The pioneer transcription factors Foxa1 and Foxa2 regulate alternative RNA splicing during positive selection in the thymus

Here we show that the pioneer transcription factors Foxa1 and Foxa2 are required to regulate RNA splicing during positive selection at the transition from CD4+CD8+ double positive (DP) to single positive (SP thymocyte). Conditional deletion of both Foxa1 and Foxa2 from DP thymocytes led to a partial arrest at the transition from DP to SP, with a reduction in the number of cells undergoing positive selection, and reduced the number and maturation of both CD4SP and CD8SP populations, with a reduction in peripheral naïve CD4+ T-cells. Foxa1 and Foxa2 were required for physiological levels of expression of several genes which encoding splicing factors (Mbnl1, Sf3b1, Hnrnpa1) in cells undergoing positive selection. Within the positively selecting CD69+DP cells, alternative RNA splicing was dysregulated leading to more than 859 significantly differentially expressed exons compared to control, with many genes important for T cell development and for RNA splicing showing differentially used exons. Overall design: Thymocyte suspensions were prepared from adult (6 week old) Foxa1fl/flFoxa2fl/fl.CD4Cre-tg+ (Foxa1/2coKO) and Foxa1fl/flFoxa2fl/fl.Cre- (control) thymus. Cells were stained with anti-CD69, anti-CD4 and anti-CD8 and facs sorted to purify CD69+CD4+CD8+ cells. RNA was extracted from these CD69+CD4+CD8+ cells.