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 mul...