TET1-mediated 5-hydroxymethylcytosine modification & airway hyperresponsiveness

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 DESCRIPTION (provided by applicant): Asthma is a complex disease characterized by airway hyperresponsiveness (AHR) and airway inflammation, which current afflicts over 300 million people worldwide. Although genetic factors unquestionably play a role in asthma, the rapid rise in asthma prevalence suggests that environmental factors likely play an equally important role. To date, epigenetic regulation is suggested to mediate, at least partly, the complex gene-by- environment interactions that can lead to asthma. Most studies to date have focused on elucidation of DNA methylation patterns in easily accessible cells including T cells and B cells to explain the immunological mechanisms driving allergen sensitization in asthmatics. Few studies have examined the epigenetic consequences of environmental triggers on lung tissues linked to chronic alterations in lung functions. We have utilized next generation sequencing techniques and proper validation assays to identify a novel set of genes which is highly enriched for Tgfb2 signaling molecules and associated with exposure to the common allergen, house dust mite (HDM), and the development of AHR through epigenetic modulation of the airway smooth muscle (ASM) cell phenotypes. In addition, we identified a novel role of the specific 5-mC dioxygenase, TET1, in the regulation of ASM function in vitro and in the development of allergen-driven AHR in vivo. Preliminarily, we demonstrated Tet1 deficiency reduced acute HDM-driven AHR in mice. Strikingly, we were able to translate the epigenetic changes in human asthmatic ASM cells; showing Tet1-mediated hydroxymethylation may influence ASM cell proliferation and contraction. Our study represents the first demonstration that Tet1 protein is regulated in the context of allergen exposure, although the mechanisms regulating Tet1 induction by allergens are unknown. Our preliminary data supported that oxidative stress generated by HDM exposure activates Tet1 and its mediated upregulation of ASM genes. Based on these novel findings, we propose the central hypothesis "HDM activates Tet1-mediated hydroxymethylation in ASM cells through regulation of redox cycling. This epigenetic reprogramming persists in ASM over long time spans, thus likely contributes to the increased ASM cell proliferation, stiffness and contractility seen in human asthma". To address these novel hypotheses, we have assembled a multi-disciplinary team of investigators with a breadth of expertise spanning the fields of epigenetics, ASM biology, pulmonology and pathobiology. First, we will determine the role of Tet1- mediated DNA hydroxymethylation in the development of the AHR phenotype in a chronic HDM-induced AHR mouse model. Second, we will investigate how HDM alters the Tet1 activity, through redox cycling of NADH in both in vitro and in vivo mouse model. Lastly, we will confirm if TET1-mediated hydroxymethylation regulates human ASM cell phenotypes. The results of the proposed experiments should provide novel perspectives on the ASM and its contributions to the excessive airway narrowing in asthma. These insights may fuel the development of new therapeutic approaches for the treatment of this debilitating disease.