Molecular basis of NFIB-Mediated Regulation of Oncogenic Transcription

The Nuclear Factor I (NFI) family of transcription factors orchestrates key regulatory programs in development, differentiation, and metabolism, with dysregulation implicated in various pathological conditions, including cancer. Among them, NFIB displays significant context-dependent roles, functioning either as an oncogene or a tumor suppressor depending on cancer type. Despite its emerging clinical relevance, the molecular basis of NFIB-mediated DNA recognition and transcriptional regulation in cancer remains poorly understood. Using HeLa cells as a model, we demonstrate that NFIB promotes oncogenic phenotypes, as CRISPR-Cas9-mediated knockout significantly impairs cell proliferation, migration, and invasion. Transcriptomic analysis reveals that NFIB regulates a cohort of cancer-associated genes, including FGFR3 and PDGFRB. To elucidate the underlying mechanism, we performed biochemical characterization and found that both full-length NFIB and its DNA-binding domain (DBD) exist as monomers in solution and bind DNA with 1:1 stoichiometry, challenging long-standing models proposing DBD-mediated dimerization. High-resolution crystal structures of NFIBDBD bound to ChIP-seq-derived DNA motifs reveal a monomeric binding mode driven by base-specific recognition of the TGGCA sequence. Mutational disruption of key DNA-contacting residues abolishes both DNA binding and transcriptional activation in luciferase reporter assays. Together, these findings define the molecular mechanism underlying NFIB-dependent gene regulation and establish a structural framework for its oncogenic activity, providing insights with potential therapeutic implications for targeting NFIB-driven cancers.