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

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-[unreadable] 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-[unreadable] 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-[unreadable] 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.