CDKN2B as a Novel Epigenetically Regulated Gene in Idiopathic Pulmonary Fibrosis

University Of Michigan At Ann Arbor, Ann Arbor MI

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Abstract

? DESCRIPTION (provided by applicant): Although the etiology of idiopathic pulmonary fibrosis (IPF) is unknown and may involve diverse mechanisms, excessive fibroproliferation remains a central hallmark in IPF pathology. Among the many cell types that play critical roles in disease pathogenesis, fibroblasts are ultimately the effector cell of fibrosis. Fibroblasts from IPF patiens have been shown by multiple investigators to exhibit a profibrotic in vitro phenotype; these observations were often made even when cells were cultured after multiple cell passages, suggesting that epigenetic modifications may be responsible for these phenotypic changes. Through a global survey of DNA methylation patterns in IPF fibroblasts, CDKN2B was discovered to be one of the most differentially methylated genes in IPF fibroblasts. Although never previously investigated in the context of IPF, CDKN2B has been shown to be highly differentially methylated in many cancers and to regulate cell proliferation and cell cycle progression in tumors. Our objectives are to determine the mechanism by which hypermethylation and diminished CDKN2B expression contributes to the activated phenotype of IPF cells, and to understand how its expression is regulated by DNA methylation, and how methylation may be dictated by the expression and actions of a long noncoding RNA (lncRNA). Our central hypothesis is that hypermethylation of CDKN2B, driven by the lncRNA CDKN2B-antisense (AS) 1, contributes to the diminished expression of CDKN2B in IPF fibroblasts, which leads to increased myofibroblast differentiation and apoptosis resistance. This project has three specific aims: 1) Determine how diminished expression of CDKN2B contributes to a profibrotic phenotype in IPF fibroblasts; 2) Determine how DNA methylation modulates the expression of CDKN2B; and 3) Elucidate the mechanisms by which CDKN2B-AS1 contributes to the DNA hypermethylation of CDKN2B in IPF fibroblasts. The biological actions of CDKN2B in pulmonary fibrosis will be explored by examining primary fibroblasts from normal lung and patients with IPF and by mouse modeling experiments. DNA methylation analysis will be performed, and the role of lncRNAs in regulating the DNA methylation of CDKN2B will be examined. This project is innovative because it seeks to understand the function, and additionally the epigenetic regulation, of a gene that may be critical to fibroproliferation, but has never been previously investigated in IPF. Completion of these studies is expected to establish the biological importance of CDKN2B to IPF fibroblast biology, and elucidate mechanisms that regulate its expression and contribute to how differential DNA methylation patterns are established. These findings will provide not only a novel gene target for future therapy, but also advance our understanding of how DNA methylation changes arise and contribute to gene expression differences in IPF.