DNA‐binding studies of FOXO1 through site‐directed mutagenesis

Abstract

Alveolar Rhabdomyosarcoma (ARMS) is a malignant, skeletal muscle tumor that occurs primarily in older children and young adults. ARMS is frequently caused by the transcription factor, Pax3‐FOXO1, which retains the full Pax3 DNA‐binding domain and a truncated FOXO1 DNA‐binding domain. While the Pax3 domain has been thought to direct DNA binding, the ability of the truncated FOXO1 domain to bind DNA has never been directly tested. The overall goal of this project is to characterize the activity of the truncated FOXO1 DNA‐binding domain to help explain how transcription factors containing FOXO1 translocations target genes. Molecular dynamics simulations predict that the truncated FOXO1 DNA‐binding domain is predicted to have a folding pattern similar to the full length domain, is stable, and maintains similar DNA contacts. In the full length DNA‐binding domain, Arg225 and Ser235 are known to be important for maintaining DNA contacts, and these residues are also predicted to be necessary for DNA contact in the truncated system. Our previous studies have shown that Pax3‐FOXO1 can bind both Pax3 and FOXO1 recognition sequences in vitro. Here, we present our studies on the different affinity of Pax3‐FOXO1 for Pax3 and FOXO1 recognition sequences as well as the effect of Arg225 and Ser235 site‐directed mutagenesis on the affinity for these sequences.