Quantitative mapping of NRF-sMAF-ARE interactions reveals biphasic regulation of NRF2-dependent transcription by MAFG.

Abstract

Transcription factors regulate gene expression through coupled protein-protein and protein-DNA interactions, yet how these interactions collectively determine the formation of NRF-sMAF-ARE complexes that support transcription remains poorly understood. Here, we quantitatively resolved the nuclear factor erythroid 2-related factor (NRF)-small musculoaponeurotic fibrosarcoma (sMAF)-antioxidant response element (ARE) network using pair-resolved NanoBRET measurements of dimerization, protein-DNA association, and complex-formation kinetics. The detected dimer set consisted of NRF-sMAF heterodimers and sMAF-sMAF dimers, whereas NRF-NRF interactions remained at baseline across the tested concentration range. Within this detected dimer set, ARE recognition depended on the specific dimer-sequence combination, with core ARE substitutions altering apparent association, dissociation, and complex stability. Order-of-addition experiments further showed that NRF2-sMAF-ARE complexes could form from pre-existing sMAF-sMAF-ARE complexes, consistent with remodeling among competing DNA-bound complexes. An experimentally parameterized occupancy model predicted that NRF-sMAF-ARE occupancy is jointly controlled by NRF abundance, sMAF abundance, and ARE sequence and, specifically for NRF2 and MAFG, that increasing MAFG would first promote and then constrain NRF2-MAFG-ARE occupancy as the balance among competing MAFG-containing complexes shifted. Consistent with this prediction, cellular reporter measurements directly demonstrated a biphasic response to increasing MAFG expression inputs, with lower inputs enhancing and higher inputs reducing NRF2-dependent transcriptional activity. Together, these findings define a hierarchical mechanism of complex formation in which partner compatibility limits the dimer species available for ARE binding, DNA sequence and kinetics shape complex stability, dynamic remodeling connects competing DNA-bound complexes, and relative NRF-sMAF abundance determines NRF-sMAF-ARE occupancy, providing a general biochemical mechanism for coupled transcription-factor complex formation.

Key Findings

• NRF-sMAF heterodimers and sMAF-sMAF dimers form the primary dimer set involved in ARE binding, while NRF-NRF interactions remain minimal.
• ARE recognition and complex stability depend on specific dimer-ARE sequence combinations, with core ARE substitutions affecting binding kinetics.
• Increasing MAFG expression exhibits a biphasic effect on NRF2-dependent transcription, initially promoting and then inhibiting transcriptional activity through dynamic remodeling of NRF2-MAFG-ARE complexes.

Clinical Significance

Citation

Wei Yongyi, Alexander Lewis, Ye Haofenget al.. Quantitative mapping of NRF-sMAF-ARE interactions reveals biphasic regulation of NRF2-dependent transcription by MAFG. Redox biology. 2026-Nov-05.