Causal variant association mechanisms in TCF21 binding coronary disease loci

TCF21结合冠心病位点的因果变异关联机制

基本信息

批准号：
10542365
负责人：
THOMAS QUERTERMOUS
金额：
$ 58.45万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-01-01 至 2024-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10542365
关键词：
ATAC-seq Affect Apolipoprotein E Binding Binding Sites Blood Vessels CRISPR interference CRISPR/Cas technology Cardiovascular Diseases Cell Lineage Cells Chondrocytes Chromatin Chromosome Mapping Complement Complex Coronary Arteriosclerosis Coronary artery Coronary heart disease Data Development Disease Disease Pathway Enhancers Epigenetic Process Etiology Exposure to Fibroblasts Funding Gene Expression Gene Expression Profile Genes Genetic Epistasis Genetic Risk Genetic Transcription Genome Genomics Goals Human Human Genome In Vitro Joints Knockout Mice Lesion Link Maps Medial Mediating Methods Modeling Molecular Mus Osteogenesis Phenotype Process Proliferating Promoter Regions Quantitative Trait Loci Research Risk Assessment Scientist Smooth Muscle Myocytes Stress Subcellular Anatomy Therapeutic Tissues Work causal variant cell dedifferentiation cell type disorder risk epigenomics experimental study gene network gene regulatory network genome wide association study genome-wide in vivo knock-down migration public health relevance response single cell analysis single-cell RNA sequencing transcription factor vascular stress

ATAC-seq Affect Apolipoprotein E Binding Binding Sites Blood Vessels CRISPR interference CRISPR/Cas technology Cardiovascular Diseases Cell Lineage Cells Chondrocytes Chromatin Chromosome Mapping Complement Complex Coronary Arteriosclerosis Coronary artery Coronary heart disease Data Development Disease Disease Pathway Enhancers Epigenetic Process Etiology Exposure to Fibroblasts Funding Gene Expression Gene Expression Profile Genes Genetic Epistasis Genetic Risk Genetic Transcription Genome Genomics Goals Human Human Genome In Vitro Joints Knockout Mice Lesion Link Maps Medial Mediating Methods Modeling Molecular Mus Osteogenesis Phenotype Process Proliferating Promoter Regions Quantitative Trait Loci Research Risk Assessment Scientist Smooth Muscle Myocytes Stress Subcellular Anatomy Therapeutic Tissues Work causal variant cell dedifferentiation cell type disorder risk epigenomics experimental study gene network gene regulatory network genome wide association study genome-wide in vivo knock-down migration public health relevance response single cell analysis single-cell RNA sequencing transcription factor vascular stress

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项目摘要

We have identified TCF21 as the coronary artery disease (CAD) associated gene mapped by genome-wide association studies at 6q23.2. By combining conditional deletion of Tcf21, smooth muscle cell (SMC) lineage tracing, single cell RNA sequencing (scRNAseq), and anatomical cellular lesion analysis in the ApoE null model, we have shown that it is upregulated in SMC to promote de-differentiation, proliferation, and migration of medial SMC into the plaque where they contribute to the protective fibrous cap. This work profiled at a single cell level the transition of SMC to a fibroblast like phenotype, creating cells that we term “fibromyocytes” (FMC). Genomic studies conducted as part of this funded work have suggested that TCF21 binds and regulates expression of a number of cooperating transcription factors (TFs) in other CAD loci to govern the SMC-FMC transition. Further, TCF21 targeted TFs in other CAD loci modulate an SMC transition to a chondrocyte-like phenotype, which is characterized by gene expression patterns typical of endochondral bone formation, producing cells we term “chondromyocytes” (CMC). These findings point to two interrelated complex gene networks that regulate SMC cell state transition as a mechanism of disease causality. Our hypothesis for this renewal application thus proposes that: disease risk associated with SMC phenotypic transition is mediated by TCF21 and related transcription factors that regulate interactive transcriptional networks constituted in large part by CAD associated genes. The primary goal of work proposed here is to further characterize these networks and define the epigenetic and transcriptional mechanisms of TF interactions that determine the CAD risk engendered by the SMC phenotypic response to vascular stress. Specifically, in Aim 1 we will conduct single cell ATAC sequencing (scATACseq) with wildtype and Tcf21 null atherosclerotic mice, as well as human coronary artery tissues, to map enhancers genome-wide that are differentially regulated in SMC phenotypic transitions, and identify TFs that bind these enhancers. In Aim 2 we will perform scATACseq and scRNAseq following CRISPR/Cas9 perturbation of identified transition TFs in a human coronary artery smooth muscle cell de-differentiation model to examine the impact of TF knockdown on transcriptional profiles and interactions of TFs linked to the FMC and CMC phenotypes. To investigate the relationship of SMC phenotype to CAD risk, we will determine how perturbation of SMC transition TFs alters accessibility at promoter regions and linked enhancer regions at CAD associated loci. In Aim 3 we will examine with molecular methods the mechanisms of epistasis and functional interactions between the TFs that primarily define the SMC transition phenotypes and identify transcriptional links to CAD. This work will thus characterize fundamental processes by which SMC TFs activate phenotypic transitions through epigenetic and transcriptional mechanisms, extending our understanding of this disease and promoting opportunities for ameliorating human CAD risk.

我们已经确定TCF21是由基因组绘制的冠状动脉疾病（CAD）协会研究在6q23.2。通过结合TCF21的条件缺失，平滑肌细胞（SMC）谱系跟踪，单细胞RNA测序（SCRNASEQ）和APOE NULL中的解剖细胞病变分析模型，我们已经证明它已在SMC中进行了更新，以促进分化，增殖和迁移内侧SMC进入斑块，在那里它们有助于受保护的纤维帽。这项工作以一个细胞水平SMC向成纤维细胞（如表型）的过渡，创建了我们称为“纤维细胞”的细胞（FMC）。作为这项资助工作的一部分进行的基因组研究表明，TCF21结合并调节其他CAD局部的许多合作转录因子（TF）的表达以控制 SMC-FMC过渡。此外，TCF21在其他CAD基因座中靶向TFS，将SMC转变为A 软骨细胞样表型，其特征是内软骨骨典型的基因表达模式形成，产生细胞，我们称为“软骨细胞”（CMC）。这些发现指出了两个相互关联的复合物调节SMC细胞状态转变为疾病卡的机理的基因网络。我们的假设因此，这种更新应用的建议：与SMC表型过渡相关的疾病风险是由TCF21和相关转录因子介导的调节互动转录网络在很大程度上由CAD相关基因组成。这里提出的工作的主要目标是进一步表征这些网络并定义TF相互作用的表观遗传和转录机制确定SMC表型反应对血管应激产生的CAD风险。具体而言，在AIM 1中我们将用野生型和TCF21无动脉粥样硬化小鼠进行单细胞ATAC测序（SCATACSEQ），以及人类冠状动脉组织，以绘制在整个基因组中受到不同调节的增强剂 SMC表型过渡，并识别结合这些增强子的TF。在AIM 2中，我们将表演Scatacseq 在人类冠状动脉中鉴定出的过渡TF的CRISPR/CAS9扰动后的scrnaseq 平滑肌细胞划分分化模型，以检查TF敲低对转录曲线的影响与FMC和CMC表型相关的TF的相互作用。调查SMC的关系表型CAD风险，我们将确定SMC过渡TFS的扰动如何改变可访问性 CAD相关基因座的启动子区域和连接的增强子区域。在AIM 3中，我们将使用分子检查方法是主要定义的TFS之间的上毒机理和功能相互作用 SMC过渡表型并确定与CAD的转录链接。因此，这项工作将是特征 SMC TFS通过表观遗传学和转录机制，扩展了我们对这种疾病的理解，并促进了机会改善人CAD风险。