Function and Regulation of MEIS1 in Vascular Disease

项目总结 血管平滑肌细胞(VSMCs)是大中型动脉的主要细胞类型。 血管疾病的表型转换，如血管成形术后再狭窄和动脉粥样硬化。 了解促进VSMC可塑性的关键因素将为下游的目标策略提供信息 影响基因表达病理变化的信号通路和核事件。Myocardin(MYOCD) 是VSMC分化状态的有效转录辅助激活因子，表达缺失预示着血管 病理学。尽管有数百篇论文描述了MYOCD在不同背景下的表达和活动，但其 在体内的转录调控几乎是未知的。我发现了一个髓样嗜热病毒的结合部位 整合位点1(Meis1)是VSMC中以前未被识别的转录因子，位于VSMC的5‘启动子区域 MYOCD，初步研究表明MYOCD基因的Meis1结合和反式激活。MEIS1减少 冠状动脉粥样硬化性血管内皮细胞及Karolinska生物库的转录图谱 动脉内膜切开术(BIKE)显示斑块中Meis1的表达也显著低于正常动脉 在有症状的患者和无症状的患者中一样。到目前为止，VSMCs中只有一份MEIS1的报告 除了肺动脉高压的描述性发现外，什么都不知道。之前的MEIS1功能丧失和 蛋白质表达研究一直很难解释，因为缺乏真正的空小鼠模型和 有缺陷的商业抗体。以下初步研究显示血管平滑肌细胞的增殖和迁移增加 随着MEIS1功能的丧失，功能获得研究揭示了收缩状态的维持。我有过 产生了一个代表第一个真正的空Meis1等位基因的通用小鼠模型，并在该蛋白上进行了检测 水平。我研究的一个重要组成部分将是使用我拥有的新的VSMC限制的CRE驱动程序 帮助确定(Itga8-CRERT2)的特征，以进行更精细的体内功能丧失研究。其他研究支持 驱动Meis1 VSMC表达的多种增强子。总体而言，前期工作支持我的 假设Meis1是一种VSMC富含的转录因子，通过 MYOCD的激活。我建议在K99/R00奖项的过程中从三个目标来检验这一假设。 K99目标1将阐明内源性MEIS1缺失的VSMC表型。R00 Aim 2将阐明 MYOCD在体内通过进化保守的Meis1反应元件的转录调控 最先进的小鼠基因组编辑技术。R00目标3将阐明Meis1在血管中的功能顺式调节组 疾病。拟议研究的完成将为MEIS1对VSMC的贡献提供新的见解 血管分化与血管动态平衡及MEIS1在血管疾病中的机制调节 在K99阶段，与多组学相关研究相关的概念和实验培训将对我有所帮助 在我的R00阶段，作为一名独立调查员继续深造MEIS1，并为我做好准备 我的第一个R01应用程序。

PROJECT SUMMARY Vascular smooth muscle cells (VSMCs), the major cell type of medium- and large-sized arteries, undergo phenotypic switching in vascular diseases such as post-angioplasty restenosis and atherosclerosis. Understanding the key players that promote VSMC plasticity will inform targeting strategies for downstream signaling pathways and nuclear events effecting pathological changes in gene expression. Myocardin (MYOCD) is a potent transcriptional coactivator of the VSMC differentiated state and loss of expression portends vascular pathology. Despite the hundreds of papers describing MYOCD expression and activity in various contexts, its transcriptional regulation in vivo is virtually unknown. I have discovered a binding site for Myeloid Ecotropic Viral Integration Site 1 (MEIS1), a previously unrecognized transcription factor in VSMCs, in the 5’ promoter region of Myocd, and preliminary studies show MEIS1 binding and transactivation of the Myocd gene. MEIS1 is reduced in human VSMCs of atherosclerotic coronary arteries and transcriptomic profiling from Biobank of Karolinska Endoarterctomy (BiKE) showed significant down-regulation of MEIS1 in plaques versus normal arteries as well as in symptomatic versus asymptomatic patients. To date, there has been only one report of MEIS1 in VSMCs and nothing known beyond descriptive findings in pulmonary hypertension. Previous Meis1 loss-of-function and protein expression studies have been difficult to interpret because of the lack of a true null mouse model and faulty commercial antibodies. Preliminary studies below show an increased proliferation and migration of VSMCs with loss of Meis1 function; gain-of-function studies reveal the maintenance of the contractile state. I have generated a versatile mouse model representing the first true null Meis1 allele and its detection at the protein level. An important component of my studies will be the utilization of a new VSMC-restricted Cre driver I have helped characterize (Itga8-CRERT2) for more refined in vivo loss-of-function studies. Additional studies support multiple enhancers that drive VSMC expression of Meis1. Collectively, the preliminary work support my hypothesis that MEIS1 is a VSMC enriched transcription factor that promotes VSMC differentiation via transactivation of Myocd. I propose to test this hypothesis in three aims over the course of this K99/R00 award. The K99 Aim 1 will elucidate the VSMC phenotype with loss of endogenous MEIS1. R00 Aim 2 will elucidate the transcriptional regulation of Myocd in vivo through an evolutionarily-conserved MEIS1-response element using state-of-the-art genome editing in mice. R00 Aim 3 will elucidate the functional cis-regulome of MEIS1 in vascular disease. Completion of the proposed studies will provide new insight into the contribution of MEIS1 to VSMC differentiation and vascular homeostasis and the mechanistic regulation of Meis1 in vascular diseases. Conceptual and experimental training related to multi-omics related research during the K99 phase will help me pursue advanced MEIS1 studies as an independent investigator during my R00 phase and position me well for my first R01 application.