures with and without EZH2 depletion, compare mouse to human differentially expressed genes For this Aim, human immortalized bronchiolar epithelial cells will be used. Differentiation will be induced in Air-Liquid-Interface (ALI) cultures after knock-down, or knock-down and rescue, of EZH2 expression. Histology and immunostaining will be used to characterize the changes in lung epithelial differentiation, which will be quantified using the Nikon NIS software. RNA-sequencing on both un-differentiated and differentiated cultures that are wild-type, knocked-down, or rescued for EZH2 expression will be used to understand the transcriptional states that are changed in response to EZH2 loss. Transcriptional changes will be compared to published COPD signatures. Single cell RNA sequencing will be conducted on fresh normal and COPD patient lung tissue before and after cell culture growth. These experiments are designed to produce quantitative data that will demonstrate in part how loss of EZH2 function, which is observed in human COPD lung epithelium, affects lung cell differentiation potentials. Both mouse and human cells will be used, and the transcriptional programs and histological similarities of both models will be compared. Though outside the scope of this proposal, future studies will examine upstream oxidative stress as a way that EZH2 activity is changed in COPD, and downstream epigenetic inhibitors that may be able to reprogram diseased epithelium back to a more normal bronchiolar state.
|Effective start/end date
|3/15/20 → 10/14/21
- National Heart Lung and Blood Institute: $23,605.00
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