Animal genomics – gene discovery and gene characterization

Sammanfattning: This thesis involves two projects. The aim in the first project was to identify genomic regions associated with spontaneous autoimmune thyroiditis (SAT), which is a hereditary autoimmune disease that affects the obese strain (OS) of chicken, an animal model for human Hashimoto’s thyroiditis (HT). In the second project, we study ZBED6, a highly conserved protein unique to placental mammals. Here we explore the functional significance of ZBED6 in general, and its effect on the regulation of Igf2 and miR483 in specific. To identify genomic regions predisposing to SAT, a nine-generation intercross between OS and their wild ancestor, the red junglefowl (RJF), was previously generated. In paper I, we developed a cell-based assay to phenotype the F9 chickens by measuring the TSH levels in their serum. We found that 1) SAT is similar to HT in the sense that the serum-TSH levels increase in affected individuals, and 2) that TSH levels in SAT-affected chickens starts to increase after 20 weeks of age. In paper II, a whole genome sequencing experiment was performed to compare a healthy and a severely SAT-affected groups of chicken. This analysis revealed 12 genomic loci to be significantly different between the two groups. In the second project, we utilized a mouse myoblast cell line, C2C12, to characterize the function of ZBED6. In paper III, we affect ZBED6 function, by either mutating its binding site in Igf2 (Igf2dGGCT), or by completely knocking it out (Zbed6-/-). Functional analysis of the mutant cells revealed that ZBED6 overexpression induces cell cycle arrest and apoptosis, that ZBED6 directly affects mitochondrial activity, and that ZBED6 in myoblast cells mainly exerts its effect through regulating Igf2. In paper IV, we use ZBED6 knock-out and knock-in mice to investigate the effect of ZBED6 on the regulation of miRNA expression. We found that ZBED6 is not a general regulator for miRNA, with the exception of miR483, which exists in an intron of Igf2. Thereafter, we generated miR483-/- cells, using the Igf2dGGCT cell line. In this analysis we found that the main function of miR483 in myoblast cells is to regulate the expression of Igf2, and that ZBED6 partially regulates Igf2 through regulating miR483.