PROJECT SUMMARY Using single-cell RNA-sequencing (scRNA-seq), we and others have demonstrated dramatic changes in cellular identities, frequencies, and molecular programs that characterize late-stage pulmonary fibrosis (PF). These advances have led to new questions regarding the timing, coordination, transcriptional regulatory mechanisms, and impact of genetic variation on the evolution of the diverse cellular pathology seen in advanced PF lungs. Utilizing transbronchial biopsy samples from subjects in our At-Risk for Familial PF (FPF) cohort, we are now uniquely positioned to investigate these mechanisms through the critical phase of early disease pathogenesis. Pilot studies in which we performed single-nucleus multiomic (RNA+ATAC)-sequencing of archival flash frozen biopsies from At-Risk for FPF subjects suggest that changes in cell-type specific gene expression (including alveolar niche factors) and chromatin accessibility (including key regulators of alveolar epithelial cell fate/identity) are detectable and may develop prior to the appearance of interstitial lung abnormalities or overt pulmonary fibrosis. Additional preliminary data reveal that a subset of genetic variants that regulate gene expression (single-cell expression quantitative trait loci, sc-eQTL) exhibit significant differences in effect size and/or direction in PF lungs compared to controls - we term these “disease-interacting sc-eQTL.” In the distal lung epithelium, disease-interacting sc-eQTL are specifically enriched within the binding motifs for a set of stress-induced transcription factors which also regulate top differentially expressed genes in KRT5-/KRT17+ basal-like cells in PF lungs. This remarkable convergence suggests that genetic variation at cell-type specific loci modulating activity of a set of stress-induced transcription factors regulates alveolar epithelial repair. We hypothesize that activation of injury-response transcriptional programs in the distal lung epithelium unmasks disease-interacting expression-quantitative-trait loci (eQTLs) to mediate dysfunctional epithelial repair and promote initiation and early progression of FPF. Our specific aims are: 1) To define the single-cell molecular programs of early interstitial lung abnormalities, 2) To determine the cell-type-specific transcriptional regulatory mechanisms through which genetic risk for FPF mediates early ILA development and progression, and 3) To examine the mechanisms through which PF-associated genetic variation regulates distal airway and alveolar epithelial repair. Integrating single-cell multiomics of pre-disease lung biopsy samples linked to future PF progression outcomes, genetic variation and patient-derived organoid models, we will investigate the mechanisms through which genetic variation interacts with injury-response transcriptional regulation to drive the early pathogenesis of PF.