Causal variant association mechanisms in TCF21 binding coronary disease loci

我们已经确定TCF 21为冠状动脉疾病（CAD）相关基因，通过全基因组定位， 在6q23.2进行关联研究。通过结合Tcf 21的条件性缺失，平滑肌细胞（SMC）谱系 在ApoE缺失的人中进行示踪、单细胞RNA测序（scRNAseq）和解剖细胞损伤分析。 模型，我们已经表明，它在SMC中上调，以促进去分化，增殖和迁移 中膜SMC进入斑块，形成保护性纤维帽。这项工作在一个单一的轮廓 细胞水平SMC向成纤维细胞样表型的转变，产生我们称之为“纤维肌细胞”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，TCF 21结合， 调节其他CAD基因座中的许多协同转录因子（TF）的表达，以控制CAD基因的表达。 SMC-FMC转换。此外，TCF 21靶向的其他CAD基因座中的TF调节SMC向TCF 21的转变。 软骨细胞样表型，其特征在于软骨内骨典型的基因表达模式 形成，生产细胞，我们称之为“软骨肌细胞”（CMC）。这些发现指向两个相互关联的复杂 调节SMC细胞状态转换的基因网络作为疾病因果关系的机制。我们假设 因此，该更新申请提出：与SMC表型转变相关的疾病风险， 由TCF 21和调节相互作用转录网络的相关转录因子介导 在很大程度上由CAD相关基因构成。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， 确定由SMC对血管应力的表型反应引起的CAD风险。具体而言，目标1 我们将用野生型和Tcf 21缺失的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，以绘制基因组范围内的增强子，这些增强子在 SMC表型转变，并鉴定结合这些增强子的TF。在Aim 2中，我们将执行scATACseq CRISPR/Cas9干扰人冠状动脉中鉴定的过渡TF后的scRNAseq 平滑肌细胞去分化模型，以检查TF敲低对转录谱的影响 以及与FMC和CMC表型相关的TF的相互作用。探讨平滑肌细胞与心肌细胞的关系 表型与CAD风险之间的关系，我们将确定SMC转换TF的扰动如何改变 启动子区和连接的增强子区。在目标3中，我们将研究分子 方法上位性的机制和功能之间的相互作用的TF，主要定义了 SMC转变表型并鉴定与CAD的转录联系。因此，这项工作的特点是 SMC TF通过表观遗传和表型转化激活表型转化的基本过程 转录机制，扩大我们对这种疾病的理解，并促进机会， 改善人类CAD风险。

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.