Novel regulator of Notch signaling in determination of left-right asymmetry durin

描述（由申请人提供）： 总结：尽管人体的外部两侧对称，但在内部器官的配置和放置中存在许多左右（LR）不对称。在胚胎发生过程中LR不对称性测定的失败导致内脏器官的位置倒置，称为异位。重要的是，超过80%的患有位置不明确的儿童（由LR不对称性确定的缺陷触发的解剖学紊乱）存在先天性心脏病的并发症，包括大动脉转位、右心室双出口、左心室双入口、房室间隔缺损和完全异常肺静脉连接。LR不对称性的确定已被证明是由各种分子和信号转导途径，包括Notch信号转导途径调节。该途径是一种进化上保守的途径，涉及发育和人类疾病的许多方面。虽然Notch信号传导介导局部细胞-细胞通讯并调节下游反应，包括细胞命运特化、祖细胞维持、边界形成、细胞增殖和凋亡，但Notch信号传导如何调控LR不对称性决定的分子机制尚不完全清楚。 最近，我们已经分离出B细胞白血病/淋巴瘤6（BCL 6），转录抑制因子，作为一种新的Notch相关因子结合蛋白质组学分析的免疫沉淀方法。我们的研究表明，Notch信号可以抑制左侧板中胚层上的左侧特异性基因Pitx 2的表达，BCL 6抑制其活性以维持LR不对称性。为此，BCL 6干扰Notch信号传导的转录复合物中Notch和共激活因子Mastermind-like 1之间的相互作用。然而，受BCL 6抑制的Notch下游基因尚未被鉴定，并且这些分子如何影响LR不对称性的确定仍然没有解决。在本研究中，我们将利用分子生物学和发育生物学的技术，包括整体原位杂交，染色质免疫沉淀和非洲爪蟾胚胎的功能获得和功能丧失的研究来解决这两个重要问题。 我们的研究将揭示Notch信号如何调节LR不对称性的新机制和见解，并加深我们对该途径的失调如何导致人类先天性疾病的理解。 公共卫生相关性： 说明：Notch信号通路已被证明在许多生物学过程中起关键作用，并且已在包括先天性疾病和癌症的人类疾病中鉴定出该信号通路的失调。在鉴定和表征调节这一途径的新因子方面的进展将导致鉴定许多负责人类疾病的基因。

DESCRIPTION (provided by applicant): SUMMARY: Despite the external bilateral symmetry of human body, there are many interior left-right (LR) asymmetries within the disposition and placement of internal organs. A failure of LR asymmetry determination during embryogenesis leads to inverted position of internal organs named heterotaxia. Importantly, over 80 % of children with situs ambiguous, an anatomical derangement triggered by the defect of LR asymmetry determination, present with complications of congenital heart disease including transposition of the great arteries, double outlet right ventricle, double inlet left ventricle, atrioventricular septal defects and total anomalous pulmonary venous connection. The determination of LR asymmetry has been shown to be regulated by various molecules and signal transduction pathways including the Notch signaling pathway. This pathway is an evolutionally conserved pathway that is involved in many aspects of development and human diseases. While Notch signaling mediates local cell-cell communication and regulates downstream responses including cell-fate specification, progenitor cell maintenance, boundary formation, cell proliferation and apoptosis, the molecular mechanisms of how Notch signaling governs LR asymmetry determination are not completely clear. Recently we have isolated the B-cell leukemia/lymphoma 6 (BCL6), a transcriptional repressor, as a novel Notch-associated factor by immunoprecipitation approach combined with proteomics analysis. Our studies show that Notch signaling can suppress the expression of Pitx2, a left-specific gene, on the left lateral plate mesoderm and BCL6 inhibits its activity to maintain LR asymmetry. For this, BCL6 interferes with the interaction between Notch and Mastermind-like1, a co-activator, in the transcriptional complex of Notch signaling. However, Notch-downstream genes suppressed by BCL6 have not been identified and how these molecules affect the determination of LR asymmetry still remains unsolved. In this proposal, we will address these two important questions by using techniques of molecular biology and developmental biology including whole mount in situ hybridization, chromatin immunoprecipitation and gain-of-function and loss-of-function studies in Xenopus embryos. Our studies will uncover new mechanisms and insights into how Notch signaling regulates LR asymmetry and deepen our understanding of how the deregulation of this pathway leads to human congenital diseases. PUBLIC HEALTH RELEVANCE: NARRATIVE: The Notch signaling pathway has been shown to play crucial roles in many biological processes and the deregulation of this signaling pathway has been identified in human diseases including congenital diseases and cancers. Progresses in the identification and the characterization of novel factors, which regulates this pathway, will lead to the identification of numerous genes that are responsible for human diseases.