Heterogeneity and Regulation of the DNA Methylome in IPF Mesenchymal Cells

项目总结 成纤维细胞是进行性肺部疾病特发性肺纤维化(IPF)的最终效应细胞 以过度产生细胞外基质和实质瘢痕为特征。与来自 来自IPF肺的非纤维化肺、成纤维细胞和肌成纤维细胞(统称间充质细胞) 患者有一种表型改变，这种表型被认为会促进和恶化肺纤维化。这种“促肝纤维化” 表型部分归因于许多基因表达的改变，但调节这些基因的机制 基因并不是完全已知的。最近的研究表明，间充质细胞也是 他们的基因表达谱具有异质性。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，但全球甲基组分析有限，其潜在的异质性 甲基组图谱从未被描述过。此外，调节DNA甲基组的机制， 尤其是在肺纤维化的背景下，这些问题没有得到很好的理解，仍然是知识上的根本差距。 这项资助的目的是评估间充质细胞DNA甲基组图谱的异质性。 在IPF肺中，确定它们如何对不同的基因表达做出贡献，并确定调节因素 并调节这些细胞中的DNA甲基化。我们将通过在单一的- 细胞水平IPF间充质细胞DNA甲基组与染色质构象的关系 和基因表达，并确定诸如僵硬和基质等微环境条件如何影响 通过DNA甲基转移酶(DNMT)、十-十一易位(TET)酶、 它们负责“去”甲基化，以及DNA甲基化的主要调节者uhrf1。我们的中央 假说是IPF间充质细胞表现出不同的DNA甲基化模式，这种模式在 在基因表达的异质性中起重要作用；这些DNA甲基组模式是 通过DNMTS、TETS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 特发性骨髓间充质细胞DNA甲基组异质性及其与染色质构象和基因表达的关系 基因表达。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 和激光捕获显微解剖。2)确定骨髓间充质细胞中DNA甲基化是如何受 转化生长因子-β1，僵硬和细胞外基质，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特定的dna模式。 间充质细胞中的甲基化。这些目标的完成将使我们深入了解 调节DNA甲基化，并通过使用表观遗传透镜，允许我们剖析影响因素 特发性肺间质细胞的异质性。最终，这些研究将促进我们对 DNA甲基化的改变有助于IPF的发病，并可作为靶向治疗的一种手段。

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.