PROJECT SUMMARY PRDMs regulate chromatin accessibility to determine alveolar maturation. Pulmonary surfactant is a complex mixture of lipids and proteins produced by AT2 (Alveolar Type 2 Cells) which is secreted into alveolar spaces to reduce surface tension and prevent alveolar collapse during the ventilatory cycle. Lack of pulmonary surfactant underlies the pathogenesis of neonatal respiratory distress syndrome (RDS) in preterm infants and contributes to the pathogenesis of acute lung injury (ARDS) in adults. The fetal lung undergoes dramatic changes in architecture, cell differentiation and gene expression in preparation for birth, increasing surfactant lipid and protein production required for postnatal lung function. While our laboratory and others have identified genes, transcriptional regulators, and gene regulatory networks controlling surfactant homeostasis, how chromatin accessibility is modulated to enable gene transcription necessary for AT1 and AT2 cell maturation and function remains relatively unexplained. Lacking are data regarding the molecular mechanisms controlling chromatin accessibility required for the activation of critical transcription factors and their targets before birth and during alveolar repair. The present application is based on our recent identification of the primary roles of PRDM3 and PRDM16 in regulation of genes critical for alveolar maturation, remodeling, and gene expression required for postnatal survival. PRDM3/16 are zinc finger, proline-rich domain-containing transcription factors with histone modifying activity regulating chromatin accessibility throughout the genome. We have shown that deletion of Prdm3/16 in fetal respiratory epithelial cells caused respiratory failure at birth, dramatically impairing surfactant protein production. We will use ATAC-Seq, Cut and Run, and RNA-Seq to identify gene loci regulated by PRDM3/16 to infer mechanisms by which PRDM proteins interact with AT2 specific genes before birth. In this application, we will test the hypothesis that PRDM3 and PRDM16 interact in transcriptional complexes regulating recruitment of histones and chromatin accessibility controlling AT2 cell differentiation and function in late gestation and during regeneration following viral-induced lung injury in adult mice. We will identify and test the functions of binding partners and transcriptional complexes mediating PRDM3/16 activity. The gene regulatory networks, physiologic and biochemical roles of PRDM3/16 on AT2 cells, alveolar structure and function will be identified in vivo and in vitro. The proposed studies represent conceptual advances regarding the molecular control of alveolar epithelial cell differentiation and function and will provide a framework for the development of future strategies to modify epigenetic landscapes controlling AT2/AT1 cell function in health and disease. Rev: 9-3-21