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

描述（由申请人提供）：基因组信息的表观遗传修饰是生物学中日益重要的领域。 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。 研究人员发现胞苷脱氨酶XAPOBEC-2（非洲爪蟾apoB编辑催化亚基2，XA 2）在非洲爪蟾胚胎中通过TGF-β信号调节因子的基因组筛选中。 在没有节点信号的情况下，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，非洲爪蟾中的XA 2和C2 C12成肌细胞中的小鼠同源物A2抑制神经细胞型TGF-β信号传导。 在非洲爪蟾中没有XA 2，左右轴不对称是异常的，并且C2 C12细胞中A2的缺乏降低了肌肉分化。 本申请的目的是鉴定与左右轴确定和肌肉分化相关的XA 2/A2的靶标。 利用非洲爪蟾和C2 C12细胞中可用的生物化学（大量胚胎材料）、基因组（基因阵列）和分子（吗啉代寡核苷酸，MO）工具，该目标将在两个目标中完成：1. A2对TGF-β信号传导的分子作用。 研究者将结合联合收割机现有的功能丧失和功能获得试剂，以确定XA 2和淋巴结通路之间的上位关系。 2. A2的目标。 他将结合联合收割机生物化学和基因组技术来检索和识别XA 2和A2靶标。 将测试候选基因与蛋白质的直接生化相互作用、其编辑活性诱导的突变以及对左右轴和肌肉分化的影响。 该项目将证实RNA/DNA编辑参与信号转导和两个主要发育过程的第一例，并将扩大我们对RNA/DNA编辑酶如何调节生物过程的理解。 人物叙述：胞苷脱氨酶是在人类病理学中日益重要的RNA和DNA修饰酶，从脂质代谢及其并发症（动脉粥样硬化）到抗逆转录病毒（HIV和相关病毒）防御和抗体产生缺陷。 研究人员发现，APOBEC 2是一种在脊椎动物中保守的胞苷脱氨酶，以前没有已知的功能，是正确定位不对称的内脏器官（左右不对称）所必需的，如心脏，肠道，肝脏和胰腺，这表明了一种新的分子机制。 此外，APOBEC 2刺激多能细胞系分化为肌纤维。 许多先天性心脏病是由人类左右方向的缺陷引起的，退行性肌肉疾病是人类病理学的重要组成部分。 因此，了解左右不对称和肌肉分化的潜在分子机制与人类病理学相关。 找到APOBEC 2靶向的基因将显着提高我们对人类病理学中越来越重要的蛋白质家族的认识。

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.