Interactions of FOXO1 Truncations with DNA

Alveolar Rhabdomysarcoma (ARMS) is a type soft tissue cancer that is mostly found in children and adolescents. It attacks the limbs, abdomen, and reproductive organs. Individuals with ARMS can have fusions between PAX3 and FOXO1 or PAX7 and FOXO1. PAX3‐ FOXO1 accounts for eighty percent of those fusions. We want to know what mechanisms are involved in the formation and advancement of ARMS disease. We know that the amino acids necessary for the FOXO1 protein to bind to DNA are also found in PAX3‐ FOXO1.This information can be useful in determining the contribution made by FOXO1in the activation of PAX3‐FOXO1 transcriptional targets. We identified six amino acids that are important for DNA binding by using a model designed to show interactions of the full length and truncated FOXO1 protein with DNA. We mutated several of these amino acids in the truncated FOXO1 DNA‐binding domain to determine if the model was correct. We expressed and purified these mutant proteins along with the wild type FOXO1 DNA‐binding domain in E. Coli. We used intrinsic fluorescence to evaluate DNA binding by these proteins. From literature, we know that the wild type protein has two DNA recognition sequences; the DBE and IRE. We have also determined that, all versions of the truncated proteins bind to more tightly to the IRE than the DBE is present. We are currently testing the effect of the mutations on the interaction with the IRE and DBE.