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.

项目概要成纤维细胞是特发性肺纤维化（IPF）（一种进行性肺部疾病）的最终效应细胞与来自的那些相比，其特征是细胞外基质产生过多和实质疤痕。来自 IPF 肺部的非纤维化肺、成纤维细胞和肌成纤维细胞（统称为间充质细胞）患者具有改变的表型，被认为会促进和恶化肺纤维化，这种“促纤维化”。表型部分归因于许多基因的表达，但调节这些基因的机制最近的研究表明，间充质细胞也是如此。 DNA甲基化的改变是一种表观遗传机制。我们和其他人已经证明了 IPF 和正常肺之间的一些差异间充质细胞，但整体甲基组学分析有限，并且它们的潜在异质性此外，调节 DNA 甲基化组的机制尚未得到表征。特别是在肺纤维化的背景下，人们对这些问题还没有很好的理解，并且仍然存在根本性的知识差距。该资助的目的是评估间充质细胞 DNA 甲基组谱的异质性在 IPF 肺中，确定它们如何影响不同的基因表达，并确定调节因素并调节这些细胞中的 DNA 甲基化，我们将通过在单一方面进行全面定义来实现这一目标。细胞水平IPF间充质细胞的DNA甲基化组，确定其与染色质构象的关系和基因表达，并确定微环境条件（例如刚度和基质）如何影响通过 DNA 甲基转移酶 (DNMT)、10-11 易位 (TET) 酶的作用进行甲基化， UHRF1 负责“去”甲基化，UHRF1 是 DNA 甲基化的主要调节因子。假设 IPF 间充质细胞表现出 DNA 甲基化的异质模式，这些模式发挥着这些 DNA 甲基组模式在基因表达异质性中发挥着重要作用；通过 DNMT、TET 和 UHRF1 的行动，我们将通过三个具体目标来检验这一假设：1) 评估。 IPF间充质细胞DNA甲基组异质性及其与染色质构象和结构的关系我们将通过首次进行单细胞全基因组亚硫酸氢盐测序来实现这一点。和激光捕获显微切割 2) 确定间充质细胞中的 DNA 甲基化如何受到影响。 TGF-β1、硬度和 ECM，假设它是通过上调 DNMT1、3a 和 TET2 来实现的。 3) 确定 UHRF1 及其与组蛋白甲基化的相互作用如何帮助建立 DNA 的特定模式完成这些目标将深入了解间充质细胞的甲基化机制。调节 DNA 甲基化，并通过使用表观遗传透镜，让我们能够剖析影响 DNA 甲基化的因素最终，这些研究将增进我们对 IPF 肺间充质细胞异质性的理解。 DNA 甲基化的改变有助于 IPF 发病机制，并可作为靶向治疗的一种手段。