Dynamic NFIX Promoter Usage Shapes Stem and Committed Myogenic States

Skeletal muscle development and regeneration rely on coordinated transcriptional programs controlled by muscle stem cells and their progeny. Nuclear Factor I X (NFIX) plays essential roles in fetal myogenesis and adult muscle regeneration. However, the mechanisms regulating its transcription and isoform expression remain unclear. Here, integrating multi-omics analyses with in vitro functional assays, we define the transcriptional and epigenetic landscape controlling Nfix expression during skeletal muscle development and regeneration. We show that Nfix promoter usage is dynamically regulated according to myogenic cell state: promoter 2 is associated with quiescent and stem-like states, whereas promoter 1 is activated during myogenic commitment and regeneration. These programs are accompanied by differential enhancer accessibility and DNA methylation changes within the Nfix locus, indicating coordinated epigenetic regulation. We further demonstrate that alternative promoter usage and exon 7/9 splicing generate distinct NFIX isoforms affecting myoblast proliferation and fusion, while transcriptomic profiling identified NFIX-dependent networks involved in muscle structure and metabolism. Overall, our findings uncover multilayered mechanisms controlling Nfix expression and identify promoter- and isoform-specific NFIX programs associated with stem, regenerative, and differentiated myogenic states. These results provide a molecular framework for future therapeutic strategies targeting NFIX in muscle diseases.