Biochemical and structural studies of NFIA and NFIC reveal a conserved mechanism for specific DNA recognition and provide insight into potential pathogenicity of disease-associated mutations

• First crystal structures of NFI-DNA complexes reveal a conserved dimerization mechanism and dyad-symmetric TGGCA(N3)TGCCA recognition. • NFI dimerization enhances both DNA-binding affinity and specificity, critical for transcriptional regulation. • Systematic evaluation of neurodevelopmental disorder-associated NFI mutations identifies potential pathogenic variants affecting DNA binding. • Provides a structural framework for understanding NFI-related disorders and guiding therapeutic development.