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

• 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
• 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

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.

项目摘要 成纤维细胞是特发性肺纤维化（IPF）的最终效应细胞，一种进行性肺部疾病 其特征是过量的细胞外基质产生和副疤痕。与来自 来自IPF肺的非纤维化肺，成纤维细胞和成肌纤维细胞（统称为间充质细胞） 患者具有改变的表型，被认为可以促进和恶化的肺纤维化。这个“促纤维化” 表型部分归因于许多基因的表达改变，但是调节这些基因的机制 基因并不完全知道。最近的研究表明，间充质细胞也是 其基因表达谱的异质。 DNA甲基化的改变是一种表观遗传机理 我们和其他人已经显示出IPF和正常肺之间的某些差异 间充质细胞，但全球甲基分析受到限制，其潜在的异质性 从未表征甲基概况。此外，调节DNA甲基团的机制， 尤其是在肺纤维化的背景下，没有充分理解，并且在知识中仍然是基本差距。 该赠款的目标是评估间充质细胞DNA甲基概况的异质性 在IPF肺中，确定它们如何促进不同基因表达，并确定调节的因素 并调节这些细胞中的DNA甲基化。我们将通过全面定义单一来实现这一目标 细胞水平IPF间充质细胞的DNA甲基甲基团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），十个易位（TET）酶的作用，甲基化 负责“脱甲基化”的甲基化和uHrf1是DNA甲基化的主要调节剂。我们的中心 假设IPF间充质细胞表现出异质的DNA甲基化模式 在有助于基因表达中的异质性方面的重要作用；这些DNA甲基团模式是形状的 通过DNMT，TET和UHRF1的动作。我们将以三个特定目的检验该假设：1）评估 IPF间充质细胞中的DNA甲基甲基异质性及其与染色质构象的关系 基因表达。我们将通过首次进行单细胞全基因组Bisulfite测序来做到这一点 和激光捕获微分解。 2）确定间充质细胞中的DNA甲基化如何受到 TGF-β1，刚度和ECM，假设它通过上调DNMT1、3A和TET2来做到这一点。 3）确定UHRF1及其与组蛋白甲基化的相互作用有助于建立特定的DNA模式 Messymal细胞中的甲基化。这些目标的完成将获得对机制的见解 调节DNA甲基化并使用表观遗传学晶状体，使我们能够剖析有助于的因素 IPF肺中的间充质细胞异质性。最终，这些研究将提高我们对如何的理解 DNA甲基化的改变有助于IPF发病机理，并作为靶向治疗的一种手段。