Function and Regulation of MEIS1 in Vascular Disease

项目摘要 血管平滑肌细胞（VSMC）是中型和大型动脉的主要细胞类型， 在血管疾病如血管成形术后再狭窄和动脉粥样硬化中的表型转换。 了解促进VSMC可塑性的关键参与者将为下游的靶向策略提供信息。 信号通路和核事件影响基因表达的病理变化。心肌素（MYOCD） 是VSMC分化状态的有效转录辅激活因子，表达缺失预示着血管 病理尽管有数百篇论文描述了MYOCD在各种情况下的表达和活性， 体内转录调控实际上是未知的。我发现了骨髓嗜亲性病毒的结合位点 整合位点1（MEIS 1）是VSMCs中以前未被识别的转录因子，位于VSMCs的5'启动子区， Myocd，初步研究显示MEIS 1与Myocd基因结合并反式激活。MEIS 1减少 在动脉粥样硬化冠状动脉的人VSMCs中和来自Karolinska Biobank的转录组学分析 与正常动脉相比，动脉内膜切除术（BiKE）也显示MEIS 1在斑块中显著下调 有症状的病人和无症状的病人迄今为止，只有一份关于VSMCs中MEIS 1的报告 除了肺动脉高压的描述性发现之外，没有其他已知的发现。Meis 1功能丧失和 蛋白质表达研究难以解释，因为缺乏真正的无效小鼠模型， 错误的商业抗体。下面的初步研究表明，VSMC的增殖和迁移增加 Meis 1功能丧失;功能获得研究揭示了收缩状态的维持。我有 产生了一个通用的小鼠模型，代表了第一个真正的无效Meis 1等位基因及其在蛋白质中的检测 水平我的研究的一个重要组成部分将是利用一个新的VSMC限制的Cre驱动程序，我 有助于表征（Itga 8-CRERT 2），以进行更精确的体内功能丧失研究。其他研究支持 多个增强子驱动VSMC表达Meis 1。总的来说，初步工作支持了我的 假设MEIS 1是VSMC富集的转录因子，其促进VSMC分化， Myocd的反式激活。我建议在K99/R 00奖项的过程中，通过三个目标来验证这一假设。 K99 Aim 1将阐明内源性MEIS 1缺失的VSMC表型。R 00目标2将阐明 利用进化保守的MEIS 1反应元件在体内对Myocd进行转录调控 最先进的小鼠基因组编辑。R 00 Aim 3将阐明MEIS 1在血管内皮细胞中的功能性顺式调节子。 疾病完成拟议的研究将为MEIS 1对VSMC的贡献提供新的见解 分化和血管稳态以及Meis 1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帮助我 在我的R 00阶段，作为一名独立的研究者，我将继续进行先进的MEIS 1研究， 我的第一个R 01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.