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.

项目总结/摘要 内皮细胞（EC）排列在血液循环系统中，属于动脉和静脉谱系。啊... 材料和静脉内皮细胞在对炎症、动脉粥样硬化和钙化敏感性方面本质上不同。 此外，在小鼠模型中，维持AV差异的遗传程序的破坏导致动静脉畸形。 脑血管畸形（AVM）是小儿中风的主要原因。从而了解基因机制 确定和维持AV差异对于更好地理解一系列人类疾病的发病机制至关重要。 紊乱动脉和静脉谱系的特化发生在血流建立之前， 认为AV差异主要受遗传控制。尽管我们做了大量的努力， 建立和维持动脉和静脉身份的分子机制仍然不完整。凹口 信号传导已被确定为对动脉分化至关重要，并且转录因子COUP-TFII 已被鉴定为对静脉分化至关重要，至少部分通过拮抗Notch信号传导。 对4种具有动脉选择性活性的转录增强子的深入研究已经产生了以下轶事信息： 它们的动脉选择性活动所需的一些特征。然而，系统的知识的原则，去- 缺乏终末动脉或静脉特异性表达。在很大程度上，这是由于低吞吐量的性质 研究这个问题的技术。 我们已经开发了两种独特的高通量方法，将克服这一障碍，并产生系统， 关于用于产生动脉或静脉选择性活性的机制的信息。一是 开发了一种高亲和力，组织特异性鉴定p300标记的活性增强子的方法， Notch靶RBPJ结合的调节元件。第二，我们开发了一种方法，通过高- 把（在一个实验中成千上万的顺序）候选增强剂的测试在一个inte- grated基因组context上下文.在这个建议中，我们应用这些进展系统地研究动静脉 分化及其受Notch信号调节的机制。 在目标1中，我们测试了可识别的转录代码驱动动脉和静脉特异性的假设， 转录增强子活性。我们将使用EC中的p300结合来鉴定候选增强子，然后 平行测试增强剂的动脉或静脉选择活性。对该数据库的生物信息学分析 具有选择活性的增强子将鉴定候选转录词典。这些预测将是 通过选择的增强子的后续密集诱变进行测试，并在转基因胚胎中进一步验证 测定。 在目标2中，我们专注于Notch信号调节RBPJ活性的机制。我们测试了- 假设RBPJ通过多种不同的Notch依赖性和非依赖性调节AV分化 机制等这一目标取决于我们在体内有效地绘制RPBJ染色质占有率的独特能力， EC。通过在Notch激活和Notch抑制条件下定位RBPJ和p300， bryos，我们将定义Notch胞内结构域对RBPJ定位和活性的影响。组合这些 在目标1中发现的动脉和静脉选择性增强剂的数据将定义动脉或静脉选择性增强剂 具有Notch/RBPJ依赖性和非依赖性活性。 该提案在新型全基因组定位和高通量增强子方面具有技术创新性 测试方法。概念创新是对动脉或静脉选择性转录的新认识， 这些新方法的应用将引起Notch信号传导的调节。 这一建议是重要的，因为它将通过填补关键的血管生成来促进我们对血管生成的理解。 我们对动静脉差异如何被指定和维持的理解存在差距。这一基本知识-- 边缘与多种人类疾病如癌症、动脉粥样硬化和炎症有关。