Transcriptional regulation of arteriovenous differentiation

动静脉分化的转录调控

• 批准号: 9376461
• 负责人: William Tswenching Pu
• 金额: $ 52.34万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9376461
• 关键词: Affect; Affinity; Alleles; Arteries; Arteriovenous malformation; Atherosclerosis; Binding; Bioinformatics; Biological Assay; Blood; Blood Vessels; Blood flow; Calcified; Cardiovascular system; Cell Line; Cells; Cerebral Arteriovenous Malformations; Childhood; Childhood stroke; Chromatin; Code; Data; Data Set; Databases; Disease; EP300 gene; Embryo; Endothelial Cells; Enhancers; Family; Gene Expression; Genes; Genetic; Genetic Transcription; Genetically Engineered Mouse; Genomics; Goals; Human; Inflammation; Informatics; Knowledge; Link; Location; Maintenance; Malignant Neoplasms; Maps; Measures; Mediator of activation protein; Methods; Molecular; Molecular Profiling; Mus; Mutagenesis; Myocardial Ischemia; Nature; Nuclear Receptors; Orphan; Pathogenesis; Pathway interactions; Pluripotent Stem Cells; Predisposition; Process; Regulation; Regulatory Element; Signal Transduction; Specific qualifier value; Specificity; Stroke; Structure; Techniques; Testing; Tissues; Transcriptional Regulation; Transgenic Organisms; VEGFA gene; Validation; Vasculitis; Veins; Venous; angiogenesis; apoAI regulatory protein-1; biochip; calcification; chicken ovalbumin upstream promoter-transcription factor; embryo cell; experimental study; gamma secretase; genome-wide; high throughput screening; human disease; improved; in vivo; induced pluripotent stem cell; inhibitor/antagonist; innovation; insight; malformation; mouse model; notch protein; novel; novel strategies; pediatric patients; precise genome editing; programs; transcription factor

Project Summary/Abstract Endothelial cells (ECs) that line the blood circulatory system belong to the arterial and venous lineages. Ar- terial and venous ECs intrinsically differ in their susceptibility to inflammation, atherosclerosis, and calcification. Moreover, disruption of genetic programs that maintain AV differences in mouse models causes arteriovenous malformations (AVMs), the leading cause of pediatric strokes. Thus understanding the genetic mechanisms that specify and maintain AV differences is critical to better understand the pathogenesis of a range of human disorders. Specification of arterial and venous lineages occurs prior to the establishment of blood flow, sug- gesting that AV differences are primarily under genetic control. Despite extensive efforts, our understanding of the molecular mechanisms that establish and maintain arterial and venous identity remains incomplete. Notch signaling has been identified as being critical for arterial differentiation, and the transcription factor COUP-TFII has been identified as being critical for venous differentiation, at least in part by antagonizing Notch signaling. In depth study of 4 transcriptional enhancers with artery selective activity has yielded anecdotal information on some features required for their artery-selective activity. However, systematic knowledge of principles that de- termine arterial or venous specific expression is lacking. In large part, this is due to the low throughput nature of the techniques that have been employed to study this problem. We have developed two unique, high throughput approaches that will surmount this barrier and yield syste- matic information about the mechanisms that are employed to yield artery or vein selective activity. First, we developed a method for high affinity, tissue-specific identification of active enhancers marked by p300, and of regulatory elements bound by the Notch target RBPJ. Second, we have developed a method for high through- put (on the order of hundreds of thousands in one experiment) testing of candidate enhancers within an inte- grated genomic context. In this proposal we apply these advances to systematically investigate arteriovenous differentiation and the mechanisms by which it is regulated by Notch signaling. In Aim 1, we test the hypothesis that identifiable transcriptional codes drive artery and vein specific transcriptional enhancer activity. We will use p300 binding in ECs to identify candidate enhancers, and then test the enhancers in parallel for artery or vein selective activity. Bioinformatic analyses of this database of enhancers with selective activity will identify the candidate transcriptional lexicon. These predictions will be tested by followup dense mutagenesis of selected enhancers, with further validation in transgenic embryo assays. In Aim 2, we focus on the mechanisms by which Notch signaling modulates RBPJ activity. We test the hy- pothesis that RBPJ regulates AV differentiation through multiple distinct Notch-dependent and -independent mechanisms. This aim hinges upon our unique ability to efficiently map RPBJ chromatin occupancy in vivo in ECs. By mapping RBPJ and p300 under Notch activated and Notch suppressed conditions in developing em- bryos, we will define the effects of Notch intracellular domain on RBPJ location and activity. Combining these data with the artery and vein selective enhancers found in Aim 1 will define artery or vein selective enhancers with Notch/RBPJ-dependent and -independent activity. This proposal is technically innovative in the novel genome-wide mapping and high throughput enhancer testing approaches. The conceptual innovation is the new understanding of artery or vein selective transcrip- tional regulation and of Notch signaling that will arise from application of these novel approaches. This proposal is significant because it will advance our understanding of angiogenesis by filling in critical gaps in our understanding of how arteriovenous differences are specified and maintained. This basic knowl- edge is relevant to diverse classes of human disease such as cancer, atherosclerosis, and inflammation.

项目摘要/摘要 血液循环系统属于动脉和静脉谱系的内皮细胞（EC）。 ar 特性和静脉EC在炎症，动脉粥样硬化和钙化的敏感性上本质上有所不同。 此外，在鼠标模型中维持AV差异的遗传程序的破坏会导致动脉静脉 畸形（AVM），是小儿中风的主要原因。因此了解遗传机制 指定和维持AV差异对于更好地了解一系列人的发病机理至关重要 疾病。动脉和静脉谱系的规格发生在建立血流之前，SUG- 病差异主要在遗传控制之下。尽管做出了广泛的努力，但我们对 建立和维持动脉和静脉身份的分子机制仍然不完整。缺口 信号传导已被确定为动脉分化至关重要，而转录因子coup-tfii 至少部分通过拮抗Notch信号传导，至少部分地被确定为静脉分化至关重要。 在对4个具有动脉选择活性的4个转录增强子的深入研究中，产生了有关轶事信息 其动脉选择活动所需的一些特征。但是，对原则的系统知识 缺乏终末动脉或静脉特定表达。在很大程度上，这是由于吞吐量低 用于研究此问题的技术。 我们已经开发了两种独特的高通量方法，可以克服这一障碍并产生系统。 有关用于产生动脉或静脉选择性活性的机制的信息信息。首先，我们 开发了一种用于高亲和力的方法，以P300标记的活动增强剂的组织特异性鉴定以及 由Notch目标RBPJ约束的调节元素。其次，我们开发了一种用于高渗透的方法 （在一个实验中，在数十万的顺序中）对候选增强子进行了测试 磨碎的基因组环境。在此提案中，我们将这些进步应用到系统地研究动脉旋转 分化和由Notch信号调节的机制。 在AIM 1中，我们检验了可识别的转录代码驱动动脉和特定静脉的假设 转录增强子活性。我们将在EC中使用P300绑定来识别候选增强剂，然后 并行测试增强子的动脉或静脉选择性活性。此数据库的生物信息学分析 具有选择性活动的增强子将识别候选转录词典。这些预测将是 通过选定增强剂的随访密集诱变进行测试，并在转基因胚胎中进行进一步验证 测定。 在AIM 2中，我们关注Notch信号调节RBPJ活性的机制。我们测试了 RBPJ通过多个不同的缺口依赖性和独立的pothesis调节了AVIATION 机制。这个目的取决于我们在体内有效绘制RPBJ染色质占用率的独特能力 ECS。通过绘制RBPJ和p300在Notch激活和抑制条件下，在发展EM- Bryos，我们将定义Notch细胞内结构域对RBPJ位置和活动的影响。结合这些 AIM 1中发现的动脉和静脉选择性增强子的数据将定义动脉或静脉选择性增强子 具有Notch/RBPJ依赖性和非依赖性活性。 该建议在新的全基因组映射和高吞吐量增强器中具有技术创新性 测试方法。概念创新是对动脉或静脉选择性译的新理解 - 这些新方法的应用将产生的调节和凹槽信号传导。 该提议很重要，因为它将通过填充关键来提高我们对血管生成的理解 我们对如何指定和维持动力静脉差异的理解差距。这个基本的知识 - 边缘与癌症，动脉粥样硬化和炎症等各种人类疾病相关。