Identification of artery- and vein-specific cis elements in the human genome

描述（由申请人提供）：内皮细胞分化和血管身份的建立对于正常和疾病情况下的血管功能至关重要。例如，在发育中的胚胎中，动脉和静脉内皮细胞的分化对于正常的血管模式和循环功能至关重要。同样，在许多先天性疾病中，血管特性的扰动可导致血管异常，如动静脉畸形。因此，了解正常内皮分化的分子基础将使我们对病理血管形成有更深入的了解，并促进不同类型血管的工程设计。虽然控制细胞分化的转录等级在其他组织中已经被广泛表征，但内皮细胞中的这种程序却鲜为人知。本应用程序中提出的研究将开始在内皮细胞的转录水平上解决动脉和静脉分化的控制。特别是，我们将确定人类基因组中负责动脉和静脉内皮特异性基因表达的顺式调控元件。这将通过全基因组鉴定一般转录调节蛋白的结合位点来完成，这些蛋白已知可以可靠地标记增强子和抑制子元件。随后的计算分析将使我们能够识别与动脉或静脉特异性基因表达相关的共同顺式调控序列。这些将为上游转录调控因子提供可能的见解。为了从功能上验证假定的动脉和静脉顺式元件的活性，我们将使用斑马鱼作为模型系统对人体元件进行体内报告分析。斑马鱼胚胎的透明性和外部发育，再加上其快速发育，将使我们能够全面确定众多动静脉限制性基因的大量顺式元件的活性。总之，这些研究将使我们能够绘制有助于动脉和静脉内皮分化和血管身份的转录调控输入。

DESCRIPTION (provided by applicant): Endothelial cell differentiation and establishment of blood vessel identity is essential for vascular function in both normal and disease settings. For example, in the developing embryo differentiation of arterial and venous endothelial cells is essential for proper vessel patterning and circulatory function. Similarly, perturbation of blood vessel identity in a number of congenital diseases can lead to vascular anomalies, such as arteriovenous malformations. Thus, understanding the molecular basis of normal endothelial differentiation would give us insights onto pathological blood vessel formation and facilitate the engineering of distinct blood vessel types. While transcriptional hierarchies controlling cellular differentiation have been extensively characterized in other tissues, much less is known about such programs in endothelial cells. The studies proposed in this application will begin to address the control of artery and vein differentiation at the transcriptional level in endothelial cells. In particular, we will identify cis regulatory elements in the human genome that are responsible for arterial and venous endothelial-specific gene expression. This will be accomplished through the genome-wide identification of binding sites for general transcriptional regulatory proteins known to reliably mark enhancer and repressor elements. Subsequent computational analyses will allow us to identify common cis regulatory sequences that correlate with artery or vein specific gene expression. These will shed possible insight onto upstream transcriptional regulators. To functionally validate the activity of putative artery and vein cis elements, we will perform in vivo reporter assays on human elements using the zebra fish as a model system. The transparency and external development of the zebra fish embryo, coupled with its rapid development will allow us to comprehensively determine the activity of a large number of cis elements for numerous arteries and vein restricted genes. Together, these studies will allow us to map the transcriptional regulatory inputs that contribute to arterial and venous endothelial differentiation and blood vessel identity. PUBLIC HEALTH RELEVANCE: Blood vessels have different functions and appearances (e.g. artery versus vein) depending on their anatomical location. These differences are apparent in early embryos and may play a role in disease settings. However, little is known about how these differences arise. In this proposal, we will focus on uncovering the signals that govern artery and vein identity.