Targets of a cytidine deaminase required for left-right axis in Xenopus

描述（申请人提供）：基因组信息的表观遗传修饰是生物学中一个日益重要的领域。胞苷脱氨酶家族的RNA/DNA修饰酶与成人的重要过程有关，但与胚胎发育无关。研究人员在非洲爪蟾胚胎中发现了胞苷脱氨酶xapobec2（爪蟾apoB编辑催化亚基2，XA2）在由结型TGF-¿信号调节的因子的基因组筛选中。在缺乏节点信号的情况下，不对称的内部器官，如心脏和肠道，是随机定向的（随机的左右轴）。初步数据表明，爪蟾中的XA2和小鼠C2C12成肌细胞中的同源物A2抑制结型TGF-¿信号传导。爪蟾缺少XA2时，左右轴不对称异常，C2C12细胞缺少A2会降低肌肉分化。该应用程序的目的是确定与左右轴确定和肌肉分化相关的XA2/A2靶标。利用在爪蟾和C2C12细胞中可用的生化（大量胚胎物质）、基因组（基因阵列）和分子（morpholino oligonucleotides， MO）工具，目标将在两个方面完成：1。A2对TGF-¿信号传导的分子效应。研究者将结合现有的功能丧失和功能获得试剂来建立XA2与节点通路之间的上位性关系。2. 确定A2的目标。他将结合生物化学和基因组技术来检索和鉴定XA2和A2靶点。候选基因将被测试与蛋白质的直接生化相互作用，由其编辑活性诱导的突变，以及对左右轴和肌肉分化的影响。

DESCRIPTION (Provided by Applicant): Epigenetic modification of genomic information is an increasingly important field in biology. RNA/DNA modifying enzymes of the cytidine deaminase family have been connected to important processes in adults, but not to embryonic development. The investigator found the cytidine deaminase XAPOBEC-2 (Xenopus apoB editing catalytic subunit 2, XA2) in a genomic screen for factors regulated by nodal-type TGF-¿ signaling in Xenopus laevis embryos. In the absence of nodal signaling asymmetric internal organs, such as the heart and gut, are randomly oriented (randomized left-right axis). Preliminary data indicate that XA2 in Xenopus and the mouse homologue A2 in C2C12 myoblasts inhibit nodal-type TGF-¿ signaling. In the absence of XA2 in Xenopus, left-right axis asymmetry is abnormal, and A2 absence in C2C12 cells reduces muscle differentiation. The goal of this application is to identify targets of XA2/A2 relevant for left-right axis determination and muscle differentiation. Taking advantage of the biochemical (large amounts of embryonic material), genomic (gene arrays), and molecular (morpholino oligonucleotides, MO) tools available in Xenopus and C2C12 cells, the goal will be completed in two aims: 1. molecular effects of A2 on TGF-¿ signaling. The investigator will combine existing loss- and gain-of-function reagents to establish the epistatic relation between XA2 and the nodal pathway. 2. identify targets of A2. He will combine biochemical and genomic techniques to retrieve and identify XA2 and A2 targets. Candidate genes will be tested for direct biochemical interaction with the protein, mutations induced by its editing activity, and effect on the left-right axis and muscle differentiation. This project will substantiate the first case of RNA/DNA editing involvement in signal transduction and two major developmental processes, and will expand our understanding of how RNA/DNA editing enzymes regulate biological processes. PEOJECT NARRATIVE: Cytidine deaminases are RNA and DNA modifying enzymes of increasing importance in human pathology, from lipid metabolism and its complications (atherosclerosis), to antiretroviral (HIV and related viruses) defense, and defects in the generation of antibodies. The investigator found that APOBEC2, a cytidine deaminase conserved in vertebrates with no previously known function, is required for correct orientation of asymmetric internal organs (left-right asymmetry), such as the heart, gut, liver, and pancreas, suggesting a novel molecular mechanism for an essential developmental process. In addition, APOBEC2 stimulates the differentiation of a multipotent cell line into muscle fibers. Many congenital heart defects are caused by defects in left-right orientation in humans, and degenerative muscle diseases are an important part of human pathology. Understanding the underlying molecular mechanisms of left-right asymmetry and muscle differentiation is therefore relevant for human pathology. Finding the genes targeted by APOBEC2 will significantly improve our knowledge of a family of proteins increasingly important in human pathology.