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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)患者死亡的主要原因是肺间质纤维化并发症。疾病(ILD)，但皮肤纤维化是导致皮肤疾病发病率的主要原因，导致皮肤毁容、皮肤疼痛和皮肤发痒。皮肤、皮肤和关节的肌挛缩。肌成纤维细胞的大量出现和扩张是主要的促纤维细胞。这是SSC皮肤病和ILD的主要发病机制之一。尽管关于肌成纤维细胞的许多研究尚不清楚。生物学在体外和在小鼠实验性肝纤维化模型中发挥作用，主要是细胞来源和分子信号的驱动。肌成纤维细胞在SSC中的分化仍然不清楚。我们最近发现了SSC的真皮细胞。肌成纤维细胞，即SFRP2--表达肌成纤维细胞和前体细胞，以及与转录组改变相关的基因。通过单细胞DNA-RNA--Sequence(scRNA--seq)进行测序，以便更好地了解的潜在驱动因素。肌成纤维细胞的分化，我们将继续研究这些基因的表观遗传学和转录调控机制。 SSC中受调控的基因包括皮肤和肌成纤维细胞。我们已经使用SERVICE分析了我们的scRNA--seq和转录组数据。开发了一种新的计算机辅助检测转录因子--转录因子相关基因的方法。在对SSC和肌成纤维细胞的scRNA--seq分析中，我们已经看到了上调的调节子。与以下三个TF相关的目标：Foxp1、NPDC1、IRF7、ZEB1、HSF1和FOSL2。这是R61第一阶段的第一个目标。我们还将测试预测的转录因子在调节真皮成纤维细胞中的肌成纤维细胞和转录组蛋白中的重要作用。来自SSC的原代成纤维细胞培养和健康的皮肤活检后，将不会再用DCAS9--CRISPRA或。 DCas9--CRISPRi和针对Foxp1、NPDC1、IRF7、ZEB1、HSF1或FOSL2的RNAs(SgRNAs)的单一指南。 Smad2或Smad3被认为是典型的转化生长因子β受调控的信号通路的阳性调控因子。然后，这些细胞将被激活。用scRNA--seq进行分析，对文库进行制备、测序、测序，并对其进行基因突变分析。表达(perturb--seq)。与未受干扰的细胞相比，受干扰的细胞表达的基因将不会更多。细胞的表型与SFRP2+成纤维细胞相同。还提供了最新的技术进步。通过DNA测序技术(scATAC--seq)，对单个细胞进行转座酶检测，以获得染色质。允许对DNA序列中的表观遗传学变化进行评估，以反映TTF对DNA具有约束力的区域。第二个目标是在第一个R61阶段，我们将分析来自健康人皮肤和活组织检查的细胞中开放的染色质成分。而在SSC中，通过SCATAC--seq.-seq.的患者的肌肉成纤维细胞中开放的染色质的高峰期将无法被进一步鉴定。与在肌成纤维细胞中开放染色质基因相比，在成纤维细胞中表达SFRP2--我们将继续关注。分析染色质和基因重塑的关系，如sFRP4基因和WIF1基因的重塑，可以显示SSC改变了基因的表达。肌成纤维细胞。我们将在开放的染色质中将预测的转录因子结合位点与转录因子的调控机制关联起来。肌成纤维细胞及其相关基因已在AIM 1中确定。在R33第一阶段中，我们将不再确认在第二阶段中的结果。 R61是一种通过过度表达转录因子来调节肌成纤维细胞基因的阶段，不能单独或以组合、基因和基因形式存在。分析其对染色质重塑、肌成纤维细胞分化的影响。