PROJECT SUMMARY Fibroblasts are the final effector cells of idiopathic pulmonary fibrosis (IPF), a progressive lung disease characterized by excessive extracellular matrix production and parenchymal scarring. Compared to those from non-fibrotic lung, fibroblasts and myofibroblasts (collectively termed mesenchymal cells) from the lungs of IPF patients possess an altered phenotype that is felt to promote and worsen lung fibrosis. This “pro-fibrotic” phenotype is partly attributed to the altered expression of many genes, but the mechanisms that regulate these genes are not completely known. Recent studies have demonstrated that mesenchymal cells are also heterogeneous in their gene expression profiles. Alterations in DNA methylation is one epigenetic mechanism that we and others have shown accounts for some of the differences between IPF and normal lung mesenchymal cells, but global methylomic analysis has been limited and the potential heterogeneity in their methylomic profiles has never been characterized. Further, the mechanisms that regulate the DNA methylome, especially in the context of lung fibrosis, are not well understood and remain fundamental gaps in knowledge. The objectives of this grant are to assess the heterogeneity of DNA methylomic profiles of mesenchymal cells in the IPF lung, determine how they contribute to varying gene expression, and identify factors that regulate and modulate DNA methylation in these cells. We will achieve this by comprehensively defining at the single- cell level the DNA methylome of IPF mesenchymal cells, determine its relationship to chromatin conformation and gene expression, and identify how microenvironmental conditions such as stiffness and matrix affect methylation through the actions of DNA methyltansferases (DNMTs), ten-eleven translocation (TET) enzymes, which are responsible for “de-“methylation, and UHRF1, a master regulator of DNA methylation. Our central hypothesis is that IPF mesenchymal cells demonstrate heterogeneous patterns of DNA methylation that play a significant role in contributing to heterogeneity in gene expression; these DNA methylomic patterns are shaped by the actions of DNMTs, TETs, and UHRF1. We will test this hypothesis with three Specific Aims: 1) Assess DNA methylomic heterogeneity in IPF mesenchymal cells and its relationship with chromatin conformation and gene expression. We will do this by performing for the first time single-cell whole genome bisulfite sequencing and laser capture microdissection. 2) Determine how DNA methylation in mesenchymal cells is affected by TGF-β1, stiffness and ECM, with the hypothesis that it does so through upregulation of DNMT1, 3a, and TET2. 3) Determine how UHRF1 and its interaction with histone methylation helps establish specific patterns of DNA methylation in mesenchymal cells. Completion of these aims will gain insights into the mechanisms that regulate DNA methylation and by using an epigenetic lens, allow us to dissect the factors that contribute mesenchymal cell heterogeneity in IPF lung. Ultimately, these studies will advance our understanding of how alterations in DNA methylation contribute to IPF pathogenesis and serve as a means of targeted therapy.

PROJECT NARRATIVE Alterations in DNA methylation, particularly within mesenchymal cells, plays a key role in the pathogenesis of idiopathic pulmonary fibrosis (IPF), but a complete analysis of the global methylome and how it varies among different mesenchymal cell populations has not been examined. This proposal will examine the heterogeneity of DNA methylomic patterns within IPF mesenchymal cells, investigate how methylation influences gene expression, and determine how the methylome is regulated by the actions of various methylation machinery. These findings will provide insight into the importance of DNA methylation in IPF and how methylation changes may be targeted for future therapy.