Examining FOXO1 and DNA Interactions through Computational and Direct Methods

Abstract

FOXO transcription factors are a subgroup of the FOX (forkhead) family and are important for regulation of cellular processes, gene expression, apoptosis, cell‐ cycle progression, and oxidative stress resistance. Several FOXO translocations have also been implicated in cancer development, with the most common being the PAX3‐FOXO1 translocation linked to the development of Alveolar Rhabdomyosarcoma (ARMS). All FOXO proteins share sequence similarity and a highly conserved DNA‐ binding domain (DBD). This DNA‐binding domain consists of three alpha helices accompanied by two winged domains; this domain binds the Daf‐16 binding element (DBE) and the insulin response element (IRE), with the crystal structure indicating that the majority of the DNA contacts are located within Helix 3. There have been reports in the literature of sequences flanking the IRE causing an increase in binding affinity; however, no mechanism has been reported to explain this effect. Using Molecular Dynamics Simulation (MDS) we observed that the number of direct interactions between protein and DNA increases over time. Interestingly, MDS showed that wing 1 has no contact with DNA at 0 ns but eventually makes contact with a region flanking the IRE consensus sequence at 10 ns. In order to verify the new interactions observed in the 10 ns model, we mutated several bases within the IRE and observed the effect of these mutations on the interaction between protein and DNA through Electrophoretic Mobility Shift Assays (EMSA) and intrinsic fluorescence. Mutation of several bases in the IRE consensus sequence as well as in the immediate 5′ and 3′ flanking regions decreased the interaction between FOXO1 and DNA. In support of our model, mutation of the new base contact identified with Wing 1 also decreased FOXO1 DNA‐binding. The truncated FOXO1 DNA‐binding domain found in the PAX3‐FOXO1 fusion was also examined by MDS, and was predicted to retain several contacts within Helix 3 as well as the newly identified contact in Wing 1. Using this information, we can further examine the DNA‐binding of PAX3‐FOXO1 to determine if the presence of flanking sequences surrounding the IRE consensus sequence also affect the affinity of the fusion protein for DNA. Knowledge of the contribution of the FOXO1 domain to PAX3‐FOXO1 DNA targeting is important for understanding the activity of this fusion protein and its role in ARMS development.Support or Funding InformationNIH SC2GM095430RCMI‐Molecular Structure and Modeling core facility (NIH‐RCMI 2G12MD007595‐06 )NIH 5RL5GM118966‐03RISE (R25GM060926)