Project Summary Asthma is a complex disease characterized by airway hyperresponsiveness (AHR), which is expected to affect 400 million people worldwide by 2025. Airway smooth muscle (ASM) cells are the primary effectors of AHR, as they exaggerate the response to bronchoconstrictor stimuli and increase ASM thickness by depositing the extracellular matrix and inducing inflammation. Targeting epigenetic changes serves as a new approach to reversing the aberrant ASM phenotypes seen in asthmatics. We previously demonstrated that global DNA hydroxymethylation mediated by α-ketoglutarate (αKG)-dependent 5-mC dioxygenase (TET1) was induced in lung tissues from mice that showed increased allergen-induced AHR. In addition, we reported a novel role for mitochondrial-specific isocitrate dehydrogenase 2 (IDH2) on regulation of ASM phenotypic genes in human asthmatic ASM cells, through alterations in αKG level and αKG-dependent TET1 activity, suggesting a possible link between cell metabolism and epigenetic regulation of ASM cell function. Preliminarily, the allergen- induced AHR and aberrant DNA hydroxymethylation patterns was abolished by a mitochondrially targeted tetrapeptide, SS-31(elamipretide, which is currently in phase III clinical trials for treating metabolic diseases). Furthermore, we showed that the interaction between mitochondria and epigenome is bidirectional. We identified increased DNA hydroxymethylation of genes involved in mitochondrial replication and transcription, which was associated with the increased AHR. Our study represents the first demonstration that TET1- mediated DNA hydroxymethylation in ASM is regulated in the context of mitochondrial function, although the mechanisms by which mitochondrial function influences the epigenetic regulation of ASM cell function, and vice versa; have not been fully investigated. Based on these novel findings, we propose the central hypothesis “Modulation of mitochondrial redox cycling and bioenergetics reciprocates with the epigenetic modifications of the ASM cell function, and ultimately modifying asthma pathogenesis”. To address these novel hypotheses, we assemble a team of investigators with a breadth of expertise spanning the fields of epigenetics, redox biology and pulmonology. First, we will determine whether modulation of mitochondrial function has an epigenetic impact on allergen-induced AHR, which can be attenuated by SS-31. Second, we will study if epigenetic regulation of mitochondrial transcription modulates mitochondrial function, which has a long-term effect on the epigenome of the ASM cells and AHR phenotype. Finally, we will confirm the mitochondrial-epigenetic interplays in the determination of ASM function utilizing clinical samples. We will apply correlation analysis of genomic profiling and measurement of mitochondrial biology to identify sets of molecular markers associated with asthma severity. Our findings should provide new evidence about the mitochondrial-epigenetic crosstal...