Abstract
ABSTRACT
Transcription factors are critical regulators of homeostasis, and disruption of their function is a common underlying mechanism in cancer. The homeobox Myeloid ecotropic integration site-1 (Meis1) and its binding partner Homeobox A9 (HoxA9) are among the most highly dysregulated transcription factors in cancer, particularly in Acute Myeloid Leukemia (AML). Upregulation of HoxA9 is observed in more than 50% of AML cases and is a predictor of poor prognosis. Meis1 forms a cooperative DNA complex with HoxA9 and is required for HoxA9-mediated leukemogenesis. However, the mechanism by which HoxA9 and Meis1 interact and form a complex with DNA is unknown. To study the molecular mechanisms behind the HoxA9-Meis1-DNA complex and support future development of inhibitors, we describe optimized procedures for the recombinant expression and purification of full-length Meis1 and HoxA9. The constructs with an N-terminal His-tag showed degradation, but the addition of a small ubiquitin-like modifier (SUMO)-fusion tag improved solubility and stability for both proteins. Conditions were optimized for SUMO cleavage to obtain tag-free Meis1 and HoxA9 with high purity. We showed that both purified proteins bound to DNA with nanomolar affinities using a fluorescence polarization (FP) binding assay. The SUMO-fusion HoxA9 showed a distinct binding profile compared to the tag-free HoxA9 and formed a cooperative complex with Meis1. The tag-free HoxA9 appeared to oligomerize under the assay conditions. To the best of our knowledge, this is the first report to visualize the HoxA9-Meis1 cooperative interaction in a high-throughput assay. Our work establishes a foundation for future studies to decipher the protein-protein interaction of Meis1-HoxA9 and the development of inhibitors.