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

Abstract

Nuclear factor I (NFI) transcription factors play essential roles in multiple aspects of nervous system development, including radial glia maturation, neurogenesis, gliogenesis, and brain morphogenesis. Numerous NFI variants have been identified in individuals with neurodevelopmental disorders, yet the molecular basis of their pathogenicity remains unclear. The absence of resolved NFI-DNA complex structures continues to impede mechanistic insights and therapeutic exploration. Here, we define the oligomeric states of NFIA and NFIC, and determine the crystal structures of the NFIC homodimer, as well as the NFIA and NFIC monomers lacking their dimerization region, in complexes with double-stranded DNAs. Structural analysis reveals the molecular mechanism underlying NFI dimerization and recognition of a dyad-symmetric TGGCA(N3)TGCCA sequence motif, and demonstrates that dimerization enhances both DNA-binding affinity and specificity of NFI proteins. The functional importance of key NFI residues and DNA bases involved in the protein-DNA interaction is further validated by mutagenesis and binding assays. Additionally, we systematically evaluate the effects of the neurodevelopmental disorders-associated NFI mutations on DNA binding of NFIA, providing insights into their potential pathogenic mechanisms. Together, our findings elucidate the structural basis of NFI dimerization and dyad-symmetric DNA recognition and highlight pathogenic variants for further mechanistic studies in neurodevelopmental disorders.

1. Introduction

The human nuclear factor I (NFI) family comprises four members: NFIA, NFIB, NFIC, and NFIX. All proteins share a highly conserved N-terminal DNA-binding domain (DBD), whereas their C-terminal transcriptional regulatory domains (TRDs) exhibit much lower sequence conservation (Figure 1A,B, and Supplementary Figure S1). In addition, alternative mRNA splicing of some NFI members generates multiple isoforms [1].

NFI proteins orchestrate cell proliferation and differentiation across multiple organ systems, and are therefore indispensable for normal tissue development. NFIA and NFIX are particularly important for the development and functional maturation of the brain, skeletal muscle, and hematopoietic systems [2–6]. NFIB plays essential roles in the formation of the brain, lung, and mammary gland [3,7,8]. NFIC primarily regulates mammary gland differentiation and tooth development [8,9]. Beyond organ development, NFI proteins also exert key regulatory functions in stem cell biology, where they govern self-renewal and lineage commitment in pluripotent, hematopoietic, and neural stem cells [10,11].

NFIA, NFIB, and NFIX are broadly expressed in the developing and adult nervous system, with particularly high levels in the dorsal telencephalon, where they function as key transcriptional regulators that drive the maturation of radial glia and their progression into neuronal and astrocytic lineages [12]. Mechanistically, NFIA expression in neural progenitors is induced by Notch signaling, directly activating glial fibrillary acidic protein (GFAP) and S100 calcium-binding protein B (S100β) transcription to promote astrocyte differentiation [4]. Fibroblast growth factor 8 (FGF8)-MAPK signaling also induces NFIA and NFIB expression, promoting the maturation of midline zipper glia and facilitating midline remodeling and corpus callosum formation [13]. Additionally, NFIA cooperates with LIM homeobox 2 (LHX2) to regulate γ-aminobutyric acid (GABA) ty