PROJECT SUMMARY The alveolus is lined by two epithelial cell types: thin gas-exchanging alveolar type 1 (AT1) cells and cuboidal surfactant-producing AT2 cells. Both are selected from a common distal progenitor by FGF, Notch, and stretch signaling. Injury in the adult lung activates AT2 into a facultative progenitor state to regenerate lost AT1 and AT2 cells. While the inductive cues that select fate have been identified, it is less clear what role – if any – repressive cues play in fate maturation. Two gaps of understanding in the lung field are 1) when and how is stemness lost after fate selection and 2) once lost, how is stemness re-accessed by AT2s after injury to regenerate the alveolar epithelium. Our recent work indicates perinatal AT2s retain stemness for weeks after fate selection, despite being transcriptionally and functionally differentiated. This proposal focuses on a putative repressor of AT2 stemness, determining its target and modes of action, whether and how it is overridden in adult AT2s to re-access stemness, and investigating the extent to which it is dysfunctional in pulmonary fibrosis - wherein aberrant AT2 states are observed. Our first aim focuses on a putative repressor of stemness we identified, the CCAAT enhancer binding protein C/EBPα. Using transgenic mouse models, we will test the requirement of Cebpa for AT2 stemness and determine whether the targeted stemness program acts in a cell autonomous or non-autonomous manner by mosaic deletion. Next, scRNAseq, ATAC- and ChIP-seq will identify genes targeted by, as well as molecules interacting with, C/EBPα. Finally, we will use organotypic culture and transgenic mouse experiments to determine whether timing of Cebpa expression is itself regulated earlier in development by the polycomb repressive complex PRC2. The second aim of the proposal is to determine whether and how C/EBPα regulation is overridden following injury to re-access AT2 stemness and promote repair. We will determine whether C/EBPα downregulation and AT2 stemness is dependent on AT1 cell death in vivo by performing genetically targeted cell type specific ablation. Finally, we will compare the spatiotemporal program of C/EBPα regulation we identify during injury and repair to the fibrotic lung and investigate whether it is dysfunctional in its associated aberrant AT2 cells. Our approach is innovative, as no transgenic mouse experiments using mosaic deletion or lineage tracing have been performed for Cebpa in the lung, nor have precise cell ablation studies been conducted in the adult lung to study C/EBPα regulation. Further, the observation of retained stemness in perinatal AT2 cells is novel and thus no investigation has implemented a multi-omics approach to understand its underlying mechanism. Finally, little is known on the role repressive cues play in AT2 cells differentiation and maturation. If successful, we will have uncovered a novel mechanism of AT2 stemness regulation for the lung field which will b...