Objective To investigate whether nuclear factor I-X (NFIX) possesses phase separation capability and to elucidate its mechanism in regulating biological functions related to craniofacial development through phase separation.

Methods An enhanced green fluorescent protein (EGFP)-tagged full-length NFIX recombinant plasmid was transfected into U2OS cells, and its subcellular localization was observed using confocal fluorescence microscopy. The protein structure of NFIX was analyzed using the Predictor of Natural Disordered Regions (PONDR), a protein analysis instrument, with a focus on identifying intrinsically disordered regions (IDRs) associated with weak interactions required for biomolecular condensate formation. A truncated NFIX-EGFP plasmid lacking the IDR was constructed, and changes in its intracellular localization were observed. Immunoprecipitation-mass spectrometry (IP-MS) was utilized to analyze differences in protein interactions with downstream factors between full-length and truncated NFIX.

Results In the nucleus, full-length EGFP-NFIX formed biomolecular condensates resembling membraneless organelles. PONDR analysis confirmed that NFIX contains an IDR (aa245-494) (IDR2), which is critical for biomolecular condensate formation. Truncation of IDR2 resulted in a diffuse protein distribution in the nucleus. IP-MS revealed that multiple proteins exhibited IDR2-dependent binding specificity and were significantly enriched in pathways related to craniofacial development, such as the MAPK pathway.

Conclusion NFIX regulates bone developmental homeostasis not only through canonical transcriptional mechanisms but also through IDR-mediated phase separation. This biomolecular condensate formation mechanism provides new insights into the pathogenesis of NFIX-related craniofacial dysmorphogenesis syndromes.