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.

全身性硬化症（SSC）死亡的主要原因是纤维化并发症，间质肺疾病（ILD），但皮肤纤维化是发病率的主要原因，导致毁容，痛苦和发痒皮肤和关节染色。肌纤维细胞的出现和膨胀基础是SSC皮肤和ILD的发病机理。虽然对肌纤维细胞有很多了解在体外和纤维化的鼠模型中生物学，细胞源和分子信号驱动 SSC中的肌纤维细胞分化仍然晦涩。我们最近确定了SSC真皮肌纤维细胞，表达SFRP2的肌纤维细胞祖细胞和相关的转录组通过单细胞RNA--测序（SCRNA-- seq）。为了了解肌纤维细胞分化我们将检查基因的表观遗传和转录控制在SSC皮肤肌纤维细胞中调节。我们已经使用Scenic，开发用于检测转录因子（TF）的计算方法 - 相关调节网络（条件）。在SSC肌纤维细胞的SCRNA - - SEQ分析中，我们看到了更新的调节与TFS：FOXP1，NPDC1，IRF7，ZEB1，HSF1和FOSL2相关。在R61阶段的第一个目标中我们将测试预测的TF在皮肤成纤维细胞中肌纤维细胞转录组中的作用。来自SSC和健康皮肤活检的原发性成纤维细胞培养物将用DCAS9-Crispra或Crispra或 DCAS9 - Crispri和单个指南RNA（SGRNA）针对FOXP1，NPDC1，IRF7，ZEB1，HSF1或FOSL2或FOSL2或 SMAD2或SMAD3作为规范TGFβ调节途径的阳性对照。然后，细胞将是由SCRNA-- SEQ分析，对基因的改变，测序和分析的cDNA文库进行了分析表达式（worturb-seq）。将TF - - 扰动细胞的基因表达与不受干扰的细胞进行比较相同SFRP2+成纤维细胞表型的细胞。最近的技术进步也提供了单细胞测定方法，用于通过测序（SCATAC --- seq）进行转座酶访问的染色质允许评估反映TF与DNA结合区域的DNA表观遗传变化。在 R61期的第二个目标我们将分析健康活检中皮肤活检细胞中的开放染色质 SCATAC和SSC患者 - seq。将鉴定出肌纤维细胞中开放染色质的峰与表达成纤维细胞的SFRP2中的肌纤维细胞祖细胞中的开放染色质相比。我们将重点关注基因的分析仪染色质重塑，例如SFRP4和WIF1，显示出SSC的表达改变肌纤维细胞。我们将在开放染色质中将预测的TF结合位点与TF调节相关联在AIM 1中鉴定的肌纤维细胞相关基因。在R33阶段，我们将确认结果 R61阶段通过表达的过表达TF，以调节肌纤维细胞基因，单独或组合，并且分析对染色质重塑的影响以及对肌纤维细胞分化的影响。