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Heterogeneity and Regulation of the DNA Methylome in IPF Mesenchymal Cells

IPF 间充质细胞 DNA 甲基化的异质性和调控

基本信息

批准号：
10584069
负责人：
STEVEN K HUANG
金额：
$ 77.5万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-02-01 至 2026-11-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10584069
关键词：
Affect Area Binding Biological Assay Cell division Cells Chromatin Cicatrix DNA DNA Methylation DNA Modification Methylases Data Disease Disease Progression Effector Cell Environmental Risk Factor Enzymes Epigenetic Process Extracellular Matrix Fibroblasts Future Gene Expression Gene Expression Profile Genes Genome Grant Heterogeneity Histologic Histones Individual Knock-out Knowledge Lung Lung diseases Lysine Mediating Mesenchymal Methylation Molecular Conformation Myofibroblast Nature Pathogenesis Pathologic Patients Pattern Phenotype Play Population Production Pulmonary Fibrosis Regional Anatomy Regulation Role Shapes Site Testing Time Transposase Up-Regulation Variant bisulfite sequencing demethylation histone methylation idiopathic pulmonary fibrosis in vitro Model in vivo insight laser capture microdissection lens methylome methylomics overexpression recruit single-cell RNA sequencing targeted treatment whole genome

项目摘要

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.

项目总结