JUNB O-GlcNAcylation-mediated Promoter Accessibility of Metabolic Genes Modulates Distinct Epithelial Lineage in Pulmonary Fibrosis [ipf_saec_bulk]

Harnessing a 3D patient derived organoid model and multi-omics approach, we provide the first inventory of the connection between metabolic alteration, chromatin accessibility, and transcriptional regulation in IPF aberrant epithelial remodeling. This remodeling is characterized by an increase in chromatin accessibility, particularly at JUNB motif-enriched promoter regions proximal to transcription start sites of metabolic and pro-fibrotic genes. Mechanistically, JUNB undergoes O-linked ß-N-acetylglucosamine modification (O-GlcNAcylation), a critical step in modulating pro-fibrotic responses to chronic injury. This modification is pivotal in fostering the emergence of aberrant epithelial basal cells in the alveolar niche, a proposed driver of IPF pathology. Our findings reveal a novel link between metabolic dysregulation and cell fate regulation at the chromatin level in fibrosis, mediated by the O-GlcNAc-JUNB axis. Overall design: To investigate the role of metabolic dysregulation in IPF basal cells, we utilized a 3D organoid system, derived from lung tissue samples obtained from IPF patients and healthy, control resections from tumor explants. We isolated EPCAM+ distal airway epithelial cells through tissue dissection and multiple sorting steps to obtain a pure epithelial cell fraction. The harvested cells were cultured in Cultrex basement membrane extract to promote the formation and growth of distal airway organoids. 6 of the 12 AOs were additionally treated with a pro-fibrotic stimuli in form of a disease-relevant cocktail (IPFrc). Finally, we performed bulk RNA sequencing and ATAC sequencing on the IPF and control AOs (n = 12 across 4 conditions).