Open chromatin and transcriptional regulation of dermal myofibroblasts in SSc

SSc 中真皮肌成纤维细胞的开放染色质和转录调控

基本信息

批准号：
9912525
负责人：
ROBERT A. LAFYATIS
金额：
$ 38.77万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-19 至 2021-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9912525
关键词：
Address Automobile Driving Binding Binding Sites Biological Assay Biology Biopsy CRISPR interference Cause of Death Cell Nucleus Cells Cellular Assay Chromatin Cicatrix Companions Complex Complication Computing Methodologies Contracture Cutaneous DNA Data Dermal Detection Diffuse Encapsulated Epigenetic Process FOSL2 gene FOXP1 gene Fibroblasts Fibrosis Gene Expression Gene Expression Profile Gene Expression Regulation Genes Genetic Transcription Goals Guide RNA HSF1 Hyperactive behavior In Vitro Interstitial Lung Diseases Joints Lung MADH2 gene MADH3 gene Methodology Modeling Molecular Morbidity - disease rate Mus Myofibroblast Nuclear Organ Pain Pathogenesis Pathology Pathway interactions Patients Pharmaceutical Preparations Phase Phenotype Plasmids Population Predictive Factor Regulation Regulon Rheumatism Role SFRP4 gene Sampling Sequence Analysis Signal Transduction Skin Source Systemic Scleroderma THBS1 gene Technology Testing Tn5 transposase Transcriptional Regulation Transforming Growth Factor beta Transposase Work activating transcription factor cDNA Library cell type chromatin remodeling cytokine insight mortality mouse model overexpression progenitor single-cell RNA sequencing skin fibrosis systemic autoimmune disease transcription factor transcriptome

项目摘要

The leading cause of death in systemic sclerosis (SSc) is the fibrotic complication, interstitial lung disease (ILD), but skin fibrosis is a leading cause for morbidity resulting in disfiguring, painful and itchy skin, and joint contractures. The emergence and expansion of myofibroblasts as the main profibrotic cell underlies the pathogenesis of both SSc skin and ILD. Although much is understood about myofibroblast biology in vitro and in murine models of fibrosis, the cell source and molecular signals driving myofibroblast differentiation in SSc remain obscure. We have recently identified SSc dermal myofibroblasts, SFRP2-‐expressing myofibroblast progenitors and the associated altered transcriptome by single cell RNA-‐sequencing (scRNA-‐seq). In order to understand the underlying drivers of myofibroblast differentiation we will examine the epigenetic and transcriptional control of genes regulated in SSc skin myofibroblasts. We have analyzed our scRNA-‐seq transcriptome data using SCENIC, a computational method developed for detecting transcription factor (TF)-‐associated regulatory networks (regulons). In scRNA-‐seq analysis of SSc myofibroblasts, we saw upregulated regulons associated with the TFs: FOXP1, NPDC1, IRF7, ZEB1, HSF1 and FOSL2. In the first aim of the R61 phase we will test the role of predicted TFs in regulating myofibroblast transcriptome in dermal fibroblasts. Primary fibroblast cultures from SSc and healthy skin biopsies will be transfected with dCas9-‐CRISPRa or dCas9-‐CRISPRi and single guide RNAs (sgRNAs) targeting FOXP1, NPDC1, IRF7, ZEB1, HSF1 or FOSL2, or SMAD2 or SMAD3 as positive controls for the canonical TGFβ regulated pathway. Cells will then be analyzed by scRNA-‐seq, cDNA libraries prepared, sequenced, and analyzed for alterations in gene expression (PERTURB-‐seq). Gene expression by TF-‐perturbed cells will be compared to unperturbed cells of the same SFRP2+ fibroblast phenotype. Recent technological advances have also provided methodology for single cell Assay for Transposase Accessible Chromatin by Sequencing (scATAC-‐seq) permitting assessment of epigenetic changes in DNA that reflect regions of TF binding to DNA. In the second aim of the R61 phase we will analyze open chromatin in cells from skin biopsies from healthy and SSc patients by scATAC-‐seq. Peaks of open chromatin in myofibroblasts will be identified and compared to open chromatin in myofibroblast progenitor, SFRP2-‐expressing fibroblasts. We will focus on analyzing chromatin remodeling of genes, such as SFRP4 and WIF1 that show altered expression by SSc myofibroblasts. We will correlate predicted TF binding sites in open chromatin with TF regulation of myofibroblast associated genes identified in aim 1. In the R33 phase we will confirm the results in the R61 phase by overexpressing TFs shown to regulate myofibroblast genes, singly or in combination, and analyzing the effects on chromatin remodeling, and on myofibroblast differentiation.

系统性硬化症（SSc）死亡的主要原因是纤维化并发症，间质性肺疾病（ILD），但皮肤纤维化是导致毁容、疼痛和瘙痒的发病主要原因皮肤和关节挛缩作为主要促纤维化细胞的肌成纤维细胞的出现和扩张。尽管人们对肌成纤维细胞了解甚多，但它是 SSc 皮肤和 ILD 发病机制的基础。体外生物学和纤维化小鼠模型、细胞来源和分子信号驱动 SSc 中的肌成纤维细胞分化仍然不清楚。我们最近发现了 SSc 真皮。肌成纤维细胞、表达 SFRP2 的肌成纤维细胞祖细胞和相关的转录组改变通过单细胞 RNA 测序 (scRNA-seq) 来了解潜在的驱动因素。肌成纤维细胞分化我们将检查基因的表观遗传和转录控制我们使用 SCENIC 分析了我们的 scRNA-‐seq 转录组数据，为检测转录因子 (TF) 相关调控而开发的计算方法在 SSc 肌成纤维细胞的 scRNA-seq 分析中，我们发现调节子上调。与 TF 相关的：FOXP1、NPDC1、IRF7、ZEB1、HSF1 和 FOSL2 在 R61 阶段的第一个目标中。我们将测试预测的 TF 在调节真皮成纤维细胞中肌成纤维细胞转录组中的作用。来自 SSc 的原代成纤维细胞培养物和健康皮肤活检将用 dCas9-CRISPRa 或 dCas9-CRISPRi 和单向导 RNA (sgRNA) 靶向 FOXP1、NPDC1、IRF7、ZEB1、HSF1 或 FOSL2，或 SMAD2 或 SMAD3 作为典型 TGFβ 调节细胞的阳性对照。通过 scRNA-‐seq 进行分析，制备 cDNA 文库、测序并分析基因的改变。 TF-扰动细胞的基因表达将与未扰动的细胞进行比较。最近的技术进步也提供了具有相同 SFRP2+ 成纤维细胞表型的细胞。通过测序转座酶可及染色质的单细胞测定方法 (scATAC-‐seq) 允许评估 DNA 中反映 TF 与 DNA 结合区域的表观遗传变化。 R61 阶段的第二个目标是，我们将分析健康皮肤活检细胞中的开放染色质通过 scATAC-seq 识别和 SSc 患者肌成纤维细胞中开放染色质的峰。与肌成纤维细胞祖细胞中的开放染色质相比，表达 SFRP2 的成纤维细胞。分析基因的染色质重塑，例如 SFRP4 和 WIF1，这些基因显示 SSc 改变了表达我们将开放染色质中预测的 TF 结合位点与 TF 调节相关联。目标 1 中确定的肌成纤维细胞相关基因。在 R33 阶段，我们将确认结果 R61 阶段通过过表达 TF 来单独或组合调节肌成纤维细胞基因，以及分析对染色质重塑和肌成纤维细胞分化的影响。