Abstract

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. An atomic view of the NFIX–DNA complex reveals recognition of the TGGCA motif and links sequence‐specific binding to the transcriptional programs governing skeletal muscle development and disease.