Cis-regulatory architecture of coronary vascular wall loci

项目摘要 冠状动脉疾病（CAD）仍然是世界范围内死亡的主要原因， 公共卫生负担。遗传和环境风险因素都有助于CAD易感性，导致 发展中国家的疾病流行率增加。全基因组关联研究（GWAS） 在30多万人的大规模荟萃分析中确定了161个与CAD风险相关的独立遗传位点。 个体然而，考虑到大多数关联位于非编码基因组区域， 将这些发现转化为生物学和临床相关的见解一直是一个挑战。有趣的是， CAD基因座上的许多候选基因被组织成新的或未知的生物学途径， 血管壁过程的失调。基因型组织表达（GTEx） 项目和斯德哥尔摩-塔尔图动脉粥样硬化反向网络工程任务（STARNET）研究 完善的候选基因调控机制，然而，特定的细胞和表型状态驱动 这些变化仍不清楚。平滑肌细胞（SMC）通常调节血管壁中的血管张力 但在疾病发病机制中起关键作用，因为它们的收缩基因程序在表型 转化为巨噬细胞样和成纤维细胞样细胞。利用表观基因组和表达数量性状基因座 我们最近鉴定了52个原代人冠状动脉平滑肌细胞（HCASMC）， 11个将血管壁过程与CAD风险联系起来的候选致病基因和机制（例如TCF 21，SMAD 3， CDKN 2B，LMOD 1）。很可能非编码调节变体通过染色质的变化发挥作用 可及性，这与转录因子（TF）顺式结合一致。通过识别这些上游TF， 我们可以开始组装靶向通路，以调节血管中隐藏的疾病风险 墙在这个提议中，我们计划在一个新的基因组中进行联合基因表达和染色质可及性QTL定位。 一个独特的队列，120例正常和患病的冠状动脉组织，来自心脏捐赠者。我们将 使用CRISPR/dCas 9扰动验证和发现GWAS基因座的功能性调控元件， SMC中的高通量表型分析。最后，我们将研究这些细胞中的顺式调控结构。 样品在单细胞分辨率和推断TF结合在表型调制。这些研究将 揭示血管壁中CAD的因果调节机制，并告知下一代 治疗或预防战略，以消除这种使人衰弱的疾病。

PROJECT SUMMARY Coronary artery disease (CAD) remains the leading cause of mortality worldwide and poses considerable public health burden. Both genetic and environmental risk factors contribute to CAD susceptibility, leading to increased disease prevalence in developing countries. Genome-wide association studies (GWAS) have now identified 161 independent genetic loci associated with CAD risk in large-scale meta-analyses in over 300,000 individuals. However, given that the majority of the associations reside in non-coding genomic regions, it has been challenging to translate these discoveries into biologically and clinically relevant insights. Interestingly, many of the candidate genes at CAD loci are organized into novel or unknown biological pathways, and point to dysregulation of vascular wall processes. Large efforts such as the Genotype Tissue Expression (GTEx) project and Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task (STARNET) study have refined candidate gene regulatory mechanisms, however the specific cellular and phenotypic states driving these changes remains unclear. Smooth muscle cells (SMC) normally regulate vascular tone in the vessel wall but play critical roles in disease pathogenesis as their contractile gene program is hijacked during phenotypic switching to macrophage-like and fibroblast-like cells. Using epigenomic and expression quantitative trait loci (eQTL) mapping in 52 primary human coronary artery smooth muscle cells (HCASMC) we recently identified 11 candidate causal genes and mechanisms linking vascular wall processes to CAD risk (e.g. TCF21, SMAD3, CDKN2B, LMOD1). It is likely that non-coding regulatory variants function through changes in chromatin accessibility, which coincides with transcription factor (TF) binding in cis. By identifying these upstream TFs at vascular wall loci we can begin to assemble targetable pathways to modulate hidden disease risk in the vessel wall. In this proposal we plan to perform joint gene expression and chromatin accessibility QTL mapping in a unique cohort of 120 normal and diseased coronary artery tissues from explanted heart donors. We will validate and discover functional regulatory elements at GWAS loci using CRISPR/dCas9 perturbation with high-throughput phenotyping in SMC. Finally, we will investigate the cis-regulatory architecture in these samples at single-cell resolution and infer TF binding during phenotypic modulation. Together these studies will reveal the causal regulatory mechanisms responsible for CAD in the vascular wall and inform next generation treatment or prevention strategies to eradicate this debilitating disease.