Mechanistic Insights Into NFIX‐Mediated DNA Recognition and Transcriptional Regulation in Skeletal Muscle

Skeletal muscle is essential for voluntary movement and exhibits a remarkable capacity for regeneration following injury. NFIX, a member of the Nuclear Factor I (NFI) family of transcription factors, plays a critical role in both skeletal muscle development and regeneration. Despite its emerging importance, the molecular basis of NFIX-mediated DNA recognition and transcriptional regulation in skeletal muscle remains poorly defined. Here, we demonstrate that NFIX promotes key cellular processes in skeletal muscle cells, as siRNA-mediated knockdown of NFIX significantly reduces cell proliferation, increases apoptosis, and impairs differentiation. Transcriptomic analysis revealed that NFIX regulates a network of genes involved in muscle metabolism, stress responses, and immune inflammatory responses. Biophysical characterization showed that NFIX exists as a monomer in solution and binds palindromic DNA with a 1:1 stoichiometry. A high-resolution crystal structure of the NFIX_DBD bound to palindromic DNA reveals a monomeric binding mode driven by base-specific recognition of the TGGCA motif. Mutations that disrupt key DNA-contacting residues abolished both DNA binding and transcriptional activation in luciferase reporter assays. Together, these findings define the molecular mechanism of NFIX-dependent gene regulation in skeletal muscle and establish a structural framework for its function, providing new insights into the potential therapeutic targeting of NFIX in muscle diseases.