PROJECT SUMMARY Pulmonary fibrosis is a chronic and intractable disease with a 5-year survival rate comparable to pancreatic or lung cancers. Deterioration of respiratory function in pulmonary fibrosis is caused by progressive replacement of normal tissue for gas exchange to dense fibrotic scar with fibrillar collagens. Pathologic fibroblasts accumulate at the sites of fibrogenesis and work as effector cells for excessive collagen deposition. Development of therapeutic strategies for targeting pathologic fibroblasts is hindered by the lack of understanding to cellular lineage and molecular detail of pathologic fibroblasts. In our previous study, we performed single-cell RNA-sequencing of normal and fibrotic lungs of mouse and human with a specialized protocol to identify all collagen-producing cells. We identified several fibroblast subsets that localize in different compartments of the lung. One of the fibroblast subsets emerge in fibrotic lungs of both mouse and human and show the highest levels of collagen gene expression and enhanced migratory capacity. These fibroblasts are characterized by specific expression of Cthrc1 (collagen triple helix repeat containing 1) and localized within fibroblastic foci of idiopathic pulmonary fibrosis, suggesting their pathologic role in pulmonary fibrosis. We recently generated and validated a novel mouse strain, Cthrc1-CreER, which allows us to specifically manipulate the pathologic fibroblast population in pulmonary fibrosis. The goal of this K99/R00 proposal is to elucidate the role and transcriptional regulations of pathologic fibroblasts in pulmonary fibrosis by using our innovative murine tools. Aim 1 (K99 phase) will reveal the role and fate of pathologic fibroblasts by ablating Cthrc1+ cells or lineage-tracing Cthrc1-CreER-labeled cells over the course of bleomycin-induced pulmonary fibrosis. Aim 2 (K99 phase) will reveal the transcriptional and epigenetic landscape of pathologic fibroblasts by performing RNA-seq, ChIP-seq, and ATAC-seq of purified Cthrc1-CreER-labeled cells at multiple time points of pulmonary fibrosis to seek master regulators for activation and deactivation. We will also seek the transcriptional regulations of CTHRC1+ cells in human pulmonary fibrosis. Aim 3 (R00 phase) will demonstrate the role of genes regulating pathologic fibroblasts by using intratracheal adoptive transfer of fibroblasts with lentiviral gene modifications and by conditionally knocking out candidate genes in fibroblasts in pulmonary fibrosis. These studies using the murine genetic tool highly specific for pathologic fibroblasts will shed light on cellular function and transcriptional regulations of pathologic fibroblasts in pulmonary fibrosis. This proposal is also designed to provide the candidate with training opportunity to obtain skill sets for murine genetic approach in search of therapeutic targets and functional genomics approach integrating RNA-seq, ChIP-seq, and ATAC- seq. The success of this project ...