Transcriptional regulation of arteriovenous differentiation

项目摘要/摘要 血管内皮细胞(ECs)排列在血液循环系统中，属于动脉和静脉系统。阿尔- 动脉内皮细胞和静脉内皮细胞对炎症、动脉粥样硬化和钙化的易感性存在本质上的差异。 此外，在小鼠模型中维持房室差异的遗传程序的中断会导致动静脉 畸形(AVM)，是儿童中风的主要原因。从而了解遗传机制 明确和保持房室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 精神错乱。动脉和静脉谱系的指定发生在血流建立之前，建议： 推测AV差异主要是受基因控制的。尽管作出了广泛的努力，但我们对 建立和维持动脉和静脉一致性的分子机制仍然不完整。凹槽 信号转导被认为是动脉分化的关键，而转录因子Coup-TFII 已被确定为静脉分化的关键，至少部分是通过拮抗Notch信号。 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关于动脉选择性的轶事信息 它们的动脉选择性活动所需的一些特征。然而，对原理的系统知识将使 终末动脉或静脉缺乏特异性表达。这在很大程度上是由于吞吐量低的性质 已经用来研究这个问题的技术。 我们已经开发了两种独特的、高吞吐量的方法，将跨越这一障碍，并产生系统- 马季奇介绍了产生动脉或静脉选择性活动的机制。首先，我们 开发了一种高亲和力、组织特异性地鉴定由p300标记的活性增强剂的方法，以及 受Notch目标RBPJ约束的监管要素。第二，我们开发了一种高通量的方法-- 在一次实验中对候选增强剂进行(在一次实验中约为数十万)测试- 严酷的基因组背景。在这项建议中，我们应用这些进展来系统地研究动静脉 Notch信号调控细胞分化的机制。 在目标1中，我们测试了可识别的转录密码驱动动脉和静脉特异性的假设 转录增强子活性。我们将在EC中使用p300结合来确定候选增强子，然后 平行测试增强剂的动脉或静脉选择性活性。该数据库的生物信息学分析 具有选择性活性的增强子将识别候选转录词典。这些预测将是 通过后续选择的增强子的密集突变进行测试，并在转基因胚胎中进一步验证 化验。 在目标2中，我们重点研究Notch信号调节RBPJ活性的机制。我们测试了HY- 假设RBPJ通过多个不同的Notch依赖和非依赖方式调节房室分化 机械装置。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 ECS。通过在Notch激活和Notch抑制条件下定位RBPJ和p300在发育EM-EMS中的作用 Bryos，我们将确定Notch胞内结构域对RBPJ定位和活性的影响。将这些组合在一起 在AIM 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依赖和非独立活性。 该方案在新型全基因组作图和高通量增强子方面具有技术创新 测试方法。观念上的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产生对Notch信号的调节和调节。 这一建议意义重大，因为它将通过填写关键字来促进我们对血管生成的理解 在我们对动静脉差异是如何明确和维持的理解上存在差距。这一基本知识- EDGE与多种人类疾病有关，如癌症、动脉粥样硬化和炎症。

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.