Molecular Basis Distinguishing the DNA Binding Profile of Nrf2-Maf Heterodimer from That of Maf Homodimer

Momoko Kimura,Tae Yamamoto,Jianyong Zhang,Ken Itoh,Motoki Kyo,Terue Kamiya,Hiroyuki Aburatani,Fumiki Katsuoka,Hirofumi Kurokawa,Toshiyuki Tanaka,Hozumi Motohashi,Masayuki Yamamoto

doi:10.1074/jbc.m706863200

Abstract

Nrf2-small Maf heterodimer activates the transcription of many cytoprotective genes through the antioxidant response element and serves as a key factor in xenobiotic and oxidative stress responses. Our surface plasmon resonance-microarray binding analysis revealed that both Nrf2-MafG heterodimer and MafG homodimer bind to the consensus Maf recognition element with high affinity but bind differentially to the suboptimal binding sequences degenerated from the consensus. We examined the molecular basis distinguishing the binding profile of Nrf2-MafG heterodimer from that of MafG homodimer and found that the Ala-502 residue in the basic region of Nrf2 is a critical determinant of its binding specificity. In Maf proteins, a tyrosine resides in the position corresponding to Ala-502 in Nrf2. We prepared a mutant Nrf2 molecule in which Ala-502 was replaced with tyrosine. In surface plasmon resonance-microarray analysis, heterodimer of Nrf2(A502Y) and MafG displayed a binding specificity similar to that of MafG homodimer. The target genes activated by mutant Nrf2(A502Y)-small Maf heterodimer were largely different, albeit with some overlap, from those activated by wild-type Nrf2-small Maf, indicating that the array of target genes regulated by Nrf2-small Maf heterodimer differs substantially from that regulated by Maf homodimer in vivo. These results suggest that the distinct DNA binding profile of Nrf2-Maf heterodimer is biologically significant for Nrf2 to function as a key regulator of cytoprotective genes. Our contention is supported that the differential DNA binding specificity between Maf homodimers and Nrf2-Maf heterodimers establishes the differential gene regulation by these dimer-forming transcription factors.

Highlights

Exploratory Research for Advanced Technology-Japan Science and Technology Corp. (ERATO-JST), Tohoku University Graduate School of Medicine, 2-1 Seiryo-cho, Aoba-ku, Sendai 980-8575, Japan
Replacing an Alanine Residue in the Basic Region of Nrf2 with the Corresponding Tyrosine Residue of MafG Converts the Specificity of Heterodimeric Binding to That of Homodimeric Binding—Our previous surface plasmon resonance (SPR)-microarray analysis demonstrated the differential DNA binding profiles of Nrf2-MafG heterodimer and MafG homodimer, which seemed to be attributed to the distinct DNA recognition modes of Nrf2 and MafG, mainly recognizing the core and the flanking regions of the MARE, respectively [14]
The induction levels of the genes belonging to this group were generally modest, probably because the Nrf2 activation domain might be disadvantageous in cooperating with transcriptional cofactors at heterologous loci different from the natural Nrf2 targets. These results clearly indicate that the profile of genes activated by Nrf2-small Maf heterodimer differs from that activated by Nrf2(A502Y)-small Maf heterodimer, suggesting that the difference in suboptimal binding to MARE-related sequences between Maf homodimer and Nrf2-small Maf heterodimer generates a significant difference in transcriptional gene regulation in vivo

Summary

EXPERIMENTAL PROCEDURES

Plasmid Construction—Previously constructed pET15bNrf2CT (Nrf fragment containing a CNC domain and bZip motif spanning from alanine 318 to the C-terminal end asparagine 597) was used to prepare the Nrf bZip domain for analyzing heterodimer binding [14]. pET15b-Nrf2CT(A502Y) was generated by replacing the alanine at position 502 with tyrosine. Protein Preparation and SPR Microarray Imaging Analysis— Fragments of MafG, Nrf, and Nrf2(A502Y) containing the bZip motifs MafG-(1–123), Nrf2CT, and Nrf2CT(A502Y), respectively, were expressed in bacteria and purified as described previously [14]. Nrf2CT (1 ␮M)/ MafG-(1–123) (100 nM) and Nrf2CT(A502Y) (1 ␮M)/MafG-(1– 123) (100 nM) were applied to the array surface in SPR buffer containing 20-mM HEPES (pH 7.9), 250-mM NaCl, 4 mM MgCl2, 10 mM EDTA, 0.005% Tween 20, and 1 mM phosphine. Immunoblot Analysis—Nuclear proteins were prepared as described before [22], and immunoblot analysis was performed using commercially available anti-Nrf (Santa Cruz, sc-722), newly generated anti-Nrf anti-lamin B (Santa Cruz, sc-6217), and anti-tubulin (Sigma, T9026) antibodies. Quantitative Real Time Reverse Transcription-PCR—cDNAs were synthesized from total RNAs prepared from 293/FLAGNrf and 293/FLAG-Nrf A502Y cells using random hexamers.

RESULTS

DISCUSSION

C Tissue factor pathway inhibitor